Southern California Joint Pole Committee Routine Handbook Of Chemistry

• Southern California Joint Pole Committee Routine Handbook Of Chemistry And Technology

Southern California Joint Pole Committee Routine Handbook 44; Directive 90 496 Cee Pdf Download; 4K Video Downloader Mac Serial Junkie; Cornell Note Taking Method Custom Pdf Generator Api; Appunti Chimica Organica Pdf. 1889: Visionary writer Jules Verne fancifully writes about engineering Earth's climate in “The Purchase Of The North Pole”. 1954: The Weather Bureau, Navy, Air Force, MIT's Institute for Advanced Study, and the University of Chicago form a Joint Numerical Weather Prediction Unit using an IBM 701 computer.

Poster Sessions

P1 A Novel Vascular Button Connection Using Combined Technologies while Allowing for Social Enrichment by Pair Housing

AJ Hehman^*1, A Zuvich¹, KA Adams², D Shuey¹

¹Toxicology, Incyte Corporation, Wilmington, DE; ²Laboratory Animal Resources, Incyte Corporation, Wilmington, DE

Traditional practice has been to single-house vessel-cannulated rodents post surgically to protect exposed exteriorized catheters. A port/protective cap model is available that allows for pair and group housing of cannulated animals and is compatible with a harness/swivel automated blood sampling system. Our goal was to develop a vessel-cannulated surgical model that could be socially housed and would be compatible with response movement caging. To do this, we modified the automated blood sample connection to be compatible with the available port/protective cap system. A sterile custom connector was designed in collaboration with an industry vendor. Animals were dual jugular cannulated using the available port/protective metal cap to allow for pair or group housing. The newly designed custom connector was primed and attached to the port after removal of the protective metal cap. The modified connector allows the standard extension line from the response movement caging system to easily connect for blood sample withdraws. Studies were conducted using our standard inhouse study design for pharmacokinetic studies with multilple blood sample collections out to 24 hours post dose. All dosing and blood sample collections were performed in accordance with an IACUC-approved animal use protocol. The end result is a connector that allows for dosing and timed collections of high-quality blood samples using a response movement caging automated blood sampling system. The modified connector further allows for enhancement to the welfare of the animals by providing social enrichment through pair and group housing without compromising the integrity of the surgical model.

P2 A Unique Blood Collection Method in a Conscious Rat

AM Walters^*1, M Hirashima¹, K Tsuyama¹, S Wakamatsu²

¹SNBL USA Ltd, Everett, WA; ²Shin Nippon Biomedical Laboratories, LTD (SNBL) Japan, Kagoshima , Japan

Pharmakokinetics and toxicokinetics are important components of drug discovery and development process. The removal of blood at multiple time points from an animal is required for these evaluations. Blood sampling can be stressful for animals because of the handling, restraint, anaesthesia, or discomfort associated with a particular technique. The jugular vein is one of a few collection sites which can be used for multiple blood collections in a conscious rat. Blood parameters, supplies, and number of personal required, along with stress of the animal were evaluated using 2 different jugular vein puncture methods. The first method requires the use of a jugular board for the blood draw and requires minimally 2 people (bleeder and holder). The rat is restrained in an unnatural position, which can cause stress. The ties used to hold the forepaws can cause damage to the limbs and nerves in this area, causing lameness. Tilting the head of the rats in the head cap can cause haemorrhage to the ears. The rat can also lose consciousness. This method is commonly used for serial blood collections in rats. The second method evaluated is a unique blood collection method called the “one man.” The rat is manually restrained and bled by one person. In preparation for this technique, rats are handled daily. The increase in handling reduces the amount of stress the animal will have while being restrained. To restrain the animal is held in a vertical position and is grasped under each axilla, using the thumb and middle finger. The pad of the forefinger is used to pull the head back. To aid in visualizing the jugular vein, the thoracic portion of the rat’s chest is shaved and wiped with an alcohol. Once the vein is located, a 25g needle attached to a 1 or 3mL syringe is inserted into the vessel and a blood sample obtained. After the appropriate amount of blood is collected, the needle is removed and digital pressure is applied. A sample size of up to 2mLs can be obtained. The restraint of the animal (tightness of the skin and alignment of the neck) is critical when learning this technique; however, it can be learned easily and because this technique does not require any specialized equipment it can easily be implemented. Hematomas have been seen on occasion with this method. One other risk of this procedure is death. This is rarely seen but can occur if the vessel is not occluded properly. While both methods produce the same quality of sample; the refinement made to the commonly used jugular vein blood collection method reduces cost in supplies, personnel needed, and the amount of stress on the animal.

P3 Table Restraint Method for Working with Awake Nonhuman Primates

AM Storm^*, A Vinar, H Eric

NIH Division of Veterinary Resources, SoBran, Inc, Bethesda, MD

The need to use physical restraint for hands-on procedures involving nonhuman primates (NHPs) is common throughout the laboratory animal community. Historically, our organization has used chemical restraint for the vast majority of these procedures. After experiencing issues related to drug tolerance, anesthesia-related physiologic complications, and the initiation of protocols requiring multiple procedures per day or multiple procedures over a period of successive days, our organization began a search for a new method of restraint which could increase efficiency, reduce or eliminate the need for anesthesia, and improve overall animal welfare. There is an abundance of information related to the various restraint devices and protocols in the literature. Some of the more common methods include chair restraint with pole and collar and the use of squeeze cages. Our organization adopted the use of a lesser known restraint device, the primate restraint table, in combination with cooperative behavioral training for a variety of hands-on procedures including blood collection and veterinary evaluations. Animals were trained to use this device by applying positive reinforcement and desensitization techniques allowing the NHPs to participate in research procedures while fully awake. This was accomplished by acclimating the NHP to the table, then progressing through a series of steps to shape the desired behavior of cooperative restraint and limb extension. Training success was measured by the number of fully acclimated animals to the restraint table prior to the initiation of medical and research procedures. The implementation of this technique has reduced the labor involved with these procedures by eliminating the time needed for anesthesia induction, monitoring and recovery, and the complications associated with the use of anesthesia. We recommend this restraint device for laboratories interested in alternative restraint methods in their research and medical procedures.

P4 Strategies to Increase Time Usage and Accessibility of Environmental Enrichment Provided to Socially Housed Primates to Improve Animal Welfare and Wellbeing

CA Carrier^*

Covance Research Products, Alice, TX

Group housing is the standard method for housing nonhuman primates at our facility. Delivering enrichment to all social group members can be difficult due to the social hierarchies. Often the dominant animal will guard and chase off other group members when enrichment is thrown in. While training can be done to combat some of this dominant behavior, we looked at other changes that could be made to improve our environmental enrichment delivery methods to large groups of primates. Simple refinements in our methods were tried to improve animals’ access to offered enrichment. The novel strategies discussed here are piñatas, game feeders on timers, and larger frozen forage blocks. Experience with primates reveals ingestible items encourage foraging and destructible items prove most interesting. As a result, selected new strategies targeted these “likes.” The most successful enrichment strategies provided the group a longer time of use, gave them something to do, and provided foraging opportunities. The longer it took to empty the item, the sooner the dominant animal moved on. This allowed other animals in the group a chance to forage. The new methods also provided more accessibility to group-housed primates especially the lower ranking members who typically only had access to leftovers. These simple and practical to employ refinements, increased duration of use, and allowed for more animals to access the environmental enrichment being offered in the group setting. As such, these methods should increase the welfare and wellbeing for low ranking animals in a group setting.

P5 Use of Hydrophobic Sand as a Refinement for Urine Collection in Mice and Rats

CA Pinkus^*1, A Pereira¹, A Nguyen², MC Debrue¹, L Garcia Menendez¹

¹Compartive Medicine, Pfizer Inc, Cambridge, MA; ²CVMET, Pfizer Inc, Cambridge, MA

Metabolic cages used to collect urine are a barren, nonenriched environment known to increase stress in mice and rats. This study looked at using hydrophobic sand in a regular disposable cage as an alternative way to collect urine from mice and rats. Water bottles were a concern due to potential leakage, so water bottles and hydrating gel were also compared in both settings. Thirty-two male C57BL/6NCrl mice and 32 Sprague–Dawley rats were chosen for the study. Each species was divided into 4 groups of 8, comparing hydrophobic sand versus metabolic cages, and water bottles versus hydrating gel in each setting. Urine was collected and volume recorded at 3, 6, and 24 hours for mice and 2, 4, and 6 hours for rats. In mice, after the 6-hour time point there was no significant difference in urine volumes collected from hydrophobic sand and metabolic cages. However, at the 24 hour-time point the volume of urine collected from the sand was significantly lower than the volume collected from the metabolic cages. In rats, significantly lower volumes of urine were collected in hydrophobic sand than in metabolic cages at all time points. A significant decrease in urine volume was found in the 2 rat groups that received hydrating gel compared to the groups that had water bottles. In mice, the provision of gel versus water bottle did not impact the urine volume. Using hydrophobic sand with mice was not as effective for maximum volume collection at the 24 hour-time point but it might be an option when shorter collection time points are required and maximum urine volume is not required. Hydrophobic sand was also not as effective for maximum volume of urine collection in rats compared to metabolic cages. Providing hydrating gel impacted rat urine volume but was found to be unnecessary since water from water bottles was not observed to be mixing with the urine. Further studies with modification to cage set up might provide higher yields of urine collection in hydrophobic sand in rats and mice.

P6 Optimization of in Vivo Blood Sampling for Pharmacokinetic Studies

ST Ulufatu, CK McCaughey*

Safety Assessment, Genentech, San Francisco, CA

Currently blood collection for preclinical mouse pharmacokinetic studies has been refined from rotated retro-orbital (RO) eye bleeds to a microsampling technique via the “whole blood” or “tail prick” method. This technique in conjunction with an automated, miniaturized immunoassay platform is now primarily employed such that we may obtain a full serum-concentration time profile from each individual mouse. By using this combination, we were able to produce a more robust data set than using the RO method and ELISA. The tail prick method involves warming the tail of a mouse and then using a 27g needle which is injected into the tail vein and quickly taken out. Approximately 20ul of whole blood is collected from the tail vein via a hematocrit glass tube and spun down after 30 minutes of clotting. The serum is collected by cutting the glass hematocrit and pipetting at least 10ul of serum. The serum is then processed with an automated, miniaturized immunoassay. Previous methods used staggered RO blood collection which combines data across animals to produce a full pharmacokinetic profile. The limiting factor with RO bleeding is the amount (∼150ul) and frequency blood is able to be collected. The microsampling technique offers us the opportunity to take a smaller amount of blood (∼20ul) at every time-point from the same mouse. In the large molecule space, the need began to arise for researching data to make direct comparisons between molecules and mouse strains, based on the need for a more predictive preclinical pharmacokinetic screening tool. Limited data existed from molecules being compared across the most common murine strains C57BL/6N, BALB/c, NU/NU Nude, CD1(ICR), and C.B. 17 SCID mice, and it was hypothesized that strain differences combined with limitations of a staggered PK approach confounded our historical data. By using an automated, miniaturized immunoassay in conjunction with microsampling for our in vivo work, we are able to drastically reduce the number of animals used on study which speaks directly to the 3Rs, and the added benefit of providing improved quality of our data by producing a more robust data set all while helping us to better understand intrastrain variability.

P7 Training Rhesus Monkeys to Walk on a Treadmill

DH Sedlak^*1,2, A Evans^1,2, M Szarowicz^1,2, E Tilmont¹, K Vaughan^1,2, J Mattison¹

¹NIA, NIH, Dickerson, MD; ²SoBran, Bethesda, MD

There has been recent scientific interest in the positive influence of exercise on health and wellbeing, including its effect on various disease states. As such, our lab has recently begun a collaborative project aimed at characterizing the progression of peripheral artery disease (PAD) and evaluating exercise as a potential intervention. This involved developing a monkey model to measure lower limb ischemia, while also implementing a novel exercise program for adult rhesus monkeys. After a thorough review of the literature and consultation with another lab, we elected to employ a treadmill to achieve exercise. Having worked with monkeys for many years, this seemed a daunting task. However, with some suggestions from the behavior staff in our facility we were able to successfully train adult rhesus monkeys to walk at varying speeds for 30 minutes per day. This was a slow process beginning with the animal sitting on the treadmill completely stopped for multiple days for short periods of time getting many treats and novel rewards for positive reinforcement. We currently have 2 study groups of stable treadmill walkers that are helping us proceed with our PAD study. Here, the pertinent steps and considerations necessary for developing a successful nonhuman primate exercise model are presented.

P8 Outreach Outside of the Box

DJ Disselhorst^*1, P Crowder²

¹Texas A&M, Ft Stockton, TX; ²Iraan High School, Iraan, TX

Typical biomedical research outreach programs involve research personnel going out to schools and giving short, impromptu presentations to science classes on what biomedical research is. Science teachers have a general understanding of the necessity of animals in research and are commonly receptive to these sessions. Although useful in getting the message out, what if the biomedical research community were to actively pursue alternative methods for getting our message out? Animal rights groups start programs in the education system through a formalized PETA cirriculum, which provides online lesson plans and the National Compassionate Classroom Report Card. Animal rights groups also target 4-H shows and high school agriculture programs such as FFA. High School FFA and 4-H youth programs are excellent starting points for biomedical research to spread the message of applicable and responsible research. Commonly thought of as livestock programs, genetics, breeding records, husbandry, and basic research concepts can be presented by incorporating biomedical research concepts into existing programs. Adjunct faculty with a background in biomedical research can be enlisted to incorporate these same concepts through junior and senior high school agriculture and 4-H programs. By requiring college-focused students to complete a research project before graduation, a sense of accomplishment is achieved, while showing how research is applicable in their projects. As most of the modern day medical discoveries are extrapolated to veterinary science, this concept can enrich an agriculture/4-H program. By displaying the posters in the school or 4-H meeting rooms, other nonscience-specific disciplines can be exposed to the responsible nature of biomedical research. We explore this approach in a FFA program and in a 4-H program.

P9 Minimally Invasive Tachypacing-Induced Heart Failure Model in Beagle

DK Hirenallur Shanthappa^*, W Sutherland, I Brun del Re, SM Davis, S Barry, M Escamilla, J Viray Jr., H Vargas, J Eswaraka

Amgen, Thousand Oaks, CA

The canine tachypacing-induced heart failure (TPHF) model has been widely used to study heart failure (HF) mechanisms and the discovery of new therapeutics for HF in drug development. The most common model of TPHF involves invasive surgical instrumentation of large deep-chested dogs (mongrel) to enable placement of a pacemaker, electrodes, and pressure-sensing catheters. This model is valuable for the acquisition of a variety of cardiovascular parameters, but significant disadvantages include prolonged postoperative recovery time, low study throughput, hardware-associated complications, extensive training of canines, and intensive veterinary care. Additionally, surgical manipulation and instrumentation creates inflammation in and around the heart that could affect cardiovascular function, ultimately resulting in low-quality data. To overcome some of these challenges, we evaluated a minimally invasive transvenous right ventricular pacemaker in Beagles. In this model, the pacemaker was inserted via a right jugular vein cut-down and secured into the right ventricular endocardial wall. Following the postoperative period, HF was induced in 16 dogs by tachypacing the animals at 240 bpm for 2-4 weeks. HF progression was monitored using echocardiography. All dogs recovered from surgery with minimal complications by day 5. At day 28, dogs had Stage C (AHA/ACC classification) heart failure as evidenced by decreased ejection fraction (< 40%), fractional shortening (dP/dt_max), increased left ventricular end diastolic volume, and increased left ventricular end diastolic pressure compared to normal dogs. Overall, findings suggest that the minimally invasive tachypacing-induced heart failure model in beagles is a refinement to the more invasive mongrel model of TPHF. This model is robust and reproducible for investigating pathogenesis of HF and screening novel therapeutics.

P10 Refining Urine and Fecal Collection Methods Using Nonstressful Acupressure during Normal Routine Handling

EP Chua^*, R Garcia-Gonzalez, J Yamada, R Scott, C Sohn

Laboratory Animal Resources, Genentech, Inc., South San Francisco, CA

Collecting urine and fecal samples from laboratory mice is often performed in support of various metabolic, biochemical, nutritional, behavioral, and physiologic studies, as well as routine diagnostic testing for animal health status. Although multiple collection techniques are available, these methods may be stressful to the animal (manual restraint), require additional sample handling (collection of urine/feces on plastic or metabolism cage), and may yield samples that are heavily contaminated. Our method was developed as an alternative nonstressful collection method of urine and/or fecal material. Use of this method has many benefits including minimal animal handling, eliminating the need of a metabolism cage, speed of sample collection, and ability to collect samples at specific time points. Using this method, the mouse remains in a lordosis-like posture. In this posture, the pelvis rises up with front of animal angling downward. Based on our experience, within a short duration of time of assuming this posture, the animal will defecate for a fecal sample. For urine collection, the bladder can be expressed by gently massaging the area over the bladder while keeping the mouse relaxed in the lordosis-like posture.

P11 A Novel Way to Reduce the Number of Swine Required to Support Trauma and Resuscitation-Focused Research

EL Dean^*, T Brown, P Barnicott, L Harris

59th Clinical Research Division, San Antonio, TX

Military surgeons and researchers seek novel approaches to mitigate morbidity and mortality of battle-field injuries and hemorrhage. We are often asked to supply large quantities of swine fresh whole blood (FWH) or blood components for transfusions to support this research. To meet the growing demand for FWH and blood components but reduce overall animal usage we developed the Nonhuman Research Subject Blood Donor Initiative (NHRSBDI). Until this development, the method for blood collection involved exsanguination of donor animals under general anesthesia with an average collection of 6 units of blood per animal. Of these 6 units only the first 3 bags are optimal for transfusion due to unpublished findings. This resulted in a high cost-benefit ratio and we saw a need to refine the blood collection process and reduce the numbers of animals needed to support trauma research. We began housing a pool of blood donor swine (>70 kg) that have blood (3 bags of 450 ml/unit) collected every 2-4 weeks depending on health status and blood parameters (suggested values: WBC 9-23k, Hg ≥ 9 g/dL, HCT ≥ 30%, pO₂ ≥ 100 mmHg, Lactate ≤ 2.5 mmol/L). Animals are under general anesthesia and on volume control ventilation during the collection process with vitals (HR, SpO₂, RR, TV, PIP, PEEP, etCO₂, %Iso, Temp., %O₂) collected every 15 minutes. Volume replacement with warmed normal saline occurs at the completion of the procedure. Once the animals recover they are housed in facility for 24-48 hours and if no complications are noted they are moved to a paddock where they are monitored daily until it is time for another donation. Animals remain within the pool until it is deemed that further blood draws could compromise animal health. We successfully removed approximately 20-30% of a swine’s circulating blood volume for every 2-4 weeks. This amount nearly doubles the single blood draw amount currently reported in the literature. The implementation of the NHRSBDI resulted in a significant reduction in the number of animals required to support trauma related protocols. Implementing this initiative on a single trauma focused research study resulted in saving approximately 420 animals. If this initiative was instituted across other facilities it could reduce the number of swine required to support trauma and resuscitation focused research, resulting in a more viable research process and enhancing accessibility.

P12 Handling Ease and Behavior Enhancement in Sinclair Miniature Swine

D Brocksmith, G Rivard^*, I Stewart, GF Bouchard, A Stricker-Krongrad

Sinclair BioResources, LLC, Auxvasse, MO

We have developed a unique behavior enhancement system program for miniature swine (MS). One of the program’s components consists of behavioral training to condition a MS to cooperate rather than experience anxiety and fear over a procedure. Here we seek to inform you about the common threats that elicit resistance-to-handling. Also, we propose positive reinforcement training (+R) to improve welfare and handling-ease. Avoid any punishments, reprimands, or corrections; and perform a routine in a highly enthusiastic and playful manner so that, when repeated multiple times, it becomes highly predictable, safe, and pleasurable for the MS. By repetition, the MS will learn the formula: your request plus its response equals its reward. If not or if the MS becomes fearful or resists to handling, then ignore the bad behavior and go back to basics. Ignoring means no looking (including direct, prolonged, or challenging eyes contact); no touching (including hitting and body pushing or blocking); no talking (including yelling, saying no or what not to do); and no moving (simply stand still and with your hands on your side). Once calm and looking for your attention, redirect the MS by requesting a previously learned basic behavior such as a target touch. Then, start the training process from a previous step when he was calm so that MS remains calm. If not, give the MS a time-out period. Before introducing a new behavior, make sure to review and reinforce all the basics and the prior lessons before starting anew. It will refresh the MS memory and give you both confidence. Teach new rewarding behaviors incrementally. All along, be careful as mixed signals, lack of reinforcement, and going too fast are the culprits of good training.

P13 Cesarean Rederivation Performed in house by Animal Care Technicians to Eradicate Opportunistic Pathogens

S Graffam, J Lorabello, JR Stathopoulos^*

Comparative Medicine Facility, Broad Institute of MIT and Harvard, Cambridge, MA

Upon opening, a new comparative medicine facility experienced a high volume of mouse import requests from affiliated institutions. A number of these institutions had less restrictive bioexclusion criteria than the standards chosen by our investigators. We sought to maintain a strict biohealth status while being able to import valuable lines of mice from other institutions. Some opportunistic pathogens that are excluded (Staph. aureus, Klebsiella spp., Helicobacter) were diagnosed positive on incoming health reports. Cesarean rederivation was chosen as the method to solve the problem. Animal care technicians were trained to perform the procedures in house. Three didactic training sessions were held with the team to develop an understanding of the technical procedures, estrous cycles, and embryo development timelines, as well as materials required. The team researched current literature and methods to develop an inhouse standard operating procedure. An experienced technician performed initial procedures. With continuing guidance, technicians quickly demonstrated proficiency in the techniques. To perform the rederivations, a colony of CD-1 outbred stocks, free of select infectious agents and parasites, were bred to use as recipient foster dams. Sequentially, donor dams were bred to give birth at e19 (CRISPR lines) and e20 (traditional genetically modified lines). Donor dams were euthanized and pups were extracted under sterile conditions. Extracted pups were warmed and then passed through an airlock to the technician to perform the fostering procedure. At wean age p21 health status is confirmed via a modified PCR rodent infectious agent panel. Upon receipt of negative results, genotyping and identification methods were performed and the progeny were assigned to a breeding colony room. To date, this method has produced a 100% success rate with 18 lines rederived. By performing sterile cesarean rederivations animal care technicians are efficiently able to eliminate known pathogens from imported colonies and maintain adherence to the bio-exclusion standards while facilitating research Importantly, this was a challenging and rewarding development opportunity for technicians to expand skillsets, while eliminating outside contract services and saving investigators valuable time.

P14 Use of a Remote Video Monitoring System as a Refinement in a Nonhuman Primate Colony Behavioral Health Program

J Santiago^*, MD Simkins

Comparative Medicine, Pfizer, Perth Amboy, NJ

Nonhuman primate (NHP) colony maintenance requires ongoing consideration of a number of factors, including the macro and micro environments, enrichment, diet, social housing, behavior, and individualized medical needs of the animals. Decisions and changes are being made on a constant basis in the best interest of both the animal and the care staff working closely with the animals. Often times when people are present in an NHP room, the animals are focused only on that person. This makes it challenging to accurately observe important interactions related to social housing, partners or pairs, or normal behavioral habits specific to each NHP. Assessing an animals’ interest in enrichment devices, hierarchical interactions within a room, and many other important behaviors that play a major role in the wellbeing of an NHP can also be obscured by the presence of staff. The key is to be able to observe the NHP with the least amount of distractions possible to encourage whatever normal or abnormal behaviors you are interested in observing. Cameras and a remote monitoring system have allowed us to keep an eye on our NHPs without added distractions and/or variables in the room. Over the past 3 years, our remote monitoring system has been used mostly for observing suspected or reported abnormal behaviors in order to gain a better understanding of how often the abnormal behavior is displayed, and whether or not the frequency is dependent on the condition of the room. Recently, we started using the remote monitoring system more often to observe animals with alopecia, animals displaying poor social and interaction skills, the progression of animals in our habituation training program, and to determine whether or not an animal is benefitting from a particular enrichment device. It has even helped us to determine the best time to successfully pair 2 NHPs s based on their cageside interactions with one another and how these interactions evolved over time. The availability of remote monitoring provides us the opportunity to observe our NHPs closely and accommodate their needs without disrupting their environment.

P15 Lymph Node Fine Needle Aspiration as a Refinement for Longitudinal Assessment of Lymphoid Tissue in Rhesus Macaques (Macaca mulatta)

JS Wood^*1, D Carnathan^2,3, A Gsrcia⁴, S Gumber⁴, C Havenar-Daughton^5,3, S Crotty^5,3, G Silvestri^2,3

¹Animal Resources, Yerkes National Primate Research Center, Atlanta, GA; ²Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA; ³Center for HIV/AIDS Vaccine Immunology & Immunogen Discovery, The Scripps Research Institute, La Jolla, CA; ⁴Department of Pathology, Yerkes National Primate Research Center, Atlanta, GA;⁵Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA

Nonhuman primates are commonly used in immunology studies. One aspect of these studies includes evaluating changes within lymphoid tissue over the course of an immunization series. There are challenges to longitudinal assessment of the immune response in rhesus macaques, including, but not limited to practical and ethical considerations restricting the number of excisional lymph node biopsies that can be collected from an individual, the impact on the immune response when draining lymph nodes are removed, and the difficulty in evaluating specific lymphoid cell subsets, particularly germinal center (GC) B cells and CD4⁺ T follicular helper cells (T_FH). Fine needle aspiration (FNA) is a commonly used diagnostic tool in clinical practice. This technique allows sampling of the peripheral lymph nodes in situ using a needle and syringe. A pilot study evaluating the utility of FNA in studying GC activity in lymph nodes was performed. Interpretable data was obtained in 76% of the FNA samples. There was close correlation of GC-B cells and T_FH frequencies observed between FNA samples and lymph node biopsies. Based on these results, lymph node FNA has been incorporated into multiple HIV vaccine studies with much success and thusly provides an alternative means of evaluating specific lymphoid cell subsets over time. FNAs were performed under injectable anesthesia and all animals received pain medication to prevent any discomfort. Since September 2014, 393 FNAs have been performed on 62 rhesus macaques. Over 97% of the samples contained usable GC-B cell and T_FH populations for flow cytometry and gene expression. The average number of cells collected was 2.5 x 10⁶ cells per FNA sample, with a range of 2x10⁴ – 4.9x10⁶. The length of time between FNA collections has varied, with a minimum of 6 days between collections. The only complication from this procedure has been minor bleeding and bruising at the collection site. FNA presents a novel way of evaluating GC changes in lymph nodes. This method allows longitudinal study of the peripheral lymph node response in a single animal, which can decrease the need for, and frequency of, excisional lymph node biopsies and lead to a reduction in the total number of animals used on certain immunology studies.

P16 Evaluation of the Effect on Heart Rate when Providing Supplemental Treats after Oral Dose Administration in the Minipig

JE Sentz^*

Surgical Services / Infusion Toxicology, Envigo, East Millstone, NJ

A critical aspect of cardiovascular (CV) safety pharmacology studies is the collection of high-quality CV data prior to dose, during dose, and post-dose administration. The minipig is extremely intelligent and every interaction is a learning process, including stressful experiences that the animal will remember and avoid in the future. Through positive reinforcement, a procedure can become less stressful for the animal. By providing positive reinforcement, such as juice, after a procedure, the animal will remember the procedure as a positive experience, thereby promoting good behavior. Minipigs are generally motivated by food and treats (e.g. apples or pears) which are routinely presented to provide positive reinforcement. However it has been observed that heart rate (HR) in minipigs increases after feeding and can remain elevated for several hours. This limits our ability to use positive reinforcement in the form of treats to promote good behavior. Apple juice has also been used for positive reinforcement; however no CV data has been assessed for the effects of apple juice on HR. The objective of this study was to assess the effect on HR when apple juice was provided immediately post dose administration. The study consisted of 2 tests. In both tests CV data was collected for 2 hours after which apple juice was presented for ∼15 secs. CV data was then collected continuously for 24 hours. In test 1 no dose was administered, only apple juice was provided. In test 2 dose (tap water) was administered via oral gavage and then apple juice was provided immediately post dose. This data was then compared to previously collected data (data obtained from the same animals after restraint in a dosing chair, using a bite bar, with/without administration of Vehicle A, and did not receive apple juice post dose). The data indicated minimal impact on HR when only apple juice was provided. When apple juice was provided immediately postdose administration, the initial change in HR was similar to the change in HR in the previously collected data. This indicates that the initial increases in HRs are due to the dose procedure, not the presentation of apple juice . We have demonstrated that reduced stress and good behavior learned by positive reinforcement can provide better overall animal welfare and subsequently lead to high quality CV data.

P17 Donating Rodents in a CRO Environment

KM Fiala^*, D Kenjebaev, CA Plews, S Snyder

MPI Research, Mattawan, MI

Donating rodent carcasses to a local nature center or bird sanctuary was initiated as a means to give added purpose to animals not selected for study. Most nature centers are not-for-profit organizations that are dependent upon donations of money and goods to stay in operation. One item that nature centers are in need of, and CROs are uniquely qualified to donate, is rodent carcasses to be given as food to their birds of prey. Once approval was given for the donations by upper management and the attending veterinarian, a committee was convened to create and implement a program that would appease all affected departments, including security, IACUC, finance, purchasing, safety, and in-life groups. The committee started by creating a title and mission statement for the program. The team then worked together to ensure all affected departments were consulted and prepared a streamlined process that would be easy to follow while allowing the maximum number of qualified animals to be donated. To qualify for donation animals must be naïve to test article administration (certain vehicles permissible as defined by the attending veterinarian) and euthanized via CO2. Within 1 month the Raptor Food Donation Program was implemented and within 3 months the first donations were submitted to the nature center. Tax-related donation forms are collected at each transaction to maintain accurate records for any deduction or other legal considerations. The program has proved beneficial in not only feeding local raptors, but also in engendering goodwill between the CRO and local communities, and helping the technical staff promote animal welfare by bringing added purpose to these lives.

P18 Refining Endpoints for a Rhesus Macaque (Macaca mulatta) Model of Tuberculosis

KE Russell-Lodrigue^*1, LA Doyle-Meyers¹, R Bickham¹, D Kaushal², JL Blanchard¹

¹Veterinary Medicine, Tulane National Primate Research Center, Covington, LA; ²Bacteriology and Parasitology, Tulane National Primate Research Center, Covington, LA

Rhesus macaques (Macaca mulatta) inoculated with Mycobacterium tuberculosis via small particle aerosol are used as models of active and latent tuberculosis. In the final stages of disease, the miliary tuberculosis produced in this model can cause severe respiratory distress and death. Veterinarians, animal care technicians, and investigators can work together to evaluate disease-related changes such as fever, decreased appetite, respiratory compromise, radiographically evident pneumonia, and increased C-reactive protein levels and use a combination of objective measurements and subjective scoring systems to determine the most appropriate study endpoint for each animal to negate the need for death-as-an-endpoint studies. This multimodal approach allows for consistency in endpoints as required for scientific vigor while maintaining the clinician’s ability to improve animal welfare by making individualized patient care decisions.

P19 Identifying Correlates Between Behavioral and Physiologic Endpoints of Mice in Infectious Disease Studies

KJ Knapek^*, K Posey-Bruner, LV Kendall

Laboratory Animal Resources, Colorado State, Fort Collins, CO

Research with select agents in mice often result in death as an endpoint, intentional or not, as identifying appropriate endpoint criteria can be difficult to establish for the various agents and mouse strains. This can have a negative impact on the animal’s welfare. Establishing a means to identify early endpoint using indicators of morbidity that can be applied to multiple agent would be invaluable for those working with infectious disease models. A behavioral ethogram was used to assess the effects of infection with Burkholderia pseudomallei (Bpd) and Bacillus anthracis (Banx). The ethogram included general activity, grooming, rearing, and orbital tightening. In addition to the ethogram, body temperatures were measured with a thermal imaging camera, and the time-to-integrate to nest (TINT) test was performed. The mice used in this study were on an existing protocol to evaluate the vaccine efficacy against Bpd and Banx. Following infection, mice were observed for 5 minutes every 6-12 hours. Mice infected with Bpd and Banx had reduced general activity, rearing and grooming, (p < 0.05), and increased orbital tightening. These behavioral observations correlated with a reduced body temperature. Bpd infected mice failed to integrate any nesting material, whereas Banx infected mice delayed integration of new nesting material to their existing nest, which in some instances took more than 24 h. The use of these observation techniques with multiple agents can assist investigators in assessing early time points for infectious disease studies. Further, it provides a means of more objective measurements, such as body temperature and integration of nesting material, that correlate to behavioral signs of disease. This provides a means to identify moribund animals which can easily be overlooked in prey species.

P20 Effects of Caretaker Behavior on Nonhuman Primate Performance

K McGrew^*

Behavior, Charles River, Houston, TX

Human interaction is an important step in gaining the trust necessary to train NHPs to cooperate. However, establishing trust can be difficult. This study examined how technician heart rate range, measured by a wrist-wearable activity tracker, correlated to how many primates would accept a hand treat (peanuts or crackers). This was then compared to the average results over several sessions. All sessions were conducted between 9 and 11 am after cleaning and feeding by husbandry. Results contradicted a hypothesis that animals responded better to calmer technicians. A slightly higher percentage (2.5%) accepted hand treats when the technician was excited (40% above resting heart rate) and a decreased percentage (-2%) accepted when the technician was calm (within 10% of resting heart rate). Normal activity range (heart rate 20% above resting) led to a -1% change, and an active state (30% above resting HR) showed no change. The test was repeated using calming exercises to lower heart rate. Lower heart rates and calm, slow movements reduced the number of animals coming up front to accept treats (-8% with female animals and -2% with males). In an effort to determine a more effective means of establishing trust, the technician repeated testing with the following conditions: spent extra time with animals before human interaction (-6% change in males, +3% change in females), “baby talk” towards animals (+2% with both sexes), and nonverbal body language (lip smacking and peering), which was the most successful strategy with females (+3%) but not with males (-2%). This data suggests NHPs are most likely not demonstrably affected in a statistically significant metric by human caretaker emotional states when heart rate is the surrogate marker for defining the emotional state. Further work is needed in this area to determine if there are other behavioral indices affected by human caretaker emotional states.

P21 Promoting Transparency: Hosting Veterinary Technicians for Research Day

KL Thompson^*, NM Gades, TR Meier

Comparative Medicine, Mayo Clinic, Rochester, MN

Historically, our institution has supported various veterinary technology training programs in the region by providing animal facility tours to students as a supplement to their laboratory animal medicine coursework. Over the years, the numbers of requests grew as word spread about the tours to other programs in the area. In order to keep up with demand, while minimizing disruption of daily activities, we elected to formalize these tours into an annual program targeted towards veterinary technology students at all 4 of the major veterinary technology programs in the region. The first program occurred in May of 2015. We had a total of 22 students and staff from all 4 of the programs in Minnesota. Due to only having 5 hours with the students, the tours had to be limited to the vivarium, with only 15 minutes dedicated to the workings of the research laboratory. We also hosted lunch with a panel of Certified Veterinary Technicians working in various areas of our research program. The technicians spoke about their positions and the students were given an opportunity to ask questions. Feedback from the first program indicated that students wanted to see more of the facilities and projects. The 2016 event was expanded to 8 hours, and included both the Rochester, MN and Scottsdale, AZ campuses. The extra time allowed for an hour dedicated to just the research laboratories besides the time spent on the vivarium tour. We had 11 students and staff who attended in Minnesota, and 13 students and staff attended in Arizona. Research Day provides ample opportunities to the students to see the roles of veterinary technicians in research and is rewarding for our employees and staff to talk about their work. It is exciting to educate new technicians on the ever growing possibilities available to them as they go through their program, graduate, and enter the world as a Certified Veterinary Technician.

P22 Dollars and Sense: When Planning a Study It Makes Sense to Know the Economic Impact of the Model You Choose

LA Knarr^*1, GJ Ochs², MJ Horn¹

¹Veterinary Sciences, Research Support, Envigo, Indianapolis, IN; ²Commercial, Envigo, Indianapolis, IN

Preclinical toxicology studies involving animal models require several factors allowing for study completion. Researchers rely on historical control databases for baseline information, and may be hesitant to consider novel models; however, other factors should be considered in model selection, such as survivability, body weight, and lesion incidence, which have animal welfare and financial implications. When planning a study, one must consider several factors when choosing the appropriate model to ensure a robust study and the ability to complete the study within budget. Additional considerations are cost of compound, vivarium space, and technician time. An analysis of 2 rat models widely used in toxicology studies was performed. The Wistar Han^® rat is widely used in Europe as the toxicology model of choice while the CD^® rat is commonly used in the United States. To allow readers to gauge economic impact of model selection between these 2 rat models, a tool has been developed to allow researchers to calculate per study cost by using various internal costs associated with these studies, including technician time, consumables, diet, bedding, and overhead. This tool will allow researchers to compare economic impact between the 2 models at various compound costs and study types by allowing the researcher to account for individual internal costs at a per study level. Briefly, with regards to body weight, at 104-wk of age, the CD^®male rat weighs 35% more than the Wistar Han^® male rat (800 compared to 594 g), and the CD^® female rat weighs 30% more than the Wistar Han^® female rat (500 compared to 385 g). Additionally, body weight begins to diverge at approximately 3 months of age in the males. Further, survivability is 32 percentage points lower (35% compared to 67%) in the CD® compared to the Wistar Han^® male and 33 percentage points lower (40% compared to 73%) in the CD^® compared to the Wistar Han^® female. Neo and nonneoplastic lesion incidence is also improved at 104 weeks of age in the Wistar Han^® compared to the CD^® male and female rats. In conclusion, the lower body weight, robust nature, and improved survival rate of the Wistar Han^® rat may translate into improved animal welfare and overall cost savings for the toxicology program. The use of this tool will aid investigators in model selection and could be considered for future studies.

P23 Goals, Achievements, and Challenges of the Institute for Laboratory Animal Research Roundtable on Science and Welfare of Laboratory Animal Use

L Anestidou^*

Institute for Laboratory Animal Research (ILAR), Washington, D.C.

Since 2014 the National Academies’ Institute for Laboratory Animal Research (ILAR) has established the Roundtable on Science and Welfare in Laboratory Animal Use to foster communication among the many constituencies with strong interests in the use of laboratory animals in research. By convening representatives from government agencies, pharmaceutical and consumer product corporations, professional societies, academic institutions, scientists, and veterinarians, the roundtable’s aims have been to stimulate dialogue and collaboration, help build trust and transparency, and provide a new pathway to promote the responsible use of animals in science. We discuss the goals of the roundtable and summarize the achievements over the past 2.5 years. Importantly, it will also analyze the challenges the roundtable has overcome and the ones still ahead.

P24 The Harm in Harm-Benefit Terminology

L Kinter³, D Johnson¹, LJ DeTolla^*2

¹Comparative Medicine, Cascades Biosciences, Sisters, OR; ²University of Maryland, Baltimore, MD; ³Green Lawn Scientific Consulting, Unionville, PA

Risk-benefit analysis is current established terminology in animal research programs to determine and appropriately mitigate potential pain and distress. Harm-benefit analysis terminology was recently introduced in animal research accreditation for the same purpose. Pain, distress, risk, and benefit are objective concepts quantifiable through clinical observation, experimental measurement, statistics, and/or probability analysis, and long used in the ILAR Guide for Care and Use of Laboratory Animals, a foundation document for design, assessment, and evaluation of animal research programs. Harm/benefit is not included in the Guide, USDA-AWA, or USPHS Policy. Harm was introduced internationally in Directive 2010/63/EU as a surrogate for “suffering, pain, and distress.” Harm is a subjective, ambiguous, and emotionally loaded term about which well-meaning individuals may reasonably disagree. When used in animal research context “harm” is jargon for pain and distress. Harm conveys inaccurate and misleading impressions that biomedical research necessarily causes substantial injury or damage to animals. Further, alternative definitions of harm include implications of moral evil and wrong-doing that should not be associated with animal research. The authors posit that harm- benefit terminology (the use of the word “harm” rather than the word “risk”) (1) contributes no advantage or improvement over risk-benefit, 2) lends support to misinformation and misdirected beliefs of nefarious conduct, 3) damages the reputation of the scientific community and recruiting of young scientists, 4) delays critical research, and 5) should not be used as alternative language for or in replacement of risk-benefit terminology in ethical considerations of animal research activities, including protocol review procedures and program accreditations and evaluations.

P25 Staffing, Salary, and Scope: Survey of Animal Care and Use Personnel

ME Luthin^*, S Shriver

Public Responsibility in Medicine and Research, Boston, MA

There are vast differences in how organizations structure their animal care and use (ACU) programs and the offices charged with overseeing institutional animal care and use committees (IACUCs). This has made it difficult for administrators to benchmark their research oversight programs in terms of workload, scope of responsibilities, and salary. In response to requests from the oversight community for this data, Public Responsibility in Medicine and Research (PRIM&R) gathered information from 3 sources. PRIM&R’s annual workload and salary survey (WLSS) asks animal care and use professionals for crucial data on workloads, salaries, and staffing patterns. On their examination applications, Certified Professional IACUC Administrator (CPIA) candidates have the option to answer a series of demographic questions, which provides critical insight into those seeking professional development opportunities. In 2016, PRIM&R conducted a job analysis survey of animal care and use professionals, which asked a series of questions about respondents’ professional roles, job functions, duties, and tasks. The information generated by these 3 resources describes the scope of the administrative structures of programs charged with overseeing the use of animals in research, as well as provides detailed insight into the responsibilities and knowledge base required of staff members. For example, nearly half of the respondents work in IACUC offices that support fewer than 3 fulltime equivalent staff members; over half of these individuals have additional responsibilities with institutional biosafety committees and institutional review boards. The majority of respondents are highly experienced, having been involved in this field for at least 5 years. These data describing overall program structure as well as profiling ACU staff will be invaluable for benchmarking analysis, identification of gaps and needs in training and staffing, and development of best practices in administering animal care and use oversight programs.

P26 Toward the 3Rs in Fish Toxicity Testing: Development of a Fathead Minnow (Pimephales promelas) Fish Embryo Toxicity

MK Jeffries^*, K Roush, JC Krzykwa, K Phillips, J Malmquist

Biology, Texas Christian University, Fort Worth, TX

A simultaneous increase in the number of fish used for toxicity testing and calls to move toxicity testing towards the 3Rs has created a need to reevaluate current test methods and identify viable alternatives. This goal of this project was to test the utility of the fathead minnow (Pimephales promelas) fish embryo toxicity test (FET) as a replacement for the currently used larval growth and survival test (LGS). The objectives of this study were to compare the sensitivity of the FET and LGS and to determine whether the inclusion of sublethal endpoints (e.g., growth-related gene expression, the incidence of developmental abnormalities, etc.) increases the predictive power of the FET. To compare FET and LGS sensitivities, estimates of acute toxicity were generated for a suite of model toxicants via the FET and LGS test. A comparison of the acute toxicity estimates generated via the FET and LGS revealed that, in most cases, the sensitivity of the FET is similar to that of the LGS. Upon FET test conclusion, several sublethal metrics were evaluated and of these growth, growth-related gene expression and the incidence of pericardial edema were found to be the most commonly altered in response to contaminants. Overall, the results of this study show that the fathead minnow FET may be a viable replacement for the LGS and that the inclusion of sublethal metrics may improve the predictive power of the FET.

P27 Creation of Epoxy Mouse Skulls to Reduce Animal Numbers for Stereotactic Surgery Practice

MM Haney^*1, C Hinkel¹, A Thiessen¹, J Allen¹, I Deninger¹, D Ohlhausen¹, T Lever²

¹Comparative Medicine, University of Missouri, Columbia, MO; ²Otolaryngology-Head and Neck Surgery, University of Missouri, Columbia, MO

Stereotactic brain surgery is an essential tool to study neurologic function in many healthy and disease states of animal models. This technique requires immobilization of the animal in ear bars and precise coordinates to be used to allow insertion of electrodes or cannulas into specific regions of the brain. Furthermore, screws and dental cement are often used to secure protective head stages to the skull. For novice users, this procedure can be technically challenging and requires a considerable amount of practice before reliable results are achieved, leading to the euthanasia of additional animals from which no data will be acquired. Our lab sought to reduce the number of practice animals needed to become proficient at stereotactic surgery. By using a resin mold kit, casting epoxy, and 1 representative mouse, we created multiple (∼20), reusable practice skulls within an hour. The process involves sacrificing a single mouse of the strain and approximate weight of future experimental mice. The head is separated from the body, the skull is cleaned thoroughly, and any excess tissue is removed. The mold is placed around the skull and removed, leaving an imprint in which casting epoxy is poured. After proper drying time, the cast is taken out of the mold and can be used for training. These practice skulls allowed lab members to practice ear bar manipulation, burr hole drilling, anchor screw placement, and dental cement application. Additionally, the practice skulls can be reused after placement in acetone to remove dental cement which does not damage the integrity of the epoxy skull. Through the use of a resin mold kit and epoxy casting material, skull replicates can be easily made for stereotactic surgery practice. These skull models drastically reduce the number of animals euthanized for practice or due to procedural error. The same materials can also be used for larger rodent species used in research, such as rats.

P28 Creating a Third-Party Risk Assessment Process

MM Perez^*, TL Condet, S Vaughn, TL Vandegiessen

Animal Welfare Compliance, Zoetis, Kalamazoo, MI

Our institution has a corporate policy that defines the approach to humane animal care and use within the facilities. This policy states we accept responsibility for conducting animal research in a humane and ethical manner and expect all colleagues to treat animals with respect. The policy also requires all third party research organizations we engage that conduct animal-based research on our behalf, including animal vendors and animal transportation companies, to work at the same standards. To ensure third-party organizations are in compliance with the policy, a risk-based process was devised and a global team formed to support the commitments described in the policy. Compliance with humane animal care and use at external facilities is necessary for animal welfare, data quality, study integrity, and regulatory compliance. We will outline the risk assessment and audit process used to ensure compliance with the policy. We will also provide a list of materials and assessment standards used, results, and a summary of the improvements in the process that came from our analysis of the results.

P29 Evaluation of Refinement Method for Mouse Identification

MJ Weinstein^*1, C Norton², D Bangari³, K Singh³, X Ying⁴, G Hunt⁵, N Campbell⁵, F Sun¹, ST Savage²

¹Pharmacology, Sanofi Genzyme, Cambridge, MA; ²IVRC, Sanofi Genzyme, Framingham, MA; ³Pathology, Sanofi Genzyme, Framingham, MA; ⁴Preclinical Safety, Sanofi Genzyme, Framingham, MA; ⁵Bio Medic Data Systems, Inc., Seaford, DE

Use of radio-frequency identification (RFID) transponder in mice is a standard practice in our animal facility. Due to concerns with RFID transponder migration or loss from improper injection techniques and also a perceived pain and distress upon injection, we proposed to refine the use of this technology in collaboration with our transponder supplier. The standard transponder provides researchers with a reliable, nonreactive, and permanent method of identification that facilitates rapid electronic data capture. To address the perceived complications associated with typical RFID transponders, we requested a smaller transponder and delivery trocar. This next generation of micro-transponder was developed by our collaborator who specializes in laboratory animal research technology. Our laboratory evaluated the animal welfare, quality of product, tissue reactivity, and longevity of the transponder in 4 of our standard mouse models. The novel transponder was implanted using a smaller 15 gauge trocar needle (previously 12 gauge). The overall transponder dimension was decreased by 60% (1.4 mm in diameter and 8 mm long). Forty mice (NOD, C57BL/6, nude, and SCID mice) were implanted and monitored for 30-90 days. During the evaluation, mice were manipulated daily to mimic study conditions, weighed, and the transponder site was frequently palpated. Upon euthanasia, the skin, subcutaneous tissues and the transponders were excised and evaluated for histopathology. Compatibility of the transponders with high-field (7 Tesla) magnetic resonance imaging and optical imaging systems was also evaluated. Less scratching at the transponder site and less struggling during restraint were observed with the smaller delivery trocar and transponder. We conclude that animals tolerated the transponders well and with increased ease of placement, there was less apparent pain on implantation. We also concluded that the smaller delivery trocar is less invasive as the reduction in delivery trocar gauge size creates a significantly smaller hole in the skin. There was also a decrease in transponder migration resulting in 50% fewer losses of the transponder, compatibility with imaging systems, and minimal tissue reactivity. The novel smaller RFID transponders represent a significant refinement in methodology, resulting in a less invasive and apparently more humane method of permanent electronic identification of animals. Additional studies to evaluate lifetime use of these novel transponders in day 0 and weanling mice are underway.

P30 Urinary Catheterization of the Female Murine Model

MM Williams^*, H Ding, M Tweedle, MV Knopp

Radiology, The Ohio State University, Columbus, OH

Contrast agents can be excellent diagnostic tools. However, there are instances where the excretion method can become hazardous or impair the quality of a diagnostic scan. This is most commonly seen in nuclear imaging of the bladder where the genito-urethral and lower gastrointestinal visualization becomes impaired and presents a spill hazard. The option to express or use a needle to drain the bladder can lead to contamination of the fur/skin, surgical theater, and an extra exposure to the handler and unfortunately does not guarantee complete removal. The procedure presented here requires a length of small tubing cut to the length appropriate for access to the imager. It is best to mark the tubing to indicate 1 centimeter of penetration. The tubing should be sterilized with ethylene oxide and lubricated prior to use. This procedure requires no metal and thus is safe for use around and MRI. The procedures used can be performed with plastics that do not fluoresce and thus are safe for optical imaging. The tubes can also be shielded with radiopaque materials to reduce exposure in a PET/SPECT. The tubing can be cut long, so no pooling occurs within the field of view. It has been noticed that the bladder does not completely drain of its own accord. Gentle pressure to the bladder should be applied just prior to scanning to assure complete evacuation. Allowing the tubing to remain during an in vivo scan would allow for much more detailed imaging of orthotopic bladder models as well as GI evaluations. The removal or reduction of bladder artifact allows for better dynamic contrast across the rest of the body. This, in turn, would improve the signal to noise ratio for faintly localized tracers. There are many advantages to adopting this as common practice prior to or during various imaging procedures. The placement of the tube is simple and the materials are inexpensive. In conclusion, the application of a urinary catheter can drastically improve the quality of data in the preclinical community without extreme expense. Further efforts will be applied to making this applicable to both sexes of mice in hopes of increasing its utility in biomedical research.

P31 Improving Sevoflurane Anesthesia with Premedication of Fentanyl-Midazolam in Laboratory Mice

M Lipiski^*1, M Arras^1,2, P Jirkof^1,2, N Cesarovic¹

¹Division of Surgical Research, University Hospital Zurich, Zurich, Switzerland; ²Neuroscience Center Zurich, University of Zurich, Zurich, Switzerland

Laboratory mice typically are anesthetized by inhalation of volatile anesthetics or injection of drugs. Using synergistic and additive effects of injectable drugs and volatile anesthetics, known as balanced anesthesia, allow for the dosages of each component to be decreased while inducing general anesthesia of sufficient depth with fewer side effects. Here we evaluate 2 balanced anesthetic protocols, a combination of Ketamine-Sevoflurane (KS) or Fentanyl-Midazolam-Sevoflurane (FMS) on their suitability for moderate surgical intervention and their impact on post-surgical recovery compared to a Sevoflurane mono-anesthesia (S). Twenty-four female C57BL/6 mice were assigned randomly to 1 of 3 protocols: S, KS, or FMS. Injectables were administered 8-10 minutes prior to induction with Sevoflurane. A sham ovariectomy was performed. A single dose of Carprofen was administered to all animals at the end of the procedure. Heart rate and core body temperature were recorded by telemetric transmitters intra-operatively and for 3 days prior and 3 days post experimental procedure. Intraoperative respiratory rate was determined by counting thorax movements. Body weight, food, and water intake were measured for 3 days prior and 3 days post experimental procedure. Measurements revealed a decrease in heart rate and core body temperature during anesthesia in both premedicated groups. Respiratory depression was evident in all groups. Painful stimuli induced respiratory alterations in the KS group but not in the FMS group. Heart rate remained stable in all groups. Time to reposture and motion was 1-2 minutes after Sevoflurane withdrawal in all groups. Heart rate was elevated in all groups for the first 12 hours following experimental procedure; changes were least pronounced in the FMS group. No changes in food and water intake were seen in any of the groups. In conclusion, single low-dose injection of Ketamine did not provide sufficient intraoperative analgesia and was not superior to Sevoflurane alone. Animals premedicated with Fentanyl-Midazolam showed a more reliable anesthesia based on responses of heart and respiratory rate to painful stimuli. A shorter and quieter induction phase and decreased negative postanesthetic effects on the heart rate were further advantages seen with FMS.

P32 Comparative Medicine Teams up with Project SEARCH

NJ Rindels^*1, N Joyce², DM Kirchner², MM Watson¹

¹Comparative Medicine, Mayo Clinic, Rochester, MN; ²Human Resources, Mayo Clinic, Rochester, MN

Project SEARCH is an internationally recognized program that was developed in 1996. The Project SEARCH High School Transition Program is a unique, business-led, 1-year, school-to-work program for students with developmental cognitive disabilities in their last year of high school eligibility. The outcome is competitive, integrated employment for each student intern. The Mayo Clinic Project SEARCH site was developed as a partnership between the Rochester Public Schools, Mayo Clinic, Vocational Rehabilitation Services, and Olmsted County Developmental Disabilities Services with support from the Project SEARCH State Leadership Team. In 2015, the first students were selected for this program, and the Mayo Clinic Department of Comparative Medicine became an enthusiastic participant. Our goal was to develop a program that would provide students a chance to develop hands-on, job-related skills and polish soft skills such as communication, problem solving, teamwork, and adaptability that are important for achieving gainful employment. We also anticipated that this program would enable our employees to work with and mentor older youth with disabilities and increase camaraderie in the workplace. During the 2015-2016 school year, we had 3 interns complete a 10-week internship in our department. Each student brought a unique skill set, positive attitude, and willingness to learn, which contributed to their personal accomplishment and departmental productivity. Our staff welcomed the opportunity to help the students throughout their internship. The interns provided an avenue towards employee engagement. Several staff members worked side by side with the students each day, mentoring and guiding them. The benefits were mutual, however, as our staff members were able to approach their jobs from a different point of view, that of a teacher. The staff also built trust amongst each other to collaborate and construct a platform for our interns to learn. Project SEARCH was an overwhelming success in our department, and is an excellent example of how organizations can participate in outreach programs as a way to support their community and enhance the local workforce.

P33 Tools of the Training Trade: Mechanics of Improved Animal Health

CR Lockworth¹, NA Monts de Oca^*2

¹MD Anderson Cancer Center, Houston, TX; ²Animal Care Operation, University of Houston, Houston, TX

A technician training program must be developed that effectively equips the technician with all of the tools necessary to provide the highest-quality of care to research animals. To prepare technicians, we have used common educational methods including didactic training and mentoring. But, we have also found the incorporation of training aids to be particularly useful implements to further augment learning during routine instruction. Moreover, aids can later function as references materials following training when needed most, while working with animals cageside. Training tools that we have developed include algorithms for rodent case management and for large animal anesthetic emergencies, which support logic and reasoning in decision-making. Additionally, we have produced a pocket reference card of all common health conditions that provide technicians with a quick and handy reference while examining animals. Finally, we developed a rodent health conditions and management poster. Posters are placed at suite level or within biosafety cabinets for continuous reference. Consistency in training with such aids, as well as the ready availability of these tools cageside, has resulted in earlier health issue identification, rapid animal treatment and disposition, and overall consistency across health activities of both husbandry and health technicians. Using these tools at 1 facility, we demonstrated a 2.5 fold increase in the identification of health issues by husbandry staff and a 14% decrease in the number of animals found dead. As a result of this training program and its implements, all members of the animal care team are increasingly empowered and the quality of animal health care has been advanced, producing better research outcomes.

P34 Disease Activity Index Scoring in Rodent Models of Inflammatory Bowel Disease

NP Allotta^*, A Miller, M Zhang

Amgen, Thousand Oaks, CA

Southern California Joint Pole Committee Routine Handbook Of Chemistry And Technology

Inflammatory bowel disease (IBD) represents a group of intestinal disorders causing prolonged inflammation of the digestive tract affecting 5 million people worldwide. The 2 most common intestinal disorders are ulcerative colitis (UC) and Crohn’s disease (CD). UC involves inflammation of the large intestine and rectum, while CD causes inflammation in any part of the digestive tract mostly affecting the tail end of the small intestine (ileum). Both UC and CD generate weight loss, diarrhea, rectal bleeding, and abdominal pain. IBD pathogenesis remains unknown; however, genetics, the immune system, and environmental factors have been linked to this disease. Animal models of colonic inflammation are a critical tool in both research and drug development. There is a large range of animal colitis models such as spontaneous colitis in susceptible mice strains or administration of dextran sulphate sodium (DSS), a colitis-inducing chemical. During the administration of DSS, a disease activity index (DAI) score is assessed to evaluate the clinical progression of colitis. To monitor the animals in the study and determine the appropriate endpoints, we established a DAI score that combined stool consistency, bleeding in stool or around anus, and health appearance of animal. The DAI scores are defined as follows: stool consistency 0 (normal), 1 (moist/sticky), 2 (soft), and 3 (diarrhea); bleeding 0 (no blood), 1 (mild), 2 (moderate), 3 (severe); appearance 0 (normal), 1 (ruffled fur or altered walking gait), 2 (moribund or lethargic). In addition to the DAI score, body weights were collected to compare weight loss from the animal’s initial weight. A DAI score of 6 and greater or 20% body weight loss resulted in euthanasia. The DAI scoring yielded strong correlation to disease progression and did not require any invasive procedures. This process provides a method to monitor animals daily and determine timely euthanasia when animals reach en points. With well trained staff and a validated model, alignment on the DAI scoring can effectively minimize pain and distress of the study animals.

P35 Ensuring That New Laboratory Personnel Stay Compliant: An Introduction to the Rules and Regulations of Animal Research

PI Mireles^*

IACUC, Northwestern University, Chicago, IL

With over 300 principle investigator laboratories the number of new laboratory personnel added to an animal study protocol (ASP) ranged from 10-30 new members a month. Some arrive with previous experience from other institutions within the US and other from institutions from other countries; however, the majority of the new laboratory members are new to working with laboratory animals. Even though they are required to take certain online courses, and a hands-on session, there still seemed to be a gap in understanding the requirements and techniques. So to fill that gap, the IACUC started hosting a monthly brown bag session during the lunch hour, inviting all the new members added to a protocol from the previous month. This ensured that they have already completed the all required training, did the hands-on requirements, and were already added to an ASP. The session is a combination a brief lecture and an interactive Q&A session that helps engage the audience throughout the session. The questions are based on the topics discussed, along with any items noticed during the semiannual inspections, PAM visits, and minor deficiencies. This program was implemented 5 months ago and so far has been a success, with a decline in minor noted deficiencies, understanding of the requirements, and has even encouraged long-time members (laboratory managers) to request an invite as a refresher for themselves.

P36 Padded Piggies to the Max

RK Hlavka^*, A Gallardo, G Moreno, C Torres

University of California—Los Angeles, Los Angeles, CA

An ongoing project required creating a stoma on Yucatan piglets, an externalized segment of the small intestine. A stoma bag for collecting intestinal fluid was secured around the stoma and bandaged with vet wrap and elastic cloth tape. Piglets were trained using positive reinforcement to walk on leashes from their cage to the clinic and stand still on the exam table for treatment. Bags were drained at least every other day and new bandages applied. Initially, no issues were anticipated, however the lab discovered stomas were healing and the opening was constricting, preventing them from moving on to the next phase of the project. In an attempt to maintain and increase the diameter of the stoma, the veterinarian recommended placing foley catheters. While this worked for the diameter of the stoma, it posed a problem for the stoma bags and bandaging. Regular leakage of intestinal fluid occurred due to the weight of the catheter in the stoma bags, soaking through the entire bandage. Because of the acidity of the fluid, piglets’ skin became scalded. Applying ointment only interfered with the bags even more and the large bandages prevented the skin from healing. In an attempt to find an alternative to the stoma bags, ultra-absorbent maxi pads replaced the stoma bags. The distal end of the foley catheter was positioned dorsal to the stoma. Gauze was placed between the catheter and the body for comfort. The absorbent side of the maxi pad was placed over the stoma and catheter. Vet wrap and elastic cloth tape was wrapped around the body covering the pad. Within 24 hours, the scalded skin had healed significantly and any leakage was quickly absorbed. The use of pads allowed ointment application to the scalded skin and reduced the width of the total bandage, allowing the skin to heal. The pigs appeared more comfortable and bandage changes were accomplished in a shorter period.

P37 Gene Expression Changes and Signal Transduction Pathway Alterations in Primary Human Oral Epithelial Cells Exposed to Smokeless Tobacco Extracts

R Rajapakse^*

University of Virginia, Charlottesville, VA

Smokeless tobacco (ST), an alternative to smoking, has gained wide popularity among tobacco users. This study is conducted to determine the time course of gene expression associated with specific signaling pathways in human oral epithelial cells after exposure to smokeless tobacco extract (STE). As an alternative to use laboratory animals as well an in an attempt to reduce use of animals for research (3R), here we conducted this study with human oral epithelial cells. A differentiated layer of epithelial cell is created as a way to mimic reasonably similar physiologic atmosphere. A dose and time-dependent response is observed for cell viability and cell proliferation assays indicating that this model system is responsive to the treatment. Expressions of 84 genes representing 18 different signal transduction pathways are quantitated. This is accomplished by using real-time polymerase chain reaction arrays at 1-hour, 3-hour, 6-hour, and 24-hour time points following exposure to STE. Changes in gene expression are observed on many cellular processes including cell cycle regulation, cell adhesion, inflammation, apoptosis, and DNA breaksdown including Akt pathway activation. Short time exposure (1 hour) leads more genes to down regulate whereas longer incubation time results in more genes up regulation. Most notable differences in the expression of genes during the course of treatment are BCL 2A 1, BIRC 3, CCL 20, CDK 2, EGR 1, FOXA 2, HOXA 1, IGFBP 3, IL 1A, IL- 8, MMP 10, NOS 2, NRIP 1, PTGS 2, SELPLG, and TNF-a. This study provides an insight on gene expression on oral epithelial cells as a result of STE exposure.

P38 An Analysis of Inspection Reports in Animal Facility Based on GLP, IACUC, and AAALAC Site Visits

S Park^*1, E Auh¹, H Han¹, J Che³, J Yun¹, B Kang^1,2

¹Department of experimental animal research, Biomedical Research Institute, Seoul National University Hospital, Korea, Seoul, Korea (the Republic of); ²Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea (the Republic of); ³Biomedical Center for Animal Resource and Development, Seoul National University College of Medicine, Seoul, Korea (the Republic of)

Our department obtained Good Laboratory Practice (GLP) certification from the Ministry of Food And Drug Safety (MFDS) in Korea in 2003, and was fully accredited by AAALAC in 2007. Since then, we have performed several inspections in accordance with GLP, IACUC, and AAALAC. According to the results of 10 GLP quality assessments, 9 IACUC visits, and 2 AAALAC inspections over the last 5 years, the total number of items noted as needing immediate remedial action was 732, 226, and 25, respectively. We classified these 983 items according to 6 categories, which were animal care and use, equipment, environment, personnel safety, facility, and drug. Indicated items about animal care and use were the highest frequency (277 of 983, 28.2%) among the categories, followed by drug (161, 16.4%), facility (157, 16.0%), equipment (156, 15.9%), environment (152, 15.5%), and personnel safety (80, 8.1%). A comparison between 2011 and 2015 reports indicated that environmental and equipment aspects decreased from 33.3% to 9.0% and 27.8% to 7.8%, respectively. But items about facility, animal care and use, drug increased from 6.3% to 27.1%, 19.2% to 28.9%, and 9.4% to 18.7%. Also, personnel safety slightly increased from 3.9% to 8.4%. Environmental and equipment aspects were found to be improved by regular inspection and postevaluation whereas items about facilities increased possibly due to a relatively decrepit facility. Also, recent social trends and guidelines associated with animal welfare seem to affect the increased animal care and drug items. In conclusion, we suggest that regular inspections and postevaluation programs are necessary not only management and improvement of animal facility but also continuous update of standard operating procedures. Therefore, animal institutes should retain the standardized staff educational and assessment program.

P39 Assessing the Influence of Different Caging Environments and Ventilation Protocols In Cynomolgus Macaques (Macaca fascicularis) Using EEG Telemetry

S Authier^*2,1, MV Accardi¹, M Pouliot¹, E Boulay²

¹CiToxLAB, Laval, Canada; ²University of Montreal, St-Hyacinthe, Canada

Environment has a major impact on animal welfare but limited tools are available to quantify animal wellbeing. Monitoring of cardiovascular parameters failed to identify beneficial effects of social housing in animal species such as cynomolgus macaque (Macaca fascicularis). This study evaluated the effect different caging and ventilation on biomarkers of anxiety in cynomolgus macaques (n = 6). Stress levels and anxiety were monitored by means of various electroencephalographic (EEG) parameters (e.g. beta power spectral analysis and individual EEG frequencies) while each macaque was either single- or pair-housed with a familiar or an unfamiliar cagemate in either an AAALAC-compliant (12 days) or ETS-123-compliant cage (12 days). On occasion, extraneous stressors were introduced (e.g. an unfamiliar staff) to determine the response to spontaneous stressors. Ventilation in the room was adjusted from 12 to 3 air changes per hour with continuous monitoring of air quality (ammonium, CO₂ and total particle) and animal welfare. Spectral analysis with quantitative electroencephalography (qEEG) demonstrated that single-housed animals displayed higher levels of anxiety biomarkers throughout the experiment but with progressive adaptation to the isolated state. At equivalent time points, pair-housed animals showed signs of decreased stress/anxiety with the presence of a familiar cagemate. An unfamiliar cagemate was associated with variable results as some animals showed a significant increase in biomarkers of anxiety and stress while others presented reduced stress with an unknown cagemate. Animals remained normal with ventilation parameters adjusted to maintain air quality but to prevent a “windshield factor” from typical high ventilation rates. Our results suggest that cynomolgus monkeys may benefit from adjusted ventilation rates. Furthermore, the data from this study suggests that qEEG can be used as a sensitive and quantitative methodology to evaluate animal welfare in cynomolgus monkeys.

P40 Cryopreservation of Mouse Two-Cell Embryos: A 3-Year Report

AS Leocádio^*, M Pereira, J Bom, A Ribeiro, M Rebelo, J Demengeot, A Pereira

Animal House Facility, Instituto Gulbenkian de Ciência, Oeiras, , Portugal

The rapid evolution of biomedical research and molecular genetics leads to an increase number and variety of new mouse strains. When no longer required, cryopreservation of mouse germplasm greatly reduces maintenance costs of live animals and it safeguards valuable lines against infectious outbreaks or natural disasters. We provide investigators the cryopreservation service of mouse 2-cell embryos mostly by in vitro fertilization (IVF). As a member of the European Consortium Infrafrontier/EMMA, we follow protocols distributed by the Consortium, including the TYH + MBCD IVF protocol developed at Kumamoto University. We started this work following this protocol, which is optimized in C57BL/6 background and implements the use of TYH + methyl-β cyclodextrin as sperm preincubation medium in addition of reduced glutathione (GSH) supplemented fertilization medium. With this method, similar fertilization efficiency rates were registered in different genetic backgrounds (24% in C57BL/6, 28% in FVB, and 22% in Balb/c). In 2014 we started to use the FERTIUP & CARD Medium, also optimized in C57BL/6, and observed a relevant increase in the fertilization efficiency rates. Analyzing the results per genetic background we found that, not only C57BL/6 (47%), but also FVB (71%) and Balb/c (36%) showed noticeable improvements. We report here the results obtained in the last 3 years, which summarize all IVF sessions done, including sessions with 0% of efficiency. In order to assess the quality and viability of the frozen material we implemented 2 quality controls: (1) embryo culture into blastocyst, done for every embryo freezing session, and (2) embryo revitalization of a 2-cell embryos straw followed by surgical embryo transfer to pseudo-pregnant recipients. Upon a viable progeny, with the expected genotype, the strain is considered secure and the live animal resource is ended. During the last 3 years, by constantly updating IVF protocols, we have contributed to a 2-cell embryo biobank of 85 secured lines, having at the moment 50 more lines in the cryopreservation process, and a total of 43,237 frozen embryos.

P41 Performance-Based Approach to Environmental Enrichment for Mice and Rats

SP Swing^*2, E Bartlett², WE Nack-Lawlor¹

¹Regulatory, Taconic Biosciences, Inc., Hudson, NY; ²Veterinary Sciences, Taconic Biosciences, Inc., Germantown, NY

An environmental enrichment policy for mice and rats should fulfill the objective of promoting animal welfare by defining the objects and/or structures to be included in the microenvironment which will encourage species-typical behaviors and engagement with their surroundings. Providing the appropriate materials to enhance the environment, rather than to create stress within the environment, is key to a successful enrichment program. While bedding and nesting material, chew blocks, and similar items are typically associated with desirable enrichment for mice and rats, differences between animal models suggest that the most effective forms of enrichment will vary within a species, and not only between different species. A performance-based, or Environmental Enrichment Efficacy Evaluation (4Es), approach offers an effective means of assessing the type of enrichment best suited to support the wellbeing of a particular model. Environmental enrichment may be characterized as ineffective where the animals ignore it, interact with it in an unproductive manner, or where the object/structure generates or increases aggressive behaviors. By designing a study wherein a different form of enrichment is offered to each study group of a particular model, observations of the animals’ interaction with and reaction to the object/structure in the cage allows identification of the best choice of species appropriate and line-specific enrichment. As a measure of desired performance, or efficacy, observation data is scored against performance criteria based on the targeted undesirable behavior with the most successful environmental enrichment resulting in reduced observations of the malbehavior. One example studied is reduced conspecific aggression, but other behaviors such as stereotypies and overgrooming will also be evaluated. These studies enable the most effective enrichment item to be used for the given line of mouse or rat, a more economical and appropriate enrichment approach.

P42 Operational Efficiency Yields a Robust Postapproval Monitoring Program

SP Swing^*2, E Bartlett², WE Nack-Lawlor¹

¹Regulatory, Taconic Biosciences, Inc., Hudson, NY; ²Veterinary Sciences, Taconic Biosciences, Inc., Germantown, NY

Post approval or other ongoing monitoring programs are among the critical activities conducted by IACUCs to ensure animal wellbeing and program compliance. The challenge is to routinely obtain the data needed to assess, and to ensure an effective review of, the animal care and use program. This balance may be difficult to strike for programs with limited resources to extend to an audit-based monitoring approach; observations of data and practices at a colony level can also be limiting in scope. Our program maintains several husbandry practices that represent departures from Guide-recommended practices. Initially, large-scale studies were conducted to obtain performance-based data for each practice departing from Guide recommendations. The performance criteria was defined for each study using the performance measures described in the Guide, as they were characterized relative to the practice in question (for example, housing density factors assessed included health, reproduction, growth, behavior, activity). Upon completion of each study and where approval to maintain the Guide-departure practice was approved by the IACUC, ongoing monitoring criteria were defined based on performance metrics equivalent to study performance criteria and routinely captured in the husbandry operations database. The IACUC has leveraged this current performance data, already recorded in the course of daily animal activities, to compare against the study data; the study data became the baseline for accepted welfare performance. Each month the IACUC is presented with a report, including graphic depictions of current performance data compared against the baseline. Having access to this volume of data in real time allows the IACUC to monitor the performance of multiple animal colonies across a variety of criteria and provides a robust assessment tool for monitoring animal welfare performance.

P43 Management of Lower Esophageal Sphincter Stenosis in a Familial Hypercholesteremic Swine (Sus scrofa)

AD Meyers^*1, AA Carter¹, AM Corn¹, P Corts¹, TM McCrum¹, PA Mount¹, K Peterson¹, MP Short¹, A Zook¹, J McGregor¹, SJ Popilskis^2,1

¹Skirball Center for Innovation, Orangeburg, NY; ²New York Medical College, Valhalla, NY

A 7-month-old female Familial Hypercholesterolemic Swine enrolled in a peripheral vascular study fed a high cholesterol diet was repeatedly noted to have undigested material beneath the cage. On exam, the animal was bright and alert with activity level and hydration status within normal limits. Initial differential diagnoses included causes of obstruction, bowel torsion, and dysphagia. Given the lack of response to diet change and medical therapy, thorough diagnostic testing was performed. When plain thoracic and abdominal radiographs were inconclusive, a barium study was performed revealing dilation of the lower esophagus and narrowing of the lower esophageal sphincter (LES) to 2.0mm. Average LES measurements for swine of this size and breed are ∼10mm. Potential causes of LES stenosis include chronic irritation from gastroesophageal reflux and achalasia, an esophageal motility disorder. Regardless of etiology, symptomatic treatment was pursued. Dilatation of the LES using a percutaneous transluminal angioplasty balloon dilation catheter was attempted. Post-dilation, the LES measured 5.6mm. This procedure along with gastroprotectant medications and modified feeding practices allowed for symptomatic improvement. Four weeks later, regurgitation increased and barium study again revealed LES stenosis. Due to the severity of stenosis, step-wise dilation of the LES was necessary. An 8.0x40mm balloon was first passed into the LES, inflated, and removed. Then, a second dilation was performed using two balloons (8.0x40mm and 7.0x40mm) in a side-by-side kissing-balloon technique. After these dilations were completed, a 30mm x 10cm balloon dilator was passed into the LES for inflation. Post dilatation, measurements of the LES reached into the normal range at 10.9 mm. While specific esophageal and LES balloons catheters are commercially marketed, they can be costly and, in our case, not kept in stock. By using available equipment and later a specific dilation balloon, we were able to maintain the animal in good condition until the scheduled study end point. This prevented the loss of valuable data and the need to enroll additional replacement animals.

P44 Use of C-Arm Clamp to Achieve Hemostasis after Percutaneous Coronary Intervention in Yucatan Miniature Swine (Sus scrofa)

AM Corn^*, P Corts, MP Short, K Peterson, TM McCrum, AA Carter, A Zook, PA Mount, AD Meyers

Scirball Center for Innovation, Cardiovascular Research Foundation, Orangeburg, NY

Swine are commonly used as models for cardiovascular disease and preclinical device evaluation. Many of these procedures require percutaneous access of the femoral artery and vein along with the need for subsequent hemostasis. While vascular closure devices are available, the mainstay of achieving hemostasis in swine remains manual compression. This can be provided by hand or by several commercially available devices designed for human patients. We evaluated a system that includes a flat metal base and a plastic, adjustable arm. A translucent plastic disc is attached to the tip of the arm. The disc is placed over the access site and lowered to induce and maintain pressure as the sheaths are removed. We used this device to obtain hemostasis of the femoral artery in Yucatan miniature swine (20 to 70kg). These animals were on an antiplatelet regimen and heparinized perioperatively to maintain an active clotting time, (ACT) greater than 250 seconds during the procedure. The sheath was not removed until the ACT drops below 250 seconds. Time of compression was based on sheath size. We have found that this device works well on animals weighing less than 50kg. As the swine grow they develop increasingly large muscles which interfere with device placement. Advantages to using the device instead of manual pressure include a more controllable and constant pressure application. The device also eliminates ergonomic challenges as well as hand fatigue. While an improvement over manual hemostasis, proper placement and monitoring of the access site is still required. We found the device led to fewer complications when used to provide hemostasis on a previously naïve vessels. Animals undergoing serial femoral vessel access tended to have an increased risk of bleeding and hematoma formation. This may be due to use of nonstandard access locations. A deeper access site requires a more exact pressure point, which the disc cannot provide as well as a finger can. Attempts to place the disc at a more proximal point usually result in bleeding due to misalignment of the pressure point or obstructed venous return due to the high amount of pressure required in that area. Overall it seems the C-arm clamp is a good replacement for manual compression only in particular circumstances.

P45 The Use of Enrichment in the Management of Endemic Trichophyton mentagrophytes in a Chinchilla (Chinchilla laniger) Colony

AE Sparks^*, DM LeMoine, JM Hickman-Davis

University Laboratory Animal Resources, The Ohio State University, Columbus, OH

Chinchillas were obtained from a local vendor for use in long-term, noise-induced hearing loss studies. Chinchillas were singly housed in solid bottom cages with aspen bedding and touch contact with neighboring animals. The colony was comprised of both genders and ranged in size from 6 to 16 animals. Chinchillas were provided a commercially available pelleted diet formulated for chinchillas and water via an automatic watering system ad libitum. Enrichment for the colony consisted of a hanging toy, a push toy, and weekly dust baths with blue cloud dust. Toys were rotated weekly and animals were given hay cubes or fresh hay daily. Routine examination identified 2 animals with patchy scaling skin lesions and fur loss on the face. Trichophyton mentagrophytes was identified by Dermatophyte Test Medium (DTM) culture and PCR of fur samples from both animals. T. mentagrophytes is the most common dermatophyte in chinchillas. Infected chinchillas show small, scaly patches of alopecia on the nose, behind the ears, or on the forefeet. Wood’s lamp examination is rarely helpful in chinchillas as T. mentagrophytes does not fluoresce under UV light. Because endemic infection of the entire colony was considered likely, treatment of all chinchillas was initiated. Systemic treatment with antifungals was contraindicated for experimental reasons therefore miconazole nitrate antifungal powder was administered in the dust bath. Medicated dust baths were initially offered for 1 hour, 3 times per week, for 12 weeks with additional application of antifungal powder directly to any skin lesion. DTM culture was negative at 6 weeks and PCR for dermatophytes was negative at 8 weeks. Antifungal powder was incorporated into the weekly dust bath enrichment to prevent future outbreaks. Due to the zoonotic nature of dermatophytosis, personnel were educated on best practices and handling techniques to reduce risk of infection and spread to other animals.

P46 Oral Analgesic Delivery Method Refinement to Improve Animal Welfare in DBA/1J Mouse Model

AC Allaire^*

Disposition, Safety and Animal Research, Sanofi Genzyme, Framingham, MA

An effort to refine delivery methods of orally administered butorphanol in the collagen-induced arthritis (CIA) DBA/1J mouse model examined an alternative approach to improve consistency in analgesic coverage. CIA triggers an autoimmune reaction causing the degradation of cartilage, hyperplasia of joint space, and turgescence of limbs varying from mild to severe. Due to disease symptoms, animals require continuous, minimally invasive pain management during a study’s in-life phase. A pharmacokinetic study was designed to evaluate the uniformity of butorphanol delivery in a liquid gel water replacement in comparison to inhouse-made butorphanol “shake” at a concentration of 1 mg/ml. Butorphanol delivered in shake must be replenished daily and delivered via petri-dish, while liquid gel water replacement can be delivered into a cage via water bottle with a volume sufficient for 4 days of animal dosing. Naïve DBA/1J mice were used to mimic the CIA model. Animals were allocated into 3 groups of 10: a non-dosed control, butorphanol delivered via shake, and butorphanol delivered via liquid gel water replacement. Animals were acclimated to a group-specific delivery method for 24 hours. Following acclimation, micro-sampling of venous blood was performed at 1-hour, 3-hour, and 6-hour post butorphanol introduction daily, for 4 days to measure long-term blood levels. Following shake administration, blood concentrations of butorphanol were variable, ranging from 4.1ng/mL (+/-6.7ng/mL) to 3210.0ng/mL (+/-3920.0ng/mL) over the 4 days studied. In comparison, the fluctuation in blood concentrations of butorphanol after gel administration was less, with a range of 4.1ng/mL (+/-8.52ng/mL) to 199.0ng/mL (+/-111.0ng/mL). The data collected demonstrates that higher butorphanol concentrations were achieved in the shake group, but the liquid gel delivery method consistently maintained a uniform dosage of butorphanol throughout the cage population. Additionally, liquid gel water replacement can be delivered up to 4 days without causing disruption to the cage. Further study in CIA studies is planned to determine efficacy of analgesic administration and to determine serum levels correspondent to clinical analgesia.

P47 Evaluation of Exhaust Air Dust PCR Testing of the Bedding Sentinel Cage Filter

NA Monts de Oca¹, L Gray², AM Williams^*2, DW Brammer²

¹Research Models—Houston, Charles River, Houston, TX; ²Animal Care Operations, University of Houston, Houston, TX

Exhaust Air Dust (EAD) PCR testing has been investigated on a variety of Individually Ventilated Cage (IVC) rack types. Previous studies suggest that cage-level filtration on some IVC racks may retard or prevent agent-associated DNA from efficiently passing into plenums thereby compromising detection of plenum sampling. Other reports evaluating 3 different rack systems suggested that the filter in the sentinel cages may serve as a better sampling medium than the actual sentinel rodent. Our facility uses an IVC rack that provides cage-level filtration; however, EAD has not previously been evaluated nor reported in this type of rack. This caging system employs the use of triangular-shaped cages and is exhausted directly into the building exhaust with an air exchange rate of 15-25 ACH at the cage level. At the beginning of a 3 month screening period, two 4-6 week old CD-1 female sentinels were placed in one cage on each of four racks. 10 cm³ of dirty bedding were transferred weekly to the sentinel cage and to another cage on each rack containing no mice. A new filter was placed in both cages on each rack and transferred to new cages as cage cleaning and bedding transferred occurred. At the end of the 3 month period, sentinel mice were evaluated by traditional screening methods and sentinel cage filters were evaluated by PCR to detect rodent pathogens. MNV and Entamoeba were both equivalently detected by both sentinel cage filters and in the sentinel mouse. Helicobacter, P. pneumotropica, P. mirabilis, Klebsiella, Spironucleus m., and Tritrichomonas were best detected using EAD PCR testing of the sentinel cage. Furthermore, the filter taken from the sentinel cage detected a substantially higher number of agents when compared to the filter from the cage that did not house sentinel mice. This finding suggests that the presence of the mice may be important to agitate and transfer material from dirty bedding onto the filters.

P48 Diagnosis and Treatment of the Mite Archemyobia (Nearchemyobia) latipilis Found in the Brazilian opossum (Monodelphis domestica)

BD Smith^*

Department of Veterinary Pathobiology, Comparative Medicine, Texas A&M University, College Station, TX

Routine testing for parasites, such as for mites and pinworms in an animal facility, is a typical part of a quality assurance program. The Guide for the Care and Use of Laboratory Animals provides for surveillance, diagnosis, treatment, and control of diseases within a vivarium, but is widely left to the discretion of the veterinarian. Species which are not widely used in the research setting also present a complicating factor as often there is a deficiency in normal values or diagnostic methods. Such was the case encountered with Monodelphis domestica, also known as the Brazilian or gray short-tailed opossum. Recently during necropsy, mites of unknown origin were recovered on histopathology of the dermis. After failure to obtain mites via tape testing, skin scraping under inhalant anesthesia was determined to be the most successful method to isolate parasites for further classification and to determine colony prevalence. These mites were found throughout the entire colony and identified as Archemyobia (Nearchemyobia) latipilis, which were recognized in 1979 when the original opossums were imported into the United States. While mites are generally host specific, there was concern for spread to other species of animals housed in the facility, including mice, rats, rabbits, and guinea pigs, however routine sentinel testing has been negative for any type of mites. Ivermectin was initially chosen for treatment to eradicate the infestation as compared to using selemectin or moxidectin, which both had variable success for mite outbreaks in mice and rats. Initial low dose oral treatment at 0.2 mg/kg in propylene glycol was not suitable to eliminate the mites, therefore additional routes of administration and dosages were employed. The route of administration did not appear to affect the success of treatment, but a topical dose of 2.0 mg/kg in either mineral oil or propylene glycol was necessary to eradicate the mites while also being less stressful, easier to administer, and did not produce any obvious toxic effects. At a 6-week recheck, all treated individuals remained negative for the mites, and treatment to the entire colony other than pregnant or lactating females was performed.

P49 Red-Tinged Urine in Arctic Ground Squirrels (Spermophilus parryii)

CP Jones^*2, DM LeMay¹, ME Maddox¹, P Austin¹, EN Yu²

¹Animal Care and Use Program, Division of Animal Care, Vanderbilt University Medical Center, Nashville, TN; ²Pathology, Microbiology, and Immunology-Division of Comparative Medicine, Vanderbilt University Medical Center, Nashville, TN

An adult, male, wild-caught Arctic ground squirrel (Spermophilus parryii) presented within days of arrival with red-tinged urine in the litter pan. Urinalysis showed no red bloods cells, no hemoglobin, a high pH, and struvite crystals. The animal was bright, alert, responsive, and hydrated, with no other abnormal clinical findings. Normal ranges are unestablished for Arctic ground squirrel urinalysis, so we performed urinalyses for the entire colony to establish baseline parameters. Samples were collected from clean, unlined pans of 5 male and 6 female individually housed Arctic ground squirrels. Individual ages were not available, but ranged from juvenile to adult. All samples were evaluated visually, by urine dipstick and by sediment microscopy. Urine color varied from pale yellow to dark orange (red-tinged). Stored samples became more red-tinged over time. All samples were negative for red blood cells and hemoglobin. Average urine specific gravity was low (1.014) and average urine pH was high (8.5 or higher). Protein was present in the urine of all animals. Struvite crystals were common, as were amorphous crystals with brown, globular material. Red-tinged urine may be a normal finding in Arctic ground squirrels kept in the laboratory, as well as high urine pH and struvite crystals. The amorphous crystals with brown globular material could account for the proteinuria and red-tinged urine color in this species.

P50 Enhancing Gene Delivery in Guinea Pigs Using an Electroporation Device

C Chiedi^*1,2, M Dillon^1,2, DG Scorpio¹

¹Vaccine Research Center, Bethesda , MD; ²SoBran, Inc, Bethesda, MD

Electroporation (EP) is an innovative biologic technique in which an electrical field is applied to cells in order to temporarily disturb the phospholipid bilayer. Increasing cell membrane permeability allows molecules like DNA or protein to be introduced into the cell more effectively. The ease of use, reproducibility, high efficiency, and low toxicity has made electroporation the method of choice for introducing many types of molecules into cells including mammalian, bacterial, yeast plant, and insect. Increasing cellular uptake is particularly important to enhance the potency of test articles such as DNA vaccines. For our institute, the standard method of delivering these test articles to guinea pigs is via a simple intramuscular injection with needle and syringe. Since investigators achieved suboptimal test results using the standard delivery method alone, electroporation may enhance this method by increasing cellular uptake, optimizing long-term gene expression. Here, we present and illustrate the steps used to prepare the animal and perform an electroporation procedure on an anesthetized guinea pig using a facility approved electroporation device. The electroporation process involves a few variables, including field strength, pulse length, and pulse shape which is species and device specific. Field strength and pulse length can be manipulated with in approved parameters in order to maximize the efficiency of gene transfer, while pulse shape is dependent on the type of pulse generator used.

P51 Whole Blood Transfusion in Mice

CB Volpe^*

Division of Laboratory Animal Medicine, University of Los Angeles—California, Los Angeles, CA

6-thioguanine (6TG) is a chemotherapeutic drug used to treat leukemia. A side effect of the agent is low blood cell counts and poor appetite. Mice receiving this treatment appear to experience the same side effects, leading to discontinuation of the study or premature euthanasia. Even with additional supportive care, the health of the mice continued to decline. To continue on the study, they needed to increase their body condition, weight, and their blood cell count. It was hypothesized that a transfusion of whole blood would increase the blood count and aid the mice with increased health and ability to thrive. We used retired breeding NSG mice as the nonsurvival donors. Blood was collected under anesthesia via cardiac puncture using a heparinized syringe. If no clots were seen, the blood was transfused into the recipient mouse via the tail vein. A total of 0.3ml blood was transfused. Immediately following the transfusion, the mice were released from restraint and monitored. Weights were taken post transfusion as a reference to their improved health. The transfused mice continued on study as their weights increased and the anemia subsided. Increased body condition and activity was seen in all recipients. Secondary transfusions have been performed with similar favorable outcomes. Whole blood transfusions in mice were performed solely as a treatment for animals receiving 6-thioguanine. All mice receiving transfusions have been able to remain on study rather than halting the treatments or having to be prematurely euthanized.

P52 Keratoacanthoma in an Adult Zebra Finch (Taeniopygia guttata)

CM Bodi^*, V Bakthavatchalu, M Siddalls, MM Patterson

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

An adult male zebra finch (Taeniopygia guttata) presented with an ulcerated cervical mass and poor body condition. Because of its declining health, the bird was euthanized and a diagnostic necropsy was performed. A 3.0 x 4.0 mm, nodular, dark brown dermal mass was attached to the ventral aspect of the neck. Otherwise there were no unusual necropsy findings. Histologic evaluation of the abnormal tissue revealed an unencapsulated, well-differentiated neoplasm disrupting the dermal architecture and elevating the overlying epidermis. The neoplasm was composed of epithelial cells arranged in cords that were separated by fibrovascular connective tissue. Immunohistochemically, the neoplastic cells stained positive for pan-cytokeratin. Based on the histologic and immunohistochemical findings, the mass was diagnosed as an avian keratoacanthoma, also known as squamous cell carcinoma of the skin or squamous epithelioma. This is the first reported case of a keratoacanthoma in a zebra finch; such tumors are rare in passerines overall. It has been speculated that various factors (e.g., skin irritation, overgrooming, low humidity) can exacerbate early lesions leading to keratoacanthoma development. Though benign, these tumors have the potential to become invasive. They should be carefully monitored and surgically removed if normal function is compromised. Keratoacanthomas should be added to the list of possible dermal lesions in laboratory-reared songbirds.

P53 Clinical Management of Immunosuppressed Swine in a Transplantation Model

AD Werts^*1, K Kolegraff², CR Moats¹, AE Goodroe¹, G Furtmuller², G Brandacher², CM Garrett¹

¹Molecular and Comparative Pathobiology, Johns Hopkins Univeristy, Baltimore, MD; ²Department of Plastic and Reconstructive Surgery, Johns Hopkins Univeristy, Baltimore, MD

Reconstructive transplantation using vascularized composite allografts (VCAs) has become a promising option for patients with amputation or other devastating injuries affecting the face, trunk, or even genitalia. Current protocols, however, require life-long immunosuppression to prevent allograft rejection, and these regimens can lead to serious complications such as infection and malignancy. To investigate novel strategies of immune modulation and the induction of tolerance, a VCA model in miniature swine has been developed which combines nonmyeloablative total body and thymic irradiation with short-term tacrolimus-based immunosuppression. Similar to severely immunosuppressed human patients, a major complication of this model has been life-threatening infections. Clinical manifestations ranging from pneumonia (Streptococcus suis, Klebsiella oxytoca, Pneumocystis carinii), sepsis (Enterococcus spp.), septic arthritis (Klebsiella oxytoca, Pseudomonas spp., MRSA) and endocarditis (Escherichia coli), to posttransplant lymphoproliferative disease (PTLD) and a wasting syndrome have all been documented within our model. Animals with PTLD and signs of wasting were confirmed to have one or more viral infections (e.g., cytomegalovirus, porcine circovirus, porcine lymphotropic herpesvirus). Infection has appeared independent of the specific immunosuppression regime used, but has been traced to a specific nidus (e.g., central venous catheters) in several instances. Pathogen screening and exclusion has proven to be challenging; many of these agents are environmental contaminants or endogenous to this particular line of miniature swine. In order to reduce the overall incidence of clinical disease, a customized preventative health and diagnostics program was established. This program includes antibiotic and antiparasitic prophylaxis, vaccination, and postoperative diagnostics aimed at rapidly linking complex clinical presentations to underlying disease etiologies. As immunosuppressed miniature swine models continue to evolve, we anticipate the clinical data established at our institution over the past half decade will serve as a unique knowledge base for medical doctors, scientists and veterinarians across the sectors of translational medicine.

P54 Sudden Death in a Black-Tailed Prairie Dog (Cynomys ludovicianus)

CM Tansey^*, JM Gary, N Gallardo-Romero, C Hutson

Center for Disease Control, Atlanta, GA

An adult, 644-gram, intact, colony housed, wild-caught male black-tailed prairie dog (Cynomys ludovicianus) was found dead during morning rounds. No previous clinical signs were reported. The animal was submitted for necropsy. Upon gross examination, the great vessels were firm and white. The stomach was adhered to the liver by fibrosis, and was thickened and firm with multifocal mucosal and serosal hemorrhages. The kidneys were shrunken with a rough subcapsular surface. Histopathology showed multifocal metastatic mineralization in multiple organs, including the great vessels and gastric submucosa. The suspected cause of the histopathologic findings in this animal was severe renal disease, which included tubular degeneration and regeneration, glomerulonephropathy, and interstitial nephritis. Differentials for these renal lesions include a chronic progressive nephropathy-like condition, bacterial infections such as Leptospirosis, and age-related degenerative changes. Metastatic mineralization is not commonly reported in the literature on Cynomys species, but should be suspected as a sequela to renal disease in animals presenting with poor hair growth, hair loss, muscle stiffness, bone deformities, nephrosis, and death.

P55 Minipig Jacket Design-Enabling Simultaneous Electrocardiogram Collections and Dermal Dose Administration

CL Savidge^*, C Artis, A Camacho, JE Sentz, M Miyamoto

Envigo, Princeton, NJ

The minipig is quickly becoming a widely accepted scientific model in toxicology studies with a cardiac component, due to the similarity between the pig and the human heart. Because of this similarity, the ability to collect continuous electrocardiogram parameters on a variety of minipig study designs is critical to the scientific community. In the past, these critical data parameters have been avoided on minipig dermal toxicity studies because the design of the jacket used to secure the electrodes in place for electrocardiogram collections would interfere with the procedure and location for dose application. The most common dose site used for dermal application on the minipig is across the dorsal area and onto the flanks of the animal, totaling approximately 10% of the total body surface area based on bodyweight. This dose site does not interfere with the placement of the electrodes for electrocardiogram collections, however the jacket commonly used to secure the electrodes and house the transmitter covers the entire trunk of the animal, thus creating the challenge of performing both dermal dosing and electrocardiogram tracings simultaneously. By modifying the jacket design to have an opening large enough to allow for the entire dermal dose site to be unaffected during the collection process, but secure enough that the electrodes stay in place, we are able to successfully add electrocardiogram collections on any minipig dermal toxicity study.

P56 Troubleshooting an Acute Complication of Cell Transfer in C57BL/6J Mice

DE Collins^*, C Fry, I Bergin, JA Nemzek

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

Adoptive transfer of autologous cells offers a unique approach to immunotherapy and is easily modeled by intravenous (IV) injection of purified cells. In IACUC-approved immunotherapy studies with fibrocytes, there were no notable complications in ICR mice after tail vein injection (1x10⁶ cells/100ul saline). However, severe respiratory complications developed after autologous cell transfer in C57BL/6J mice. In total, 6 out of 21 (28% loss) mice decompensated within 2-3 minutes of injection, displaying dyspnea and recumbency that required euthanasia. A comprehensive literature review revealed reports of dose-dependent pulmonary thromboembolism in the pulmonary capillaries and arterioles of mice after IV administration of mesenchymal stem cells and collagen respectively. Fibrocytes are similar in size to mesenchymal stem cells with a range of 15-20 uM and also secrete collagen in culture. The lungs of some animals (n = 3) were submitted for histopathology and special staining. Thromboemboli were observed affecting at least 30% of pulmonary arterioles. The thromboemboli contained fibrin, evidenced by blue staining with PTAH. Pink granular material suggestive of platelet, cellular, or granular collagen debris was also present, but organized collagen was absent, as indicated by a lack of blue staining with Masson’s trichrome. The histopathology suggests that degenerate cells or extracellular matrix debris may lodge in pulmonary arterioles, triggering acute thrombosis. The reason for strain differences in the occurrence of this complication is unknown. Several measures were put in place to ameliorate these adverse effects, including improved positive isolation of fibrocytes from primary cultures, extra filtration steps, and doubling of the injection volume. These combined changes reduced mortality to 2 out of 26 animals (7.6% loss) in subsequent experiments. The distribution of this information may help other labs experiencing similar problems, as adoptive transfer injection associated mortality is not widely covered in the veterinary or laboratory animal literature.

P57 Facial Abscesses in NOD-SCID Mice Weaned from a Breeding Female with Abscessing Pyelonephritis

DM LeMoine^*, C Keller, S Lewis

The Ohio State University, Columbus, OH

Eleven NOD-SCID mice in a breeding colony managed by a single principal investigator presented with subcutaneous nodules affecting the muzzle, mandible, and submandibular regions. The colony was housed in 9 sterile individually ventilated cages, provided irradiated rodent chow and reverse osmosis water ad libitum, and all husbandry activities were conducted within a disinfected biosafety cabinet. Cytology, culture, and sensitivity from 1 mouse led to a diagnosis of abscesses containing enrofloxacin-sensitive Staphylococcus sciuri. Evaluation of the breeding records revealed that all affected mice had been weaned from a single founder cage. A second founder trio and its offspring comprised the unaffected half of the colony. Enrofloxacin treatment of all affected cages was initiated, but all treated animals (n = 13) were subsequently euthanized due to lack of response to therapy. S. sciuri was cultured from lesions in all affected mice. All tissues from the affected founder female and representative tissues from one of the affected offspring were submitted for histopathology. Findings in the founder female included maxillary tooth root abscess, focal abscessing myositis of the epaxial muscle, unilateral abscessing pyelonephritis, and bilateral otitis media. Lesions in the offspring mouse were limited to multifocal subcuticular abscesses on the head. The simultaneous presentation of several mice with facial abscesses has never been seen at our institution. Staphylococcosis is considered an opportunistic infection predisposed to by compromised immune status, skin wounds, and trichotillomania. At presentation, none of these mice had clinical signs suggesting a route of exposure. S. sciuri is a ubiquitous organism that has been isolated from healthy laboratory mice, but has also been implicated in human and swine diseases. We suspect that a contamination event caused disease and bacterial shedding in the founder female, leading to colonization and disease in most of the cohoused animals. Our standard husbandry practices limited the infection to animals originating from that single cage, but the source of exposure remains unclear. Constant adherence to aseptic technique by all personnel is critical for maintaining the health of immunocompromised mouse strains.

P58 Endometriosis Associated with a Malignant Tumor in a Rhesus Monkey (Macaca mulatta)

EH Curran^*, F Connor-Stroud, S Wilson-Cox, A Gsrcia

Yerkes National Primate Research Center, Atlanta, GA

A 15-year old female rhesus macaque (Macaca mulatta) was presented for physical examination in March 2014 due to abdominal bruising and petechiation on her forearms and legs and an enlarged firm uterus. Based on clinical findings endometriosis was diagnosed. Complete blood count and chemistry were within normal limits; however, the coagulation profile showed an increased partial thromboplastin time. A few months later, the animal was reevaluated due to dysmenorrhea and recurrent abdominal bruising. She was started on meloxicam for potential discomfort and medroxyprogesterone to regulate her menstrual cycles. The animal was euthanized and submitted for postmortem examination in August 2015 at the completion of study. Postmortem examination revealed extensive adhesions of the abdominal viscera. A “chocolate cyst” was noted in the omentum and several dark to light red masses were present on the uterine serosa. On cut section, the uterus had multiple small white nodules. Sections of the uterus and associated tissues were submitted for histologic evaluation. Histologically, the myometrium had multifocal cystic areas and severe proliferation of round to oval cells intermixed with bizarre multinucleated giant cells, degenerative neutrophils, and necrotic debris. The neoplastic cells were predominantly round and moderately pleomorphic with vesicular round nuclei and eosinophilic cytoplasm with distinct borders. Similar neoplastic cell aggregates were observed within the endometrium and adjacent tissues. Many glands were severely dilated and contained an amphophilic secretory material. Immunohistochemical analysis of tumor cells was positive for vimentin and cytokeratin. Malignant transformation of endometriosis in humans has been described in numerous case reports and literature reviews. Approximately 1% of women with endometriosis have lesions that undergo malignant transformation. In nonhuman primates, endometriosis is relatively common but malignant transformation is rare. To the authors’ knowledge this is the first report of malignant neoplasia associated with endometriosis in a rhesus monkey.

P59 Comparison of Intravenous Anesthetic Protocols for Chemical Restraint in Multiple Macaque Species

EA Nunamaker^*2, LC Halliday¹

¹Biologic Resources Laboratory, University of Illinois at Chicago, Chicago, IL; ²Animal Care Services, University of Florida, Gainesville, FL

Adequate monitoring during anesthesia in Old World primates is critical for adjusting and maintaining proper anesthetic depth. It is essential to have an understanding of both the plane of anesthesia, as well as the maintenance of adequate tissue perfusion during anesthesia. Furthermore, anticipating the deleterious effects of an individual agent in a given species helps minimize complications associated with anesthesia. Opting for combinations of inhaled and/or injectable anesthetic agents provides flexibility to tailor anesthetic protocols to specific animal health and experimental needs. Thirty-four macaques (10.4 ± 4.6 y), male and female, Macaca fascicularis, Macaca mulatta, and Macaca radiata undergoing routine dental prophylactic cleanings were enrolled in the study to compare continuous infusions of propofol or ketamine and midazolam (KM) to inhaled isoflurane. Animals were randomly assigned to treatment groups and the average anesthetic duration was 53.1 ± 17.5 m. The effects of species, sex, body weight, body condition score, age, and anesthetic agent on extubation time, recovery time, heart rate, temperature, and blood pressure were evaluated using a repeated measures mixed model. There were no significant differences between the anesthetic groups on either extubation (14.0 ± 11.9 m) or recovery times (37.2 ± 17.3 m). Heart rate was significantly affected by the interactions between time and anesthetic agent, sex, age, and body condition score. Heart rate was highest over the anesthetic period in animals receiving KM. Body temperature was significantly affected by time and the interactions between time and anesthetic agent and time and species, trending to be highest after 15 minutes of anesthesia in animals receiving KM. Mean arterial pressure was significantly affected by time and the interactions between time and anesthetic agent and time and body condition score. Mean arterial blood pressure was highest over the anesthetic period in animals receiving KM (107.8 ± 25.5 mmHg), compared to isoflurane (66.8 ± 21.5 mmHg) and propofol (49.5 ± 20.6 mmHg). The results highlight the benefit of tailoring an anesthetic regimen to the specific animal and experimental needs to minimize the negative physiologic effects of anesthetic agents.

P60 A Prospective Analysis of Causes of Hind Limb Paralysis in Research Mice (Mus musculus)

E Bryant^*, C Autieri, JG Fox, S Muthupalani

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Hind limb paralysis is an infrequent cause of euthanasia in research mice and there is a paucity of studies in the literature describing the pathologic basis of this clinical condition. In mice, acute hind limb paralysis can occur from various causes, including spontaneous or experimental tumors (metastatic or primary) involving the spinal cord or the brain, infectious processes, traumatic, degenerative or toxic neuropathies, or anomalous causes. To better elucidate the etiopathogenesis of sporadic hind limb paralysis in mice, we undertook a multiyear intrainstitutional study to clinically identify and perform a detailed pathologic evaluation of all mice exhibiting signs of hind limb paresis and/or paralysis. During this ongoing study of nearly 30 months, we included all mice with a clinical presentation of hind limb paralysis, regardless of sex, strain, age, and manipulation status. We have evaluated 20 mice with clinical signs of hind limb paralysis and 5 mice with no signs of hind limb paralysis. The various causes of hind limb paralysis in these 20 mice were the following: metastatic lymphoma involving the thoracolumbar spinal cord (n = 8), spinal cord sarcoma (n = 2), known history of injection of orthotopic tumors (n = 3, two of which had adenocarcinoma involving the spinal cord, and the third had a poorly differentiated sarcoma), vertebral body and spinal cord abscesses (n = 2, presumed bacterial), multifocal necrohemorrhagic and degenerative myelopathy of undetermined cause (n = 2), acute neutrophilic myelitis (n = 1, etiology unknown), polyarteritis involving multiple visceral vasculature as well as the intercostal vessels supplying the spinal cord and nerve roots (n = 1), and a focal epidermoid cyst causing spinal cord compression (n = 1) . Additionally, immunohistochemical analyses are being performed to further classify the neoplasms involving the spinal cord. The data from this diagnostic study increased our understanding of spinal paralysis in various strains of mice and its underlying etiopathogenesis.

P61 An Approach to Spontaneous Cardiopulmonary Arrest in Nonhuman Primates

E Bryant^*, JG Fox, RP Marini

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Spontaneous cardiopulmonary arrest is a rare but serious potential problem in nonhuman primate research colonies. Although primates have been used (albeit infrequently) as models of resuscitative therapy following induced arrest, occasionally veterinary personnel are required to respond to an unexpected spontaneous cardiopulmonary arrest. Animals undergoing sedation or anesthesia are at higher risk for adverse events, given that the agents used to achieve sedation and anesthesia have the ability to depress pulmonary and/or cardiac functions. Nonhuman primates are often sedated for routine procedures, such as physical examinations, and therefore have a higher risk for adverse events than other species that do not have to be sedated for handling. At our institution, we have devised a coherent, straightforward flow chart detailing the sequence of events that should take place following an arrest, based on recommendations from the Veterinary Emergency and Critical Care Society RECOVER documents and human pediatric guidelines. The flow chart is included in all rooms that have the greatest potential for a spontaneous cardiopulmonary arrest, such as areas in which primates are sedated for physical exams, surgical preparation rooms, and operating rooms. We have also developed a standard operating procedure for training research and veterinary staff to respond immediately and efficiently following an arrest. In addition, we have developed a protocol for stocking and maintaining our emergency supply cart with relevant pharmaceuticals and supplies, such as endotracheal tubes and catheters of various sizes, manual resuscitation self-inflating bags, and other relevant emergency supplies. As part of this emergency supply cart, a short, coherent pictorial guide depicting the locations of various items contained in the cart is included. The goal of this management initiative is to decrease morbidity and mortality of primates undergoing sedation or anesthesia for routine procedures by implementing protocols to achieve a fast and efficient response in the event of a cardiopulmonary arrest.

P62 Low Reproductive Performance Associated with Mycobacterium Infection and Egg-Associated Inflammation and Fibroplasia in a Zebrafish (Danio rerio) Colony

GJ Barnett^*, J Rodenfels, SR Compton, CJ Zeiss

Comparative Medicine, Yale University, New Haven, CT

A colony of zebrafish (Danio rerio) experienced decreased reproductive performance and increased incidence of coelomic distension in females. Two adult females were initially submitted for necropsy, which revealed egg-associated inflammation and fibroplasia, with acid-fast rods in retrocoelomic tissue and ovaries. Forty-seven adult female zebrafish from the colony were subsequently submitted for necropsy and PCR diagnosis of Mycobacterium species. Histopathology revealed two distinct patterns of intracoelomic disease: 1) extensive fibro-granulomatous sheets scattered throughout the coelom with multiple, discrete granulomas containing acid-fast rods and 2) oocyte death within the posterior coelom, accompanied by variable oviduct obstruction. Granulomatous inflammation at the interface between live and degenerate oocytes was noted; however, these regions did not contain acid-fast rods. While these histopathological changes were distinct from each other, clinical presentation was identical between all affected animals. Ovarian acid-fast rods were found in 4 out of 6 individuals that were PCR-positive for Mycobacterium chelonae/abscessus in ovarian tissue (the two species of Mycobacterium were indistinguishable by PCR). Liver PCR identified M. chelonae/abscessus in 2 animals despite absence of pathology in extraovarian tissues. Neither acid fast rods nor inflammation was noted in the liver or other tissues. To investigate the source of Mycobacterium, multiple components of the housing system were tested by PCR, and a hose containing recirculated, filtered water prior to its introduction into the system was found to be positive for M. chelonae/abscessus. Failure of routine decontamination of embryos prior to introduction into the colony was ruled out as a source of bacterial seeding into the colony. To our knowledge, this is the first time that the combination of egg-associated inflammation and fibroplasia and Mycobacteriosis has been observed to have contributed to low reproductive performance discovered in a zebrafish colony. These data indicate that 2 distinct syndromes, egg-associated inflammation and fibroplasia and ovarian Mycobacteriosis may contribute to a single, clinically indistinguishable syndrome of decreased reproductive performance.

P63 Cranial Vascular Hamartoma in a BALB/c Mouse

HR Holcombe^*, V Lau, P Chamberlain, JG Fox, V Bakthavatchalu

Massachusetts Institute of Technology, Cambridge, MA

Thirty 7-week-old female BALB/c mice were obtained from a commercial vendor on March 8, 2016. A mass was noted on the top of the head of 1 of the mice 6 days later. The mouse was unmanipulated, and the investigator elected to euthanize the animal without additional diagnostic testing other than necropsy. Grossly, the cranial mass was observed to be firm, red, roughly spherical in shape, and measuring approximately 9 mm in diameter. Histologic examination demonstrated that the mass originated between the meninges and the skull. The connective tissue stroma overlying the cranium was disrupted and expanded by encapsulated densely packed bundles of spindle cells separating blood-filled vascular channels. The vascular channels were lined by endothelium and surrounded by smooth muscle and collagen bundles. Based on these findings, the mass was determined to be a vascular hamartoma. The primary differential would be hemangioma; however, hemangiomas lack all of the supporting structural elements of a normal blood vessel. Vascular hamartomas are developmental malformations that result in overgrowth of disorganized mature vascular tissue. Spontaneous hamartomas are rarely reported in rodents, with only a single report of a vascular hamartoma in the uterus of a Sprague–Dawley rat. Although hamartomas tend to be benign and are typically considered incidental findings, the size and location of the mass in this mouse made the animal unsuitable for research purposes.

P64 Developmental Abnormalities in Zebrafish (Dana rerio)

JR Owiny^*

Colorado State University, Fort Collins, CO

Zebrafish (Danio rerio) are an increasingly important model organism for a wide range of basic science and disease studies. One investigator has a small zebrafish facility containing 8 racks housing about 250 tanks primarily to generate embryos for developmental studies. The fish are on a recirculating system with deionized, charcoal-filtered and ultraviolet-sterilized water. The system adds bicarbonate and a sea salt mixture to maintain a pH of 7.5-8 and conductivity of 300-1200 µS. Water temperature is held at 23-28°C. A staff member setting up fish for spawning reported to the veterinary staff that several fish appeared to have reddening of their branchial region. An examination by a veterinarian revealed that the fish were behaving normally and water quality was within normal limits. Affected fish were distributed over 5 tanks, and were transgenic on a WIK background. The majority of affected fish had lesions on the left opercular region, although a couple had bilateral lesions. Three fish were submitted for necropsy, and the remaining affected tanks were taken offline. Differential diagnoses included trauma, possible developmental abnormalities, neoplasia, poor nutrition, branchiatis, or an infectious case. A preliminary pathologic diagnosis included unilateral chondro-osseous malformation of the operculum in 2 fish. The third fish had mild opercular lymphocytic dermatitis. The lesions were suspected to be congenital and may represent a phenotype associated with the background or transgenic manipulation of the fish.

P65 Constrictive Tail Lesions in Multiple Litters of Gray Short-Tailed Opossum (Monodelphis domestica)

JM Criley^*, J Ludwig, LJ Goodly

University of Illinois, Urbana, IL

A breeding research colony of approximately 100 adult gray, short-tailed opossums (Monodelphis domestica) is maintained for biomedical research projects focused on developmental biology. The colony is maintained in individually ventilated cages designed for rats in an AAALAC-accredited animal facility. Temperature and humidity ranges are recorded daily. Animals are housed on recycled paper bedding with nesting jars and shredded paper towel nesting material and fed a commercial diet supplemented with treats. Over an 8-year time span, 8 litters of Monodelphis presented with annular tail constrictions resulting in inflammation and necrosis distal to the lesions. Animals presented with lesions between 1 and 2 months of age and typically not all individuals in a litter were affected. Response to topical lanolin therapy varied. Lesions resolved with mild scarring or with sloughing of the distal tail. The histologic changes included acanthosis and hyperkeratosis with extensive edema similar those described for ringtail in rats. The etiology of the condition is unknown but may have environmental, genetic, and/or nutritional factors as suggested in rodent ringtail.

P66 Routine Cage Wash Cycle Does Not Clear DNA From Cage Lid Filters When Tested By Polymerase Chain Reaction

JS Hubbard^*, P Chen, K Boyd

Vanderbilt University Medical Center, Nashville, TN

Common mouse parasites in modern laboratory settings include Myobia musculi, Rhadfordia affinis, and Myocoptes musculinus (mouse fur mites) and Syphacia obvelata and Aspicularis tetraptera (mouse pinworms). A recent dual infestation of mouse fur mites and pinworms led to an evaluation of current practices regarding PCR testing for these parasites. These organisms are routinely detected by microscopic examination or by PCR analysis on samples collected directly from the mouse. However, recent studies advocate PCR testing of environmental samples such as plenum swabs and cage lid filters in place of animals. Currently there is limited information surrounding the effect that standard cage wash has on DNA degradation or clearance from housing material and how this affects PCR results. To test the efficacy of a routine cage wash cycle on DNA clearance from cage lid filters, we collected filter samples from fur mite and/or pinworm positive cages (n = 5) pre- and post-cage wash. Standard cage wash at our institution is defined as an 8-minute cycle which reaches a minimum temperature of 180°F and includes an alkaline wash/rinse cycle followed by an acid wash/rinse cycle. All samples were submitted to a commercial diagnostic laboratory for PCR fur mite and pinworm analysis and pre- and post-cage wash results were compared. Post-cage wash results varied from positive (n = 2), negative (n = 2), to inconclusive (n = 1). There were also 2 cases in which different agents were identified on the post-wash filter when compared to the same filter pre-cage wash. Our results show that a routine cage wash cycle does not degrade or clear DNA from the cage lid filter. This information must be taken into consideration when designing surveillance testing schemes and interpreting PCR results using cage lid filters.

P67 Observation and Intervention of Stereotypic Behaviors in Raccoons (Procyon lotor) in a Laboratory Animal Setting

JM Jarrell^*, AE Sparks, S Lewis

University Laboratory Animal Resources, The Ohio State University, Grove City, OH

Standard housing systems, environmental enrichment, and species-specific natural behavior is well established in common laboratory animal species. When animals that are not routinely used in a research setting are introduced, these principles can become challenging. We received 10 purpose-bred raccoons (Procyon lotor) from a commercial furrier. Upon arrival, one raccoon was observed engaging in a repetitive locomotion stereotypic behavior. Briefly, the raccoon would stand on its hind limbs and “weave” in a vertical pattern. This behavior persisted despite the provision of physical, food, and human-interaction environmental enrichment. Within a few weeks, the raccoon in the adjacent cage appropriated the behavior. In an attempt to ameliorate the behavior, an ethogram-based direct observation scoring system was developed to assess which raccoons were thriving in the environment and which were not adapting as readily and/or displaying stereotypic behaviors. This scoring system provided objective information on which animals to house adjacent to one another: high (positive adaptation) and low scoring (negative adaptation) animals. After 1 week, the positive animal’s score decreased slightly as it appropriated the stereotypic behavior, but the low scoring animal’s number increased, indicating less frequent stereotypic behaviors. This trend continued through week 2 of the study, but by week 3 a significant decrease to complete absence of stereotypic behaviors observed in both animals which has been maintained. To the author’s knowledge, this is the first report of stereotypic behaviors of Procyon lotor in a laboratory animal setting.

P68 T-cell Lymphoma in a Gilt (Sus scrofa)

JA Scholz^*1, TR Meier¹, C Frisk¹, J Brazzell²

¹Department of Comparative Medicine, Mayo Clinic, Rochester, MN; ²Marshfield Labs, Marshfield, WI

A 1.5-year-old transgenic gilt with a targeted disruption of the alpha 1, 3 galactosyltransferase (GGTA-1) gene presented for thin body condition, lethargy, and decreased appetite. Prior to presentation, the gilt had no major health issues and was experimentally naïve; a recent change in her environment occurred a week earlier when the animal’s pen mate was moved to a different room to farrow. On physical examination, the gilt was noted to have increased respiratory effort with normal heart rate and body temperature. Primary rule-outs included pneumonia and gastric ulceration, and the animal was started on a proton pump inhibitor and given a long-acting cephalosporin. Food supplements and electrolyte water were offered for supportive care. While the gilt initially showed signs of improvement, condition declined over the next 4 days. The respiratory rate persistently increased and muffled heart and lung sounds were noted on the ventral left side. Due to poor health and lack of response to treatment, euthanasia was elected. Significant gross findings included bicavitary effusion with fibrinous serositis; collapsed lungs; ulceration of the esophagus and pars esophagea; multifocal firm white to light yellow hepatic and splenic nodules; markedly enlarged cervical and thoracic lymph nodes; and a pericardial abscess. Histology of the lymph node masses was consistent with high-grade lymphoma. Immunohistochemistry of the neoplastic cells displayed strong positivity for CD3 while neoplastic cells were diffusely negative for CD20. In addition, septic peritonitis, epicarditis, and thoracic abscessation was observed and were suspected to originate from bacterial translocation from the esophageal/gastric ulceration. Although lymphoma is considered to be a common neoplasm of domestic pigs, it is very infrequently reported in the laboratory animal literature. Lymphoma should be considered as a rule-out for respiratory and nonspecific clinical signs in research pigs.

P69 Spontaneous Urinary Bladder Leiomyoma in a Rhesus Macaque (Macaca mulatta)

KE Scott^*, G Frydman, JG Fox, V Bakthavatchalu

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

A 19-year-old female Rhesus macaque (Macaca mulatta) involved in neuro-cognitive research for 7 years underwent a routine necropsy after a terminal intracranial viral injection procedure. She was socially housed and fed a commercially available standard monkey diet supplemented with fresh fruit and vegetables. Gross examination revealed a diffusely thickened bladder wall and a soft circumscribed dermal mass proximal to the left stifle joint. Histopathological examination of the urinary bladder revealed leiomyoma, characterized by the adventitia expanded by an encapsulated benign neoplasm. The neoplasm was composed of well-differentiated neoplastic spindle cells arranged in fascicles. The neoplastic cells stained positive for vimentin, smooth muscle actin, and desmin and negative for S-100 and pan-cytokeratin. The macaque lacked clinical signs of dysuria, pollakiuria, macroscopic hematuria, discomfort, or pain. The soft tissue mass proximal to the stifle was diagnosed as a lipoma. In humans, urinary bladder leiomyoma is 3 times more likely to occur in females as in males and account for less than 0.43% of all bladder tumors in humans. This is the first report of urinary bladder leimyoma in a rhesus macaque.

P70 A Novel Approach to Intravenous Access for Sampling and Administration in the Axolotl (Ambystoma mexicanum)

LD Cacioppo^*, K Novak-Janus

Research Animal Resources Center/SMPH, University of Wisconsin, Madison, WI

Axolotls (Ambystoma mexicanum) have long been known as a model organism for regeneration due to their ability to replace and repair lost limbs and damaged organ systems. In order to elucidate the mechanism of this ability and aim toward translational therapies, scientists have incorporated axolotls into studies using stem cells, epigenetics, and endocrinology, among others. Although axolotls are unique models for many biomedical research studies, their usefulness can be stifled by difficulties with in vivo blood sampling and drug and experimental compound administration. Here we describe a novel approach to intravenous access in the anesthetized axolotl using the external jugular vein. Our technique demonstrated consistent and repeatable collection of blood samples of scientifically useful quantities. Methylene blue injection using the technique verified extensive perfusion of the dye throughout organ systems and peripheral tissues, indicating the technique can also be used for intravenous injection. Use of this technique results in reliable collection of undiluted blood samples and may be useful for administration of medical treatments, experimental compounds, and contrast agents.

P71 Which Nest Is Best? Nesting Material Improves NZW Transgenic Rabbit Breeding Colony Kit Survival Rates

LA Burlingame^*1, S Thurston¹, J Song², JL Lofgren¹, P Lester¹

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Int Med-Cardiovascular Medicine, University of Michigan, Ann Arbor, MI

Providing adequate nesting material is essential for rabbit kit survival. In our New Zealand White, transgenic, breeding colony, does were previously provided a double cage and nesting material 1 week prior to parturition, including cage board, ad lib hay, approximately (∼) 10 cups(c) crinkled paper, and a partial enclosure under a shelf (hutch). The kit survival rate at 2 weeks of age was 48%. We hypothesized increasing the amount of nesting material in addition to a nesting box would increase the temperature of the nest and increase the kit survival rate. Six nesting groups were evaluated over a 6-month period, which included double caging, cage board, ad lib hay, a hutch and nesting material (∼18c of crinkle paper, woodchip, or a mixture of 12c crinkle paper and 6c woodchip) with or without a nesting box (NB). The internal temperature of the nest and cage was recorded 3 days post parturition. The woodchip with no nesting box group was discontinued due to poor nest quality. Data analysis from the remaining nesting groups, spread evenly over 35 kindlings, demonstrated higher survival rates at 2 weeks of age than historic controls: crinkle paper + NB (92%), crinkle paper only (87%), crinkle paper + woodchips + NB (83%), woodchips + NB (82%), crinkle paper + woodchips (74%). All nesting groups had significantly higher internal nest temperature than cage temperature demonstrating thermogenic properties of all nesting materials evaluated. However, crinkle paper, both with or without a nesting box, provided the highest internal nest temperatures amongst the groups, 81°F and 83°F respectively. Interestingly, presence of a nest box did not make a significant difference in internal nest temperatures. This study demonstrates using ∼18c of nesting material with or without a nesting box will increase survival rate compared to providing ∼10c crinkle paper and no nesting box. Crinkle paper (18c) in particular, with or without a nesting box, produced a significant increase in both survival rate and internal nest temperature.

P72 Development of a Vascular Access Port Model in Woodchucks (Marmota monax)

MA Batchelder^*1, HA Dulac¹, S Aborn², G Marks¹, J Brennan¹, KD Walton¹

¹Veterinary Sciences, Bristol-Myers Squibb, Wallingford, CT; ²University of Connecticut, Storrs, CT

A colony of woodchucks was scheduled to go onto a study which involved repeated IV dosing and multiple blood collection time points. Some of the animals were wild-caught, most were aggressive, and all were challenging to handle or restrain for venipuncture. Therefore, all vascular access procedures were performed under anesthesia. Because of the frequency of access required for the study, we decided to place vascular access ports to allow dosing and collecting blood without anesthesia. We incorporated knowledge and experience obtained inhouse from implanting ports in other species and information from collaborators who worked with ported woodchucks to develop a successful Woodchuck Vascular Access Port model. We placed arterial and venous ports in 70 woodchucks with patency extending up to 23 months. We will provide an overview of the surgical procedure and some unique aspects of this procedure in woodchucks.

P73 Apoptotic Enterocytes Misidentified as Entamoeba in Gnotobiotic Mice with Clostridium Toxin-Mediated Dysbiosis

ME Keith^*1, C Besch-Williford², I Redelsperger³, H White¹, C Cumo¹

¹Molecular and Diagnostic Analysis, Taconic Biosciences, Rensselaer, NY; ²Pathology, IDEXX BioResearch, Columbia, MO; ³Veterinary Sciences, Taconic Biosciences, Rensselaer, NY

Unassociated, gnotobiotic Swiss Webster females presented for routine necropsy examination with mild rectal prolapse and irritation of the perianal area. Upon dissection, gas-distended ceca were noted. Cecal contents had an abnormally thin consistency, with sparse, granular material. Wet mount examination showed round organisms, approximately 10 microns in diameter, with a small indistinct nucleus. PCR testing of feces was negative for Entamoeba spp. Four additional mice from the same isolator were submitted for histologic analysis of the digestive tract, wet mount analysis, fecal float analysis, and to confirm negative PCR results for Entamoeba spp. Wet mounts of cecal and colonic contents were positive for the same round organisms 10-12 microns in diameter, which contained 1 or more condensed nuclei and did not display pseudopodia formation or evidence of motility. Fecal flotations were negative for protozoal cysts, and fecal PCR results were once again negative. The GI tracts had a normal appearance at time of necropsy. Histopathologic examination revealed mild to moderate typhlocolonic mucosal hyperplasia with mucosal edema and neutrophilic infiltrates. Mucus, bacteria, and numerous clusters of sloughed, degenerate enterocytes were present in the lumen of affected gut segments. Subsequent testing identified Clostridium perfringens in fecal samples from this isolator. Given the small size of the observed organisms and the negative PCR results, the organisms observed were most likely sloughed, apoptotic enterocytes. These were likely observed due to C. perfringens toxin-mediated enterocyte injury, with compensatory crypt epithelial hyperplasia. While luminal enterocyte apoptosis occurs as a normal part of mucosal replacement, the level is normally low enough that degenerate cells are not identified in wet mount analysis. Shifts in enteric microbiota (as occurs with dysbiosis due to overgrowth of certain Clostridial species) can alter enterocyte turnover and result in an increase in sloughed enterocytes found in the GI lumen. Lack of motility, indistinct nuclei, and granular cytoplasmic appearance are features that may be used to distinguish these degenerate enterocytes on wet mount exams from Entamoeba and prevent misidentification.

P74 Management of a Colony of Mice with Motor Neuron Disease

MC Dixon^*

Neurology, Drexel University, Eagleville, PA

Managing a colony of transgenic mice with various motor neuron disease presents a variety of complications. The 2 mouse models discussed are the SOD1 G93A model of ALS and the G59S-hDCTN1 mouse model of a mutation in the dynactin complex (commonly referred to as Dynactin mice). The backgrounds of these mice are either C57BL/6, SJL/J, or B6SJL. Due to the nature of these neurodegenerative diseases, the animals face challenges such as muscle degeneration, weight loss, paralysis of either a hind or front limb, and grooming problems. To remedy challenges pertaining to muscle loss and weakness, soft food in the form of gruel is provided to the animals. The gruel diet also provides the animals with water in the event they are too weak to posture to drink from the water valve. Once animals are placed on a gruel diet, they are monitored daily for disease end stage. End stage for the SOD1 mice qualifies as paralysis of one hind limb. End stage for the Dynactin mice qualifies as not being able to right themselves after 30 seconds when placed on their back. Grooming also is an issue for these mice towards their end stage. They have more discharge from their eyes and trouble cleaning their eyes due to muscle loss. For this problem, the animals’ eyes and faces are cleaned as needed using an artificial tears solution. Another eye problem, pertaining mostly to the Dynactin mice, are corneal ulcers. These ulcers tend to show up later in life for these mice (>1 year of age). For treatment, triple antibiotic ointment is applied to the eye daily. Most of the time the ulcer will not completely dissipate, however it will stop worsening. Most of these animals with corneal ulcers remain on triple antibiotic ointment treatment for the rest of their lives. If the ulcer continues to worsen (reddening, swelling) euthanasia is recommended by the veterinary staff. Another symptom typical for the Dynactin mice is hepatomegaly and/or splenomegaly. These issues usually affect mice over 1 year old. Frequently, the liver and/or spleen will have multiple tumors upon necropsy. Due to the constant care these animal models require, future researchers should be mindful of these points should they choose to work with these animals.

P75 Early Detection of Gastrointestinal Hemorrhage in Swine (Sus scofa)

MP Short^*, AA Carter, AM Corn, P Corts, TM McCrum, K Peterson, A Zook, PA Mount, AD Meyers

Cardiovascular Research Foundation, Orangeburg, NY

The overall health and wellness of animals is of the upmost importance in a research environment. Unfortunately, an internal ailment is more difficult to recognize as opposed to an external injury. This is especially true of gastrointestinal ulceration in swine where sudden death is a common presentation. As a cardiovascular model, pigs are often placed on antiplatelet and anticoagulation therapies to prevent device thrombosis and mimic clinical situations. These study requirements along with perioperative stress, fasting, and the coadministration of nonsteroidal anti-inflammatory drugs can exacerbate the risk of gastrointestinal hemorrhage. Clinical signs of gastrointestinal hemorrhage may include lethargy, generalized pallor, pale mucus membranes, and darkening of stool; however these manifestations are only noticeable after significant blood loss has occurred. We aimed at using a commercially available fecal occult blood test (FOBT) for earlier detection of gastrointestinal hemorrhage. Within the past calendar year, we screened 21 swine using this method. These FOBTs are noninvasive, easy to use, and can be run inhouse yielding results in seconds without specialized equipment. As a result, the time and expense of sending fecal samples to an external diagnostic lab is bypassed. Of the two common FOBTs, guaiac and immunochemical, the guaiac FOBT is more readily available therefore this is the test we choose. The guaiac FOBT is able to detect a low amount of blood, if present, 2-4 mL per 100g of feces. The reliability and use of guaiac FOBTs has been questioned in literature with studies showing a decrease in its positive predictive value and with the ever increasing availability of immunochemical FOBT. However, in our facility the benefit of low cost, rapid screening has prevailed as we are routinely able to screen animals or groups of animals who are at high risk for multiple days to ensure there were no false negatives. When working with animals, such as swine, that have a propensity for gastrointestinal ulceration, a guaiac FOBT is a simple, cost-effective first step in determining if hemorrhage has occurred.

P76 Effect of Ketamine Sedation on Maternal and Fetal Heart Rate in the Common Marmoset (Callithrix jacchus)

MA Burns^*, MT Lieberman, L Wachtman

Division of Comparative Medicine, Massachusetts Institute of Technology, Somerville, MA

The common marmoset (Callithrix jacchus) has grown in popularity as a research model due to the high reproductive efficiency, small size, and ease of handling of the species. Our institution maintains an active breeding colony of marmosets with approximately 40 infants born per year. Estimation of parturition date is important for management of marmoset breeding colonies. While correlations between uterine size and fetal biparietal diameter (BPD) with gestational age have previously been described, current advances in ultrasound and transducer technology offer the opportunity to refine existing data. Normal fetal heart rates (FHR) in marmosets have not been established and offer a rapid, noninvasive method for evaluating fetal health or potential distress. Additionally, the effect of the commonly used sedative ketamine hydrochloride on maternal and fetal remains undefined in marmosets. We hypothesized that because ketamine is able to cross the placenta, FHR would be affected during ultrasound exams performed under ketamine sedation. We performed weekly abdominal ultrasounds using 22-30 MHz linear probes on 5 pregnant marmosets at various stages of gestation until delivery. Dams were manually restrained and given positive reinforcement training during ultrasounds, excluding scheduled semiannual examinations performed under ketamine sedation (25-30mg/kg via intramuscular injection). Measurements acquired included uterine diameter, uterine lumen diameter, fetal BPD, FHR, and maternal femoral pulse rate (MPR). The date of parturition was set at 145 days for purposes of matching dams for gestational stage. Mean and standard deviations of FHR acquired from conscious and ketamine-sedated dams were 217 + 43 beats per minute (bpm) and 172 + 27 bpm respectively. Mean and standard deviations of MPR were 334 + 40 bpm and 425 + 26 bpm respectively. Both FHR and MPR were significantly lower under ketamine sedation compared to values obtained from awake ultrasound examinations (Mann-Whitney; P < 0.01). In conclusion, ketamine sedation can decrease FHR, which may obscure signs of fetal distress. Ultrasonagraphy of pregnant dams habituated to manual restraint is recommended for accurate assessment of FHR.

P77 Sustained-Release Buprenorphine Improves Postsurgical Clinical Condition and Does Not Affect Survival and Cytokine Levels in a Murine Model of Polymicrobial Sepsis

NL Herndon^*1, S Bandyopadhyay², E Hod², K Prestia³

¹Institute of Comparative Medicine, Columbia University, NY, NY; ²Department of Pathology and Cell Biology, College of Physicians and Surgeons, Laboratory of Transfusion Biology, New York, NY; ³Division of Comparative Medicine, New York University Langone Medical Center, New York, NY

Cecal ligation and puncture (CLP) is a commonly used technique to study sepsis in the murine model. Due to the invasiveness of the procedure and the impact on animal clinical condition, many IACUCs require analgesics. However, some analgesics have immunomodulatory effects and can hinder research outcomes. We sought to determine the effect of buprenorphine hydrochloride (Bup HCL) compared to sustained-release buprenorphine (Bup SR) on the inflammatory cytokines, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6, animal clinical condition, and overall mortality in a murine CLP model of sepsis. We hypothesized that there would be no difference in the tested cytokine levels, mortality, or animal clinical condition in mice dosed with either Bup HCL or Bup SR analgesics following the CLP procedure. Male C57/BL6 mice were separated into Bup HCL or Bup SR treatment groups and each mouse had the identical CLP procedure performed. Mice assigned to the Bup HCL group were dosed with 0.1 mg/kg Bup HCL intraoperatively. Additional doses of Bup HCL were given at 6, 12, 24, 36, and 48 hours. The Bup SR group was dosed with 1 mg/kg Bup SR intraoperatively, followed by equal volumes of saline at the same time points as the Bup HCL group. MCP-1 and IL-6 levels were measured at 24 hours post procedure and were not significantly different between the 2 groups. Mortality over the duration of the study was not significantly different between the 2 treatment groups. Mice were observed twice daily for clinical condition scoring by the same blinded investigator for the duration of the study. An abbreviated version of a validated behavioral scoring system was used to create a clinical condition scoring system, integrating physiologic and behavioral parameters. Mice were first observed from a distance in their home cage and assessed for movement, interaction with conspecifics, posture, and amount of time spent focused at the surgical site. Following the initial observation, mice were observed immediately following the opening of the home cage lid to evaluate responsiveness to cage opening, grooming, and respiratory effort. Finally, mice were evaluated for reactiveness and exploration while being handled and a clinical exam was performed. The results showed a significant decrease in behaviors of perceived pain in mice in the Bup SR group at 12 and 24 hours postoperatively compared to the Bup HCL group. Because of the lack of significant effect on MCP-1 and IL-6 cytokine levels and mortality, and the superior analgesic effects of Bup SR, we recommend the use of Bup SR in the murine CLP model of sepsis.

P78 Listeria monocytogenes in a Wild-Caught Indoor-Housed Black-Tailed Prairie Dog (Cynomys ludovicianus)

NL Lukovsky-Akhsanov^*, BL Skinner, G Lathrop, JR, j hayes, J Ritter, M DeLeon-Carnes

Center for Disease Control, Atlanta, GA

An indoor and group-housed adult, prairie dog (Cynomys ludovicianus) was found acutely obtunded, exhibiting a Schiff-Sherrington posture. During an examination, body condition score was 4.5 out of 5, rectal temperature 86.4°F, and paddling in all limbs began. Euthanasia was elected due to rapid decline and poor prognosis. Necropsy findings included abundant adipose stores, segmental mineralization of the aorta, renal cortical cysts, and a nodular mass attached to the cecal serosa. Histopathology revealed extensive neutrophilic and mixed inflammation with microabscesses in the brainstem, midbrain, cerebellar white matter, and associated leptomeninges. There was also mixed perivascular inflammation, neutrophilic vasculitis, and fibrinoid vascular necrosis in affected regions. Scattered gram-positive short bacterial rods were present amidst neutrophilic infiltrates, and were highlighted by immunohistochemical assay for Listeria monocytogenes. Molecular testing of DNA extracts from the brain tissue was also positive for L. monocytogenes. At this time, the source of infection is unknown, and all other animals in the colony appear clinically normal to date. Testing of the animals’ commercial and supplemental enrichment feed for Listeria was performed by a government laboratory with negative results. Raw food sanitization practices have been enhanced to include washing fresh fruits and vegetables in a 200ppm chlorine solution before rinsing with potable water. It is possible that infection may have been latent, with reactivation at time of clinical presentation. Our facility sources wild-caught prairie dogs from the Southwestern United States. This animal had been housed in our facility for 7 years. Animals are evaluated twice daily by trained animal care staff and receive a physical examination annually. L. monocytogenes is a gram-positive bacterial rod of public health significance, as it is a zoonotic agent capable of causing severe clinical illness and death in humans. More commonly isolated from ruminant species and occasionally birds, this is the first case report in this species. Public health awareness of the potential for listeriosis in prairie dogs is important, as these animals may be kept as household pets.

P79 Clostridium difficile-Positive Mice: Two Barriers and Two Outcomes

PL Roesch^*1, SP Swing³, A Ericsson⁴, C Besch-Williford², RS Livingston²

¹Molecular and Diagnostic Analysis, Taconic Biosciences, Rensselaer, NY; ²IDEXX BioResearch, Columbia, MO; ³Veterinary Sciences, Taconic Biosciences, Germantown, NY; ⁴Mutant Mouse Resource and Research Center, Metagenomics Center, University of Missouri, Columbia, MO

The first clinical case was reported as a female NOG mouse with dried perianal feces, hunched posture, and a rough hair coat. At necropsy, yellow viscous fecal matter was noted in the cage and at the anus. Grossly, the colon and cecum were enlarged and edematous and contained gas and semi-formed mucoid feces. Over the next several days, many more cages in this location were identified with sick and dying animals exhibiting loose stool. Note that most animals housed in this location were severely immunodeficient lines lacking B, T, and NK cell production. Feces and blood were collected for culture and PCR. In addition, tissues were collected for histopathology. Blood cultures were positive for Lactobacillus murinus, a member of the Altered Schaedler Flora, and fecal cultures were positive for Clostridium difficile. Histopathologic exam of the cecum and colon revealed mucosal changes consistent with the presence of a toxin-producing bacteria. As a proactive measure, all other locations housing NOG mice were tested for the presence of C. difficile by both culture and PCR. Surprisingly, another location was positive for the organism, but no clinical signs were evident. This led to the hypothesis that differences in the gut microbiome may be pivotal to the clinical outcome. To test this hypothesis, fresh fecal samples were collected from both sick and healthy animals housed in multiple locations and sent for microbiome analysis. Results demonstrated that the animals from the location exhibiting severe clinical signs had a relatively simplified microbiome while the location where animals remained healthy had a more diverse microbiome that included the presence of Lachnospiraceae, a family of bacteria often associated with colonization resistance. These results suggest that the composition and diversity of the intestinal microbiome of immunodeficient mice play a significant role in protection from dysbiosis due to C. difficile and the development of subsequent clinical signs.

P80 Enrofloxacin Site Reaction after Subcutaneous Injection in Chinchillas (Chinchilla chinchilla)

RM Burns^*1, EL Sheldon¹, EL Horn-Ranney², MC Rodriguez¹

¹Eastern Virginia Medical School, SoBran Bioscience, Norfolk, VA; ²Tympanogen, Inc., Norfolk, VA

Enrofloxacin (ENR) is a common antimicrobial used in the laboratory animal science field. ENR is a fluoroquinolone antibiotic with broad spectrum for both gram-negative and -positive bacteria. It is available for oral administration and for intravenous, intramuscular (IM), and subcutaneous (SQ) injections. ENR is a favorable off-label choice for exotic and laboratory animal species. A Chinchilla (Chinchilla chinchilla) project was approved for the use of ENR SQ with a dosage of 10mg/kg and a concentration of 2.27%. Injections were given SID for 5 to 7 days, alternating injection sites on each side of the back. There were no signs that the ENR could have been tainted and no cultures were taken from the vials used. Fifteen out of the 38 treated chinchillas (39.5%) showed an inflamed reaction of the skin at the injection site. The lesions started as round and red in color with inflammation and subsequent hair loss at the injection site. The lesions resembled a hematoma and in some cases ulcers. Lesions ranged from a few millimeters to about 1 centimeter in diameter. Immediate treatment was provided using topical antimicrobial ointment (triple antibiotic) and wound dressing powder. Complete resolution took an average of 21.5 days (6-45 days). Biopsies and histopatholoy of injection sites were ordered after the project was completed. The pathology report indicated the presence of healed skin lesions with decreased hair follicles supporting a clinical diagnosis of a local tissue reaction of the injected solution. Similar injection site reactions have been described as transient, localized tissue reactions resulting in necrosis, for both SQ and IM injections in horses, cattle, pigs, rodents, and rabbits. To our knowledge this is the first time it is reported as described in chinchillas.

P81 Development of a Pain Scoring System for Ruminants in Biomedical Research

RA LaFleur^*, J Izer, R Wilson

Pennsylvania State University, Hershey, PA

Effective pain management is integral to the use of animals in biomedical research and laboratory animal medicine. A common assumption is procedures which are painful to humans will also be painful to animals. Although this principle is helpful in determining which procedures will require analgesics, additional challenges lie in determining if the analgesic regimen is effective and when pain medications can be discontinued. In order to overcome these challenges, it is important to consider species-specific indicators of pain and distress. Ruminants manifest pain in subtle ways and some biomedical protocols may require ruminants to be housed in a nontraditional environment which makes typical measures of pain in these species such as lameness and flock behavior difficult to evaluate. Several publications exist on pain evaluation for ruminants but these focus on lameness or pain following castration or tail docking in a farm environment. Therefore, we have developed a pain scoring system specifically designed for ruminants in biomedical research. The pain scoring system uses behaviors that can indicate pain in ruminants such as bruxism, restlessness, excessive vocalization, kicking at the abdomen, and abnormal posture combined with objective measurements such as heart and respiratory rate. After the score is generated, a decision tree is used to help guide further action. Sheep in our facility were scored at least twice daily in the immediate postoperative period and the system was effective in identifying several animals with increased pain scores that benefited from administration of additional analgesics. We believe this pain scoring system is an easy to use tool which can guide veterinary and animal care staff in the assessment and alleviation of pain in ruminants used in biomedical research.

P82 Blood Volume Measurement in Rhesus Macaques by¹⁸F-Albumin and PET Imaging

RA Byrum^*1, S Chefer¹, J Solomon², D Thomasson¹, P Sayre¹, J Seidel^1,5, R Reeder¹, K Hagen¹, F Basuli³, R Swenson³, X Zhang³, PB Jahrling^1,4, RF Johnson⁴, M St Claire¹

¹Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD; ²Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., NCI Campus at Frederick, Frederick, MD; ³Imaging Probe Development Center, National Heart, Lung and Blood Institute, National Institutes of Health, Rockville, MD; ⁴Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD; ⁵Molecular Imaging Program, National Cancer Institute, National Institutes of Health, Bethesda, MD

Currently, multiple methods are available to measure blood volume (BV) using dyes or serum albumin labeled radioisotopes. These methods require repeated blood sampling over an extended period of time. Use of ¹⁸F labeled serum albumin (¹⁸F-ALB), a novel blood pool imaging probe, measures BV without repeated sampling. Since the probe is retained stably (1 hour post-injection) in the circulatory system after intravenous injection, use of this tracer as a positron emission tomography (PET) blood-pool imaging agent is feasible. In this pilot study, we evaluated ¹⁸F-ALB application for BV measurement in nonhuman primates by PET. Three male Rhesus monkeys (Macaca mulatta) were injected intravenously with ¹⁸F-ALB (0.37–0.48 mCi/kg) and underwent up to three whole body ¹⁸F-ALB-PET and CT scans using Gemini PET/CT scanner (Philips Healthcare). The whole body volume of distribution (V_T) was calculated using PET imaging as the ratio of total ¹⁸F-ALB radioactivity in the body to the ¹⁸F-ALB concentration in the left ventricle (LV), V_T=Total ¹⁸F-ALB / LV ¹⁸F-ALB, with the concentration in the left ventricle representing arterial blood concentration. The V_T was also evaluated by assessing the concentration of ¹⁸F-ALB in venous blood samples collected at the end of PET scans by scintigraphy. ¹⁸F-ALB concentration in the LV determined by PET was in agreement with that in venous blood samples assessed by scintillation counter (r²=0.87, P = 0.0003). The V_Ts obtained from three NHPs were 74.7 ± 4.9, 71.9 ± 2.1, and 66.1 ± 1.1 ml/kg when using PET and 68.2 ± 5.9, 76.3 ± 8.8, and 62.6 ± 11.3 ml/kg when using scintigraphy of venous blood samples. These data are in agreement with previously published BV estimates in rhesus macaques. In this study, the total BV in NHP was determined for the first time by PET imaging. The data suggest that detection of changes in BV using ¹⁸F-ALB PET could be useful for assessing animal health and disease progression in animal models of disease.

P83 Managing Abnormal Behavior in a Rhesus Macaque (Macaca mulatta) through an Individualized- and Multifaceted-Approach Focusing on Preventative Treatment

RJ Mistretta^*, M Carey, B Sullivan

Animal Behavior and Enrichment, BIOQUAL, Inc., Rockville, MD

Management of abnormal behavior through an individualized and multifaceted approach focusing on preventative treatments can be used in an attempt to reduce or eliminate instances of severe self-injurious behavior (SIB) in nonhuman primates. A 15-year-old rhesus macaque (Macaca mulatta) designated L862 with a history of performing severe SIBs required additional behavioral intervention to increase the animal’s wellbeing and study availability. L862 had a medical history including 11 instances of SIB requiring veterinary attention from 3/12/2008 to 12/10/2012. Through detailed observations of possible triggers for abnormal behaviors an individualized Abnormal Behavior Management Plan (ABMP) was created. The management plan focused on transitioning the animal’s treatment methods from reactive (responding to SIB events such as medications, e-collar, etc.) to preventative (avoiding SIB events such as positive human interaction, positive social group construction, etc.). Solutions were designed to alleviate each hypothesized trigger for these behaviors and continuous evaluations were performed in an attempt to track progress and continuously improve the ABMP. It was hypothesized that through detailed individualized behavioral management, focusing on preventative treatment, the triggers for abnormal behavior could be dealt with and the instances of self-injurious behavior would be reduced. The statistical data obtained from comparing the instances of SIB prior to the implementation of the ABMP and after the implementation of the ABMP displayed significantly less SIB (P = 0.01, α=0.05). The p value correlates with both the observed decrease in percent chance of SIB post plan implementation from 14% to 5% and the observed reduction of abnormal behavior during human interaction sessions from 11% to 0%. The average months between major SIB events was increased from 5.0 months to 13.3 months with the current count at 31 months and counting since the last event. Through an individualized and multifaceted approach to behavioral management it has been possible to eliminate the occurrence of abnormal behaviors such as self-injurious and self-directed behaviors in the case of L862.

P84 Evaluation of Trimethoprim-Sulfamethoxazole in Medicated Gel Formulation in Mice

S Satheesan^*, TW Adamson, R Ermel

City of Hope, Duarte, CA

The use of immunocompromised and transgenic mice has increased the risk of bacterial infections; however individual dosing of medications for prolonged duration is not always a feasible option in laboratory rodents, particularly when a large number of animals need treatment. The administration of antibiotics, and in general medications, as oral formulations is desirable as it results in decreased handling and less stress to the animal while also saving time for research and animal care staff. Recently, there have been many gel products that are available to facilitate the delivery of oral medication and provide supplemental support to laboratory animals. In our study, we assessed the efficacy of a commonly used broad-spectrum antibiotic combination trimethoprim–sulfamethoxazole (TMP-SMX) when administered in a medicated gel formulation. C57BL/6 mice received medicated gel for up to 7 days, and daily medicated gel consumption was measured by weighing the gel cup for each cage of mice for the course of the study. There were 4 experimental groups (4 animals each), each receiving the medicated gel for a different length of time: Group 1–24 hours, Group 2–48 hours, Group 3–72 hours, and Group 4–96 hours. At the end of the predetermined administration period, blood was collected (AM) to measure the antibiotic plasma concentrations. These levels were then compared to control groups that received a fresh cup of medicated gel daily. Plasma concentrations of TMP-SMX were well above the MIC₉₀ values for common pathogenic bacteria in all groups, with the levels varying from 4.41 ± 4.0 µg/mL to 23.58 ± 6.4 µg/mL. Our results indicate that the consumption of TMP-SMX medicated gel by C57BL/6 results in therapeutic plasma concentrations of the antibiotic with a recommended changing frequency of every 3-4 days.

P85 Real-Time Induction of Specific Fractional Flow Reserve During MRI Perfusion Studies Using a Programmable Infusion Pump and Data Acquisition System

SM Kozlov^*1, GW Daniel², JL Taylor¹, A Arai²

¹NHLBI/ASR, National Institutes of Health, Bethesda, MD; ²Advanced Cardiovascular Imaging, NHLBI, Bethesda, MD

Fractional flow reserve (FFR) is defined as the ratio of blood pressure distal to a coronary artery stenosis divided by the pressure proximal to the stenosis during maximum coronary blood flow as typically induced by adenosine. In order to validate quantitative cardiac perfusion measurements using cardiac MRI (magnetic resonance imaging) in the presence of experimentally controlled coronary artery stenosis and different severities of FFR, a system was needed that would respond to the changing aortic pressure during the stenosis to maintain the required distal coronary pressure in Yorkshire pigs (Sus scrofa domesticus). The experimental coronary artery stenosis was induced during pharmacological vasodilation with adenosine. A programmable pump was paired with a data acquisition system to create a real-time feedback system to inflate or deflate a balloon occluder around the left anterior descending artery (LAD). The data acquisition system was used to simultaneously and continuously measure the aortic pressure and the distal coronary artery pressure (24G catheter). The desired FFR was programmed as a separate channel and calculated as a percentage decrease of the aortic pressure. A series of event triggers and digital outputs from the data acquisition system combined with a custom program regulated the rate of infusion or withdraw of the syringe pump to achieve the desired reduction in FFR as part of a continuous feedback loop system. The syringe pump is connected to the balloon occluder which is around the LAD to maintain the desired stenosis that will provide the desired FFR during the acquisition of the perfusion scan. The end result is a precise stable method to maintain a partial occlusion of the LAD during the acquisition of cardiac perfusion with contrast enhancement. The system has successful used in over twenty simultaneous MRI and FFR experiments.

P86 Combined Inapparent Zoonotic Diseases in an Awassi Ewe (Ovis aries): Implications for Health and Biosecurity Management

SM Prattis^*

Agriculture, American University of Beirut, Beirut, Lebanon

Sheep (Ovis aries) have historically served as experimental subjects. As a result of their physiologic parameters, ovine models have been developed to study orthopedic disease models, fetal physiology and surgical intervention, lipid metabolism, feeding behavior, germplasm cryopreservation, drought and temperature adaptation, and to serve as sentinels of environmental chemical disease onset. It can be difficult in locations outside of the United States to obtain animals from flocks that have been raised using standard laboratory husbandry and management principles, and that are specific-pathogen free in health status. The most common local sheep breed in Lebanon is the Awassi sheep, which are drought-resistant, nomadic, fat-tailed sheep that originated in the Syro-Arabian desert of southwest Asia. In this study a thin Awassi ewe was identified with a body condition score of 2 that was detected at shearing. The ewe was then sacrificed and necropsied. This ewe was born and reared onsite as a part of a 75-head Awassi sheep and Baladi goat mixed herd in residence on a farm that also had previously housed dairy cattle and poultry. Mycobacteria avium subspecies paratuberculosis was identified, and acid fast (+) organisms were present in intestinal lesions. The Rose Bengal assay detected a Brucella-positive reaction in the ewe only, which was accompanied by suspect liver lesions. We also identified Echinoccocus granulosa, Clostridium perfringens, Staphylococcus, and Streptococcus species, and parasitic skeletal muscle cysts most consistent in appearance with either Toxoplasma gondii or Neosporum caninuminfection. Molecular PCR amplification for Torque Ten Virus, Blue Tongue Virus, Epizootic Hemorrhagic Viral Disease, and Foot and Mouth Disease was performed on the ewe and herds and was negative. We review the disease implications for public health and biosecurity management on an experimental farm. To date implementation of routine governmental test and eradication programs and consistent border control is limited in developing countries and those with contiguous ongoing conflict zones.

P87 Echocardiography Screening of Nonhuman Primates with Asculted Heart Murmurs

TA Swanson^*1, H Pisharanth², B Morenko Campbell¹

¹Comparative Medicine, Pfizer Worldwide Research, Groton, CT; ²Animal Resources Core, Nationwide Children’s Hospital, Columbus, OH

Cardiac auscultation of nonhuman primates (NHP) during a routine physical exam may sometimes reveal a heart murmur. The presence of a murmur must be taken into consideration when assessing the animal’s suitability for toxicology and safety pharmacology studies. Echocardiography of these animals often does not reveal any valvulopathy or other cause of the murmur. We theorized that this finding, which can be transient and/or positional, is physiologic in origin in our NHP population. Our goal was to perform a thorough auscultation and echocardiography exam, including B-mode images of heart, pulse wave, and color Doppler analysis, on animals with audible murmurs to generate a knowledge base on the incidence, relevance, and possible causes of the murmurs. In our study to date; NHPs with a grade 3 or higher murmur had echocardiographic evidence of a valve insufficiency either in the mitral or tricuspid valves. Animals with a grade 2 or below often have transient murmurs that may be positional or due to other factors (hemoconcentration or cardiac implant), but no evidence of insufficiency was observed in this group. Murmurs with grade 2 or higher were also noted to have statically significant greater left ventricular diameter. As a result of our findings, we propose that any animal with suspected murmur be screened by echocardiography, and those found to have documented valvular insufficiency not be used in studies with cardiac endpoints. In addition, normal nonhuman primates have a wide range of functional cardiac parameters according to previously published information. We found that our animals diagnosed with murmurs were well within those normal ranges, specifically ejection fraction and fractional shortening.

P88 Ultrasound Monitoring of Fetal Guinea Pig (Cavia porcellus) Development for Maternal-Fetal Vaccine Programs

TA Swanson^*1, D Church¹, P Lewis¹, HR Devantier¹, D Yates¹, I Scully²

¹Comparative Medicine, Pfizer Worldwide Research, Groton, CT; ²Vaccines Research, Pfizer, Pearl River, NY

Guinea pigs (Cavia porcellus) continue to evolve as a vital species supporting research involving maternal-fetal disease transmission. Maternal immunization to infer passive protection to the fetus or neonate is gaining momentum in the field of vaccines research and development. The ability to serially monitor fetal development in a noninvasive, nonstressful manner could serve to provide data supporting a correlate of protection in maternal vaccine efficacy studies. The guinea pig was specifically chosen as the most appropriate small animal species to support this type of model development due to the length of gestation (68 days), as well as being closely translational in implantation process (interstitial) and placentation type to human which has a significant impact on maternal/fetal antibody transfer. Guinea pigs were mated and shipped on embryonic day 1 (e1). Ultrasound imaging was performed 2-3 times each week using light manual restraint. We found no ultrasound evidence of pregnancy prior to day e17. On or after e17 we were able to track development of individual fetuses over the length of the development cycle. Heartbeats were visible and measureable after day e19. Specific morphologic parameters including heart rate, crown-rump length and brain/skull size were serially monitored, as well as resorbing embryos and still birth rate, to establish baseline data on normal fetal development and pregnancy outcomes in healthy, unchallenged animals.

P89 Repeated Tail-Tissue Sampling Does Not Affect the Growth of Mice versus Single Sampling

TS Highbaugh^*1, KL Stewart¹, A Dawson¹, AL Fletcher¹, M Suckow²

¹Freimann Life Science Center, University of Notre Dame, Notre Dame, IN; ²University of Minnesota, Minneapolis, MN

The use of tail biopsy samples for the genotyping of genetically engineered mice (GEM) is common among research laboratories. For the wellbeing of the animal, the age of the animal and the sample size must be considered. Because it is a common occurrence for researchers to request additional tail tissue to perform the genetic analysis, we wanted to determine if it is beneficial to the animal to have 1 large biopsy or a small biopsy with a second biopsy later, if required. We also wanted to ascertain at which age the procedure causes the least detriment to the growth of the animal. For this study 2 inbred mouse strains, FVB and C57Bl/6, 8 mice per gender per strain for a total of 48 mice, including the control group, were used. Mice were subjected to either a single biopsy of 5 mm or 2 biopsies, both 2 mm in tail tip length, that were taken several days apart. Single biopsies were taken from mice at either days 5, 10, or 21. For mice that underwent dual sampling, biopsies were performed on days 5 and 21, days 10 and 21, days 10 and 28, and on days 21 and 28. Animals over the age of 14 days were anesthetized with Isoflurane for the procedure. Mice were weighed at 1 week of age through 16 weeks of age. We chose to use the weight gain comparison to ascertain the wellbeing of the animals because it is a noninvasive measurement that does not interfere with physiologic parameters in the neonatal mice. The comparison of the weekly weight gained over the 16-week period revealed no significant variation of weight gain for all treatment groups, suggesting that dual small tail-tissue sampling through at least 4 weeks of age is not detrimental to the growth of the animal when compared to mice that underwent a single, larger sampling of tissue.

P90 The Use of an Antibacterial Cleanse and an Antifungal Corticosteroid in Resolving Ulcerative Dermatitis Cases

VN Frazier^*, J DeGraff

Department of Laboratory Animal Research, Duke University, Raleigh, NC

Antibacterial cleansing treatment chlorhexidine followed by an antifungal corticosteroid ointment has been a common treatment for many years to manage ulcerative dermatitis (UD) on research mice. Recently clinical veterinarians suggested terminating use of chlorhexidine and replacing with a cleansing treatment better suited for the facility’s needs. A new therapy of an antibacterial cleanse containing active ingredient hypochlorous acid, followed by standard application of antifungal corticosteroid ointment containing active ingredients nystatin, neomycin sulfate, thiostrpton, and triamcinolone acetonide was suggested for treatment of UD. To determine effectiveness of the new treatment plan husbandry staff scored all UD cases found in C57BL/6 research mice, or mice with C57BL/6 background, immediately upon discovery. UD scoring was based on a standard scoring system creating a calculated severity score percentage derived from scoring the scratching number, character of lesion, length of lesion, and regions affected. Higher scores reflect more severe cases. The effected mice were then treated by gently cleansing the ulcerated area with antibacterial-soaked gauze followed by a thin layer of antifungal corticosteroid ointment. This treatment was performed daily and cases were scored weekly or when considerable improvements were seen. During the trial period UD cases were diagnosed and treated. Of these cases, cases originally scored at above 40% were closed and considered completely healed by a clinical veterinarian within a 2-week time. Some cases originally scored above 60% healed in a longer time period (4 weeks). Based on this information, we can confidently conclude that early diagnosis of UD, treating with an antibacterial cleanse followed by antifungal corticosteroid ointment does effectively resolve clinical cases of ulcerative dermatitis within a reasonable time period.

P91¹H NMR-Based Metabolomics Profiling to Investigation of Metabolism Changes in Urine and Serum of Cynomolgus Macaques (Macaca fascicularis) Subjected to Air and Road Transportation

X Pan^*, Y Chang, X Zeng

Shanghai InnoStar Bio-Tech Co., Ltd., Shanghai, China

Stress is increasing markedly in modern society. Many types of physiologic and socioeconomic stresses have adverse effects on human health. We aimed to study the effects of transport stress on cynomolgus monkeys and molecular changes after transport. This study included 6 cynomolgus monkeys that were subjected to road and air transport for 8 hours from Hainan to Shanghai. To assess alterations in metabolite levels in the serum and urine due to combined transport, we applied a proton nuclear magnetic resonance spectroscopy (¹H-NMR)-based metabolomics strategy in conjugation with the orthogonal projection to latent structure with discriminant analysis (OPLS-DA) method. Serum and urine samples were collected during pretransport, arrival, and recovery. NMR-based metabonomics revealed that serum levels of lactate, alanine, glutamate, glutamine, pyruvate, myo-inositol, creatinine, and urea were higher posttransport, while serum levels of isoleucine and proline (P < 0.05) were lower after transport. Additionally, marked changes in urine metabolites, including increased levels of lactate, glycoproteins, pyruvate, citrate, choline, creatinine, α-ketoglutarate, taurine, and allantoin, and decreased levels of isoleucine, leucine, creatine, myo-inositol, 3-hydroxybutyrate, and hippurate were observed after 24 hours recovery compared with 1-week recovery. These novel findings indicate that cynomolgus monkeys subjected to transport stress have a distinctive metabolic status, including altered energy metabolism, amino acid metabolism, lipid metabolism, and perturbations in gut microbiota. The information obtained is important for prevention or intervention of transport stress to improve health and welfare.

P92 A Simple and Reliable Protocol for Induction of Superovulation in New Zealand White Rabbits

X Peng^*

Comparative Medicine, Penn State University, Hershey, PA

Genetically modified rabbits, especially transgenic rabbits, have been increasingly used as animal models for a variety of human diseases and as bioreactors for production of pharmaceutical proteins. To date, the majority of transgenic rabbits have been produced by microinjection of a DNA construct into the pronucleus of fertilized eggs at single cell stage. Therefore, the first step for production of transgenic rabbits is the capability to obtain enough fertilized eggs via hormone induced superovulation. Numerous protocols have been reported to induce superovulation by using pregnant mare serum gonadotropin (PMSG) or follicular stimulating hormone (FSH) with various doses, injection route and time points. However, these protocols have shown some disadvantages, such as significant number of non-responders, wide variation in number of oocytes produced by individual rabbits, large number of abnormal eggs unsuitable for injection, and requirement of multiple injections (FSH, twice a day for 3 days). Here we report a simple and reliable protocol for inducing superovulation in New Zealand White (NZW) rabbits. The rabbits were given a single intramuscular injection of PMSG (120 IU) at 1:00PM on day 1, followed by mating and a single intravenous injection of human chorionic gonadotropin (hCG, 150 IU) at 1:00PM on day 4. Fertilized eggs at single cell stage were harvested at 9:00AM on day 5 for microinjection. In this study, a total of 358 fertilized eggs were collected from the PMSG and hCG injected rabbits (n = 11) with 21 to 54 eggs from different rabbits (32.5 + 3.0). Results of this study indicate that a stable induction of superovulation with a reasonable number of eggs can be achieved by this simple protocol.

P93 Factors Contributing to Successfully Pair-Housing Adult Male Rhesus Macaques (Macaca mulatta)

AA Smith^*1,2

¹Production and Population Health, Royal Veterinary College, Potters Bar, United Kingdom; ²Center for Comparative Medicine, Massachusetts General Hospital, Boston, MA

Pair housing nonhuman primates in the laboratory setting is considered a best practice and is widely accepted as a means to better animal welfare and psychologic wellbeing. However, few investigations on methods and approaches to overcome the specific challenges of introducing adult male Macaca mulatta have been accomplished. An online survey targeting individuals directly involved in pairing Macaca mulatta was distributed to determine which factors are perceived to be the most critical when selecting adult male rhesus to introduce, and which methods are associated with a greater rate of successful long-term pairs. The survey was sent to approximately 187 individuals at 93 different institutions in 5 different countries (USA, Canada, UK, Germany, and China) with a survey response rate of 15% completed and 37% partially completed. Survey users, 17 out of 24, reported that identifying primates having different ranks was an important selection criteria and that dynamic, larger cages increased their perceived rate of successful pairings. No significant correlation was evident between the cage sizes used for introductions to the rate of successful pairs formed. Methodical stepwise introductions were the most commonly implemented practices (78%), using quad cages (25%) giving new pairs horizontal and vertical space. Users perceived lip smacking and mutual grooming as successful prognostic indicators, but lunging and the presence of food aggression were reported as negative prognostic indicators. The results from this survey are intended to aid institutions in achieving pair-housing success of adult male-male rhesus macaques by consolidating best practices from laboratories in multiple countries.

P94 Bridging Nonhuman Primate Husbandry and Research Workflows with Task-Specific Information Tools

AW Legasse^*2, GD Lawrence¹

¹Director’s Office, Oregon National Primate Research Center, Portland, OR; ²Oregon National Primate Research Center, Portland, OR

Hardware and software technologies are developing at a ferocious pace. Servers procured today are twice as fast as those deployed in 2010, while the latest software technology allows some applications to be developed, deployed, and in production in as little as two weeks. While these changes are tremendously promising, dividends for the quality and efficiency of work remain stubbornly unremarkable in many biomedical research institutions, where monolithic record systems constrain access to, interaction with, and benefits from, institutional data resources. Putting a new face on these systems, one more tailored to user needs versus developer expedience, promises to unlock substantial gains in productivity and job satisfaction. We set out to test this by developing a set of task-specific computational tools according to what was required by users performing those tasks. We targeted the intervention at an especially complex workflow spanning laboratory, clinic and husbandry domains in nonhuman primate (NHP) pathobiology involving 700 animals, 40 IACUC protocols, 60 experimental schedules, with more than 100 individuals involved at some point in mission-critical work. We asked workers to describe their needs at the individual and task levels, and then used state-of-the-art programming technologies to rapidly build computational tools that did precisely what they had described. This is in contrast to conventional approaches that train the user to apply an institutional system in very particular and inflexible ways of working, where there is a cost of training, and a relatively larger software-development expense that comes with maintenance and upgrade cycles in complex institutional systems. We expected that tools developed in this way, embedding the user’s insight and understanding of their work, would also dramatically enhance efficiencies, work products and job satisfaction. Over the course of a few weeks we deployed a collection of tools to “scaffold” the pathobiology workflow, refining them in an evolutionary way based on experience in use. Initial results indicate a savings of almost 30% in the cost operations (3.2 FTE), along with reduction in the frequency of irregularities, error and “stress events”.

P95 Naked Mole Rats (Heterocephalus glaber): Short-Term Housing and Husbandry of a Nontraditional Animal Model

A Gallagher^*, C Sims, DL Coble

Unit for Laboratory Animal Resources, The Ohio State University, Columbus, OH

Naked mole rats (Heterocephalus glaber) present unique housing and husbandry challenges when maintained in the biomedical research setting. Naked mole rats belong to the order Rodentia and family Bathyergidae which is characterized by small infraorbital foramina into which the masseter muscle does not enter. Naked mole rats are particularly resistant to neoplasia which makes them useful for research on aging, metabolic regulation, development, analgesia, and behavior. A novel caging system was created using repurposed plastic feed bins (24” x 16” x 15”) and a filter paper lid. Each system contained 3 socially compatible male naked mole rats for an IACUC-approved study. Enrichment toys were provided to replicate the tunnel systems, toilet, and sleeping areas used by these animals in the wild and to provide opportunities for chewing. Initially, aspen wood shavings were used for bedding, but this was replaced with a paper bedding following clinical observations of dermatologic changes on the limbs and tails. Cages were changed every 2 weeks with spot changes occurring twice weekly. Development of per diem costs, maintenance of temperature and humidity, determination of floor space allotment, and provision of an optimal diet were addressed for these naked mole rats. Our facility charged a per diem similar to conventionally housed rats to maintain the naked mole rats. The temperature of the animal room was maintained at 85 ± 4° F and the animals’ primary enclosures were misted with reverse osmosis (RO) water twice daily to maintain appropriate microenvironment humidity. Sweet potatoes soaked in water were provided as the primary diet and water source, but animals were also supplemented with additional low calcium fruits and vegetables. In summary, we describe techniques used to overcome the unique husbandry and housing challenges when maintaining naked mole rats in a biomedical research facility.

P96 Environmental Monitoring to Support Extended Cage-Change Frequency in Individually Ventilated Mouse Cages

AM Swanson^*, JW Grimm

Psychology, Western Washington University, Bellingham, WA

We measured temperature, humidity, and ammonia levels inside individually ventilated mouse cages (IVCs) to support extending cage cleaning to every 14 days using compacted cellulose 1/8” bedding. Different social housing densities (1, 3, 6, and dam with litter) were studied with C57/B6 mice of both sexes. Routine cage changes are required in order to limit ammonia levels within the mouse microenvironment, but the frequency of which can negatively affect both animal and human welfare. Cleaning less frequently can help improve negative stereotypic behaviors in rodents and limit staff exposure to allergens, ergonomic injury risk, and zoonotic diseases. Most cage change intervals have been set based on visual cues indicating a dirty cage and the odor of ammonia, which humans can detect at 0.4 ppm, far below the suggested maximum within the mouse microenvironment. This suggests that it may be possible to extend the time between cage changes without exposing animals to high levels of ammonia. Previous literature supports the ability to extend mouse cage changes up to and past 14 days while housed in IVCs, dependent on the type of bedding substrate used, and amount of air changes per hour. For this study, 50 ppm was the maximum level of ammonia allowed. The highest average ammonia level observed during the study was 5 ppm in the cage housing a dam and litter. It was determined the cage change frequency for all mouse cages within the facility could be extended to every 14 days and possibly even longer. There was a significant (P < 0.5) effect of ammonia level variation by condition and all results were found to be well under the allowed maximum of 50ppm. The next step is to conduct a similar study examining current environment conditions within rat IVCs.

P97 Novel Wild Squirrel Tree Hideaway to Aid in a Research Setting

AS Glenn^*

Comparative Medicine, University of Maryland, Baltimore, MD

We have designed a novel hideaway for wild-caught Gray Squirrels (Sciurus carolinensis) to try and replicate a more natural setting while using them for research. We sought to design a structure that would replicate a natural tree with a nesting hole. What we designed was a user-friendly structure that could go through the cage washer after each use and with slight modification could be used as a cat perch once the squirrel study had concluded. We designed our structure using PVC material that was easy to cut and simple to put together. The trunk of the tree was made with 4” PVC that was connected to an “H” base also using PVC material. The H base was also constructed using 4” PVC with flat end 4” caps for stability. We overlaid the trunk of the tree using corrugated black drainage pipe to allow the squirrel to climb up the smooth PVC surface and added ¾” pipes to the trunk as branches. This allowed the squirrel to climb up to the top of the tree with ease. At the top of the tree we attached a nesting box to give the squirrel a hide-a-way as was needed for our study. Once released into the secure room the squirrels immediately climbed the tree and went into the nesting box. The squirrels were allowed to come and go into the nesting box as they willed and were seen climbing down the tree to roam around the secure testing room. We suggest that this design is successful as a new tool in keeping squirrels stimulated in a more nature environment during testing in a research facility.

P98 Study Considerations for Vibration-Induced Behavior Changes in Mice

A Garner^*, RP Reynolds, JN Norton

Division of Laboratory Animal Resources, Duke University Medical Center, Durham, NC

Minimizing noise and vibration in animal facilities is a concern of critical importance. Previous studies have shown that vibration in particular can have a wide range of adverse effects in rodents including impaired immunity, altered neuroendocrine function, changes to cardiovascular parameters, and decreased reproductive efficiency. Decreases in reproductive efficiency can heavily impact the viability of breeding colonies and research results. Despite documented adverse effects, there are no established vibration exposure limits for rodents. Therefore our goal has been to determine vibration thresholds at which female C57Bl/6 mice show alterations in normal behavior. Prior to being exposed to vibration, mice were first acclimated to the testing environment to ensure they were reacting to the vibration and not the novel environment in which they were tested. Mice were video recorded immediately after being placed in a cage on the shaker and at various time points up to 24 hours. Behaviors demonstrated in video recordings from each time point were reviewed, scored, and categorized as active, inactive, or maintenance every 10 seconds during a 10-minute period. Within the first 3 hours mice exhibited significantly more active behaviors when compared to both inactive/maintenance behaviors. By 6 and 24 hours inactive/maintenance behaviors were more frequent but the difference was not statistically significant. Once an adequate acclimation period was established mice were then exposed to vibration at frequencies between 30-100 Hz and accelerations between 0.1-2.5m/s². Mice did not demonstrate a behavioral change until the acceleration of vibration was 2m/s² or above. These results suggest that a longer acclimation period may be needed for behavioral testing relative to acclimation times noted in the literature and that relatively high acceleration of vibration may be necessary to cause behavior changes in mice.

P99 Let’s Get Certified! Keeping Employees Motivated and Driven

AA Gyles^*, M Anderson, K Lucas, P Trice, L Keener

SoBran Inc, Bethesda, MD

The laboratory animal science industry offers a variety of avenues for career growth and employee development. Management faces the challenge of continuously motivating their employees to achieve AALAS certification, which will assist the employee to be equipped with the appropriate qualifications for advancement within the field. Many institutions promote and, in many cases, require certification from AALAS to qualify for promotional opportunities. Based on our pass/fail stats we felt the need to assist employees by providing an inhouse AALAS certification training program. Developing an option for making AALAS certification achievable for each employee is a useful strategy in promoting career growth and advancement. The first step in creating a program is developing a syllabus that will maximize each student’s capabilities, identify subject experts to provide the training, identify any potential adult learning challenges, and develop a schedule that is feasible with a convenient location. We’ve found that smaller inhouse AALAS certification preparation courses are more desirable as they create more employee engagement and interaction. Incorporating various tools such as chapter quizzes, crossword puzzles, and games into the training syllabus greatly assist with the connection of a variety of learning styles. These tools maintain employee interest and reiterate information learned that may have otherwise been forgotten. One-on-one review/training should be offered to individuals that are perhaps struggling with reading and technical comprehension. Providing this attention provides the employees with a sense of comfort, thus creating self-confidence and a drive to succeed. As a result, employees are inspired and empowered. They are gaining knowledge, enjoying the training, and achieving AALAS certification. It’s paramount in understanding that each student is different and training options should be flexible to achieve maximum success. The inhouse AALAS training program has increased our staff pass/fail rates by 20.2%.

P100 Electronic Rodent Colony Management Made Simple

A Dollar^*, M Rammling

Insourcing Solutions, Charles River Laboratories, Orlando, FL

In many laboratory animal facilities the task of rodent colony management is the responsibility of the animal care staff (ACS). When commercially available electronic systems are not in place, manual means to create and exchange colony management information, such as new litter logs, wean reports, and service requests can cause excessive paper trails and delays. Having an efficient documentation process in place is crucial to ensure successful colony management. Our program has streamlined the process by creating an electronic New Litter Log form (eNLL) which can easily be implemented in other animal facilities to enhance the service provided to the principal investigator (PI), research staff, and veterinarian. The processes of handwritten documentation and manual distribution via mailboxes for new litter logs, wean reports, and service requests, was eliminated. Instead, an eNLL form in PDF format was created to facilitate these processes electronically and directly from the animal holding room via laptops (wi-fi or ethernet). This simple PDF form can easily be customized to any site-specific needs and processed via any email provider or even integrated into a web application such as a content mangement system, which enables additional features on workflow management. In order to further expedite the distribution of colony management information via the email application, our program has developed standardized eNLL email formats, along with task specific email distribution lists. By developing this system and creating the eNLL form, the rodent colony management flows with greater ease for the ACS, PI, research staff, and veterinarian. The results are ease in reporting, consistency in delivery, remote access via electronic library, faster turnaround time, paper reduction, and most importantly, happier animals.

P101 Comparison of Individual Ventilated Cage Rack Cleaning Methods: Tools for Exhaust Air Dust Testing

A Dodelet-Devillers^*1,2, J Canale², CL Perkins³, A Jimenez², J Gourdon², KS Henderson³

¹Medecine Veterinaire, Universite de Montreal, Montreal , Canada; ²Comparative Medicine and Animal Resource Centre, McGill, Montreal, Canada; ³Charles River Laboratories, Wilmington, MA

Rodents housed in individually ventilated cages have been routinely monitored for pathogenic agents by the use of soiled bedding sentinels. Wanting to take advantage of the recent advances in exhaust air dust testing as an alternative to sentinels unfortunately left us with historical fur mite results that do not reflect the current state of our animal facility. In this study we tested 4 different cleaning methods (rack wash only, mechanical wash only, mechanical and rack wash, and mechanical with a bleach solution and rack wash) to best determine the most effective way to remove residue fur mite DNA, bacterial DNA (Pasteurella pneumotropica) and rodent nonspecific DNA. We tested by PCR assay before cleaning and after cleaning, and determined that all 4 ways were effective in removing fur mite DNA. A mechanical wash with a bleach solution alongside rack wash was most effective to eliminate the dust containing Pasteurella pneumotropica and rodent DNA. With this knowledge, we are now able to fully implement an environmental health monitoring program.

P102 Comparative Analysis of 2 Health Monitoring Programs in an IVC Mouse Facility: PCR versus Conventional Screening

K Schlarmann², MC Pils¹, H Riedesel³, B Pasche^*2

¹Mouse-Pathology, Helmholtz Centre for Infection Research, Braunschweig, Germany; ²Central Animal Facility, Helmholtz Centre for Infection Research, Braunschweig, Germany; ³Central Animal Facility, University Medical Center Goettingen, Göttingen, Germany

Health monitoring programs using sentinels are being criticized for their inability to detect pathogens with low submission rate. Here we report our practical experience with a PCR-based health monitoring in 2 separate barrier facilities, 1 of which is operated as a specified and opportunistic free (SOPF)-breeding unit with about 5,000 cages. The other one with about 1,000 experimental cages is run under incomplete SPF conditions with Helicobacter, Norovirus, and Pasteurella pneumotropica being tolerated. Both facilities are fully equipped with IVCs. During this study the animals’ health status was monitored quarterly by PCR of pooled environmental swabs taken from exhaust prefilters of all IVC blowers and fecal samples taken from 26 dirty bedding sentinels, which are kept in pairs of 1 immunodeficient (B6.129S7-Rag1^tm1Mom/JHzif) and 1 immunocompetent CD1 (Crl:CD1(ICR)) female used for 6 months. These results were compared to those of the conventional health monitoring using serology, bacteriology, parasitology, and pathology of 160 CD-1 dirty bedding sentinels which were shipped quarterly to an external diagnostic lab. Whereas in the SOPF breeding unit no mouse pathogens could be detected, all the above mentioned tolerated pathogens were found in the experimental unit with both methods. The presence of ß-hemolytic Streptoccoci, Staphylococcus aureus, Klebsiella oxytoca, Proteus mirabilis, Helicobacter spp., and Pasteurella pneumotropica could be identified with environmental swabs but not in fecal samples. The PCR-based health monitoring significantly reduces the number of sentinels and cuts the overall costs by half. It is an excellent method to monitor the health status of barrier maintained animals and shows an increased sensitivity compared to conventional methods. The reduction of the number of live animals necessary for examination is an active contribution to animal welfare and the principle of the 3Rs.

P103 Assessment of Drinking Water Quality in Storage Water Bottles and Carboys in the Vivarium

W Gately^*

Comparative Medicine, Pfizer, Andover, MA

Providing high-quality drinking water is essential to both the welfare of laboratory animals and the scientific integrity of studies. Provision of water in bottles continues to be the most common method of water delivery in rodent colonies. We provide high-quality, filtered reverse osmosis (RO) water to our animals in glass water bottles. RO employs an ultrafiltration membrane through which water flows under pressure. Up to 99% of inorganic compounds including toxins, pyrogens, heavy metals, and microorganisms are removed from the filtered water. The filtered water is collected into bottles, some of which are used same day. The remaining bottles and carboys filled with water are stored for use over the next 7 days. The Guide requires that periodic monitoring of water be done for pH, hardness, and microbial or chemical contamination, but does not state how long different types of clean water (RO, tap, etc.) can be stored safely. We routinely store RO water in glass bottles for up to 7 days, though the basis for 7-day storage rule for water used in our rodent colonies is unclear. To determine if this 7-day rule is a reasonable expiration date for RO water stored in glass bottles and carboys, we measured pH, Relative Light Units (RLU), and bio burden on a weekly basis for 4 weeks. RLU numbers started to spike on day 14 of testing, though bio burden numbers remained at 0, and we saw only minimal changes in pH. Based on the results of this study we haven’t made any changes to our water bottle storage practices.The data that was collected is on par with what we do currently on site now. We are currently revaluating the data and study to determine if it can be duplicated.

P104 Reducing Aggression in Mice with Addition of Cage Dividers

BR Tallent^*1,2, J Lifshitz^1,2, T Parekh¹

¹Child Health, University of Arizona College of Medicine, Phoenix, Phoenix, AZ; ²Translational Neurotrauma Research Program, Barrow Neurologic Institute at Phoenix Children’s Hospital, Phoenix, AZ

Mice fight for hierarchical dominance, resources, territory, breeding, or aberrant behavior. Reducing aggression has long been the goal of many institutions and investigators. The advent of environmental enrichment has helped, but is often limited due to cost, housing, or research parameters. The end result of fighting is injury, which requires isolation of animals, thereby increasing costs, with an impact on research studies. Further, animal models of traumatic brain injury (TBI) have led to increase in aggressive tendencies in mice, particularly following the stresses associated with cognitive and motor function testing. Wherein behavioral assessment and stress are both key components of our paradigm, our goal is to develop a method of reducing aggression that does not involve isolation of animals, drugs, or other behavior modifying techniques that push responses away from “normal.” Additionally, the solution cannot incur undue costs or work for laboratory animal staff. Therefore, we conducted preliminary observational studies of group-housed mice within standard disposable mouse cages with the addition of custom built dividers. Mice cohorts were videotaped and reviewed for quantity and severity of aggressive behavior, in comparison to cages without dividers. Marked reductions in aggressive behavior were observed in cages containing dividers. The cage dividers, which provide burrows and common access to food/water for mice, are a simple, cost-effective means to reduce aggression in group-housed mice. Overall, animal welfare is predicted to benefit from compartmentalization within a single cage, without obscuring observation or restricting access to food/water.

P105 Evaluating the Quality of Chlorinated Water for Use with Severely Immune-Compromised Mice

BP Long^*, R Diamond, KA Adams, D Shuey

Incyte, Wilmington, DE

The industry standard to provide adequate, sterile drinking water to severely immune-compromised mice requires multiple steps of water and equipment preparation. This includes ultra-filtering water supplied to the animal facility, water acidification (pH ≤ 3), filling the water bottles, followed by autoclaving the batch to be supplied to the animals. This is the current process used for NSG mice housed in our animal facility. The NSG mice are our most sensitive strain susceptible to disease caused by organisms considered to be commensal in immune-competent mice, and we wanted to explore a more simplified and refined process for supplying water to our NSG mice. This would be a 1-step process of chlorinating water (5 to 6ppm) in a sealed drinking pouch thus replacing the acidification process. We first tested the sterility of the unused acidified/autoclaved water and unused chlorinated water pouches. Test results proved both waters to be sterile and appropriate for NSG mice. We next tested used acidified/autoclaved water and used chlorinated pouches to determine if sterility was maintained with normal use Sterility was maintained in both samples. Our test results indicate that the refined process of chlorinated water pouches could provide an appropriate water source for NSG mice.

P106 Big Efficiency Gains in Census Management with Radio Frequency Identification Technology

CD Ranns^*1, PA Sinnett-Smith³, RL Menner², BM Stewart²

¹Comparative Medicine, Pfizer, Inc, Cambridge, MA; ²Business Technology, Pfizer, Groton, CT; ³Comparative Medicine, Pfizer, Sandwich, United Kingdom

Census management can be a time-consuming process for operations personnel in large vivarium facilities. We partnered with colleagues in the Business Technology department to find a more efficient solution for census management to improve accuracy and compliance, decrease the resources required to manage the census, and fully integrate with the existing animal ordering and stock management system already in place. The first step was to reevaluate and reengineer the census business process establishing a standardized process across all sites. Radio Frequency Identification (RFI) technology was adopted as the primary tool of the process. All cage cards now include an embedded chip and antennae which contain animal and cage identification data. Partnering with a third party vendor, disposition boxes were created with radio frequency identification scanners which read the chip on cage cards deposited in them after the animals are euthanized at the end of a study. The dataset created from the scanner is sent to the animal ordering and stock management system where the euthanized animal cages are removed from the census hourly. Using the disposition boxes reduces recording a disposition from 84 seconds per cage to 1 second per cage, a significant time savings for operations personnel. Cage counts in the vivarium are captured using hand-held radio frequency identification readers which substantially reduce the time spent collecting this data compared to hand count or optical bar code scanners previously used by Comparative Medicine. An automated report then compares the cages scanned with the active cages in the animal ordering and stock management system, highlighting any variance. Comparative Medicine personnel can review the report, assess reasons for the variance, and take direct action to remediate variances in the report by clicking on the appropriate button. The report automation was able to reduce reconciliation time by 54% and continues to improve as experience with the tools is gained.

P107 Yellow to Green: Exploring the Possibility of Composting Soiled Animal Bedding

M Stoltz¹, C Paulson^*1, JD Reuter¹, E von Bleichert³, K Ramirez Aguilar², M Lapham³

¹Office of Animal Resources, University of Colorado—Boulder, Boulder, CO; ²CU Green Labs, University of Colorado—Boulder, Boulder, CO; ³Environmental Health and Safety, University of Colorado—Boulder, Boulder, CO

An academic institution has a campus-wide initiative to promote sustainability through CU Green Labs (CUGL). CUGL uses a team approach to minimize the use of energy, water, and material goods without compromising research integrity or safety. In January 2015, CUGL, collaborating with Environmental Health & Safety (EH&S) and the Office of Animal Resources (OAR) initiated a feasibility study to assess the practicality of composting soiled animal bedding. It was estimated that vivaria, supporting a modest cage population of 4,000 rodent cages, generated around 52 tons of soiled animal bedding annually (mainly hard wood shavings). Propitiously, the institution already had an arrangement with a composting facility to receive compostables generated on campus. Samples of soiled bedding were submitted for review to ensure compliance with local, state, and federal regulations. Biologic, chemical, and other hazards were assessed for compost compatibility by the IACUC and EH&S during protocol review as these items must be segregated into different waste streams. Next, we established performance metrics to identify biocompatible collections bags, determine how many bags of soiled bedding were generated daily and traced OAR workflow routes, including compost holding areas. These factors were not found to be obstructive of our goals. The animal care staff were trained on segregating compostables as it was crucial that bio/chemical hazards were sent out via independent waste streams. Plans are underway for modification of loading dock areas to expand waste holding capabilities, which was the greatest challenge because of the cost, restricted space, and impact to various groups. Bedding ultimately will be sent to an anaerobic digester, which will convert the waste into useable products such as renewable natural gas, high value organic amendments, and fertilizer grade products. Establishing a program to recycle material animal waste requires a team effort from multiple departments, vivarium staff, and institutiuonal support. It is the goal of this program to reduce the environmental impact of vivaria waste and minimize use of municipal landfills by incorporating animal bedding into the current campus compost waste stream.

P108 Health Monitoring Programs for Zebrafish (Danio rerio) by PCR Testing

CL Perkins^*1, C Lawrence², KS Henderson¹

¹Charles River, Wilmington, MA; ²Boston’s Children’s Hospital, Boston, MA

Zebrafish (Danio rerio) are used as models for research in genetics, disease, toxicology, and behavior studies. To maintain research integrity and reproducibility, health monitoring programs should be implemented for zebrafish colonies so that they are consistent with standard practices for other laboratory animal models. In our study, a simulated health monitoring program using environmental samples and fish from a tank rack at an academic institution was evaluated using qPCR assays for the detection of 17 pathogenic bacteria, parasites, and viruses. Each fluorogenic real-time PCR assay was designed to be specific for the target agent based on sequences available in GenBank or sequencing performed in our laboratory. Internal controls for nucleic acid isolation efficiency and PCR inhibition were included in the protocol. Baseline PCR testing was performed on a subset of the sentinel fish population, prior to introduction into the system, and all results were negative. The remaining fish were added to standard sentinel tanks. After 6 months exposure to the effluent water in a recirculating aquaculture system (RAS), qPCR testing was performed on pooled sentinel fish, as well as pooled moribund fish, detritus, and biofilm sample types from the same RAS. Detritus and biofilm samples were submitted from both the sentinel tank and the pooled system tanks for separate evaluation. The parasite P. neurophilia was detected in the moribund and sentinel fish only, and was not detected in any environmental sample. The DNA copy number detected for P. neurophilia was approximately one log10 higher in the moribund fish than the sentinel fish. The environmental samples were preferential for detecting Mycobacterium, especially M. abscessus, which was only detected in the detritus samples. M. fortuitum was detected in all sample types, but M. chelonae was detected only in the sentinel fish, detritus, and biofilm from the sentinel tank. The DNA copy number for the Mycobacterium spp. was consistently the highest in the detritus sample from the system tank. This study data supports that a health monitoring program for zebrafish via PCR should include sentinel fish, moribund fish, and detritus samples for the most comprehensive analysis of a given RAS.

P109 Transcending Challenges during a Departmental Restructure: Fostering Autonomy and Communication through Technician-Lead, Cross-Functional Teams

CM Curci^*, KM McDonald

Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA

Our organization recently restructured, resulting in partially hybridized cagewash, animal care, and veterinary technical positions. Potential benefits included enhanced integration between units, increased efficiency, and development opportunities for staff through diversification of skill sets. However, employees vocalized fear of changing performance expectations and reporting lines. Six months post restructure, in our largest site (N = 40), we observed decreased morale; 21% of staff actively engaged in extra-departmental job searches. We also observed a decline in efficiency. In response, we surveyed staff. Positively, 79% reported they intended to stay with the department for greater than 2 years, and 87% felt there was good team environment. However, staff reported dissatisfaction with time management between units, 51% felt overworked, and 48% felt a need for improved communication from management and generalized ambiguity. To address staff concerns and improve understanding of restructure goals, we implemented a 6-week project, using 5 cross-functional teams, comprised of cagewash, animal care, and veterinary technical staff. Technicians recently delegated leadership positions in the restructure were assigned as team captains. Captains lead the groups and facilitated charter goals including attendance, efficient scheduling, volunteering, providing positive feedback to others, and generating ideas for process improvement. Incentive for participation was based on a point system, and teams were provided weekly standing updates. The project improved assignment volunteering from 59% before implementation to 95% post implementation. Staff was more efficient despite increased volunteering. There was 1 resignation during the project, but otherwise we observed no increase in overtime. Staff was more integrated, as 60% independently reported an appreciation for another unit’s responsibilities. Lastly, staff was more autonomous and communicative, leading to improvements such as sanitization scheduling between cagewash and animal care. The project successfully facilitated job comprehension and autonomy, but final surveys revealed a need for additional efforts to improve bilateral communication between staff and management.

P110 Comparison of Vaporized Hydrogen Peroxide and Paraformaldehyde Gas Decontamination on High-Efficiency Particulate Air Filter Banks

CM Brown^*1, J Gibbs Erwin¹, S Crossfield²

¹Animal Resources, University of Alabama at Birmingham, Birmingham, AL; ²Occupational Health and Safety, University of Alabama at Birmingham, Birmingham, AL

Decontamination of high-efficiency particulate air (HEPA) filter banks is required for high-containment facilities. Filter bank decontamination may be performed prior to HEPA filter change in response to a spill of infectious agent outside primary containment or other reasons based upon a risk assessment. When decontaminating with paraformaldehyde gas or vaporized hydrogen peroxide (VHP), a complete kill of the organisms on the biologic indicators must be documented 3 consecutive times to be accepted as the approved method of decontamination. Biologic indicators were placed in 3 locations inside the filter bank: the supply port, downstream side of HEPA filters, and downstream side of HEPA filter at the exhaust port. Fourteen decontamination cycles were run; 7 using vaporized hydrogen peroxide and 7 using paraformaldehyde gas. Six of the 7 VHP runs were successful as evidenced by the biologic indicators, which showed no growth for 7 days. The failure was attributed to a faulty biologic indicator, where the positive control did not grow. All 7 of the paraformaldehyde runs failed. After the initial failure the concentration of paraformaldehyde was increased and a blower was introduced to the system to circulate the gas through the filter bank for different periods of time. All of the biologic indicators grew within 48 hours. Consistently demonstrating 100% kill of the biologic indicators is required for HEPA filter change. Of the 2 methods evaluated, vaporized hydrogen peroxide was shown to be the most consistent and efficient method of decontamination. Paraformaldehyde gas failed to achieve decontamination of the HEPA filters, thus making it unacceptable for use in our facility.

P111 Cage Card Wars

CL Yates^*1, S Lunt^2,1, E Jackson¹

¹Laboratory Animal Resource Center, Southern Methodist University, Dallas, TX; ²Office of Research and Graduate Studies, Southern Methodist University, Dallas, TX

On a daily basis, animal care technicians and other vivarium users navigate a multitude of animal holding room and cage-specific requirements. These requirements can range from biosafety-level designation, nonstandard diets, and specialized husbandry to environmental enrichment for study-specific cages. Additionally, breeding projects can require supplemental cage-level tracking and documentation. It is critical that visual identification and labeling systems are developed to effectively communicate these requirements. Through the implementation of these systems inconsistencies are reduced, communication is improved, and efficiencies in animal care and disease model management in any animal care and research program can be gained. Our facility has developed a systematic cage card program to eliminate overuse of cage cards and provide organization and conformity. This system consists of a series of 1’’ x 1’’ adhesive, colored labels, various colored 4’’ x 5’’ and 3’’x 5’’ transparent cards, and card stock of 2.5’’ x 5’’ standard facility card, 3’’ x 5’’ breeder cage card, and 3’’ x 5’’ sick and dead animal notices. Each rodent housing cage is furnished with either a standard facility card or a breeder cage card. Additionally, every rodent housing cage is supplied with a 4’’ x 5’’ clear transparent overlay. The transparent overlay serves a place holder to affix the labels. The facility issued cage cards serve as a helpful reference source for obtaining information about an individual cage. The colored overlays assist with providing more of a comprehensive understanding of what should be focused on during the daily health monitoring process. Lastly, the labels affix to the transparent cards to provide direction for specified instructions. All components of the cage card notification system that is in place at our institution work in a collaborative effort to aid in seamless communication of special instructions, treatment regimens, and post surgical and breeder cage requirements at the cage level.

P112 Husbandry and Clinical Management of a Unique Species: The Ossabaw Swine

C Sims^*

The Ohio State University, Columbus, OH

The domestic pig is one of the most valuable animal models used in biomedical research today because of its biologic, physiologic, and anatomic similarities to human beings. The Ossabaw pig breed is biologically unique having been shaped by natural selection in an isolated island environment. They possess a “thrifty” gene which enables individuals to store significantly higher amounts of body fat compared to other domestic pigs. This gene enables them to survive when food is scarce, but also predisposes them to developing metabolic syndrome. This adaptation makes them a useful animal model for human metabolic syndrome and its associated disease processes. In a research project with Ossabaw swine, pigs were fed a high-fat diet to induce metabolic syndrome over an extended duration (∼17 months). Multiple husbandry and veterinary challenges were encountered during the study period. The clinical problems associated with the study-induced obesity which required additional management included: skin fold dermatitis, loose stool, laminitis, and possible neurologic effects. Maintenance of the pigs for an extended duration was also problematic as the growth of their canine teeth or “tusks” mimic that of their feral cousins. Both sexes are born with deciduous canine teeth and permanent tusks erupt at about 7 to 13 months of age. To prevent injury to both humans and animals alike, a routine teeth-trimming program was implemented to manage the length of the tusks. In summary, long term maintenance of Ossabaw swine on a high fat diet results in multiple husbandry related and clinical challenges; however, appropriate management of this valuable animal model can be achieved with diligent care.

P113 Successful Housing of Pigmy Rattlesnakes (Sistrurus miliarius) in a Laboratory Setting

DM LeMoine^*, CL Freed, ML Holding

The Ohio State University, Columbus, OH

Laboratory housing of any species must take into account the physiologic and behavioral requirements of that species, personnel safety, and research needs. Housing of venomous snakes in particular requires consideration of specific environmental requirements, proper handling, safety precautions, and emergency plans. Our institution has maintained a group of 13 pigmy rattlesnakes (Sistrurus miliarius) for venom composition research, requiring the establishment of an IACUC-approved satellite housing space. Prior to housing venomous snakes, drains and heating units were covered with screens and a floor sweep was attached to the bottom of the door to prevent snakes’ egress from the laboratory should they escape their primary enclosures. A relationship with the local emergency room was established, including the development of an emergency SOP. Following these preparations, wild-caught gravid females were obtained and adults and their offspring have been successfully maintained for 8 years. Snakes are individually housed on newspaper with pine bark bedding which was spot changed as needed. Standard plastic reptile cages with opaque walls and a padlocked sliding transparent front were sanitized monthly. The room temperature is maintained at 70-80°F year-round with a 12:12h light cycle and full-spectrum lights. Bedding is misted as needed to maintain optimum humidity at the cage level. Fresh water is provided ad libitum and, as part of the study design, snakes are fed frozen-thawed mice, lizards, or frogs sourced from a collaborator once weekly. Hides or exam glove boxes are provided for additional shelter. For personnel safety, snakes are transferred into a secure plastic holding container during routine husbandry. Snake hooks, tongs, and transparent restraint tubes are used for restraint and handling is restricted to trained individuals with a minimum of 2 people present during any procedure. Only 1 enclosure unlocked at a time. Multiple levels of security restrict access to the housing space to approved personnel only. There are specific risks associated with housing venomous species, however this pigmy rattlesnake colony has been maintained with minimal clinical concerns, no observable behavioral abnormalities, and no injuries to personnel.

P114 Disposable Water Delivery Systems Role in Contingency Planning

DK Joseph^*, J McNair, JM O’Donnoghue, ED Allen, LJ DeTolla

Comparative Medicine Program, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD

Contingency and disaster planning in accord with the USDA APHIS directive and Office of Laboratory Animal Welfare is an essential component of an animal care and use program that should account for all aspects of animal health and wellbeing. In the face of any disaster, 1 of many contingencies is provisions for uncompromised and uncontaminated water. Water is delivered to research facilities through a municipal supply or well reliant on utilities like power to render water potable and to convey water to the site where it is consumed. Furthermore, research facilities equipped with water treatment and purification systems also rely on site utilities to further refine and treat water and to bring water to the point of use. Laboratory animals maintained on automated watering systems and/or water bottles and the associated equipment used to process water delivery systems are affected by utilities outages. Despite possible provisions for emergency power to water delivery and processing systems, there are many times limitations on the duration emergency power may be sustained. For example, fuel-driven emergency generators may be operational for a limited duration in the absence of fuel or unless the fuel supply is re-established or replenished. Our institution’s contingency plan provides for the use of disposable water delivery systems that provide a viable solution for water delivery under these conditions. Automated watering systems and bottle filling equipment can be easily adapted to feed water from an elevated container relying simply on the transfer of water from the disposable water delivery system and gravity. Producing and maintaining a sizable inventory of water is a practical contingency approach and one that can be used to deliver water to all species regardless of the native water delivery system used.

P115 Moving Mice from Static Caging to IVC: Effects on Microbiome, Breeding Performance, and Blood Chemistry

DR Goulding^*1, PH Myers¹, CA McGee¹, S Hackney¹, J Locklear², SD Peddada³, DM Kurtz², TL Blankenship¹

¹Veterinary Medicine Section, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC; ²Quality Assurance Laboratory, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC; ³Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC

Our facility has migrated from static microisolation cages to individually ventilated cages (IVC) and it was of interest to determine if changes in physiologic parameters might occur. We evaluated differences in mice housed in the two types of caging as well as differences in mice born in one type of caging and moved to the other. Fecal microbiome, breeding performance, blood chemistries, and complete blood counts (CBCs) were the parameters selected for evaluation. Twelve breeding pairs of 8-week old C57BL/6 mice each were set up in static cages and in IVC. Offspring were weaned into their respective caging types. Two weeks following weaning half of the litters born into static were moved into IVC and half born into IVC were moved into static. Breeding pairs remained together until 3 litters were produced. Litter to litter interval and litter size at weaning were documented. At 6-weeks of age 8 males and 8 females from each group were selected from the first litters and euthanized and blood collected for serum chemistries and CBCs. Feces were collected from all cages for microbiome composition 3 weeks after migration from one cage type to the other. No significant differences in litter size at weaning or interval were observed between or among the groups. A significant increase in Lactobacillus spp. in feces of mice housed in the IVCs compared to the static cages was observed; however, the relative abundance in both groups was considered too low to be biologically relevant. Male mice housed in ventilated caging had slightly elevated alkaline phosphatase. Hematocrits were significantly higher in both sexes that had their caging type switched from static to IVC compared to mice that remained in static cages. Significantly fewer monocytes in both sexes were observed in those that were moved from static to IVC compared to mice that remained in static cages. Although only a few differences in blood chemistries and counts were observed, these differences may be significant depending on the research goals and should be considered before moving animals into a new caging type. These findings further suggest that the fecal microbiome and breeding performance are minimally impacted by caging type or whether mice are moved into a different type of cage.

P116 Single Technician Setup and Husbandry Of Germ-Free Mice Housed in a Positive-Pressure Caging System

DM Urbonas^*, J Alderman

Immunobiology, Yale University School of Medicine, West Haven, CT

Conventional flexible film and semi-rigid isolators require a great deal research space. Labor costs and lead time to ready an isolator for occupancy are significant. Positive-pressure caging offers a revolutionary alternative, as the set up and husbandry of the cages can be can be performed by a single technician. Reprocessing of the cages can be done in a single day, significantly reducing the lead time necessary for study setup. Cage components are washed, double-wrapped in a surgical pack style, autoclaved using the facilities established gnotobiotic cycle, and assembled under a biosafety cabinet that has been pretreated with a concentrated solution of chlorine dioxide. Rodent chow is processed in the wire bar assembly of the cage bottom. Packs containing empty water bottles and sipper tubes are also delivered into the BSC. Flasks of water and bagged HEPA filters are submerged in the sterilant before entering the BSC. One sampling cage, including forceps and sample collection tubes, is included with every cage change event to allow for cage sampling. With all cage components under the BSC, a sealed container of germ-free mice is submerged in sterilant and placed into a running biosafety cabinet. The technician then dons sterile surgical attire and begins to assemble the cages including installation of a HEPA filter in the cage lid. Prior to securing the lid assembly, mice are transferred into the cage bottoms. The cages are visually inspected for proper closure, removed from the BSC, and docked on the rack. For cage changing, soiled cages are removed from the rack, submerged in sterilant, and entered into the BSC where clean equipment had already been introduced. The technician dons sterile surgical attire and uses the packs to perform the cage change and sampling. New cage bottoms containing bedding, food, and enrichment are changed out weekly. Water bottles are changed every 2 weeks. Cage lids with a new HEPA filter are changed every 4 weeks. Soiled cages are broken down and processed through a tunnel washer. During this evaluation, cages were sampled and changed weekly. PCR analysis of the fecal pellets indicated our ability to maintain the germ free environment after 7 cage changes. An alternative to conventional flexible film and semi-rigid isolators this system offers a time saving, staff conserving, cost effective alternative for conducting both short and long term gnotobiotic studies.

P117 Collaboration and Innovation: Developing a Device to Help Track Mice after Blood Sampling/Dosing

MF Lazar, D Forteza^*

LAR, Merck, Kenilworth, NJ

Dosing and/or bleeding multiple rodents occupying a single cage can prove challenging, especially if the animals are not individually identified. Investigative staff may resort to shifting mice to an empty cage. If it is a clean cage this could result in extra labor and materials and be impractical if the animals are dosed daily. If transferred to a cage or container previously occupied by other mice on study, this could increase the risk of potential cross-contamination, may be stressful to the animals, and could therefore negatively impact study results. It is for these reasons that a multifunctional team consisting of veterinarians, animal care management, investigative staff, and bioelectronics engineers was put together to develop a solution to this problem. Based on group discussion and feedback from investigator needs, the team designed a device that would be used to divide the home cage into 2 separate spaces. Investigators could start their dosing/bleeding procedure with all mice on one side of the divider and then move them to the other side after the procedure, all within the home cage. Design parameters included lightweight and sanitizable material, as well as a way to keep it in place on the cage. Bioelectronics engineers recommended building usable prototypes via a 3D printer to be evaluated by the team and tested by a select group of investigators. After a series of design modifications and retesting by investigators, the final design consisted of a divider made of stainless steel that would hang from the cage top via rails that fit on the cage lip. The collaborative team effort resulted in a novel device that met the objectives and was readily accepted by the investigative staff.

P118 Pinworm Syphacia sp. Decontamination through Efficient Team-Based Action Plan

P Kathy, MF Lazar, D Forteza^*

Laboratory Animal Resources, Merck, Kenilworth, NJ

Historically our facility has been rodent pinworm Syphacia sp. free. All incoming rodent vendor health reports are reviewed prior to receiving animals into the facility. Unfortunately, due to a break at a vendor facility, we received 21 suspect positive rodent shipments over a 3-month period. A task force was immediately assigned for the development of an action plan to address the potential impact of pinworm eradication from our facility. The action plan was developed by representatives from the affected departmental areas coming together to assess the problem, develop solutions, and communicate to all key stakeholders the steps to be taken on executing the plan. The action plan allowed us to identify the affected areas by assessing initial housing of suspect positive animal orders, animal transfers, and analysis of staff traffic flow patterns. At least 6 of the orders were found to be positive, impacting 7 rooms over 2 floors. Rodent tape test, fecal samples, and environmental swabs were taken to confirm the presence of pinworms. Decontamination procedures were established for caging, scientific equipment, and the overall facility. The action plan was presented to the research community to demonstrate an approach that would have minimal impact on their research. The implementation of the action plan was successful in eliminating pinworms form the facility. Since pinworm eggs can remain viable for long periods in the environment, the rodent colony continues to be monitored through tape and fecal test to ensure eradication. At the 20-month time-point the facility remains pinworm free.

P119 A Custom-Devised Catheter System and Platform Collecting Apparatus Refines Nasal Wash Procedures in Ferrets

DA Pfefferle^*, J Hess, J Lucas

Vivo, Battelle Memorial Institute, Columbus, OH

Nasal washes are routinely conducted to detect viral load shedding, antibody titers, or other analysis. Historically, a standard intravenous catheter is used to conduct the nasal washing, requiring that the sharp stylet be removed by hand prior to conducting the procedure. This posed an unneeded sharps hazard, created excess sharps waste, and required increased labor time in prepping the catheter. Increased handling of the catheter also creates a greater potential for decreased sterility of the catheter prior to the procedure. A needleless catheter was sought to increase safety and sterility and to decrease waste and labor. A commercially available option was not found so facility staff worked with an outside vendor to develop a needleless catheter. Additional refinements to the nasal wash procedure included designing a custom elevated and easily sanitizable platform and sample cup holder to be used during the nasal wash procedure to aid with sample collection. The ergonomic design of this set up helps the technician position the ferret so sample collection can be obtained comfortably and naturally for both handler and ferret. The use of the needleless catheter was evaluated by several staff where it was appreciated that the catheter did not need to be disassembled prior to use and performed in the same manner as the traditional catheter. No negative impact was seen in the nasal wash return volumes. The needleless catheter is a custom order item so special attention must be paid when preparing for a study which uses nasal washing. While there is a minimal cost increase for this special unit, the added benefits to using a needleless catheter over the traditional catheter cancel out the added expenditure. The needleless catheter stays in the sterile package for an increased length of time leading up to the procedure. Staff labor is more efficient by negating the need for disassembly and preparation of the catheter for the procedure. Less waste is deposited into a sharps container. And most importantly, an unnecessary health hazard to staff has been eliminated since no sharps are used in the procedure.

P120 Husbandry of Wild-Caught Arctic Ground Squirrels (Spermophilus parryii)

DM LeMay^*1, J Farley¹, BS McGinnis¹, CP Jones¹, EN Yu¹, ML Riess²

¹Division of Animal Care, Vanderbilt University, Nashville, TN; ²Anesthesiology and Pharmacology, Vanderbilt University Medical Center, Nashville, TN

Arctic ground squirrels (Spermophilus parryii) possess unique hibernation physiology that lends itself to biomedical research, but are not available from a commercial vendor for study in the laboratory setting. Our institution was presented with the challenge of developing a husbandry plan for this unique wild-caught species in our research facility. We based our husbandry plan on contributions from the researchers, animal care staff, leadership, and collaborative information from other institutions that have housed these animals in the past. A modified rabbit rack with water bottles and automatic watering were utilized to individually house the animals. A high-fat rodent diet was chosen as the main nutrition source. Enrichment provided included crinkle paper bedding, a hide tube, toys, and fresh produce and treats on a daily basis. Pan liners were changed twice per week, and racks were sanitized every 2 weeks. To simulate the natural decline in daylight hours wild Arctic ground squirrels experience as summer ends, the light:dark cycle was initially 12:12 hours, and then each day the lights were turned off 8 minutes earlier than the day before. While at our research facility, the Arctic ground squirrels gained weight, acclimated to housing and husbandry procedures, and utilized environmental enrichment devices provided. We were able to successfully house this wild-caught species by modifying our current equipment and husbandry processes.

P121 Managing and Monitoring the IVC Microenvironment: Sensor Placement Differences and Suggested Best Practices

J McNair^*, DK Joseph, JM O’Donnoghue, ED Allen, LJ DeTolla

Comparative Medicine Program, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD

The use of building automation systems (BAS) to manage heating, ventilation, air conditioning, electrical, lighting, safety, and other systems affords institutions the ability to effectively and centrally monitor and manage while also affording control of energy costs without sacrifice to human comfort and to control the laboratory animal macroenvironment. BAS monitoring and managing laboratory animal room systems are usually based on strategic and engineered placement of individual sensors as a single point of reference. However, managing these systems at this level does not necessarily reflect the microenvironmental conditions within the animals’ primary enclosures, particularly temperature and humidity. In 2014, Veterinary Resources rolled out a standalone wireless environmental monitoring solution to monitor, report, and alert husbandry management personnel of real-time temperature, humidity, and lighting values and critical deviations. Placement of temperature sensors at strategic locations within the secondary enclosure intended to be representative of intracage temperatures resulted in disparate results compared to BAS values. Further evaluation of temperature sensor placement within occupied and unoccupied intraventilated rodent housing systems (IVC) as well as IVC exhaust plenums revealed temperatures higher than reported at the room level and different than those values reported by the BAS. Studies were also carried out to assess the comparative temperature measurements between occupied IVC cages and their associated exhaust plenums. Results revealed temperature values were highly correlated and indicates sole reliance on BAS and secondary enclosure values may not represent the best solution for monitoring and managing microenvironmental temperature and humidity. The study also indicates placement of sensors in IVC exhaust plenums is equally representative of occupied intracage microenvironmental values and may be the appropriate basis for managing and monitoring these conditions.

P122 Labor-Cost Analysis associated with Disposable Water Delivery Systems: A Matured Assessment

ED Allen^*, DK Joseph, J McNair, JM O’Donnoghue, LJ DeTolla

Comparative Medicine Program, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD

A medical school introduced a disposable water delivery system for laboratory mice in 2004. Extensive time and motion studies were carried out to define labor costs specifically associated with the existing water delivery technologies at the time (both automated watering systems and water bottles) and to establish a comparative cost basis. The same principles of labor-cost analysis were applied to establish true labor costs associated with the new delivery system. This analysis revealed the fully loaded labor cost, inclusive of inhouse production, transportation to the use locations, and waste disposable yielded a labor cost of $0.005/cage/day. Since 2004, there have been substantive process changes and program enhancements that warranted revisiting a labor-cost analysis. Local and remote water storage strategies were changed to address the assurance of a continuous and available water supply. The dynamics associated with early cage retirement prior to the IACUC-approved 14 day cage change interval, changes in housing densities, and expansion of genetically altered mouse lines were reviewed. An increase in the number of breeding cages generated a need for additional cages. Husbandry staff cage intervention to mitigate unsatisfactory micro-environmental deviations and a host of other unaccounted for factors were identified. The results of this cost analysis revealed inconsequential increased labor costs translating to a revised true cost/cage/day. The focus of this publication is to show all labor costs associated with a mature program using a disposable water delivery technology, how those costs were captured, and inclusion of all labor costs associated with the use of this system. The potential for other peer institutions to adopt this solution is evident in the queries we have received. This analysis and use metrics ultimately may benefit those institutions, gaining insight into true labor costs of a relatively young disposable water delivery solution.

P123 A Little Warmth is Good for Nude Mice

SC Nance^*, JM O’Donnoghue, ED Allen, T Coksaygan, LJ DeTolla

Comparative Medicine Program, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD

In an ever evolving industry of managing animal research facilities one of the greatest challenges faced is minimizing disruption of mice between cage-changing cycles to prevent the introduction of variables on research outcomes. Currently, most intraventilated caging (IVC) systems allow for a 14-day cage changing cycle at which feed and water last for 14 days in a cage of 5 adult mice. However, athymic nude mice (NU(NCr)-Foxn1^nu) consume more food and water due to higher metabolic demand under ambient temperature (70-74 °F). The need of replenishing feed and water between cage changes may cause interference of intracage microenvironment and introduce unnecessary stress to animals. Husbandry management hypothesized that housing NU(NCr)-Foxn1^nu mice at their thermoneutral zone of 84-86°F will not only decrease their feed and water intake, but also reduce the frequency of replenishing feed and water between cage change. NU(NCr)- Foxn1^nu mice were acquired from an inhouse breeding colony. Using sterile caging on an IVC system, the mice were housed in autoclaved cages, complete with bedding, diet, and nesting material and provided with hyperchlorinated water pouches. The study was carried out over the course of a 4-week period with cages changed every 14 days. One set of mice were housed at ambient room temperatures (72 ±2°F) and another set were housed at the lower threshold of their thermoneutral zone (82 ±2°F). Food and water consumption was measured every other day. Athymic mice housed under ambient temperatures consumed an average of 15 gr. of diet/mouse/day and 12 ml. water/mouse/day lasting only 7 days and mice housed under thermoneutral conditions consumed an average of 10 gr./mouse/day and 8 ml. water/mouse/day, comparatively reducing both food and water consumption. NU(NCr)-Foxn1^nu mice housed at thermoneutral temperatures eliminated the need to replenish food and water and eliminated interference of intracage microenvironment by normalizing their feed and water intake. A little warmth can go a long way.

P124 Suitable Identification Method for Transgenic Hemophilia Type A Mice

J Forget¹, L Bernier¹, EE Arlund^*2

¹Charles River, Senneville, Canada; ²Somark Innovations, Germantown, NY

Reliable identification of laboratory animals is critical to safety assessment study conduct to generate quality data. Various identification methods are available, including ear tags, manual tattoos, and radio frequency identification devices (RFID); however, most of these are not suitable for transgenic Hemophilia Type A (HA) mice as this model has less than 1% of normal factor VIII activity and exhibits prolonged clotting times. Minor lesions induced by ear tags, RFIDs, or manual tattoos have resulted in excessive blood loss leading to death within few hours. An automated tail tattoo system was evaluated for its suitability. The system provides a controlled depth of needle penetration and delivery of the ink to the mid dermal layer of the skin. Injury to blood vessels is avoided thereby alleviating risk of hemorrhage in these hemophilic mice. This method was evaluated on 210 transgenic HA mice that were 6 to 7 weeks of age at time of identification. Animals were observed for up to 7 weeks during which time there were no clinical observations associated with the identification procedure, confirming absence of substantial damage to blood vessels at the set depth of penetration. Legibility of the tattoo was maintained throughout this 7-week period. The automated characters of the LabStamp system also eliminated risks of ambiguity in characters which may exist with manual tattoo inscriptions. This method was therefore considered to provide an appropriate identification method for use with transgenic HA mice.

P125 Microbial Contamination of Purified Rodent Diets

F Adsit^*, J Locklear, TE Whiteside, T Ward, DM Kurtz

Quality Assurance Laboratory, NIEHS, Research Triangle Park, NC

The term “purified diet” can be misleading to the consumer, in particular, to laboratory investigators lacking a working knowledge of the laboratory animal food industry. Some may assume by the term purified that these diets are free of extraneous chemical and microbial contamination. However, a purified diet is defined by the American Institute of Nutrition as diets that are formulated with a more refined and restricted set of ingredients. Such ingredients frequently include isolated proteins (casein), refined carbohydrates (sucrose and corn starch), and oils (soybean oil). While this definition assures chemical purity, it does not address microbial purity. These diets are often not amenable to being autoclaved due to the labile nature of their ingredients, and some investigators avoid the use of ionizing radiation out of concern for chemical changes that may result from that process (e.g., lipid peroxidation). Thus, it is incumbent upon the investigator to understand what microbial agents may be present in unsterilized purified diets that may be undesirable for consumption by their specific animal model. As an example, investigators respiratory biology may wish to avoid the introduction of species such as Klebsiella pneumoniae to their animal colonies. We examined a number of commonly used purified diets by enrichment in Rappaport-Vassiliadis broth to obtain microbial isolates after streaking for isolation on Brilliant Green agar. The type of microbial agents most often detected were then identified via sequencing and represented at least 5 different families with Enterobacteriaceae being most commonly encountered, including documented opportunistic pathogens.

P126 Factors Affecting the Vocational Calling of Laboratory Animal Care and Research Employees

GP Boivin^*1, RJ Markert²

¹Laboratory Animal Resources, Wright State University, Dayton, OH; ²Internal Medicine, Wright State University, Dayton, OH

A survey was conducted to determine the factors affecting the vocational calling of laboratory animal care and research workers. The survey was comprised of 56 questions in 4 groups: passion, job stability/happiness, work volition, and demographics. We hypothesized that individuals who worked in the field a longer time, were older, had higher education levels, were involved with AALAS, and in higher positions in their organization were more likely to indicate a calling to the laboratory animal care field. We also hypothesized that job satisfaction and classifying one’s job as a calling were positively related to organizational support and work volition. Forty-four percent of respondents categorized their work as at least partially a calling. Those working at a higher level in the position of laboratory animal technician and in the organization were more likely to view their work as a calling. Increasing education level was related to work being a calling. Overall, vocational calling was associated with higher pay, but technicians were the only subgroup where calling and higher pay were significantly related. Vocational calling and job satisfaction were significantly associated with organizational support. For our sample of workers in the animal care field other factors analyzed were not related to work being considered a calling. Leaders in the field of animal care may find our survey results valuable as they strive to adapt their organization’s structure to the perceptions of their workforce with regard to their sense of calling.

P127 The Improvement, Consideration, and Suitability of Nonhuman Primates Caging

H Jung^*1, K Roh¹, S Park¹, E Auh¹, J Yun¹, H Yu², B Kang^1,3

¹Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea (the Republic of); ²Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea (the Republic of); ³Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea (the Republic of)

Our institution provides infrastructure and support for a large number of biomedical research projects using nonhuman primates (NHP) as models for human disease. We introduced NHP cages according to the Guide in 2009. In 2010, an updated edition was published and standard of floor area was changed from 0.54 m² to 0.56 m². In addition, some problems and necessity of improvement were found in old cages in convenient aspect. At first, the new cages was designed and manufactured to meet the revised guideline by increasing frame length from 0.54 m² (710 mm by 760 mm) to 0.58 m² (740 mm by 790 mm) that provided the expansion of floor area. Furthermore, we additionally installed new functions for user convenience and animal enrichment as follows. The height of perches was lowered 100 mm from the floor considering the species-typical behavior and sitting height. The sitting height is approximately 50 cm in 8kg NHPs. Also, width and number of perches were modified from 130 mm (2 bars) to 56 mm (3 bars) for preventing from tangling tether lines. A newly small door in the front side facilitated convenient approaching and the blood collection for researchers. Removable holder for heat lamp or fluid in the side frame increased efficiency at the space arrangement additionally. Trimmed edges in the floor panel facilitated water cleaning and sanitation for eliminating excrement. Visual barriers which were printed with trees and forest for optical stimulation affected social housing and naturalistic environmental enrichments. Above changes were based on the compatibility between old and new design frame because compatibility of components are convenient in inventory management and minimized additional cost in cage maintenance aspects. Although improvement in NHP cages was primarily due to revised guideline associated with floor area, it also enhances animal wellbeing, research convenience, and cost-efficient management.

P128 Practical Experiences with PCR-Based Health Monitoring Compared to Classic Methods

H Riedesel^*, A Brockmann, V Reupke, J Sichelstiel, G Hindermann

Central Animal Facility, University Medical Center Goettingen, Goettingen, Germany

A reliable health monitoring program (HMP) is essential for the health maintenance of rodent colonies in lab animal facilities. In the past 2 decades this has mainly been achieved by screening dirty bedding sentinels for rodent pathogens by classic serological, microbiological, and parasitological methods. The validity of dirty bedding sentinels, especially in rodents housed in individually ventilated caging (IVC), is being critically questioned in some recent studies, which clearly show that the transmission rate for some important pathogens is poor. Furthermore, such a sentinel program requires a large number of animals and is therefore space consuming, costly, and a potential animal welfare issue. During the past few years PCR-based health monitoring methods (PCRHM) have been developed which open up new possibilities. Last year it was decided to change our dirty bedding sentinel HMP for a PCR-based, sentinel-free program in our rodent facility consisting of SPF-barriers and several conventional units with an average daily census of about 30.000 rodents. Mice were mainly kept in IVC and rats in open caging. The housing units differ regarding the health status: conventional units are positive for Norovirus, Helicobacter spp., Pasteurella pneumotropica and gut protozoa, while another one unit harbors MHV. All these pathogens which formerly have been detected by classic methods, are now easily and regularly identified by PCRHM in pooled environmental swabs (dust from the exhaust ventilation pipes or exhaust prefilters of the IVC blowers) and in pooled animal swabs (fecal pellet, oral, anal, and fur swabs). This new HMP results in a reduced work load, significantly lower costs, and saving of more than 600 sentinel animals per year which allow more than 150 free cages to be made available for scientific use. In conclusion, PCRHM presents an efficient, fast, and reliable means for routine health monitoring in rodent facilities. It is a contribution to the 3Rs in terms of replacement of all sentinel mice, a methodical refinement, and significantly reduces the costs of a HMP. Furthermore, a kind of standardization of health monitoring can be achieved if a specific profile is being used which will facilitate animal exchange between different institutions.

P129 Technical Support for the Maintenance of a Guinea Pig (Cavia porcellus) Myopia Model Colony

JA Stewart^*, AD Pucker, JM Hickman-Davis

The Ohio State University, Columbus, OH

Proper management of a guinea pig (Cavia porcellus) colony to produce and sustain pups for myopia induction experiments is vital for success. Some concerns with producing the model included the need for pigmented eyes, source of breeders, number of breeder pairs, average litter size, level of noise exposure, design and maintenance of myopia induction hoods, additional technician training, and type of bedding used. Pigmented eyes and a susceptibility to myopia are essential for the model. Guinea pigs were obtained from 3 separate sources (government, private institution, and commercial breeding facility) however only 2 of the sources showed a susceptibility to myopia. Animals were housed in commercially available caging with aspen bedding and automatic water systems. Enrichment consisted of music, a hut, and a toy with the addition of a novel item every week. Nutritional enrichment was provided daily in the form of fruits or vegetables according to a varied schedule. Approximately 3 females per source were set up for breeding with a consistent average litter size of 3.6 ± 1 for all strains. Pup mortality was < 1% for the private institution and commercial source animals; however, mortality for the government strain was 38%. The high mortality rate was attributed to music played in the room, as once it was discontinued, the mortality rate decreased dramatically. To generate myopia, a soft, cloth hood was designed to hold a lens over one eye. Hoods were applied to pups only at 24-72 hours after birth. Pups were acclimated to hoods for 1 day prior to lens placement. Hoods did not interfere with normal movements and did not appear to disturb pups or sows. Low dust crinkle paper was used for bedding following hood application to prevent foreign particles from being trapped under the lens. Lenses were cleaned once every 24 hours for the 8-day study timeline. In addition to basic husbandry, technicians were given special training to give technical support for hood management. Technical support above husbandry needs for this colony amounted to 2-3 hours per week including weekends and holidays. The successful identification of susceptibility to myopia in two of the guinea pig strains is the direct result of careful collaboration between the researchers and animal care team.

P130 A Novel Design for Minipig Protective Jackets

J Lovaglio^*1, K Thrall², K Tyrrell³

¹Rocky Mountain Laboratories, Hamilton, MT; ²SNBL USA, Everett, WA; ³Pacific Northwest National Laboratory, Richland, WA

Miniature pigs are a common large animal model used for a wide variety of research projects ranging from toxicology to surgical models to wound healing. Many of these projects require the use of garments or other means to protect critical study components, such as drug application sites, surgical sites, and/or bandages from animals that are known to rub on cage bars, cagemates, and any other items in their environment. A number of techniques have been used over the years for such studies, and many with great success. When presented with a project that necessitated the bandaging of minipigs and protection of those bandages for several months, we embarked on developing a method that would allow us to complete the project with the least impact on the minipigs. A multitude of bandage fixation and protection methods were tried, however, all failed for 1 reason or another. Thus, we set out to design a new protective jacket. After careful review of the positive and negative attributes of available techniques, several prototypes were developed and tested until a satisfactory design was discovered. The new jacket is simple in design and construction, is machine washable, accommodates some variation in body conformation, incorporates regular and stretch fabric and Fabric hook-and-loop fasteners closures, and is worn with a collar. The novel design allowed us to exquisitely protect the bandages of the animals on study (up to 75 days) with very little impact on the everyday activities of the minipigs. Additionally, animals quickly acclimated to wearing the collars and jackets. We believe this novel design may be useful for other research projects faced with similar challenges.

P131 Decontamintion Validation of Duct Work Associate with Biosafety Level 3 Labs

JW Sweeley^*

University of Georgia, Athens, GA

Decontamination is vital to the operation of Biosafety Level 3 (BSL-3) facilities. Multiple proven decontamination methods are available, each having their own pros and cons. Validating that these decontaminations are successful is easy with the use of biologic indicators (BIs) placed throughout the decontamination space. However, some spaces such as the duct work from the room to the associated high efficiency particulate air (HEPA) filters can pose a problem when attempting validation. Validation for this space is needed if any work or repairs are need for the duct work. Our instiution has developed a novel way to achieve this validation. During the preparation of the space for decontamination BIs are placed within the duct work using a flat drain auger. First, the length of the duct work from the room to the HEPA bank is determined. Second, BIs are placed at a distance of every 10 feet on the flat drain auger. The flat drain auger is then placed into the duct work until it reaches the HEPA bank. The room and duct work are then decontaminated using vaporized hydrogen peroxide (VHP). This is accomplished by inputting the VHP directly into the room through ports in the mechanical area and exhausted through a decontamination port at the terminal end of the duct work, right before the HEPA bank. After the decontamination is complete the BIs are retrieved and incubated to determine the successfulness of the decontamination. This method has been used several times with great success. Having validation that the duct work has been successfully decontaminated allows work and repairs to be done in between studies.

P132 Single Chickens Need Enrichment

JA Blair^*

Office of Animal Resources, University of Missouri—Columbia, Fulton, MO

Maintaining the physiologic needs of our chickens, even though they are single housed, is vital to the overall health of the chickens. At our facility, due to the need of the study, animals must be singly housed and enrichment is vital to maintaining the health of these birds. Identifying the materials to be used was determined by choosing sanitizable and safe items, which could easily be purchased at a local store, and food items chosen were already approved for chicken consumption in our facility. The goal was to encourage more foraging behavior with positive interaction, which were criteria considered a success. Three variations were made with the materials chosen and purchased. Two were used with food, while the third was visual. The first 2 were plastic bottles with holes cut in it, which food material could be pecked out of the inside. The third had glass marbles in a plastic bottle of water. During observations, the food had the most interaction and had the most success. The chickens seemed to look forward to the treats after a few times of being offered. The nonfood set-up was a nice change to a hanging mirror and will continue to be used within the enrichment rotation. The chickens were observed interacting with it, but did not have the level of anticipation as the food/forage enrichment did. Overall, the added enrichment for our chickens was a success based on the interaction by the chickens and materials were easy to aquire.

P133 The “Baa”sics of Sheep Flocking

JL Volkmann^*, C Evans, CA Buckmaster

Center for Comparative Medicine, Baylor College of Medicine, Houston, TX

When sheep are pair-housed in standard stalls grazing and ruminating behaviors are limited, relative to what is seen typically in natural settings when sheep are in groups of 5 or more. We converted a large rodent room into a flock room used to socially house all of our sheep in an effort to stimulate more normal flock behaviors in our animals. Constructions costs for this project were minimal and included mounting hay bale bags, swine scratchers, and feed and water containers on the wall to maximize square footage for roaming and grazing. A livestock gate was installed a few feet from the entrance to provide a small ante space for caregivers to observe animals before entering their living space. Over the past 13 months, we have observed an increase in walking while grazing and ruminating behavior for our six sheep that are currently housed in the room. Sheep usually ruminate while resting on their sternum with one or both front feet tucked under their bodies. When pair-housed our sheep stood while ruminating, but are now lying down for long periods of time in the flock room. By taking a large open floor plan and housing 6 sheep together we are able to improve their wellbeing and make them better research models.

P134 Got Milk? A Guide to Effective Milk Extractions in Mice

JL LeGrand^*1,2, S Himmelfarb¹, T Pilant^2,1, Y Belkaid¹

¹National Institutes of Health—LPD , Bethesda, MD; ²SoBran Inc., Bethesda, MD

Passive immunity is the short-term immunity that results from the introduction of antibodies and/or immune cells from one person or animal to another. In nature, this occurs from mother to neonate during breastfeeding and across the placenta during fetal development. However, it is not well understood what types of cells are involved in this process. In order to analyze the cellular contents of breastmilk and its role in passive immunity, we have developed a technique that allows us to obtain a high yield of breastmilk from a mouse with minimal discomfort for the animal. Here, we show that using oxytocin and by taking advantage of the natural progressive milk development cycle we can optimize milk output collection. Furthermore, we also found that using a 20uL pipette with tip gave us the perfect amount of suction with minimal cell disruption during this collection method. Now that we have developed this technique, we can collect measurable volumes of milk to be studied and gain further insight into how passive immunity is passed in the breastmilk to offspring and the physiologic consequence of this transfer to short term and long-term immunity.

P135 Implementation of a Gnoto/Axenic Facility: A Case Report

J Bom^*1, K Berman², A Ribeiro¹, M Pereira¹, AS Leocádio¹, M Rebelo¹, J Demengeot¹

¹Animal House Facility, Instituto Gulbenkian de Ciência, Lisboa, Portugal; ²Baylor College of Medicine, Houston, TX

Our germ-free (GF)/axenic facility started in 2005 and features rigid walls isolators for axenization and maintenance of mutant and wild-type mouse strains of different genetic backgrounds. The facility is open to the international community through the EMMA/Infrafrontier consortium. In 2013, a gnotobiology facility was implemented for experiments with GF animals. Each cage is a microisolation, allowing multiple studies on the same rack, providing full bioexclusion for maximum animal protection, excluding cage-to-cage contamination. SOPs and GF sentinels were implemented to validate our working system. These animals are manipulated exactly the same way as experimental animals, and their health status is checked monthly for quality control. Experimental animals are microbiologically tested in the beginning of the experiments and the respective control animals at the end. The use of this system for gnotobiology experiments allowed free space in the isolators and expanded the number of GF animals produced. Currently, the gnoto/axenic facility is running with 7 isolators, 2 systems, and is breeding 4 different axenic strains. The facility has a capacity of 400 deliverable animals and at least 2 EMMA/Infrafrontier research projects concluded per year. Axenization of new strains is performed by c-section rederivation and adoption by GF foster mothers, with a high rate of success. All isolators follow a health monitoring program that consists of microbiology testing in house once a month and a FELASA annual panel every 6 months. Major concerns are to avoid contaminations and technical problems, such as isolator breakdown. To prepare for such situations, strains are always housed in 2 isolators. In addition, 1 isolator is always kept as a backup for speed expansion of the affected colonies. The gnoto/axenic facility has highly specialized and dedicated personnel. It is integrated in the animal house facility, which allows resource optimization such as equipment (sterilizers, washing machines), basic supplies (diet, bedding, and beverage), human resources, and general facility workflow (staff and animals circulation).

P136 Using a Novel Disposition Box to Improve Daily Accuracy of A Vivarium Census Database

JA Maher^*1, JP Rodriguez², SA Mischler¹

¹Comparative Medicine, Pfizer, Inc, Pearl River, NY; ²Charles River Laboratories, Wilmington, MA

Animal research facilities need accurate and timely information about their animal population. This information is obtained via censuses, where a physical count is made first of the cages/animals in the facility, and then the results of the count are used to update a previously existing census database. The database is accurate on the day of the census, but in subsequent days it may differ from the real population by as much as 10 to 15%. As cages/animals are euthanized, these changes are not updated in the database due mainly to constrains on the researchers’ time. We sought to solve this problem by automating the database update process at euthanasia time by using a “disposition box,” which is a plastic box with an RFID scanner at the top and a slot through which the cage cards were fed. As the animals were euthanized, the cage card was fed through the box slot, the cage/animal identification was read by the RFID scanner, and the information was passed to a communications circuit at the bottom of the box. A program then passed the animal/cage card information to the database, which marked that animal/cage as euthanized. It was theorized that these real-time updates would reduce the differentials between the census database and the real vivarium population, improving the accuracy of the database. We now have data for 9 weeks (March and April of 2016) of field usage of these disposition boxes and compared it to data from the same period in 2014 and 2015, when no disposition box was in use. Median PD values (number of cages/animals dispositioned at census time as a percent of the total number of cages in the Vivarium) were 9.1% for 2014, 7.7% for 2015, and 2.7% for 2016. Data to date indicates that using disposition boxes significantly improved the accuracy of the census database.

P137 Intracage Ammonia Levels in Cotton Rats Cages: Effects of Cage Density, Age, and Bedding Sterilization

JA Maher^*1, JP Rodriguez², J Phillips¹, SA Mischler¹

¹Comparative Medicine, Pfizer, Inc, Pearl River, NY; ²Charles River Laboratories, Wilmington, MA

Cotton rats are useful models for the study of respiratory diseases, but they are high strung and difficult to handle during routine procedures such as cage changes. Reducing the frequency of cage change (initially 2 times per week) could reduce the stress on the animals and the handlers. No intracage environmental quality information, however, was readily available for cotton rats to help make this decision. We conducted a series of studies to obtain such information using intra cage NH3 concentration as the criteria of environmental quality. We studied the effects of the number of cotton rats per cage (2 or 3) and/or their age (7, 14, 18, or 22 weeks at the start of the test). Ammonia levels were measured daily for 7 days with a Drager x-am 7000 meter equipped with an NH3 sensor calibrated to detect levels between 0 and 200 ppm. The measurements were done by introducing the sampling probe through the automatic watering system port of the cage in test 1, and through the cage’s lid water port in the other tests. NH3 production rate was lower (P < 0.001) in cages with 2 animals than in those with 3 rats ((<20 ppm versus ∼60 ppm at test’s end). Older animals had higher initial ammonia levels (P < 0.01) but the rate of NH3 production with time was similar for all ages. No adverse histopathological findings were observed in the nasal tissues of the rats. Study 3 sought to determine if using irradiated bedding would slow down the production of ammonia in the cage but no differences were found due to the use of sterile bedding. The results of our studies indicated that if cotton rats were housed 2 per cage, the cages could be changed once each week without adversely affecting the animals.

P138 Slip Lead Program for Positive Reinforcement Training

J McMahon^*, SS Rapa, S Rubino, B Deschenes, R Flores, K Noorbehesht

Pfizer, San Diego, CA

The benefits of physical activity are evident in most pet dogs and many enjoy going for a walk. Walking offers the dog an opportunity to exercise, reduce stress, explore, gain mental stimulation, and it promotes a sense of bonding with the handler. With this in mind, we proposed to broaden our canine environmental enrichment program and mimic the benefits of dog walking by introducing slip lead training within the boundaries of our research environment. A slip lead was selected over a leash and collar to eliminate the need for collars on dogs. A shaping plan was developed, with the final behavior requiring the dogs to accept lead placement, walk with minimal tension to anteroom, get on a scale and wait, then get off the scale when released and walk back into kennel with minimal tension. The project was piloted with 1 group of 6 dogs and 1 group of 10 dogs. One-hour sessions were scheduled over 2 weeks so each dog received approximately 6 to 10 minutes of training daily. Of the 2 scenarios, we found it most beneficial to work with the dogs in groups of 6 providing them with 10, 10-minute sessions. The cage cards of dogs that completed training were marked with orange dots to differentiate trained and untrained animals. The shaping plan implemented was effective for training most of the dogs in the pilot. Slip lead training has improved ergonomics for our canine technicians by decreasing the need for lifting and carrying dogs. It has provided a stimulating environment for the canines and increased 1:1 time with the handler. Several apprehensive dogs were also noted to be more confident following training. The results of this pilot indicate that slip lead training is a valuable addition to an environmental enrichment program for canines. An added benefit is improved ergonomics for the comparative medicine staff and the animal users.

P139 Optimization of Blood Sampling from the Rabbit Pinna

KA Hough^*1, IM Washington^1,2

¹Magee-Womens Research Institute, Pittsburgh, PA; ²SoBran BioScience, Pittsburgh, PA

Rabbits are used for blood sampling in toxicology, pharmacokinetic, and antibody production studies due to ease of handling, ample blood volume, and accessible pinna blood vessels. This IACUC-approved work provides a comparative study of methods used for blood sampling from pinna vessels to help researchers optimize blood sampling in rabbits. Adult (∼3kg; N = 24) female New Zealand White rabbits (Oryctolagus cuniculus) were treated subcutaneously (SC) with ketamine, xylazine, and glycopyrrolate for a surgical implant study. The right pinna was treated topically with lidocaine/prilocaine and photographed. Rabbits were treated for 10m with 0.5mg/kg acepromazine (ace) SC, heat (∼90°F), or no treatment, and the pinna rephotographed. After topical alcohol and povidone-iodine, a 24G catheter was placed in central auricular artery or lateral marginal vein. Dressing (Tegaderm^®, Hypafix^®, or Moleskin^®) was affixed over catheter. Time to withdraw 2mL blood was recorded and catheter flushed with 1mL heparinized saline. At 6 hours and 24 hours, catheter persistence, blood draw, and dressing adherence were noted. Results showed more pronounced pinna vessels after heat or ace versus no treatment, more rapid 2mL arterial blood draw after heat versus ace or no heat, and much more rapid arterial versus venous blood draw. Catheter persistence varied with dressing used, with dressing 1 > dressing 2 > dressing 3 at 6 hours and dressing 2 > dressing 1 > dressing 3 at 24 hours. Most catheters drew blood at 6 hours, but few at 24 hours due to clots in catheter stem or hub. Dressing characteristics varied. Dressing 1 was transparent, flexible, difficult to affix, and edges rolled. Dressing 2 was translucent, flexible, easy to affix, and remained adhered. Dressing 3 was opaque, less flexible, easy to affix, but edges detached. In summary, heat produced the optimal combination of visible pinna vessels and rapid arterial blood draw. Dressing 1 and dressing 2 were optimal for visibility and adherence, respectively. Our comparative data on treatments, vessel types, and dressings provide information intended to help optimize blood sampling in research rabbits.

P140 Reducing Social Housing Complications in Adult Female New Zealand White Rabbits (Oryctolagus cuniculus) through Application of Male Rabbit Urine

KM McDonald^*, K Hoffmann

Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA

In accordance with the Guide, social animals should be housed with compatible conspecifics. However, species, sex, and age greatly influence socialization success and injury risk. Illustrating these challenges, when socially housing adult female rabbits at our institution, we observed a discouraging 12% separation rate, due to fight wounds, over a 6-month period. Subjectively, we noticed greater success with female groups when they were housed proximal to a male. We hypothesized that like some rodent species, male urine and its associated sex hormones, may affect female rabbit behavior. To evaluate the effects of male urine, we assessed research naive, 2-year old, female, New Zealand White rabbits (N = 36). The animals were singly housed at the vendor, and upon arrival at our facility, were socially housed in pens (25 sq. ft.), in groups of 4. No intervention was given to the control room (n = 16), and in the treatment room, rabbits (n = 20) were marked with 0.5 ml of male rabbit urine, immediately before socialization. To evaluate the frequency of dominant and affiliative behaviors, we video recorded the control and treatment rooms for one hour during initial housing, and at one 1 post arrival. We also measured fecal cortisol levels that corresponded to stress before shipment, and 2, 7, and 10 days post arrival. Our results indicate no significant difference in cortisol levels between the groups over time. However, the urine marked group engaged in significantly more affiliative behaviors such as laying with others, both at intake (F(1, 35) = 2.629, P = 0.015) and 1-week post arrival (F(1, 19) = 14.420, P = .001). Contrarily, the control group exhibited significantly more dominant behaviors at intake, such as mounting (F(1, 35) = 5.577, P = 0.027), and incurred significantly more injuries than the urine marked animals (Mann-Whitney U=79.0, n₁ = n₂ = 35, P < 0.05) over 2 weeks. Compared to none of the urine marked animals, 37.5% of the control were removed from groups due to fighting, suggesting that male rabbit urine application may improve social housing success with sexually mature females.

P141 The Effects of Socialization and Playtime on Antibody Production in Long-Term, Single-Housed New Zealand White Rabbits

K Wearsch^*, P Lincoln, J Villano

Unit for Laboratory Animal Medicine, University of Michigan, Westland, MI

Rabbits are used in biomedical research as an animal model for various physiologic and immunologic processes. Until its 2011 edition, the Guide did not address the behavioral and psychologic implications of single housing in rabbits. The most recent Guide indicates that single housing of social species may only be justified based on experimental requirements or veterinary related concerns. Thus, animal care and use programs are faced with the challenge of meeting this standard vis-à-vis minimizing unwanted effects on research studies. Many investigators have had singly housed rabbits prior to the establishment of the new guideline, and older rabbits that have not been exposed to socialization may not interact well with other rabbits thus posing a dilemma. In this retrospective study, 5 adult female New Zealand White rabbits used for antibody production that had been housed alone for 4 years since young were introduced to one another for pairing attempts and supervised playtimes. The process rendered no successful pairs for long-term housing but allowed weekly group playtimes in a large open pen with dividers. Supervised socialization attempts and playtimes that presumably caused stress in these rabbits did not adversely alter antibody production, with titer values consistently staying at or above 10⁵. To conclude, if rabbits cannot be socially housed, then open pen playtimes can serve as an alternative source of enrichment without affecting antibody production. Socialization and playtimes can then be adopted into the animal care program for single-housed rabbits.

P142 Validation and Refinement of Sanitization Practices in a Facility Housing Ferrets

K Noorbehesht^*, JM David

Comparative Medicine, Pfizer Inc., San Diego, CA

Sanitizing animal housing and equipment is essential to securing the health and wellbeing of laboratory animals. The sanitization process for ferret racks at our institution includes an initial 10-minute soak with hot water, followed by 5 minutes of scrubbing, and finally, a 45-minute rack washer cycle using 180F water, an acid descaler, and an alkaline detergent. We evaluated the procedure to determine if any steps in the process could be modified to improve efficiency, or conversely, if steps needed to be added to achieve satisfactory levels of cleanliness. The sanitization process was assessed using an ATP luminometer, a device that has been successfully used at other facilities, and Replicate Organism Detection And Counting (CONTACT PLATES) plates, the industry standard for sanitization monitoring. We were able to significantly reduce our rack washer cycle from 45 to 35 minutes and eliminate usage of the acid descaler while still demonstrating Colony Forming Unit (CFU) and Relative Light Unit (RLU) values consistent with levels considered sanitary by industry standards. Additionally, by cross validating RLU readouts with CFU counts from the CONTACT PLATES plates, we are now able to evaluate a variety of our sanitization processes quickly and objectively using the luminometer. Our improved rack sanitization process may provide a useful baseline process for facilities currently housing, or planning on housing, ferrets in the future.

P143 Routine Sanitization Scheduling Inhibits Psocid Population within Biologic Safety Cabinets and Animal Transfer Stations

KA Bozek^*1, CC Sem¹, MC Debrue²

¹Comparative Medicine, Pfizer, San Diego, CA; ²Comparative Medicine, Pfizer, Cambridge, MA

Biologic safety cabinets (BSCs) and animal transfer stations (ATSs) are an essential part of research facilities striving to maintain a Biosafety Level 2 (BSL-2) environment. With the recurring receipt of animals and other miscellaneous products packaged in paper-based containment, the opportunity for nuisance such as Psocoptera to infiltrate is frequent. Psocoptera is an order of insects commonly known as booklice that primarily feed on starch based household items like grains, wallpaper glue and book bindings. Psocoptera thrive in conditions higher than 50% humidity, and desiccate in warm and dry conditions. As per the Guide for the Care and Use of Laboratory Animals, animal facilities should maintain a humidity level between 30-70%. When room humidity is higher than 50%, ATS or BSC filters can provide a steady breeding ground for these pests. The ATS is where our psocid infestation occurred. To help with the psocid remediation, we lowered our humidity set point from 50% to 40% within the room. To resolve our psocid infestation, we implemented a weekly disinfection process of the internal pieces of the ATS or BSC which includes a thorough vacuum detail and sanitizing with Accel TB. The psocid lifecycle is usually completed within 1 month and adults can survive up to 6 months. Once the eggs are laid they will begin to hatch in 1-2 weeks and development from egg to adult takes approximately two weeks. The weekly sanitization breaks the psocid lifecycle by exterminating the female psocids before they can lay eggs, which are resistant to traditional means of sanitization. By maintaining this process, we have not had further infestations. Psocoptera are manageable with proper sanitization practices.

P144 Assessment and Modification of a Mechanical Restraint Device for the Prevention of Genital Manipulation in Singly Housed Male Cynomolgus Macaques (Macaca fascicularis)

KR Lambert^*1, A Dean², SS Lankford², JK Williams², E Mitchell¹

¹Animal Resources Program, Wake Forest School of Medicine, Winston-Salem, NC; ²Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC

In the laboratory environment, jackets have been utilized for approximately 4 decades for the protection of surgical sites, indwelling catheters, and skin graft locations. While the use of a skirt attached to a jacket is mentioned in the literature as early as 1995, the amount of available information on its use, function, design, assessment, and modification is lacking when compared to the use of a jacket alone. In order to successfully create a primate animal model for the regeneration of the lower urinary tract, it was necessary that male cynomolgus macaques (Macaca fascicularis) wear a jacket with skirt postoperatively. The use of the jacket with a skirt was to prevent access of the surgical site and inadvertent damage or early removal of the urinary catheter. Each animal was fitted with a commercially available nylon mesh jacket. A plastic skirt was attached to the outside of the jacket to prevent self trauma or manipulation of the genital area, urinary catheter, and/or surgical sites. Each animal was allowed a period of at least 5 days acclimation with the jacket/skirt combo prior to surgery. During this acclimation period, each animal was closely monitored to ensure the device posed minimal stress and impact on daily behaviors. The acclimation period allowed us to identify and address challenges the animals may face postsurgically. Some of those challenges include the prevention of dermatitis or excoriations, varying sizes of individuals requiring jacket/skirt application, the ability to assess surgical sites, and administering injectable medications once in the animal was placed in the jacket and skirt. With slight modifications based on individual challenges, we believe the jacket and skirt provides an effective means of mechanical restraint postsurgically that still allows the animal to have adequate use of his environment and the ability to engage in most species-typical behaviors.

P145 Husbandry of Laboratory-Housed Egyptian Fruit Bats (Rousettus aegypticus)

KM Pincolini^*1, CE Ferrecchia¹, H Chum¹, C Roberts^1,2

¹Office of Laboratory Animal Care, University of California, Berkeley, Berkeley, CA; ²University of California, San Francisco, San Francisco, CA

We house a variety of unique species, each requiring specific husbandry, enrichment, and safety protocols. While housing of bats in zoological parks is quite common, but new to us, we developed standards specifically for housing a wild-caught breeding colony of bats in an indoor laboratory facility. The Egyptian fruit bats (Rousettus aegypticus) that instigated these protocols are used in research studies investigating the neural basis of complex spatial behavior, the neural basis of social behavior, and vocal communication and learning. Developing daily husbandry practices that allowed for effective sanitation but minimized the potential impact on breeding and research were essential. During the initial quarantine period, the unknown health status of the colony necessitated the implementation of added personal protective equipment as well as rabies vaccination and safety training for all personnel. Furthermore, transitioning a group of bats from the wild to a laboratory setting involved providing environmental enrichment that promoted species-typical behaviors, such as foraging, in order to maintain a lean, freely flying, breeding colony. Examples of current enrichment devices include metal parrot skewers for fruit, nonhuman primate Prima-Hedrons, and faux leaf fencing. Thus far, the fruit bat colony has thrived under these conditions, with a very promising breeding season in progress. As the field of bat research continues to grow, the lessons learned through development of husbandry, enrichment, and safety protocols presented here will prove valuable for other institutions housing bats in a laboratory setting.

P146 Evolution of a Nonhuman Primate Enrichment Program

KI Graika^*1, D Toomey¹, T Rodriguez¹, NA Monts de Oca², M Cabrera¹, CR Lockworth¹

¹Department of Veterinary Medicine and Surgery, MD Anderson Cancer Center, Houston, TX; ²Charles River Laboratory, Houston, TX

We continuously strive to improve animal welfare and wellbeing and are committed to producing high-quality research, which can only be achieved through physiologically and psychologically sound research subjects. For many years, we maintained a nonhuman primate (NHP) enrichment program that met minimum standards. Common provisions for occupational enrichment, produce, and documentation were followed. Nevertheless, after examining the program, it became clear that although we were meeting basic requirements, there were numerous opportunities for development and improvement within the program. With effort and innovative ideas gathered from within our team and from colleagues at nearby institutions, we were able to dramatically transform our program into one that more closely meets the behavioral needs of the NHP colony under our care. The support of our innovative program was only possible through the addition of infrastructure necessary to sustain its additional requirements. To ensure success of this new and complex primate enrichment program, we implemented new processes and resources in order to ensure its functioning without increasing personnel. We were able to do this by investing in significant upgrades and additions to our enrichment preparation facility to meet our requirements of space, ergonomics, and versatility. Additionally, we developed an efficient logging system, coded calendars to quickly and efficiently manage tasks, and visual standard operating procedures for enrichment preparations and personnel training support. This foundation permitted the augmentation and enhancement of the entire behavioral management program at our facility. We were able to increase options and provide novelty, standardization, and structure by implementing a rotating system for the management of the occupational, nutritional, and sensory enrichment devices. Moreover, we expanded our social enrichment, positive reinforcement training, and behavioral monitoring programs. Together, all of these changes resulted in a more complex, yet efficient program, and most importantly, improved animal wellbeing for our NHPs.

P147 HERBS LAW: An Animal Health Assessment Method

KM Vera^*

Alcon, Fort Worth, TX

While performing daily animal observations, animal care technicians look for any abnormality to assess animal health and wellbeing. Assessing the same animal every day can make the abnormal seem normal, and undesired behaviors may be accepted as personality traits. Some years ago during a facility inspection, an animal was observed to have buphthalmia, an enlargement of the eyeball, but had no record of this being reported despite daily health observations. This resulted in a recommendation to better improve our program for daily animal observations. In response, HERSLAW was created. HERSLAW (head, eyes, respiration, skin, limbs, appetite, waste) is an acronym used to describe an animal health assessment method for technicians to use while making daily animal observations. This acronym guides the animal care technician to ask certain questions when looking at particular areas of the body. Is the head tilted? Are the eyes fully open and clear? Is the breathing normal? What is the appearance of the animal output and is it a normal amount? In recent years more focus to capture behavior as a significant indicator of health has prompted modification of HERSLAW to HERBS LAW (head, ears/eyes, respiration, behavior, skin, limbs, appetite, waste). In addition to the incorporation of ears, we also added behavior. Behavior can be a significant indicator of potential abnormal health issue that is not visually apparent. For example, repetitive rubbing of the same spot on the arm could be indicative of a laceration they are trying to rub away. Some things to ask are: Are they spinning or eating feces? Are they biting themselves? These are examples of abnormal behaviors that should not be accepted as personality but reported for further assessment. Since the implementation of the HERSLAW method, we have not received any further recommendations for improvement regarding our daily animal health observations. In addition, this method has helped to refine our process and improve animal welfare by ensuring that all animals across the vivarium are being examined in a uniform consistent manner.

P148 Down and Dirty with Rotifers: Daily Care to Support a Zebrafish Colony

KM Burritt^*, S Lowry, CA Johnson

University Animal Care, University of Arizona, Tucson, AZ

One of the main concerns when it comes to caring for any animal colony is providing healthy and nutritious food. The majority of feeds are standardized diets that are manufactured in a manner that ensures the highest quality available. These diets are an efficient and cost-effective way to feed most colonies. This is not the case when beginning a larval zebrafish colony. It has been shown that providing live feed rotifers between the ages of day 5 to 30 results in bigger, healthier zebrafish, and more fish survive to adulthood. While providing rotifer diets has shown these positive outcomes, it also comes with more time spent caring for the live feed. We have developed a streamlined process to ensure a healthy, disease-free source of rotifers are always available. Here we show the innovative husbandry care for the rotifer colony. Daily husbandry care activities include a 30% water exchange in each tank and 2 daily feedings, as well as maintaining a marine mix water supply at an appropriate salinity and pH. A constant supply of 15 ppt marine mix water is maintained by testing the pH with a test strip and using a refractometer each time a new batch of water is mixed up to ensure the correct salinity. Weekly husbandry care includes samples sent to pathology, where the total number of rotifers are counted, as well as the number of active rotifers and the number of rotifers bearing eggs. Other tasks such as changing rotifer floss and watching for low-performing air stones are also crucial to keeping a rotifer colony excelling. The floss placed in the tanks catches unwanted contaminants and extra, uneaten food. Once an air stone becomes clogged and begins to restrict the airflow, the level of oxygen in the tanks depletes, killing off many rotifers. When either of these are not watched, it could lead to a complete loss of the colony. To aid in keeping the colonies healthy, a biweekly tank change is also in effect. Also, every 4-6 weeks, a new rotifer colony is started, ensuring there is always a healthy growth in our rotifer culture.

P149 Extended Use of Nylon Rodent Chews Resulted in Cost Savings with No Detrimental Effects in Sprague–Dawley Rats

L Danner^*, R Varada

Comparative Medicine, Pfizer Inc, Andover, MA

Environmental enrichment for laboratory animals has beneficial effects on their welfare and health. The data generated from environmentally enriched animals has been shown to be less variable and of higher quality. Rodents rely in large part on their olfactory cues to navigate in their natural environment. Enrichment devices such as nylon chews and wood blocks have been shown to reduce anxiety and stress in rodents. In this study we evaluated whether the use of a commercially available nylon rodent chew can be prolonged without affecting the health of the rats. Sprague–Dawley Rats (5-6 week old) were pair-housed in cages with a loose animal bedding made of alpha cellulose and ad libitum diet with portion-controlled nesting material. We provided the chew (one/cage) to 3 groups each containing 8 cages (n = 16 rats) and assessed the chews at 4-, 8- and 12-week intervals for their bacterial load, debris accumulation, and percent usage. We also examined rats for their CBC/chemistry at the above intervals and measured their body weights weekly. The bacterial load as well as debris accumulation on the chews peaked at 4 weeks and dropped to lowest levels by 8 weeks. The percentage use of the chews as measured by their weight peaked at 12 weeks. In addition, we saw no significant changes in RBC, WBC, liver enzymes, and body weights. Therefore, we suggest that chews can be safely used for extended periods up to 8 weeks as enrichment devices for rats.

P150 Did Your Brooder Fail You? Get the Pail and Have Happy Quail!

LN Ward^*, T Hanna, M Quinn, A Jackovitz

Army Public Health Center, Bel Air, MD

Complications from housing Japanese quail (Coturnix japonica) in galvanized steel brooders led study staff to brainstorm and develop an alternative housing option. Disadvantages to the galvanized steel brooders included poor visibility of the chicks, and since the flooring was constructed of a wire mesh material, there was potential for chick/personnel injury. “Quail pails” were constructed from 54-gallon tote boxes, transforming the plastic storage solution into a free-range chick set-up. Modifications included drilling holes in the totes to accommodate a carboy for delivery of drinking water and replacing the solid plastic lid with mesh for increased air circulation. Advantages to these pails included easy observation of the chicks and soft bedding was used as alternative to wire mesh to prevent injury to chicks/personnel. Since their development, quail pails have housed nearly 500 chicks. No animal welfare or husbandry issues occurred during their use.

P151 Sterilization Methods for Flexible Film Isolators Used for Housing Gnotobiotic Mice

Y Gonzalez², JM Koontz¹, CL Horton³, BC Dancy¹, JA Lewis¹, LM Caffo^*3

¹U.S. Army Center for Environmental Health Research, Fort Detrick, MD; ²Excet, Inc., Fort Detrick, MD; ³Oak Ridge Institute for Science and Education, Fort Detrick, MD

Gnotobiotic animals have become an increasingly popular research model, yet standardized methods and procedures for animal husbandry have not been established. We have assessed and developed methods to sterilize and maintain gnotobiotic conditions within flexible film isolators. A combination of sterilization methods was required. Caging materials, rodent food, water, and all necessary experimental tools and reagents were autoclaved or chemically sterilized to ensure elimination of exogenous microbes prior to transferring materials into the isolators. Gamma irradiation and electron beam irradiation processes were employed for items that were not compatible with liquid or vapor contact. Modifications to gaskets, tubing, transfer cylinders, and sleeves were made to reduce chances of breaches within the isolators. Vaporized hydrogen peroxide (VHP) and chlorine dioxide were compared for use as cold sterilants. While both VHP and chlorine dioxide effectively sterilized the isolators, we determined that, due to isolator material incompatibility with VHP, chlorine dioxide was the more appropriate sterilant for flexible film isolators. While carrying out sterilization protocols, occupational exposure levels of VHP and aerosolized chlorine dioxide were found to be well below permissible exposure limits for both chemicals. Twenty-four hours following chlorine dioxide fogging of the isolators, a mold trap consisting of autoclaved food, caging material, and sterile water was placed in the isolators for sterility testing. Sterility of the isolators was verified by culturing swabs of the isolators to confirm that sterility had been achieved. Flexible film isolators present operational challenges that semi-rigid and rigid isolators do not, but do offer advantages in terms of cost and ease of adaptability. Generating reliable, quality data from gnotobiotic mouse colonies is only possible when sterility is properly maintained. Matching the sterilization technique to the isolator and material type is crucial to success, and methods presented here help to standardize and optimize workflows in these isolators. Research was conducted in compliance with all Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

P152 The Establishment of Specific Pathogen-Free Ducks in China

L Han^*1, J Han², L Qu¹, H Chen¹

¹Harbin Veterinary Research Institute, Harbin, China; ²CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources, Institute of Animal Science, CAAS, Beijing, China

Ducks are one of major domesticated poultry species for meat, egg, and feather production. Thus duck diseases are of great concern to duck production industry, especially after the birds have been confirmed to be a combine host for highly pathogenic avian influenza virus which can spread from nonpathogenic ducks to humans. Furthermore, several duck and human vaccines are produced and qualified by using the regular duck eggs which may carry pathogens. Therefore, specific pathogen-free (SPF) ducks are urgently required both for avian disease research and biologic products. In 2005, hatching eggs of great parent generation of Shaoxing White Shell I ducks were introduced for developing SPF ducks in an isolated environment. The ducklings and adult ducks were kept in duck special isolators with HEPA all through their lives and fed with radio-sterilized feeds and acidified drinking water. Sera were regularly detected at a 3-month interval and serum antibody-positive individuals were weeded out. After have being bred and purified for successive 8 generations, the duck population is proved to be free from 10 major pathogens including duck plaque virus, duck hepatitis virus (DHV), avian influenza (AIV), Salmonella pullorum, Pasteurella multocida, Newcastle disease virus, reticuloendotheliosis virus, avian reovirus, mycoplasma, and avian adenovirus group III. Meantime, 2 genetic lines of B and Q were selectively bred according to the phenotypes of egg shell colors (white or cyan) as well as genotypes at 18 genomic microsatellite markers. Four families, named as B1 to B4, were specifically selected following selective mating among homozygote birds screened from genomic sequences of a transporter associated with antigen processing gene located within major histocompatibility complex region. Animal experiments showed that B2 family was more susceptible to DHV and B3 induced higher AIV HI antibody titers than the other 3 families. At present the scale of breeder duck stocks are about 500. We believe that these unique SPF duck genetic materials enrich laboratory animal resources and provide valuable standard animals for avian science research.

P153 Enhancing Husbandry and Laboratory Personnel Safety by the Creation of a Dedicated Animal Safety Coordinator

L Steiner^*, VA Hill, RC Dysko

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

Health and safety issues are often identified during facility inspections performed by the IACUC and AAALAC. The Occupational Health Department (OHD) at a large, decentralized institution is often unable to provide routine visits to all locations to ensure a robust health and safety program for all individuals who work in the animal research facilities across campus. In an effort to enhance the occupational health and safety component of the animal care program (ACP) and to more directly address and prevent health and safety concerns, the centralized husbandry unit created the position of Animal Safety Coordinator (ASC). The ASC serves as a liaison between the ACP and the institution’s OHD. This person advises facility supervisors/managers on various health and safety issues but is not an official inspector. As such, interactions are primarily collegial and collaborative, and not punitive. Duties include (but are not limited to) assessment of appropriate use of biologic indicators, ensuring appropriate placement and flushing of eyewash stations, sanitation validation of both mechanical cage washing and hand washing of supplies, annual verification of animal transfer stations to ensure correct operation for personnel protection, and monitoring Personal Protective Equipment (PPE) for both effectiveness and application. Previously, these tasks had been occurring as separate functions over several different job classifications. By assigning these responsibilities to a single individual, we have centralized these roles and have a dedicated staff member to prioritize the occupational health and safety component within the ACP. The most important aspect of the ASC is that the ACP now has a person who goes into the facilities on a regular basis and interacts with the staff to support safety and look for areas of risk. The OHD creates the policies and enforces them in a formal capacity. The ASC is their partner in the field to understand how the policies are being applied and where the institution has inconsistencies or liability. After 6 months of full-time activity, our ASC has contributed to long overdue improvements to the program along with a much more efficient and effective means of addressing health and safety concerns.

P154 Can Type of Environmental Enrichment Help Reduce Food Grinding in Mice?

LH Webb^*, A Pacio

Research Triangle Park, Mispro Biotech Services Corporation, Durham, NC

We questioned if different types of environmental enrichment can affect or reduce food grinding in research mice. Food grinding is a common problem observed among CD-1 mice in our animal program. It occurs when rodents chew food pellets, but do not consume them, resulting in excessive food particles (orts) in the cage bedding. Food grinding leads to more frequent cage changes attributing to increased animal stress, as well as higher food and management costs. The hypothesis is that food grinding occurs due to boredom, composition of diet, or lack of natural behavior opportunities. According to previous published studies conducted to evaluate if the causes of food grinding were behavioral or nutritional deficits, it was concluded that change in diet or additional nutritional enrichment (i.e. sunflower seeds) was most effective at reducing overall food grinding behavior. However, as is the case in most animal studies, change in diet or added edible enrichment is considered an unacceptable study variable. In our animal program, a modification to environmental enrichment is the initial approach to address stereotypical behaviors. This study compared mice known for grinding (CD-1 male and females) with mice not known for grinding (C57BL/6 males). Mice were given a different enrichment type each week, starting with a single product followed by combinations of products for 1 week each. Food consumption, individual body weight, and visual ort rating scale were measured over the 13-week study. Preliminary data results suggest that a single enrichment product offered has little to no impact on food grinding behavior. Mice had increased weight gain when offered nesting enrichment as compared to enrichment that promoted chewing behaviors, which resulted in weight loss. This suggests that, when given a single product, mice prefer nesting enrichment versus chewing enrichment as represented by increased individual body weights, but has mixed results on reducing food grinding behavior. Overall, this study demonstrates environmental enrichment that promotes nesting behavior in mice can be used to help decrease food grinding behavior leading to improved animal welfare and wellbeing while decreasing management costs.

P155 A Novel Mouse Individually Ventilated Cage Design Demonstrating Extended Cage Change Interval, Reduced Airflow, and Compensation of Modeled Water System Leaks

L Kramer^*, M Bailey, M Semenuk

Lenderking Caging Products, Millersville, MD

A novel individually ventilated cage (IVC) design, where air flows through the bedding, was used in a study of female C57BL/6J mice. The study used 27 cages containing 5 mice per cage, and, another 9 cages containing 2-timed pregnant mothers and their litters. Cage change intervals were extended past 21 days using 30 air changes per hour (ACH). Slow and fast water system leaks were simulated by spraying 5 mL and 30 mL of water (respectively) on the bedding through a sampling port. The serology panel was completely negative. Blinded histopathology samples of the lungs and turbinates in the nasal passages were all within normal limits with 1 exception in the control group which showed slight inflammation in the lungs, but no necrosis. This included pups reared their entire lives under this novel airflow schema. Ammonia concentration data was gathered using a Matheson Kitagawa gas sampling system through the sampling port located 1 inch above the bedding in the cage front. Histology and serology data demonstrate that the novel airflow design did not compromise mouse health under an extended cage change interval. Water bottle leaks garnered surprising results of lower ammonia concentration as compared to positive controls.

P156 Effects of Partial Bedding Changes on Inter-Male Aggression in Mice

G Bhade, LV Kendall^*

Laboratory Animal Resources, Colorado State University, Fort Collins, CO

Group housing of male mice can lead to increases in aggressive behavior, and cage cleaning is a source of disturbance that may exasperate this problem. This study tested if differences in bedding composition affected stress levels of mice. This could potentially develop new cage-cleaning methods that lower stress levels of laboratory mice which can aid other research projects in which stress is a variable that needs to be controlled. Sixteen cages in 4 groups were made with fresh bedding, food, and water. Five male mice were placed into each cage. Behavior was observed daily for the next 2 weeks. Aspen chip bedding was changed at the beginning of the third week using the same cage. The first group received 100% fresh bedding; the second received 25% old bedding and 75% new bedding; the third group received 50% old bedding and 50% new bedding; and the fourth received 75% old bedding and 25% new bedding. Behavior was observed daily for the following week until the next bedding change. Mice then received new cages with fresh bedding for 1 week then the study repeated in a cross over design so each group received each bedding treatment. Individual instances of sexual aggression (SA), nonsexual aggression (NSA), vocalization (V), bar gnawing (BG), and bare spots in bedding (BS) were recorded over a 30-minute period. There was no difference in the SA observed the day of the change; however, during the week, the SA was significantly greater in the mice that received 100% fresh bedding in a used cage (p < 0.01). There was no significant difference in the other behaviors observed. It was noted as the male mice aged there was a significant increase in SA and NSA (p < 0.01) regardless of the amount of partial or fresh bedding used. These results demonstrate the current cage cleaning method (new cages and fresh bedding) leads to fewer instances of SA, NSA, and BG than reusing the cage with complete or partial bedding changes.

P157 The Effects of Lower Protein Diet on Growth, Survivability, and Lesion Incidence in Aged Male Sprague–Dawley Rats

MJ Horn^*1, BD Mickelson²

¹Veterinary Sciences, Research and Support, Envigo, Indianapolis, IN; ²Teklad, Envigo, Madison, WI

Two-year toxicology studies and some types of age-related research involve maintenance of cohorts of animals for extended periods of time. Survival above a specific threshold at study completion is desirable and may be required for regulatory reasons. Diet restriction is one method used to increase survival; however, this is resource intensive and raises animal welfare considerations. Another approach to consider is the use of a lower-protein, lower-energy density diet. This study used a cohort of 200 male Sprague–Dawley rats maintained on a 14% protein diet from 8 to 104 weeks of age. Body weight was monitored monthly while survival was reported weekly. At the culmination of the 2-year study, animals were submitted for necropsy including histopathology of select organs. Neoplastic and nonneoplastic lesions were characterized, and blood and urine were analyzed for complete blood count/serum chemistry and urinalysis, respectively. Data from the study were compared to data collected in a previous study where male Sprague–Dawley rats were maintained on an 18% protein diet under similar conditions. One hundred and four-week survivability was numerically but not statistically improved in animals maintained on 14% (68.5%) compared to 18% (63.3%) protein diet. Additionally, body weight was decreased by nearly 120 g (20% body weight reduction) at 104-wk of age (P < 0.05). Thymus, spleen, liver, heart, testes, epididymis, and brain tissue weight (normalized to brain tissue weight) were significantly different due to diet (P < 0.05) at 104 weeks. Neoplastic lesion incidence was also reduced with the 14% protein diet in both pituitary gland and pancreas. In conclusion, the use of a lower protein/lower energy density diet decreases body weight, may enhance survivability, and reduces neoplastic lesion incidence without the use of diet restriction in long-term studies. This is a viable and simple option for long-term diet maintenance of outbred male Sprague–Dawley rats.

P158 Cross-Fostering Mice to Produce “Clean” Colony for Research

ME Elliott^*

Office of Animal Resources, University of Missouri, Columbia, MO

Cross-fostering is a technique that may be used to eliminate certain diseases in research rodents that may be difficult to eradicate by medication alone. When a principal investigator has a need to establish a new colony in a higher health status (cleaner) vivarium, cross-fostering is an accepted process to eradicate certain diseases, including Helicobacter sp. In simple terms, cross-fostering is the removal of pups from the biologic dam shortly after birth and placement of the pups with a surrogate dam, another nursing dam that is free of the disease requiring elimination. The process requires careful timing to ensure the foster attempt is successful. Close observation of the breeding female for a vaginal plug is first needed to determine the exact due date of the pups. Subsequently, a surrogate dam that will deliver pups a day before the infected dam must be available or purchased. Using a surrogate that is a different color than the original mouse is helpful in being able to differentiate pups. Following deliberate protocols during transfer of pups from their dam to the surrogate is necessary to ensure disease control and acceptance of the foreign pups. For example, making sure there is no bedding or nesting material on the pups before moving them to the surrogate’s nest and having the surrogate urinate on the new pups helps the acceptance process. Following a successful transfer of pups, weaned animals should be tested to determine that the cross-fostering process eliminated the agent of interest. We have used cross-fostering successfully in starting a “clean” colony free of Helicobacter sp. numerous times.

P159 Implementing a Sanitization Verification Program for PI Testing Chambers and Surgical Equipment

M Nigro^*, LS Bird

Rutgers University, Piscataway, NJ

ATP (adenosine triphosphate) testing is being used on an increasingly routine basis for testing the effectiveness of cleaning in animal rooms and cage washer sanitized items in our facilities. Our department wanted to expand this practice to other areas of live animal use, as well. A large portion of the research conducted within our university is behavior based, which requires the use of different behavior chambers, devices, and equipment. Many scientific lab staff also perform surgical procedures with mice and rats within their lab’s benchtop space, as opposed to inside the animal facility. In compliance with SOPs, each lab is required to maintain the cleanliness of their equipment both in between each animal and at the completion of their work. In order to verify that their equipment used for live animals is being cleaned and sanitized thoroughly, our department developed a sanitization verification program. The program needed to be accurate and reliable but also timely for both the veterinary technicians performing the verification and the scientific staff. Our veterinary technician team began the process by taking an inventory of the scientific staff’s behavior and surgical equipment. Based on this information and further discussion with our veterinary staff, we decided to biannually sample 10-25% of each type of equipment depending on the total amount. We use the values preprogrammed into the ATP device for our numerical result and any result higher than 150 (“good”) is an automatic fail. Our veterinary technicians communicate with the PIs and scientific staff to schedule the initial testing date, provide sanitation policy information, and to answer any questions. If results are unsatisfactory, a training session is organized for the PI staff, which includes education on acceptable solutions, contact time, and cleaning technique. Once a satisfactory result is achieved, the items tested are added into our regular biannual testing schedule.

P160 Constant Antibiotic Treatment Reduces Breeding Prosperity of MyD88/TRIF Double Knockout Mice in a Specific Opportunist Pathogen-Free Mouse Facility

U Heise¹, K Schlarmann², B Pasche², H Riedesel³, MC Pils^*1

¹Mouse-Pathology, Helmholtz Centre for Infection Research, Braunschweig, Germany; ²Central Animal Facility, Helmholtz Centre for Infection Research, Braunschweig, Germany; ³Central Animal Facility, Universitätsmedizin Göttingen, Göttingen, Germany

MyD88 knockout mice are deficient in the recognition of bacterial and viral infectious agents due to the lack of toll-like receptor (TLR) signaling. It is widely assumed that these mice cannot be bred and maintained without constant antibiotic treatment because of their susceptibility to infections. However, the need of this chronic antibiotic treatment has not been demonstrated for Specific Opportunist Pathogen-Free (SOPF) housing conditions. We have transferred several MyD88 knockout mouse strains to our SOPF mouse facility. Breeding pairs received constant antibiotic treatment (trimethoprim-sulfonamide). Twenty-five to 50% of the breeding females died due to endometric/urogenital infections with Enterococcus faecalis. The isolated Enterococcus faecalis strain was highly resistant against various antibiotics. In order to investigate the possible protective effect of the antibiotic treatment, 3 groups of breeding pairs (n = 6 females) of the MyD88/TRIF double knockout strain (B6.129P2-Myd88^tm1Aki-Ticam1^tm1Aki) were constantly treated via the drinking water with placebo, trimethoprim-sulfonamide, or enrofloxacin. The study was setup double-blinded: neither animal care takers nor veterinarians were aware of the group-assignment. Surprisingly, blinded clinical and pathohistological assessment revealed an increase of endometritis in trimethoprim-sulfonamide treated animals. The difference in enrofloxacin and placebo treated groups was not significant. These data suggest that the antibiotic treatment with trimethoprim-sulfonamide is deteriorating the health status of the animals and promoting endometritis in breeding MyD88/TRIF ko females. Enrofloxacin treatment did not display any advantage compared to placebo. In order to decrease the risk of further resistance development, the application of antibiotics needs to be reduced. Moreover, antibiotic treatment effects the microflora and might thereby influence experimental results. Thus, we conclude that the constant antibiotic treatment of MyD88 mouse strains should be omitted in SOPF facilities.

P161 Temperature/Humidity Data Logger during Animal Exportation/Transportation

MC Rodriguez^*, R Cook

Eastern Virginia Medical School, SoBran Bioscience, Norfolk, VA

Transportation within or between institutions is a common procedure where laboratory animals are routinely removed from their highly controlled macro and microenvironments. Controlled environment, including stable within acceptable ranges for temperature and humidity (T&H), are important and regulated conditions. However, T&H are rarely monitored closely by shipping couriers despite the possible adverse physiologic effects when basic parameters are lost. Our institutional exportation SOP now requires a T&H data logger (DL) to monitor environmental parameters during any exportation. We use a data logger that looks like an ordinary USB memory stick. With the help of a customized casing we are able to place the device inside the primary transport enclosure. The DL provides continuous monitoring and recording of T&H during the transit of our sensitive packages and can be set to users preference on how often data is obtained, acceptable ranges for parameters, and even an audible alarm for when ranges are breached. Once a shipping container arrives to its destination, the logger is shipped back to us in a self-addressed envelope. The DL allows us to verify the T&H inside the cage during transport and confirmed that it was maintained within acceptable levels. This helps us asses the quality of the courier service and inform the receiving institution of any situation. In addition, the audible alarm could alert the driver when deviations occurs. The only drawback to this DL is the lack of real-time monitoring or distance alarm. Technology offering text or email alerts exist, as well as GPS and video. Institutions have to study the feasibility of such considerably more expensive equipment.

P162 Effect of Wheel and Bearing Types on Initial Pulling and Pushing Forces on Ventilated Racks

GK Palma^*1, B Becker¹, SJ Graciano¹, SC Adams¹, JD Reuter^2,1, M Leblanc¹

¹ARD, Salk Institute, La Jolla, CA; ²Office of the Vice Chancellor for Research, University of Colorado—Boulder, Boulder, CO

Mouse colonies in barrier facilities are often housed on ventilated racks that can weigh over 1.000 lbs and must be pushed and pulled repeatedly to accomplish normal tasks. Selecting the right rack wheels is often overlooked and may result in work-related injuries. Using a digital force gauge, we evaluated the effect of different wheels and bearings on the initial push and pull forces to displace ventilated racks. Three wheels of increasing hardness were evaluated: 1) 95 durometer Shore A with roller bearings (soft 95); 2) 70 durometer Shore D with ball bearings (Hard 70) and, 3) 75 durometer shore D with ball bearings (Hard75). Casters were identical on all racks and wheels had the same diameter and thickness. Experimental conditions were strictly standardized. Forces were applied 3 times at waist height in a horizontal plane with a constant floor slope (<1° variation) on an epoxy-coated floor and with an initial wheel position aligned to the applied force. Overall, none of the racks required an initial force exceeding the Liberty Mutual Research Center standards for workers of both sex. Except for pushing with Hard 75 wheels (R²=0.61, p<0.0001), the initial push/pull forces were not correlated with the number of cages on the rack even though cages can represent over 50 percent of the total rack weight when fully occupied. Push forces were significantly lower than pull forces except for Hard 70 wheels. The hardness of the wheels and bearing types significantly influenced both initial forces. Pull/push forces with Soft 95 were significantly higher than with Hard 70 and Hard 75 wheels. We also observed that any damaged wheels, floor imperfections or objects impeding rack movement could greatly influence initial pull/push forces. Using the same standard conditions on racks equipped with Hard 75 wheels, we evaluated the effect of 2 floor types, namely smooth epoxy and rough methyl methacrylate (MMA) coated floors on both initial forces. Pull forces were similar but there was a tendency for greater push forces on the MMA floor (NS). Overall, these results stress the importance of carefully choosing wheels to minimize initial pushing and pulling forces and prevent work related injuries.

P163 A Case Study on Implementing an Animal Management Software

D Latterich^*, E Schouten, L Belen, JD Reuter, M Leblanc

ARD, Salk Institute, La Jolla, CA

Electronic systems are commonly used to streamline animal management. Some institutions choose in house developed software while others purchase off-the-shelf solutions, but all face similar implementation challenges that require careful planning. Our department operates over 45,000 ft² of vivarium including 15,000 mice IVC and supports 40 labs and over 700 animal users. In 2015, we purchased and implemented an electronic system to manage animal ordering, bar code census, and recharges. We identified 3 critical areas that would impact the success of implementation: user adoption and training, system performance, and internal support resources. Because users are diverse with different levels of computer skills, we estimated a prolonged implementation timeline lasting at least 1 year. We adopted a phased approach, rolling out the animal-ordering module first to accustom users to the system and gradually added the remaining functions with extensive training at each phase. Second, we strove to use as closely as possible out-of-the box functions and minimize custom changes. It lessened the burden of database maintenance and occurrence of glitches as well as streamlined software upgrades. We also built interfaces with the human resources and finance department databases to automate data flow. Furthermore, we conducted exhaustive testing on each function to minimize system kinks and met individually with lab managers and end users to identify process conflicts and make changes early. Third, an effective technical support team including members from the vendor and various company departments was assembled to coordinate efforts throughout the process. In retrospect, the phased approach was strategic to secure user confidence in the new software. Significant time spent on system testing paid dividends and minimized hiccups. While the project team could not anticipate all facets of system or user issues, the company and vendor allocated the necessary resources to resolve ad hoc problems and support users throughout the process. In conclusion, having a collaborative team of key stakeholders, proper training resources, and a customized plan were essential to the successful implementation of the software.

P164 Reduction of Animal and Technician Stress via an Efficient, Simple Method of CO₂Euthanasia for Mice

MM Trogdon^*1, JW Hensley¹, GE Kissling², K Laber³

¹Animal Resources Section, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC; ²Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC; ³Office of the Chief, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC

With the continued high use of genetically altered mice, the need for euthanasia of nondesirable genotypes persists. To accommodate the large numbers of rodents used and to maximize available space, institutes are using individually ventilated caging (IVC) systems. Following recommendations that rodents should be euthanized in their home cage whenever possible, we have come up with a simple, efficient method of delivering CO₂ that reduces not only the procedure time, but the stress experienced by both the mice and the technicians. Using a manufacturer’s IVC transport rack, and building on the use of our multiport Plexiglas manifolds, we used a horizontal multiport manifold obtained from a home brewing supplier, some flexible polymer tubing, and stainless steel sipper tubes cut from bottle caps. Removing only the water bottle from cages on the transport rack, we were able to simultaneously euthanize 5 cages of rodents by delivering a concentrated distribution of CO₂ in compliance with AVMA guidelines, and caused no visible change in animal behavior or animal activity. In addition, we reduced the overall time of the euthanasia process by at least 2 minutes per cage. For an 80-cage transport rack, this equates to a reduction of approximately 30 minutes.

P165 Preclinical Immuno-Oncology Models—Husbandry and Housing Considerations

MM MacBride^*

Taconic Biosciences, Hudson, NY

The growth in immunooncology research, in which the body’s own immune system is recruited to fight a tumor, has prompted a shift in preclinical animal models used for oncology research. While traditional cell line xenografts in immunodeficient models are still widely used, they are generally not appropriate for immunooncology studies as they lack critical immune cell types required for immunotherapy efficacy. Immunooncology researchers have turned to 2 model systems: syngeneic tumor models in immunocompetent inbred strains and mice engrafted both with a humanized immune system and a tumor. Facility managers and veterinarians involved in these types of studies must consider various factors. For syngeneic models, what health standard is acceptable or preferable? Understanding the microbial status of inbred mice used and being consistent is critical. Inbred mice with higher health statuses are now readily available, and these should be housed in immunodeficient or high-barrier rooms rather than conventional rooms in order to maintain that health status. Humanized models are severely immunocompromised mice reconstituted with a human immune system that has functional limitations. As the base model is so immunocompromised, they are exquisitely sensitive to opportunistic agents and require special housing and husbandry, including very strict bioexclusion processes. All materials must be sterile and aseptic technique must be used. Robust health monitoring is critical to distinguish between infectious processes and graft-related issues. The severely immunocompromised base models used for humanization may be susceptible to health problems caused by agents not on typical exclusion lists, so veterinarians must keep an open mind during diagnostics. Pooled fecal samples are tested monthly for bacterial agents, and sentinels which have been exposed to dirty bedding from humanized mice are used for health testing. We use both immunocompetent C3H, as well as humanized mice which have not engrafted sufficiently as sentinels. While humanized mice are not suitable for serology, using them as sentinels provides a 3Rs benefit as mice that would otherwise be discarded have a further value, and they may be more sensitive for parasitology and microbiology.

P166 Framework to Mitigate Complications Resulting from the Relocation of the Administrative Support Area Out of a Vivarium

MW Brunt^*1, J Ferguson², M Zoethout², R MacQueen², ME Fowler¹

¹Campus Animal Facilities, University of Guelph, Guelph, Canada; ²Animal Facility Support, University of Guelph, Guelph, Canada

Laboratory animal facilities cannot function without significant support from personnel that do not directly interact with the animals. Any disruption to this requisite support has the potential to profoundly impact operations in a manner that is both expected and unexpected. Indoor air quality is a prominent concern in laboratory animal science with research animals and workers in animal facilities both participating as stakeholders of this ongoing discussion. Our administrative support area had experienced ongoing air quality concerns since 1974. In 2012, in an effort to address the accumulating and significant health and safety concerns, personnel with offices in the administrative area were provided with alternative space in another building on campus. The administrative area was the main entry point and communication hub for the animal facility. Its relocation had the potential to cause logistical complications surrounding facility access, security, communication, and the ordering of products and animals. Eight weeks before the planned relocation, biweekly meetings were scheduled and included administrative, technical, veterinary, and facility management staff to develop action plans to mitigate any disruptions that could be foreseen. Once the relocation occurred the importance of continued biweekly meetings became apparent to ensure emerging issues did not progress into functional obstacles. Two months after the move the biweekly meetings reduced to monthly meetings. Due to the proactivity and innovation of the Campus Animal Facilities and Animal Facility Support team, meetings are currently held on an ad hoc basis. The initiation of a “grand rounds” that includes administrative, technical, veterinary, and facility management staff has allowed for ongoing face-to-face dialogue between colleagues working in different buildings and provides an effective forum for resolving logistical issues on an ongoing basis.

P167 Subfloor Radiant Heat: A Novel Approach to Animal Warming

MA Gregory^*, B Gien

Surgery, Envigo, Indianapolis, IN

Maintenance of normal body temperature minimizes cardiovascular and respiratory disturbances caused by anesthetic agents and is of particular importance in small animals where the high ratio of surface area to body weight may lead to hypothermia. Therefore, to improve surgery outcomes, supplementary heat is provided to animals during and after surgery. We recently began searching for an alternative to our current system. There are many different methods to provide supplemental heat, but most are not designed to provide heat support for prolonged surgeries and recoveries. We were also looking to provide a heat gradient during recovery so the animals can escape the heat if they wanted to, but this is difficult to do with most of the available products. Noisy motors, leaking pads, and connections have been a constant issue when using our current heating source. We decided to approach the problem from a new direction using a radiant heat product designed for under-floor heating sandwiched between 2 sheets of clear acrylic. The acrylic provided support and protected the heating element, as well as making it sanitizable. We connected the heating element to a programmable thermostat which allowed us to set a schedule and monitor the temperature. The width of the heating element, combined with the overall width of the acrylic, provided a wide thermal gradient, allowing the animals to find a place to feel comfortable. When compared to our current heating source the initial cost savings are not substantial, but the lower maintenance cost and longer life of this new heating source allows savings to continue to grow year after year. The enhanced monitoring and scheduling capabilities, worry-free operation, and better in-cage environment make this an excellent solution for peri and postsurgical animal warming.

P168 The Use of Braided Cloth as a Pig Enrichment Device

KR Lambert, MG Kimmer^*, E Mitchell

Animal Resources Program, Wake Forest School of Medicine, Winston-Salem, NC

Pigs are highly intelligent animals that need lots of stimulation in their environment to meet the species behavioral needs. Despite stereotypes, pigs prefer interacting with devices that are relatively clean and free from fecal material. Hanging enrichment devices is a good strategy to provide pigs with the opportunity to interact with clean devices in a laboratory environment. In developing a toy rotation for pigs housed at our facility, we studied the use of a braided cloth hanging on the inside of the pig enclosure as a potential enrichment device for laboratory pigs. Each singly housed pig was provided with a braided cloth hung on their enclosure every morning and the cloth was removed in the afternoon. Observations occurred twice per day: once in the morning after device presentation and once in the afternoon prior to device removal. We found that most of the pigs were interested in the devices when first placed on the enclosure, but interactions typically decreased by the afternoon. On average, the pigs spent approximately 37% of their time interacting with the devices in the morning, and only 9% of their time in the afternoons. We also found that the interactions with the braided cloth device appeared to decrease over the course of the week with repeated exposure. On average, the animals spent 78% of their time interacting with the devices on day 1, but only 18% of their time interacting with the devices by day 5 in the observations directly after provision. Even though the device did not maintain novelty for long, it still received more interactions from the animals than the other cage enrichment we regularly provide. A challenge we faced included finding the right thickness for the braid to prevent easy destruction and encourage use, and this is a focus for ongoing studies.

P169 Novel Enrichment for Sheep (Ovis aries) Used in Biomedical Research

MR Lowery^*, P Vertz

Teaching and Research Animal Care Services, University of California, Davis, Davis, CA

Sheep (Ovis aries) are an excellent research model, as the anatomy and physiology have been well defined. Sheep have been shown to recover quickly from general anesthesia and invasive surgery. We regularly house sheep that are used in biomedical research. The animal care program’s mission is to ensure humane care and use of animals in teaching and research and to assist with the advancement of scientific knowledge and the future of research goals set forth. Environmental enrichment enhances animal wellbeing by providing animals with sensory and motor stimulation. Traditional enrichment techniques for sheep include providing a “buddy” sheep so their social needs are met. While group-housing sheep and providing forage material meets most of their needs, not all sensory and motor stimulation needs are being met by just these items. Normal grooming behavior in sheep includes rubbing or scratching themselves on areas such as trees, fences, or walls. This behavior is especially popular with sheep that shed their wool each year, which is the typical breed of sheep that we house and maintain. With this in mind, we designed a novel enrichment device that we call a “sheep scratcher.” We used ordinary material such as angled broom heads that were installed in metal frames that mounted to the walls of sheep pens. There was an initial adjustment period for some sheep to become comfortable with the new item in their pen, but after this initial adjustment period these scratchers became heavily used by all of the sheep in our facility. These scratching units give the sheep a chance to engage in species-specific behavior in addition to their interactions with other sheep. This low-cost and easy-to-manage enrichment method has exceeded our expectations and given our sheep a better environment to thrive in. This enrichment device would also work for other livestock species that have the same behavior of rubbing and scratching on trees or fences.

P170 Scrub Access and Availability Project

D McNeill¹, L Schliep¹, NM Gades^*1, J Benedetto², C Ehrler¹, S Moe¹, J Posvenski¹, R Waldron¹

¹Comparative Medicine, Mayo Clinic Arizona, Scottsdale, AZ; ²University of North Carolina—Chapel Hill, Chapel Hill, NC

Our department was not meeting its goal of a complete set of scrubs for every DCM employee every day of the week. The Guide states that soiled attire should be disposed of, laundered, or decontaminated by the institution. We reorganized scrubs by size, creating a color-coded reference chart. Scrub disposal bins were numbered, and soiled scrubs counted at the end of each day. Clean scrubs were counted upon receipt, and compared with the vendor count sheet. Scrub-need worksheets were placed in the locker rooms and were completed by research staff and DCM employees. Data were collected from May 2 through August 1, 2014. An online survey of scrub users was completed. Results showed a significant difference in the number of clean scrubs received compared with what was listed on the vendor count sheet, with the difference ranging from -5 to 30; tops and bottoms were counted separately and categorized by size. The scrub need worksheet identified a lack of 2X large scrub tops (n =3), 2X large bottoms (n =1), and X-small bottoms (n = 1). The scrub survey identified that scrubs tops were worn more often than bottoms. We identified 2 critical areas for improvement in our system. First, the need to reconcile the vendor count sheet with the actual number of scrubs received. Secondly, the need to order more tops than bottoms, based on usage. We continue to do periodic monitoring of the scrub inventory using the tools developed above.

P171 Making an Animal Facility Emergency Response Plan Integrated, Dynamic, and Accessible

NR Young^*1, P DeLuca³, GP Langan^1,2

¹Animal Resources Center, The University of Chicago, Chicago, IL; ²Surgery, The University of Chicago, Chicago, IL; ³Emergency Management, The University of Chicago, Chicago, IL

The Guide requires animal facilities to have an emergency response plan (ERP) and for the plan to be integrated into the overall institutional emergency management program. The importance and functionality of these plans becomes evident during the response to an emergency. There are several key components to a successful emergency response plan. The ERP has to be able to easily evolve with changes in the animal program and it needs to be frequently tested and modified. In addition, the plan needs to be readily accessible during an emergency. To address these important factors, we developed a new ERP program to improve the functionality of our plan. The improvements included the use of even minor events as drills of the ERP with subsequent dissemination of lessons learned to all members of the program. Quick action guides were developed and posted in several locations which highlight the most important information to know during an emergency. In addition to the hard copy ERP, an ERP dashboard was created on commercially available project management software to allow more accessibility and ease in updating. This new system has improved the efficiency and effectiveness, as well as increased the number of people who are able to respond to emergency situations.

P172 A Novel Pin Hole Sipper Tube for Mouse Water Bottles

DW Mallon¹, NS Linde^*2, JD Chinchilla², TJ Plemons¹, J Raber¹

¹VRRS, National Eye Institute, Montgomery Village, MD; ²Priority One Services, Alexandria, VA

With the goal of transitioning to a 14-day rodent cage change schedule, one of the biggest challenges was ensuring that a cage of adult mice at maximum density, 5 in this case, would have enough ad libitum water to last 14 days. We measured the volume of water used daily with our current water bottle and sipper tube system in multiple cages with 5 adult mice over the course of 14 days. We found that we needed to reduce the amount of water wasted when the cage was moved or animals played with the sipper tube. In addition, we needed to increase the capacity of the bottle from 16 oz to 18 oz to ensure enough water was always available. Working with some of our vendors, one vendor created a 3 inch long sipper tube with a 1/32nd inch diameter pinhole and while another increased the water bottle capacity by 2 oz while maintaining a proper fit in the cage. Upon arrival, we tested each sipper tube pinhole for uniformity by using a NIST traceable pin gage. After we verified the desired tube specifications, we slowly implemented the new system; rack by rack and room by room over the course of 3 months all the while monitoring results during a 14-day cage change. In conclusion, we are able go 14 days between cage cages, reducing labor costs and improving animal welfare with a 90% reduction of wet or saturated cages due to water loss or rodent manipulation of the sipper tube.

P173 How Routine Husbandry Can Provide Opportunities for Socialization and Bonding in a Minipig Facility

ML Salerno, B Grambo, BH Jasmin, N Navratil^*

Marshall BioResources, North Rose, NY

Socialization enhances animal welfare, strengthens the human-animal bond, and reduces stress experienced by both animals and caretakers. Socialization at our facility begins at birth, and minipigs receive positive human interaction throughout their entire time in our facility. As a breeding facility with thousands of animals to care for, we also need to balance the need for socialization with the other husbandry and related tasks we must perform each day. Therefore, we have implemented ways to incorporate positive human interaction into our routine husbandry procedures. This includes physically entering the animals’ space and interacting with the minipigs when we perform simple daily tasks such as feeding, washing, and weighing of the animals, as well as when we apply topical treatments. We use clickers at feeding time, and we also provide positive and reinforcing touches to our sows before and during parturition, and to the litters soon after birth. Combining socialization with routine husbandry tasks creates positive benefits for both the animals and the staff without requiring many extra resources.

P174 Breeding Mice and their Various Changes with Aging: Toward Clarifying the Mechanisms of Senescence

N Ogiso^*

Laboratory of Research Animal, National Center for Geriatrics and Gerontology, Obu-city, Aichi, Japan

Our facility has kept many naturally aged animals (mice and rats) for gerontology and geriatric studies. If a scientist conducts a study with these aged animals, it is important to know what changes will occur in them with aging. However, a clear criterion for defining of aged mice has not been established. In this study, we evaluate various characteristics as senescence indicators in naturally aged mice kept in our facility. Four-week-old male and female mice (C57BL/6N) were obtained from Japan SLC and were kept over their lifetime. Physiologic (measurement of body weight, food consumption, and survival rates), behavioral (the rotarod tests were conducted every 3 months), and morphologic analyses (autopsy and histologic examination) were performed. Additionally, the intestinal flora of mice was also examined. Body weight peaks at 18-19 months old in male (approximately 46.0g) and at 14-16 months old in female (approximately 35.0g) mice, and there is no significant changes up to 24 months old. Survival rates start to decline in both sexes at 18 months old and show a similar decrease to that in the other research institutes. Rotarod performance peaks at 3 months old in male (208±61.4 sec) and at 6 months old in female (235.3±75.1 sec) mice and then continues to decline. Male mice are more strongly affected by body weight in aging (R = -0.76, p < 0.01), while female mice have large variation in their performance. Enlarged seminal vesicles in male mice or splenic tumors were found in approximately half of dead animals at autopsy. Mice with abnormalities in various organs are mostly over 20 months old. Additionally, our results show that some alteration of the intestinal flora may occurs at around 12 months old. Various age-related changes (such as the motor performance, the intestinal flora, or pathologies) are found in 12-20 months old B6N mice, therefore a possible turning point of senescence in this strain could occur during these ages. We will focus on these specific life phases and continue to analyze various parameters in detail.

P176 Mobile Data Collection Workstation: Design and Implementation

P Sherfinski^*1, M Peterson², R Smith³, J Johnston⁴

¹Small Animal Toxicology, MPI Research, Mattawan, MI; ²Purchasing, MPI Research, Mattawan, MI; ³Information Technology, MPI Research, Mattawan, MI; ⁴Equipment Compliance, MPI Research, Mattawan, MI

Data collection is the core of any nonclinical study work. Providing researchers an efficient workstation for electronic data collection is a must. Often, the workstation is adapted from a cart with an entirely different purpose. Our facility has used a media cart designed for a television and retrofit to contain our required components. We determined this construction left many characteristics desired for our end users, maintenance personnel, and for our management. The cost of maintenance of the workstations was too costly in both labor and components. The existing design was difficult to clean as the materials and configuration often prevented more industrial means of cleaning. The layout was inefficient, and there was not enough workspace where appropriate and excess space outside of the reach of the end user. Instruments used on the workstation were poorly secured, leading to damage. Several common repairs to the computer or cart components were time consuming. In addition, the configuration of the workstation required the repair technician to either bend down or crouch while providing service. After researching appropriate materials stakeholders discussed the issues with the current design. The positive features of the previous design were discussed and a new design was drafted. A modular configuration was adopted to address several of the issues of the previous design. After having the design fabricated, the workstation was sent out for pilot work with the end users. They provided feedback to improve the scale platform, an accessory work surface, and the placement of the transponder reading system. We made changes and the workstation was sent out for more testing. After months of operation the redesigned workstation was determined to have the features and functions necessary for additional deployment. The following improvements were accomplished by the design. The workstation can now be quickly separated from the electronic components and the remainder processed through cage wash equipment. Several components of the workstation were replaced to keep maintenance costs down. The retention of the instruments was reengineered to limit risk to those devices. The size of the workstation was reduced to make transportation and use in tight quarters more efficient. The configuration in conjunction with components reduces time during maintenance and reduces the need for maintenance staff to bend down or crouch while performing repairs.

P177 Efficacy of Supplemental Heat Options for Supportive Care in Flooded Mouse Cages

P Chamberlain^*, JS Kilpatrick, KE Scott, JG Fox

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Flooding is a common problem when housing rodents and can occur in caging using either water bottles or automatic watering systems. Depending on the severity of the flooding and duration of time before the mice are removed from the flooded cages, supportive care may be required for full recovery. One of the most common methods of supportive care is to provide supplemental heat to the mice once they are placed in a dry cage; however, dedicated cage heating devices are cumbersome and inconvenient to use in a large vivarium setting. Our facility commonly uses 2 methods to provide supplemental heat: placing an air-activated chemical hand warmer under the cage or placing nitrile gloves filled with hot-water under the cage. Our rational for taking these procedures were that hot-water filled gloves increase the ambient temperature in the cage more quickly, while hand warmers provide a more sustained heat source, and that a combination of the two will provide an immediate and sustained heat source for the rodents. In this study, 6 full cage setups with hygrometers placed inside were placed in a change station in an empty animal room for 18 hours. Temperature and humidity within the cage setups were recorded every 30 minutes for 2 hours before the heat sources were added. Three treatments of 2 ventilated cages (n = 6) each were tested. The treatments included 2 hand warmers placed underneath the cage on one end (n = 2), 2 hot-water filled gloves placed in an empty cage bottom with the home cage placed on top of the gloves (n = 2), and a combination using both hand-warmers and hot-water gloves (n = 2). Temperature and humidity within the cage setups were recorded every 30 minutes for 4.5 hours. Initial data (n = 6) revealed that all treatments provide at least a 10 degree Fahrenheit increase in temperature for up to 4.5 hours. Treatments with hot-water gloves had the highest temperature at the 30-minute time point while hand-warmers alone maintained a higher temperature at 4.5 hours. The results indicate that hand-warmers are the best option for providing consistent supplemental heat as supportive care over a 4.5 hour time period. In future testing, the type of bedding will be examined for effect on ambient temperature.

P178 The Process of Animal Diet Harmonization: Big Trouble following Good Intentions

PF Pohlig^*, B Zevnik

In Vivo Research Facility, CECAD, University of Cologne, Cologne, Germany

An animal facility network consists of several animal housing units managed by 3 site managers. The network has heavily intensified its efforts to identify synergies. In the past we developed structural concepts for centralization and training, mutual exchange of SOPs, implementation of database exchange, and identifying common vendors for technical equipment maintenance and diet. Before this, every institution has negotiated their own conditions leading, for example, to the use of 3 different mouse diets in the various facilities. Unfortunately, this is accompanied not only with financial but also scientific discrepancies. Mice exchanges are performed regularly between the different institutions; however problems can arise without a standardized mouse feeding. We therefore decided to start a process of harmonization to find an appropriate standard diet suitable for use in every animal facility on the campus. During an extensive assessment process, different diet distributors were invited to discuss diet compositions, logistical issues, and financial aspects. Furthermore, food acceptance tests were performed with the mice, followed by handling tests. Other considerations included figuring out the practicability during daily use such as autoclaving and development of dust and handling inside the barriers. Finally, a joint decision was made for one company for the production and delivery of the animal diet. Of course, the diet change needed to be performed in close cooperation with the scientists. Additionally, in parallel to the change, our facility started a study to prove how the change of diet would affect mouse holding and breeding. However, after the first trial period of about 3 months, information was derived from the study and from investigators feedback about negative effects on breeding performance and offspring survival in wildtype and transgenic mouse strains, all on C57/Bl6 background. This resulted in an, at least temporary, a switch back to the former diet and a second refined study, which is currently ongoing. We would like to present this extensive evaluation process, highlight difficulties, and discuss best procedures for change management processes in different institutions.

P179 Retirement for Research Nonhuman Primates

R McAndrew^*1, J Lingo VanGilder², K Carter³

¹Bioengineering, Arizona State University, Gilbert, AZ; ²Phoenix Children’s Hospital, Phoenix, AZ; ³Mount Sinai Health System, New York, NY

Though retiring lab animals to a sanctuary is a great way to acknowledge their service and express gratitude for their contributions to the scientific community, this can often be difficult undertaking. Aside from the considerable costs associated with residential animal care, researchers are faced with selecting a suitable facility for providing for the wellbeing of the animal for the remainder of its life, often under the potential threat of backlash from animal rights groups. For some species, such as mice, rabbits, dogs, or cats these challenges are allayed by their status as companion animals and a more widespread integration throughout society. Sanctuaries exist for these animals outside the realm of research. In contrast, nonhuman primate (NHP) adoption must contend with higher costs for care and limited range of choices to provide it. The Research Animal Retirement Foundation was created to provide the necessary financial resources for NHP retirement initiatives, as well as advocating for a paradigm shift in perspectives on end-of-career options for these animals among the research community at large. We have applied for nonprofit status in Arizona, and will begin fundraising efforts once our application is accepted. Once established, investigators from around the country will be able to apply for funding to be used for the retirement of their research animals. Selection criteria will be based on the health and behavior of the animal and potential for positive outcomes. In addition to providing direct financial support, our efforts will also be focused on outreach to the science community as well as the general public, promoting positive attitudes toward research that upholds the ethical responsibility to provide animals with a life outside the lab.

P180 An Assessment of Nesting Material and Ocular Health in Nude Mice

RL Kitsberg^*, C Hedrick, S Lewis

University Laboratory Animal Resources, The Ohio State University, Columbus, OH

Nesting material is a basic environmental enrichment requirement for mice at our large academic institution. Providing the appropriate type of nesting material for various strains of mice can prove challenging when attempting to maintain a level of consistency within multiple large facilities. Previously, commercially available compressed cotton squares have been used as the nesting material of choice and used with ¼” corncob bedding in individually ventilated caging systems. With this standard cage setup, a higher incident rate of nude mice were reported with varying degrees of conjunctivitis, blepharitis, and/or debris in either 1 or both eyes. It was hypothesized that this higher incident rate was due to small dust particles from the nesting material and that nude mice lack of eyelashes to assist as a barrier against it. To test this theory, 162 cages of nude mice were provided either a single compressed cotton square or 8 grams of brown crinkled paper, which is reported by the manufacturer to have little to no dust. Each cage of nude mice were individually evaluated every 2 weeks for 12 weeks and given a score of 0 to 3 with 0 having no issue and 3 presenting swelling, redness, and ocular debris. Data shows that there is a fewer number of ocular health concerns (70.98%) for the nude mice that received the crinkled paper. Following further investigation regarding nesting material and nude mice an institutional change may be needed to prevent and reduce ocular health concerns in nude mice.

P181 A Novel Method to Provide Thermal Heat Support to Primates

CA Carrier, R Samaniego^*

Covance Research Products, Alice, TX

Thermal heat support is needed for many reasons when providing care for debilitated animals. Some examples are thin or dehydrated animals, newborn animals, or animals recovering from anesthesia or surgery. We provide hospital care for nonhuman primates, many of which need thermal support at some point during their treatment. Many different types of heating methods have been used in the past such as electric blankets, warm-water recirculating blankets, individual incubators, and forced-air warmers. However, each type has particular pros and cons when working with primates. Manufactured incubators are ideal because they are generally made with smooth nonporous materials and offer temperature, humidity, and oxygen control. These incubators also provide secure containment for nonhuman primates. Space and cost was a factor when we considered the number and type of incubators needed in the hospital. To take maximum advantage of our forced-air warmers, an inhouse incubator was made out of recycled 30-gallon drum barrels in a PVC frame. A forced-air warmer is attached to the unit and simultaneously warms the four animal enclosures. Another inhouse unit was made out of 55-gallon drum barrels for larger animals. The units are small enough to move, easy to clean, and 2-4 animals can be housed in them and receive thermal support. It has been a cost-effective efficient means to provide thermal support to our primates in rooms not designed for banks of recovery cages.

P182 The Feasibility of Introducing High-Quality Nesting Material to Mouse Colonies

R Gainer^*, JM Cadillac, E Dohm, SC Cartner

Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL

This study explored the feasibility of implementing easy-to-use, preportioned shredded paper nesting products that provided 2 different substrates, and could readily be integrated into husbandry practices. It was thought that cost would be prohibitive, since cost is typically a stumbling block when considering changes to husbandry practices. Unfortunately, the cost of a preportioned shredded paper nesting pack (preportioned pack) was nearly twice the amount of the compressed cotton squares currently being used, suggesting the only way a switch could be made would be to raise per diems, which was not an option. The compressed cotton squares were added to every clean cage prior to autoclaving and distribution to the animal housing room. The provision of autoclaved pre-portioned packs was instead moved to the animal housing room, so that only cages lacking enrichment were provided with new packs. Cages that already had a shredded paper nest at cage change simply had the nest transferred to the new cage, thus avoiding the expense of providing new enrichment every cage change and reducing the cost to equal or less than the compressed cotton squares. Introduction of the preportioned packs began in one animal room, and then expanded to 1 room per building. This provided time for adequate staff and investigator education. Campus wide implementation is now underway. It was determined that the preportioned packs do not need to be replaced more frequently than once every other cage change. In some cases, staff report not needing to replace the pack for up to 6 weeks. In addition, the total number of clinical cases significantly decreased for both overall case reports and specifically fighting cases by as much as 50% or more in the 9 months since implementation. Investigators have also anecdotally reported improvement in reproduction. We determined it is possible to switch to a high-quality nesting material with an equal if not lower cost than the original enrichment. Moreover, dramatic decreases in the overall clinical cases and specifically fighting cases were seen in the colonies observed.

P183 Do Your Employees Feel Stressed and Underappreciated?

RA See^*1,2, C Mathieu¹, S Paynter^1,2

¹The University of Chicago, Chicago, IL; ²Charles River, Chicago, IL

Our animal resources center (ARC) consists of several types of employees: cage wash technicians, facility attendants, animal care technicians, special services technicians, supervisory staff, administrative staff, certified veterinary technicians, and veterinarians. All work and interact with each other and with investigators on a daily basis. All are faced with a wide variety of issues every day. Whether customer service, husbandry, or special services, they must deal with stress on a regular basis. The mission of the ARC is to take care of customers and animals. Who is taking care of employees? One way to address the issue was to develop an employee program that builds a culture of recognition and that yields higher levels of employee engagement, retention, and satisfaction. It is easy to lose sight of the workforce and expect employees to just perform without question. But in reality, in order for employees to be successful and feel happy performing their duties, they must be recognized for the work that they do. We believe that happy or satisfied employees are more likely to perform at a high level. An effective recognition strategy helps create employees who are more productive, more creative, and more customer service oriented. Small tokens of appreciation and kind words go a long way in thanking employees for their dedication and hard work. It is also a reflection of the unit’s overall culture. If a unit professes that people are its greatest asset, it should also be ready to invest, recognize, and reward people in visible ways. Based on this information, we came up with the RAT Pack (Recognize Acknowledge Treat). Once implemented, we were able to receive feedback showing a significant increase in employees’ attitudes and morale. In addition, the RAT Pack is particularly appreciated because it is composed of employees who have input in figuring out ways to recognize their peers.

P184 Swine Swaddlers

RD Moore^*, L Roberson

Office of Comparative Medicine, University Of Utah, Salt Lake City, UT

The swine model is frequently used in research for different studies such as new instrumentation, implants, and organ functions. The pig is an intelligent animal, as well as strong and stubborn. Protecting a surgery or implant site can be challenging. Originally using commercially available stretch covers for this purpose, we found that trying to put the animals head and limbs into premade outlets were difficult, especially with a 60kg+ swine. We needed to find a similar, but easier method of clothing these animals. We created what we call “swine swaddlers.” We omitted the outlets for the legs and head, and sewed on straps to either buckle or Fabric hook-and-loop fasteners the clothing in place. One fastening point is around the neck to keep the clothing secure, and one around the abdomen to keep it across the back of the swine. The swine swaddler kept a postoperative/bandage site clean and dry on the swine model, while eliminating extra stress and restraint, providing comfortable clothing for the animal and less time and staff for placement.

P185 Transmission of Pseudoloma neurophilia in Zebrafish (Danio rerio) When Using a Mass Spawning Chamber

SM Peneyra^*4,5, J Cardona-Costa^4,5, J White^4,1, C Whipps², ER Riedele³, NS Lipman^4,5, C Lieggi^4,5

¹Laboratory of Comparative Pathology, The Rockefeller University, Memorial Sloan-Kettering Cancer Center, and Weill Cornell Medical College, New York, NY; ²Center for Applied Microbiology, SUNY-ESF, Syracuse, NY; ³Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY; ⁴Tri-Institutional Training Program in Laboratory Animal Medicine and Science, The Rockefeller University, Memorial Sloan-Kettering Cancer Center, and Weill Cornell Medical College, New York, NY; ⁵Center of Comparative Medicine & Pathology, Memorial Sloan-Kettering Cancer Center, and Weill Cornell Medical College, New York, NY

Pseudoloma neurophilia, a microsporidium that primarily infects neural tissues, is one of the most common pathogens in laboratory zebrafish. Infected fish can transmit P. neurophilia vertically; however, horizontal transmission during spawning is more common. It is unclear if risk of parasite transmission is different when spawning zebrafish in individual spawning tanks as compared to mass spawning chambers, and if this risk is correlated to the number of spawning events. In addition, disinfection protocols have not been evaluated to determine if any could decrease or eliminate the risk of transmission following spawning. The present study was conducted to evaluate P. neurophilia transmission from a known infected colony (prevalence = 52%) to uninfected fish and their offspring when using 2 different spawning apparatuses (n = 30 per group). Infected and uninfected fish were allowed to spawn in either a mass spawning chamber or breeding tanks for 1 or 3 spawning events. Uninfected fish and offspring were evaluated for infection 15-weeks later by histopathology and Luna stain. In addition, we sought to develop an effective protocol for disinfecting the mass spawning chamber between uses to eliminate P. neurophilia spores. Devices were either allowed to dry for 1 hour or were disinfected with one of the following protocols for 10 minutes after infected zebrafish spawned for 1 hour (n=5 per method): reverse-osmosis water, 90-110 ppm bleach (pH = 6.8 - 7.5), 1.0% Virkon Aquatics, 75 ppm Wescodyne, or 175 ppm Wescodyne Plus spray. Results demonstrated increased risk for transmission to uninfected fish when spawning occurs in a mass spawning chamber (100% infected after 3 spawning events), as compared to the other groups (0-7% infected). The parasite was not detected in offspring from these spawning events regardless of spawning device or number of spawning events. The Wescodyne Plus spray, bleach soak, and Wescodyne soak were 100% effective at eliminating P. neurophilia spores as determined by PCR following spawning. It is important to prevent or minimize spread of this pathogen as there is no treatment and infections can cause morbidity and mortality with the potential to confound research results.

P186 Use of Hand Warmers and a Portable Heater to Provide Supplemental Heat in an IVC System

S Hackney^*1, A Dickerson¹, DR Goulding¹, PH Myers¹, CA McGee¹, J Locklear², TL Blankenship¹

¹Veterinary Medicine Section, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC; ²Quality Assurance Laboratory, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC

Supplemental support is an important aspect of laboratory animal medicine. Our facility has recently transitioned from a rodent static caging system to a ventilated caging system which has required us to evaluate how supplemental heat was provided to rodents. The objective of this study was to evaluate more efficient ways to provide supplemental heat to cages in a ventilated caging system. Air-activated hand warmers, both 12 and 24-hour, were applied to the bottoms of occupied cages as they hung in place on the rack and intra cage temperature levels were evaluated. We compared this to static microisolation cages on a cart which had a 24-hour hand warmer or a microwaveable heat pad placed underneath. Additionally, we evaluated the efficacy of a portable heater placed inside a small animal room (cubicle room) to warm the ambient and intra cage temperatures. Heat from both types of hand warmers did not significantly raise intra cage temperatures when applied to the bottom of the cages or when placed in a static environment. The heat from the hand warmers seemed to dissipate too quickly to be of any benefit. The microwaveable heat pads did significantly raise temperatures in a static environment. The portable heater raised the intra cage temperatures significantly on a single- sided rack inside a cubicle room; however, there was temperature variability between the top and bottom rows. The ventilated cages can go static for up to 3 days without adverse effects on the mice. We determined that when needing to provide supplemental heat to a few cages, the microwaveable heat pad would be the preferred choice over the hand warmers. When a large number of cages require supplemental heat, a portable heater placed inside a cubicle appears to be a viable option although cages would be limited to the top 7 rows of the rack to minimize the impact of temperature variability. Temperature evaluations would also be necessary at different facilities to account for unique HVAC operating parameters.

P187 A Unique Barricade Solution to Stop Flooding Conditions

SM Saverino^*, K Silva, M Murchison, BR Therault

Animal Resource Center, University of Chicago, Villa Park, IL

Flowing water in an animal housing facility may occur due to faulty equipment, animal mischief, human error, or environmental factors. While some potential overflow water situations may be identified when only small puddles are on the ground, others can consist of unrestricted flowing water creating flood situations. Regardless of the size, all facility water situations need to be handled in a prompt, timely fashion. Often unique circumstances resulting in free water require a bit of creativity in finding prompt, innovative solutions. Our campus has encountered animal facility errant water on numerous occasions arising from a variety of causes. Typically, the influx of water stops after only a few minutes and facility staff are quickly able to clean up the water and associated soiling. On one recent occasion, however, one of the pipes in the maintenance space cracked and water was slowly flowing into the hallways of the vivarium. Once the water reached the hallway, it flowed directly into laboratory spaces storing delicate electronic equipment. With no sandbags on hand, a swift solution was needed to control the flooding while campus maintenance crews worked to find a resolution short of widespread water shut-off. Technicians quickly gathered available supplies: a pair of used, discarded scrub pants, zip ties, and a bag of pelleted paper bedding and got to work. The ends of the pants were twisted and tied off using zip ties and then filled with the absorbent bedding material. Once they were filled, the waste was similarly secured. The flood barrier pants were placed in front of the maintenance spaces out of which the water was flowing and continued to absorb the water for several hours while the cause of the flood was being resolved. Having the flood barrier pants in place allowed our technicians to continue with their work throughout the day, requiring only periodic checks and twice-daily swap-out to ensure the flood pants were continuing to control the errant waters. This technique has subsequently been used on numerous occasions throughout all of the campus animal holding facilities during times of flood situations and has helped to reduce costs in labor and equipment damage.

P188 Occupational Health and Animal-Pathogen Status Are Maintained with Decreased Use of Personal Protective Equipment in 2 Rodent Facilities

SJ Danon^*1, D Coomber², M Lavin³, M France⁴

¹BRC, Faculty of Medicine, UNSW, Sydney, Australia; ²Faculty of Medicine, UNSW, Sydney, Australia; ³UNSW, Sydney, Australia; ⁴France Consulting, Haberfield, Australia

A center operates 2 specific pathogen-free (SPF) rodent facilities that followed the same husbandry procedures but had operated with different procedures for use of personal protective equipment (PPE). PPE procedures in Facility 1 were based on historical convention and included mandatory use of hair nets, surgical masks, shoe covers, gowns, and vinyl gloves. In Facility 2, choice of PPE were based on evidence-based risk to staff and animals and involved only gowns, nitrile gloves, enclosed shoes, and optional P2 face masks. Both facilities complied with Australian animal research regulations, and while both met the required levels of pathogen exclusion and containment, there was a need for operational uniformity. we sought to implement the same PPE requirements across both facilities. Risk analyses were conducted in relevant aspects of facility operation and included consideration of biologic hazards, allergens, cytotoxic hazards, noise, and radiation exposure. The most appropriate PPE was matched to each risk based on Standards Australia criteria, animal research legislation, and scientific literature. Results of the analysis were communicated to all stakeholders and changes in PPE implemented on an agreed date. Live sentinel screening programs, conducted every 3 months, monitored animal health status 1 year prior to, during the PPE change, and for a further 18 months after the change in PPE. The risk analysis indicated that the PPE regime in Facility 1 was unlikely to make an effective contribution to the protection of users or animals and may even increase the risk of pathogen spread. For general operations, the only PPE considered essential were those used in Facility 2. Other PPE were required on a case-by-case basis. Live sentinel testing conducted prior to, during, and post PPE change showed no change in disease status in either facility. The benefits of removing unnecessary PPE included less confusion among users who worked in both facilities, significant cost saving, reduced risk of pathogen spread, and better protection of facility users while still maintaining SPF status.

P189 Using Positive Reinforcement Training with Dogs Makes the Job Easier

SA Iliff^*, J Amador, L Zacniewski, GM Savastano

Merck & Co., Inc., Kenilworth, NJ

Implementation of positive reinforcement training (PRT) programs can be beneficial in research settings, reducing the need for manual restraint or anesthesia and enhancing personnel and animal safety, which can decrease stress for animals and staff. PRT was employed with a colony of beagles to gain their voluntary cooperation on husbandry procedures. An animal behaviorist provided an overview and demonstrated paws-on training. One technician was selected to train existing pairs of run-housed beagles. Three desired behaviors were selected: animal comes to trainer, sits, and returns to kennel. Verbal commands and criteria were established and animals were trained 5 days per week for 5 minutes per animal per session. Implementation of PRT led to all beagles enrolled in the program successfully exhibiting behaviors on command. The dogs’ cooperation has enhanced staff satisfaction and made the job of cleaning easier and more efficient. Additional benefits include exercise for the dogs and more social interactions with other dogs and staff. The dogs are currently being trained on behaviors that will assist in obtaining weights and transferring to kennels for room sanitation. This expansion of the dogs’ repertoire is based on their recognition and response to the foundational behaviors.

P190 Development of Personal Protective Equipment Requirements for Handling Animals Exposed to Hazardous Chemicals and Biologic Agents

TE Noe^*, BS Blank, A Brubaker, K Yager, B Reddyjarugu, B Collins, EK Daugherity

Cornell University, Ithaca, NY

There is no specific regulatory guidance to govern institutional requirements for personal protective equipment (PPE) choice when handling animals exposed to hazardous chemicals and biohazards. Researchers are usually well-acquainted with the chemicals and biologic agents they are using, but animal husbandry staff and weekend veterinary care staff are less likely to be aware of the specific risks associated with individual agents. Little is known about the risks associated with animals excreting chemical compounds after exposure. We are standardizing hazardous agent signage at the animal housing room level and developing requirements for appropriate PPE when handling mice exposed to hazardous chemicals and biologic agents. Initial user feedback revealed user fatigue and overheating with our initial PPE choices (one yellow spunbonded polypropylene gown worn under one blue polyethylene-coated polypropylene spunbonded gown), resulting in reduced user compliance. We set out to establish comfortable alternatives that minimize the risk of exposure to different types of liquid hazards by physically testing the permeability of various protective gown/sleeve combinations using a commercially available UV-fluorescent liquid. One standard yellow spunbonded polypropylene gown with white impermeable nonwoven spunbonded olefin sleeves provided maximal user comfort and appropriate liquid barrier protection in 2 events determined to carry the highest risk of liquid exposure when handling animals (mouse urination and liquid smear). Engineering standards currently in place in our animal rooms (handling animals exposed to biohazardous and chemical agents in biosafety cabinets, housing animals in individually ventilated cages) should absorb remaining risks not addressed with PPE. Overall, functional performance of specific PPE used in hazardous agent studies should be evaluated prior to use. Additionally, PPE standard operating procedures should take into account ease of use for the animal handler to ensure proper compliance.

P191 The Effects of Mainstream Media on Public Perception of a Baboon Research Program

TJ Holbrook^*

Comparative Medicine, The University of Oklahoma Health Science Center, Newalla, OK

This study investigates the effects of mainstream media on public perception regarding a university’s baboon research program. A survey was administered with participants recruited using the researcher’s network of contacts. The survey contained 2 actual mainstream media clips covering the baboon program. One clip portrayed the program positively and the other negatively. Participants viewed each clip then answered questions immediately after related to the baboon program. The survey was counterbalanced. Results of a mixed model ANOVA revealed a main effect for both media type and a main effect for media presentation order on public perception of the program. There was not a significant interaction between media type and media presentation order. Based on the results from this research, I suggest the university employ crisis management strategies and techniques in order to mitigate further damage caused by the negative exposure regarding the baboon program. Although the decision to phase out the baboon program has been made, the process could take several years to accomplish. University officials must recognize that the media and animal rights groups are likely to continue to monitor the baboon program, and there is continued risk for media exposure concerning the baboon program and the university’s other lab animal research programs. It is important to keep in mind that a snowball sample was used. Participants were not randomly chosen so the results may not be representative or generalizable of the whole population. The participant sample was limited. Only the researcher’s contacts were used so it is possible that some of the participants may have had previous knowledge of the crisis situation prior to taking the survey which could have influenced their perception on the situation prior to taking the survey. There was only 1 media clip used for each slant of media coverage. The positive media clip only used some of the image restoration strategies. It is unclear if results would replicate for other image repair strategies.

P192 Programmatic Design of a Safety Program at a Primate Center

T Fountain^*

Yerkes National Primate Research Center, Atlanta, GA

There are numerous challenges to address in an occupational health and safety program when working in a primate research facility. Hazards are present in every area ranging from animal care, facilities management, as well as research labs. These hazards need to be identified both from the research aspect as well as the daily operations of managing an animal facility. In order to achieve this, it is necessary to have an effective safety program. We have developed a comprehensive safety program managed within the Safety Office which is located onsite at the facility. The office is comprised of a registered nurse who serves as the Safety Officer; the occupational health manager; the training and compliance coordinator; the containment manager; and the biohazard waste technician. The ability to have the occupational health, training, compliance, and other aspects of a safety program located within the facility serves to remove barriers to safety and moves the center towards an effective safety program to meet the unique challenges of working in research, particularly with nonhuman primates. Other methods in support of an effective safety program at the center include the development and maintenance of standard operating procedures, a safety committee with representatives serving from various departments and a quality assurance program. In addition, the Safety Officer serves on several University committees, such as IBC and IACUC. The structure of this safety program facilitates interdisciplinary collaboration with personnel who become invested in this program, which ultimately promotes the incorporation of safety in all aspects of the center.

P193 A Novel Cage Retrofit Design to Allow Social Housing of Rabbits

S McBride^*, M Whalen, WR Kingsbury, JA Scholz, C Frisk, TR Meier

Comparative Medicine, Mayo Clinic, Rochester, MN

According to the Guide, social animals must be socially housed unless there is a veterinary or study exemption. At our institution, rabbits were housed in 6 bank racks with 5 square foot cages that did not allow social housing. We wished to modify our existing cages to allow social housing and avoid the cost of purchasing new cages. Our goal was to develop a system with a gate that could be opened for social housing and easily closed when needed. The gate mechanism needed to be independent and not directly attached to the cage so the cage could be disassembled for cleaning. The mechanism for opening, closing, and securing needed to be simple for ease of operation while minimizing parts needed for future maintenance or repairs. Finally, the type and size of the socialization opening needed to be appropriate for a wide range of rabbit sizes. We designed a triangular (“A”) shape gate mechanism that slides between 2 side-by-side cages. We decided upon a 7-inch opening and worked with a local metal fabricator to design a prototype that could easily be slid into place using existing frame structure of the rabbit rack. Two prototypes were made: one with a 7” round hole and one with a 7” square hole. We piloted the protypes with pairs of adult New Zealand White and Dutch Belted rabbits. Both breeds of rabbit were easily able to use 7” hole with no preference for the square hole or the round hole. We chose the square hole because it would be easier and more cost effective to fabricate. The 1 modification that was made after the pilot study was the addition of the 1” holes to the sliding gate so that if the rabbits needed to be housed individually housed, they could still have visual contact without nose contact when it was in the closed position. We are now successfully using the cages to house social pairs and trios of rabbits. The cage modifications were a key component in developing our rabbit social program while providing a budget-friendly solution.

P194 Differences in Humidity Readings across 3 Common Testing Devices

ZK Dietz^*, K Clark, A Rodecap, N George, WA Hill

Animal Care Unit, University of Kansas, Lawrence, KS

According to the Guide, the acceptable range of relative humidity is 30% to 70% for most mammals. Situated in Lawrence, Kansas we experience a relatively dry climate with hot summers and cold winters. Macroenvironmental humidity within animal facilities exhibited seasonal variability and values recorded from in-room digital hi-lo hygrometers were frequently below the range recommended by the Guide as noted on accreditation and IACUC semiannual evaluation reports. We sought to determine for our IACUC the accuracy of digital hi-lo hygrometers compared to centralized environmental monitoring systems (CEMS) and the actual microenvironmental humidity experienced by pair-housed CD-1 mice static and individually ventilated cages (IVC). Humidity data was collected using 3 separate methods, an in-room digital hi-lo hygrometers, intracage humidity data loggers, and CEMS, from February through April 2016, in 3 rooms in 2 separate buildings. Humidity was recorded once daily for digital hygrometers and every 6 hours for other methods. Data was analyzed using a 1-way ANOVA. Data showed a significant difference between digital hygrometers (M= 30.1%, SD=2.2) and CEMS (M= 42.7%; SD=0.8; P = .0001). In addition, the microenvironmental humidity measured in static cages was significantly higher (M= 69.9%; SD=1.7) than that measured by the CEMS (M= 43.2%; SD=0.28; P = .0001). Intracage IVC humidity (M=45.8%) was significantly lower than that of static cages (M=69.95%; SD=1.7; P = .0001). Microenvironmental humidity experienced by mice in static caging was higher than relative humidity of the secondary enclosure and micoenvironmental humidity experienced by mice in individually ventilated caging more closely approximated the relative humidity of the macroenvironment. Based on these findings we recommended that use of in-room digital hi-lo thermometers/hygrometers be discontinued in areas where CEMS existed.

P195 Genotyping of Cryopreserved Mouse Lines

A Navis^*, E Kalkbrenner, PL Roesch, AV Perez

Taconic Biosciences, Rensselaer, NY

Minimizing animal use is an essential component of laboratory animal research. For mouse lines that are not in use, freezing sperm samples of the line for later recovery is an attractive way to reduce animal usage. While this method is a powerful way to minimize animal use, it also introduces a number of challenges for later studies. One primary challenge is identifying the genomic characteristics of these lines prior to their recovery. For live animals, tissue samples are easy to obtain. However, for frozen lines, critical information may be out of date or incomplete, adding uncertainty to a costly recovery procedure. Here we describe a method for analyzing the genotypes of sperm samples to improve quality control. The method is a direct comparison of 3 different loci in the cryopreserved sperm samples, archived tissue samples of known genotype, as well as samples from the background control strains (B6 and FVB). To validate the method, we genotyped 5 sperm samples and 3 related tissue samples using real-time PCR. Each sample was tested for the SNP markers CRB1 and PDE6B, and for the presence of a Cre-Luciferase transgene. We used this information to confirm that the sperm samples carry the desired transgene and that they contained markers from the proper background. Our genotyping successfully identified the genotype of the frozen samples and live tissue. This method can provide valuable quality control information for frozen sperm and could also be extended to analyze other aspects of the line including the background strain. This analysis improves quality control procedure for existing and future cryopreserved lines to help prevent the generation of excess research animals while also avoiding the duplication of costly work.

P196 Comparison of Animal Welfare Issues and Vaccinia Virus Expression Kinetics of NanoLuc and Firefly Luciferase Marker Genes in TC-1 Tumor-Bearing Mice

RS MacGill, AR Alfaro^*, J Lallibert, NC Peterson, S Robbins

Laboratory Animal Resources, MedImmune, Gaithersburg, MD

Bioluminescence has become a valuable tool for measuring in vivo endpoints such as PK/PD relationships, tumor growth, and metastases. One advantage of bioluminescence imaging is its application to longitudinal studies using nonlethal quantitation, thereby greatly reducing intraanimal variability and the number of animals to be euthanized for these types of studies. Here we report on the comparisons of vaccinia virus (VACV) expressing a new bioluminescent protein, NanoLuc (VACV-NL), or firefly luciferase (VACV-FL) in regards to technical feasibility and vector expression kinetics following intratumoral injection into TC-1 tumors, a murine model of human papilloma virus-associated cancers. We measured transgene retention at the site of injection and expression kinetics following intratumoral injection of VACV-NL or VACV-FL into TC-1 tumors. TC-1 tumors were established by injection of 20,000 viable cells into the left footpad of C57Bl/6 mice. Initial comparisons between IP and IV administration of NanoLuc’s furimazine substrate found IP administration was lethal in a portion of the mice, and resulted in nonspecific abdominal luminescence. IV administration of furimazine was well tolerated and the bioluminescent signal was localized to the tumor. However, NanoLuc bioluminescent imaging was complicated due to the short transition period between IV administration of furimazine and the start of imaging (<1 min.), and great care had to be taken to avoid excessive heating while dilating the tail veins. Repeat IV administration via the tail vein was marginally well tolerated, but often resulted in scarring and sometimes loss of the tail. In contrast, bioluminescent imaging of VACV-FL was technically uncomplicated. In direct comparisons between VACV-NL and VACV-FL delivered at 1e7 plaque forming units intratumorally, similar expression kinetics were noted. However, the VACV-NL had greater half-life relative to VACV-FL. This is most likely due to the greater half-life of NanoLuc relative to firefly luciferase. These results indicate bioluminescence imaging is a suitable means to monitor VACV expression in oncology studies. Firefly luciferase was technically easier to use, had fewer animal welfare concerns, and is likely the most relevant for studying vector expression kinetics.

P197 Early-Life Stress Alters Correlations between IL-6 and Depression Behaviors

A Hicks-Nelson^*, B Nephew

Biomedical Science, Cummings School of Veterinary Medicine, North Grafton, MA

Postpartum depression, anxiety disorders, and disabling mood disorders are increasingly common and impacting mothers and their children more frequently than ever. However, most depression research in animals only includes males. This limitation has led to significant gaps in our knowledge of these disorders. Recent studies from other laboratories indicate the significant role of the immune system in what our laboratory has shown to be transgenerational behavioral effects. Naturally occurring increased levels of IL-6 in mice have been associated with stress susceptibility, leading to a depression-like phenotype following social stress. The current study investigated the role of cytokines in the effects of chronic social stress (CSS) on rat F1 offspring behavior. The CSS model of postpartum depression and early-life stress depresses maternal care in F0 generation dams using an intruder male to antagonize the dam, simulating neglect and establishing a cycle of deficient social and maternal behavior in the F1 and F2 generations. We hypothesized that impaired social behavior of F1 juveniles would be associated with increased peripheral IL-6 concentrations. There was a strong correlation (r² =.86, p < 0.001) between social investigatory bouts and IL-6 for control juvenile F1 females that was absent in their CSS counterparts. Similarly, there was a moderately strong correlation between IL-6 and both self-grooming frequency (r² =.46, P = .04) and duration (r² =.49, P = .03) only in the juvenile F1 control females. In conclusion, CSS disrupts the robust associations between social- and depression-associated behaviors and basal IL-6 concentration seen in control F1 juvenile females. This correlation, while not yet a direct cause-and-effect relationship, further advances the immune system as a reservoir of targets for future therapies for mood disorders and postulates that IL-6 may be involved in stress associated social impairments.

P198 Turkeys as a Model for Flexor Tendon-Related Research

AM Vrieze^*1, A Kadar², R Reisdorf¹, A Thoreson¹, S Moran², P Amadio¹, C Zhao¹

¹Orthopedic Surgery, Mayo Clinic, Rochester, MN; ²Mayo Clinic, Rochester, MN

Canines are commonly used for flexor tendon-related research, however there are ethical considerations associated with their use as they are companion animals. The chicken is another model commonly used, having a major disadvantage regarding size that prevents performing complex surgeries on. We hypothesized that the turkey will be a suitable large animal model for flexor tendon research, and designed an in vitro comparative trial to evaluate all models. We compared the canine, chicken, and turkey model to the human using the following cadaver parts: third digit from the dog forepaw, the third digit from human hands, and the third digit of turkey and chicken feet. Comparing all 4 species anatomy, structure, and biomechanical properties by means of the digits range of motion, work of flexion, tendon excursion, and by a simple suture pull out strength.The anatomy and structure comparisons identified the turkey superiority to the canine and chicken in several aspects. The turkey digit was much larger than the chickens and it was not webbed like the canines. Work of flexion for the turkey was similar to human and canine. The cross-sectional area, and the gliding resistance of the turkey tendon was much closer to the humans than its counterparts as well. The canine had a significantly higher simple suture pull out strength; this was explained by the fibrocartilage abundant in the canine tendon, different than human and turkey. The turkey had a significantly lower range of motion than human. We were able to confirm our hypothesis that the turkey flexor tendon is similar to human in tendon size, shape, and biomechanical properties; only faltering when it comes to arc of motion. In vivo studies will need to be conducted to determine if the turkey model is suitable in terms of husbandry and tendon healing.

P199 Identifying Canine Flexor Tendon Repair Failure on a Pressure Sensing Walkway

AM Vrieze^*1, R Reisdorf¹, A Thoreson¹, P Amadio¹, S Moran², C Zhao¹

¹Orthopedic Surgery, Mayo Clinic, Rochester, MN; ²Mayo Clinic, Rochester, MN

Using canine model for flexor tendon-related research is common as the anatomic, structural, and biomechanical properties of the canine flexor tendons are comparable to humans. Developing novel techniques to evaluate digit function following flexor tendon repair or graft surgeries in live dogs versus sacrificing is crucial. It will decrease the number of animals used by longitudinally evaluating the digit functions, and also provide accurate force distribution, time, and pressure values of individually treated digits of the paw. The aim of this study was to evaluate dog digit function associated with flexor tendon repair on a newly developed pressure walkway system. The second and fifth flexor digitorum profundus (FDP) tendons from 11 dogs were transected at the proximal interphalangeal joint and repaired with modified Kessler technique. The pressure sensing walkway was used to assess the paw pressure while walking before repair surgery and 14-21 days post repair. Dogs were pretrained to walk on the system; a camera captured their movements enabling identification of each cycle of gait. At least 10 passes were collected on the walkway per visit and each pass yielded up to 2 gait cycles. Due to active motion, the repairs ruptured, which was confirmed 42 days postoperatively in all 11 dogs. The peak pressure, average pressure, and contact area of the surgical second and fifth digits were decreased compared to the second and fifth digits of the nonsurgical paw measurements. However, the central pad pressure increased in the surgical paw compared to the nonsurgical paw, which indicated that dynamic pressure was altered due to FDP tendon repair failure. The new pressure walkway system is a valuable tool when assessing the paw and digit function during the dogs’ gait cycle, which would be beneficial outcome measurements for other flexor tendon related research using the canine model.

P200 Inhibin Antiserum for Superovulation of Genetically Engineered Mouse Lines

A Dasari^*

Product and Program Management, Taconic Biosciences, Rensselaer, NY

Assisted reproductive technologies have become indispensable for efficient production and management of mouse and rat breeding colonies. Recent advances in superovulation technique, coadministration of Inhibin anti-serum+equine chorionic gonadotropin (IASe), followed by human chorionic gonadotropins (hCG) at 48-hour interval reported to have increased the oocyte yields by 3-4 times from prepubescent wild type C57BL/6 mice. However, to date there has been very little data on using IASe for superovulation of genetically engineered mice (GEM) and its application in a large-scale rederivation of GEM lines. Female mice from at 3-4 weeks age primed for superovulation with standard (5 IU of PMS and hCG, 48 hours apart per animal) or IASe (0.2 ml of Hyperova and 7.5 IU of HCG, 48 hours apart per animal) methods were compared for oocyte yields/oocyte donor, fertilization rate, and development to term. The oocytes generated by IASe method were fertilized in vitro following the standard IVF protocols in mouse, and the 2-cell embryos generated were implanted into the oviducts of 0.5dpc pesudopregnant recipients for in vivo development to birth. IASe method produced a significantly higher number of oocytes per oocyte donor than standard superovulation method. IASe method improved the overall efficiency of rederivation by increasing the numbers of embryos and live pups generated per oocyte donor used in IVF. Improvement in efficiency of rederivation of mice through IVF-ET technologies reduce the number of oocyte donors required. In summary, we believe IASe in combination with meticulous planning and execution of large scale derivations will help us with striking a balance between our commitment to 3R, and high-quality services to researchers.

P201 Improving the Collection Accuracy of Common Intramuscular Injection Sites for Histopathology in Rabbits

AL Doan^*, M Ashley, B Harmsen, C Hollinger, K Nelson, DVM, PhD, DACVP

MPI Research, Mattawan, MI

Intramuscular (IM) dose sites are commonly used for vaccine safety studies because of rapid and uniform absorption of test compounds. Rabbits are a common model because they mount a robust immune response to vaccine administration and are large enough to test full human doses. Dose sites are commonly assessed histopathologically and ideal histopathologic specimens have the complete local tissue anatomy including skin, subcutaneous tissue, and muscle in a single plane, allowing pathologists to most reliably assess test compound effects. However, collection of consistent high quality IM injection sites for histopathology in rabbits is impeded by the loose attachment of rabbit skin to the underlying muscle, resulting in unacceptable margins of error for IM dose site collection with routine procedures. We hypothesized that improved postmortem skin immobilization and specimen collection methods would improve the accurate collection of rabbit IM dose sites for histopathology. Use of multiple sections through externally marked injection sites improved histopathology evaluation, but was insufficient. Multiple methods of skin and muscle apposition were attempted, including suturing, pinning to a cork board surface, and stapling. Pinning the excised muscles and skin to a cork board prior to fixation or suturing muscle to overlying skin had suboptimal outcomes, with tissue deformation and suture dehiscence upon fixation, and excessive use of technician time. Stapling the skin to the underlying muscle was the most effective methodology in terms of time and consistency, albeit at an increased cost in materials. Studies using the stapler method in conjunction with exact anatomic localization of injection sites at dosing had significantly (p < 0.05) improved recovery of injection sites upon histopathological evaluation when compared to other methods. Tissue immobilization by securing skin and muscle edges with a surgical stapler at all cut margins, in conjunction with multiple section sampling, results in more accurate and complete sectioning for histopathology evaluation of IM dose sites, and is associated with decreased overall use of resources including technician time. The instituted measures also help ensure consistent and accurate collection of IM injection sites, and support high-quality tissue assessment.

P202 Assessment of Buprenorphine Sustained-Release Efficacy in a Rabbit Survival Surgical Model

A Zanetti^*, S Putta, C Franciozi, S Louie, M Humayun, D Casebolt

Department of Animal Resources, University of Southern California, Los Angeles, CA

In rabbits, buprenorphine instant release (IR) is commonly used to alleviate pain, especially in surgical models. However, maintenance of IR therapeutic levels requires redosing which significantly increases unnecessary animal handling, restraint, and noncompliance issues postoperatively. We evaluated the most effective dose of sustained-release (SR) formulation of buprenorphine in rabbits, as indicated by adequate drug plasma concentration and satisfactory analgesia, thus improving the standard of care. We hypothesized that single subcutaneous administration of 1 of 3 SR concentrations (SRLow-0.06 mg/kg, SRMedium-0.15 mg/kg, SRHigh-0.3 mg/kg; n = 3 males per group) would provide up to 72 hours of pain relief and more predictable behavior and pain associated parameters than IR (0.05 mg/kg BID) in a rabbit knee defect model. To evaluate pain and associated parameters, body weight, fecal output, grimace, and lameness scales were used to score pain based on daily assessment obtained before and within 72 hours after surgery. Results showed there was no skin reaction to any of the tested SR concentrations. Postoperative assessment also revealed no significant changes in body weight across groups. Daily fecal output was higher in rabbits treated with SRLow and SRMedium than IR and SRHigh. Single right limb lameness and signs of pain (grimace scale) were only observed in rabbits treated with IR (24 hours) and SRLOW (24-48 hours). Consequently, only rabbits treated with SRLow received meloxicam (0.3 mg/kg PO SID) within the first 48 hours. Pain was not observed in SRMedium and SRHigh group. As a conclusion, these preliminary results indicate that 0.15 mg/kg of SR buprenorphine (SRMedium) provided effective analgesia and safely manages pain in rabbits enrolled in the selected surgical model.

P203 Targeted Buprenorphine Sustained-Release Analgesia for Guinea Pigs

A Zanetti^*, S Putta, S Louie, D Casebolt

Department of Animal Resources, University of Southern California, Los Angeles, CA

Sustained-release (SR) formulations of buprenorphine were recently introduced into the veterinary market to minimize redosing, restraint, and noncompliance issues. Therefore, studies addressing the SR efficacy, safety, and pharmacokinetics (PK) in laboratory species are becoming common within the laboratory animal community. In guinea pigs, however, such studies remain lacking. In an attempt to fulfill the gap, we assessed the PK of buprenorphine SR (0.15-0.6 mg/kg SQ once) and instant release (IR-0.025-0.1 mg/kg SQ BID) in 8 (4 males and 4 females) healthy Hartley guinea pigs using a validated liquid chromatography with tandem mass spectrometry (LC-MS). At the same time, we also monitored animals for safety based on physiologic parameters (i.e. weight, heart rate, respiratory rate, and body temperature) and the presence of potential adverse effects (i.e. anorexia, injection site reaction) using noninvasive methods. According to the results, physiologic parameters were within normal range with less than 10% body weight loss observed in all guinea pigs during the first 2 days post buprenorphine administration. Pharmacokinetic analysis of IR and SR revealed the time to maximum concentration (T_max) to be around 1 hour. At this time point, the maximum concentration of the drug in the plasma of healthy guinea pigs was 2.41±0.56 ng/ml for IR and 1.7±0.13 ng/ml for SR. Twelve hours post administration, the plasma concentration of drug was 0.35±0.07 ng/ml (IR) and 1.06±0.57 ng/ml (SR). Buprenorphine SR remained within the detectable range for at least 72 hours. All together, SR (0.3 mg/kg SQ once) provided sustained levels of the drug in the plasma of healthy guinea pigs for up to 3 days with no major adverse effects to the animals. Further investigation is needed to determine if this SR concentration can be safely used to alleviate pain associated with survival procedures involving guinea pigs.

P204 Development and Validation of a Bleeding Time Assessment Model in the Nonhuman Primate

AM Lelkes^*1, J Sheehan¹, T Ramani¹, K Conde-Knape², T Ziegelhofer¹

¹Envigo, East Millstone, NJ; ²Johnson and Johnson Innovation, London, United Kingdom

Bleeding time assessments are used in drug development to determine the efficacy, safety, and dose response of an anticoagulant. Many animal models have been described in literature; however there are surprisingly few studies on suitable methodologies in nonhuman primates. Whereas using a template bleeding method in the skin may seem straightforward, it has been shown to be unreliable for drugs that do not interfere with platelet function such as Factor Xa inhibitors. This project aimed to develop and validate a reliable and reproducible bleeding time assessment model in nonhuman primates for the investigation of factor Xa inhibitors. Apixaban (0.1 and 1 mg/kg) was used as a positive control to investigate internal organ bleeding techniques. Organ bleeding requires a complete coagulation cascade response and agents that interfere with coagulation factors such as factor Xa inhibitors (Apixaban) can be better studied using an internal organ bleeding methodology. The liver was selected due to ease of access, consistency of bleeding, and ability to quantify total amount of blood loss. An incision of 2mm x 10mm was made in the right liver lobe using a scalpel blade. Using filter paper and gauze, timed blood collection from the incision site was performed until no further bleeding was observed. Weights of the filter paper and gauze were also taken to record volume. A positive control pilot study with 4 adult male cynomolgus macaques showed significant differentiation of bleeding time between saline and Apixaban. In the main study, 3/sex cynomolgus macaques per group (saline, 0.1 mg/kg Apixaban, 1 mg/kg Apixaban), were used to evaluate the feasibility of the selected liver bleeding method. The liver methodology developed resulted in fairly consistent bleeding times that allowed for discrimination between 2 different dose levels of Apixaban, a Factor Xa inhibitor. It allowed for bleeding to be recorded in a quantifiable way by recording both time and volume of blood loss. This experiment resulted in a robust model for evaluating bleeding time at different dose levels of a Factor Xa inhibitor in nonhuman primates.

P205 Optimizing Inhouse Sample Collection and Processing for Identification of the Mouse Pinworm Aspiculuris tetraptera

AE Goodroe^*2,1, VK Baxter¹, J Watson^2,1

¹Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Research Animal Resources, Johns Hopkins School of Medicine, Baltimore, MD

Aspiculuris tetraptera continues to be a problem in rodent vivariums partly due to difficulties in parasite detection. While PCR testing is highly sensitive, it is expensive and does not always provide immediate results, so many institutions rely on passive fecal flotation as a quick inhouse exam for diagnosing A. tetraptera infections. In order to increase the sensitivity of this test, we examined multiple parameters to determine the optimal test protocol. A 30-minute soaking period prior to a 15-minute fecal flotation time allowed fecal pellets to soften and facilitated efficient egg isolation. We also evaluated the impact of time of day, sample size, age, sex, and social housing on egg isolation. No evidence of cyclical egg shedding was found, and while larger fecal sample sizes did not result in more eggs isolated, incidence of false negative exams was reduced. The most eggs were isolated from 8- and 12-week-old mice, and as mice aged, numbers of eggs isolated declined. Overall, sex and social housing did not impact the number of eggs isolated. Finally, examination of multiple diagnostic tests (fecal flotation exam, direct examination of cecal and colonic contents, and fecal PCR) revealed no single test was definitive, indicating multiple tests may be required to successfully screen mice with low pinworm burdens. These findings provide guidance in sample selection, collection, and processing to efficiently detect A. tetraptera.

P206 Automated Blood Collection for Arterial Blood Gas Sampling in Unrestrained Monkeys

D Hopper⁴, BM Kemmerling^*1, P Kruzich⁴, L Kinter⁵, T Riggs³, K Bigge⁶, S Kurtz¹, C Kreilein³, N Suttles², M Shelton³

¹Veterinary, Bioanalytical Systems, Inc., Mt. Vernon, IN; ²Animal Care, Bioanalytical Systems, Inc., Mt. Vernon, IN; ³Clinical Pathology , Bioanalytical Systems, Inc., Mt. Vernon, IN; ⁴Toxicology , Bioanalytical Systems, Inc., Mt. Vernon, IN; ⁵GLP Scientific Consulting, Unionville, PA; ⁶Chemistry, Bioanalytical Systems, Inc., Mt. Vernon, IN

Measuring the impact of drug-induced changes in respiratory function and acid-base status requires serial monitoring of blood pH, pCO₂, PO₂, and HCO_3.. Blood sampling during standard cardiovascular radio-telemetry studies is less than optimal due to repeated disturbance of subjects. This study assessed the feasibility of serially collecting respiratory blood gases, pH, and HCO3 in cynomolgus monkeys with automated blood collection and to detect acute acidosis and alkalosis in conscious monkeys under cage conditions similar to radio-telemetry studies. Four tethered cynomolgus macaques (2/sex) were administered IV infusions of saline (0.75 mL/kg bolus plus 0.125 mL/kg/min for 20 min), NH₄CL (250 mg/kg over 20 min), HCO₃ (1680 mg/kg over 20 min), and remifentanil (12 µg/kg bolus plus 2 µg/kg /min for 20 min) in a modified Latin Square design wherein each animal received test article on different days. Serial carotid arterial blood samples (400 µL) were withdrawn by programmed automated blood collection (Culex-L ABC) at baseline, then from 5 minutes to 360 minutes post-infusion via a vascular access port. Samples were collected and analyzed for pCO₂ and pO₂, pH, and HCO₃ (RapidPoint 405 Analyzer)._. Baselines values for all parameters were within normal ranges. NH4Cl produced metabolic acidosis (pH -0.2 units, HCO₃ -50% by 5 min) followed by compensatory respiratory alkalosis. NaHCO₃ produced rapid metabolic alkalosis (pH +0.25 units, HCO3 +100% by 5 min) followed by compensatory respiratory acidosis. Remifentanil produced rapid respiratory acidosis (pH -0.21 units, HCO3 -15% by 5 min) followed by recovery with dissipation of respiratory depression. This study demonstrated the feasibility to collect serial respiratory blood gas, pH and HCO3 concentrations under experimental conditions compatible with cardiovascular radio-telemetry, and thereby to combine ICH S7A core battery respiratory and cardiovascular function assessments within a single study while supporting the 3R’s of animal research by decreasing animal use.

P207 Corynebacterium bovis Infection of Immunocompromised Mice Variably Impacts Tumor Xenografts

CM Santos^*1,2, A Chandler^1,2, A Valdivia^1,2, P Villanueva^1,2, S Camargo^1,2, T Williams^1,2, C Surratt^1,2, M Ross^1,2, S Montgomery^1,3, D Darr¹

¹Lineberger Comprehensive Cancer Center, University of North Carolina—Chapel Hill, Chapel Hill, NC; ²Division of Laboratory Animal Medicine, Chapel Hill, Chapel Hill, NC; ³Department of Pathology and Laboratory Medicine, Chapel Hill, Chapel Hill, Chapel Hill, NC

Corynebacterium bovis (C. bovis) is a common, gram-positive bacterium frequently encountered in research vivaria, particularly in those housing immunocompromised mice. Although nonpathogenic in immunocompetent animals, C. bovis clinically manifests as a hyperkeratotic dermatitis in naïve, immunocompromised mice. While it is often anecdotally reported that C. bovis infection decreases xenograft tumor take rates and subsequent tumor growth profiles in nude mice, published data on this as well as in other immunocompromised strains is lacking. To determine if C. bovis infection had an effect on tumor take and growth rates, we subcutaneously inoculated commonly used carcinoma cell lines (MDA-MB-231 [human breast adenocarcinoma] or SKOV3 [human ovarian clear cell adenocarcinoma]) into either nude mice (Crl:NU(NCr)-Foxn1^nu) or NSG (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ) mice with or without active C. bovis infection. C. bovis status was determined using a combined skin/buccal swab prior to cell inoculation and at the study endpoint. C. bovis-positive and -negative colonies were housed in separate rooms and C. bovis status was evaluated weekly by exhaust air swabs. Tumor presence and growth was monitored twice weekly for 60 days using caliper quantitation. For nude mice, both tumor take and growth rates for both tumor cell lines were significantly reduced in C. bovis-positive mice in comparison to C.bovis-negative mice. However, for NSG mice, which are more severely immunocompromised, tumor growth and take rates were not significantly altered by C. bovis status. Our study provides evidence that C. bovis infection can negatively impact carcinoma xenograft models, but that the impact varies across strains, likely due to the nature of immunocompromise. Our findings have important implications to the impact of C. bovis in tumor xenograft studies and management.

P208 Identification of Novel Murine Astrovirus in Taiwan

C Su^*1, C Wan^1,2

¹Graduate Institute of Molecular and Comparative Pathobiology, National Taiwan University, Taipei, Taiwan; ²Animal Resource Center, National Taiwan University, Taipei, Taiwan

Mouse astrovirus was first identified in mice in 2011. Lately, the surveillance data have indicated that murine astroviruses (MuAstV) are highly prevalent in laboratory mice in U.S. and Japan. However, the existence of murine astroviruses and prevalence of infection are still unclear in laboratory mouse colonies in Taiwan. In this study, identifying the MuAstV-like virus in Taiwan and surveying the infection status in laboratory mouse colonies were pursued. First, the fecal or tissue samples from different strains of mice of various breeders and research institutes (n = 9) in Taiwan were screened for MuAstV by RT-PCRs. Molecular surveillance results showed that 57.9% mice were positive for MuAstV, and 7 out of 9 facilities (77.8%) were contaminated. Comparison with the RdRp sequences of MuAstVs revealed that the isolates identified in our study (MuAstV-NTUs) were closely related to the previously-published MuAstVs with 92.4%∼97.4% similarity. One MuAstV-NTU isolate was molecularly characterized and designated MuAstV-NTU1. Comparison of the nucleotide and protein sequence alignments revealed that the MuAstV-NTU1 was different but closely related to the previously characterized murine astrovirus and suggested to be a variant of the same virus species. The present study has demonstrated a distinctly high infection rate of MuAstV in the laboratory mouse colonies in Taiwan, and MuAstV-NTU1 is currently the only variant characterized molecularly outside the USA.

P209 A Comparison of 2 Buprenorphine Quantitative Methods

CA McGee^*1, F Lih², PH Myers¹, DR Goulding¹, TL Blankenship¹

¹Veterinary Medicine Section, Comparative Medicine Branch, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC; ²Mass Spectrometry Research and Support Group, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC

The analgesic buprenorphine is commonly used to alleviate pain in laboratory rodent models undergoing surgical procedures. Many dosages, routes, and frequencies have been described. The therapeutic levels of buprenorphine in mice have been reported between 1ng/ml–8ng/ml depending on the level of pain associated with the procedure. Two primary methods have been identified in the laboratory animal science literature for measuring buprenorphine in serum. A commercially available forensic enzyme linked immunosorbent assay (ELISA) has been described to quantitate buprenorphine; however it is marketed as only a qualitative assay. Liquid chromatography-mass spectrometry (LC-MS) is the gold standard of buprenorphine quantification. We compared these 2 methodologies for accuracy and precision in detecting buprenorphine concentrations in the serum of mice receiving either standard buprenorphine HCl (Bup) or a commercially available sustained release buprenorphine (SR-Bup) product. A single dose of Bup or SR-Bup was administered by the subcutaneous route. Mice were euthanized at designated time points over 24 hours and blood was collected. Each sample collected was used for both the ELISA and LC-MS assay. We also measured buprenorphine metabolites in each sample by LC-MS. The ELISA data had greater variability than the LC-MS data. By comparing the LC-MS buprenorphine both with and without the combined metabolites to the ELISA data we determined the ELISA detects the glucuronides which can falsely elevate the level of buprenorphine. While the glucuronides have been described as biologically active the antinociceptive effect has been reported to be significantly less compared to buprenorphine when evaluated by tail flick latency or tail withdrawal tests. The ELISA may, therefore, give a false representation of analgesic level.

P210 Challenges in Establishing Enhanced Gut Microbial Diversity in Laboratory Mice

DR Montonye^*1,2, C Smith², A Ericsson^3,4, CL Franklin^3,4

¹Comparative Medicine, University of Missouri, Columbia, MO; ²Veterinary Pathobiology, University of Missouri, Columbia, MO; ³Mutant Mouse Resource and Research Center, University of Missouri, Columbia, MO; ⁴MU Metagenomics Center, University of Missouri, Columbia, MO

Mice are widely used in biomedical research, and optimizing translatability of mouse models to recapitulate human disease processes is highly desired. It has been speculated that the diversity of the gut microbiota (GM) of laboratory mice has decreased over the past several decades through practices such as rederivation that were designed to render research mice free of opportunistic and overt microbial pathogens. Unfortunately, this lowered GM diversity may not accurately replicate the GM diversity seen in the human population. As differences in microbial diversity have been shown to influence disease phenotypes of rodent models, it is conceivable that producing mice with a microbial diversity that resembles that in humans could result in more translatable model phenotypes. We sought to establish laboratory mice with a more diverse GM than is currently available. To this end, 40 mice from 2 local pet stores were obtained and the GM characterized using next generation sequencing. This analysis showed that these mice had significantly higher GM diversity than contemporary laboratory mice with an average 74.8% increase in the number of operational taxonomic units (OTUs). As expected, these mice were also contaminated with a variety of pathogens including, but not limited to, Mycoplasma pulmonis, mouse hepatitis virus, mouse parvovirus, mouse rotavirus, fur mites, pinworms, and Cryptosporidium sp. To rid mice of these pathogens, the following strategies were employed: treatment with fenbendazole (in feed for 5 months), moxidectin (twice topically 8 weeks apart), azithromycin (50 µl orally once daily for 4 weeks), cessation of breeding, and test and cull. Following this regimen, 5 mice were rendered free of adventitious pathogens. Unfortunately, analysis revealed that the number of OTUs decreased by 65.5% and the GM closely resembled that of contemporary laboratory animal mice. These findings demonstrate how quickly and dramatically a diverse GM can be lost using common procedures designed to eliminate pathogens. Moreover, these highlight challenges faced in creating a more translatable GM in laboratory mice.

P211 Liquid Microsampling in Rats and Mice

DN Bilgo^*1, PM Mielke¹, A Ledvina², S Ploch³, C Scheuerell⁴, L Geiger⁵

¹Animal Operations, Covance, Madison, WI; ²Bioanalytical, Covance, Madison, WI; ³Toxicology Operations, Covance, Madison, WI; ⁴Associate Pharmacokineticist, Covance, Madison, WI; ⁵Metabolism, Covance, Madison, WI

Popularity of the dried blood spot method has declined somewhat due to various regulatory and technical challenges. Consequently, the use of small liquid samples (microsampling) has been gaining popularity regarding serial sampling in mice and for nonclinical toxicokinetic (TK) analysis. The benefits of microsample collections include the reduction of total number of satellite (toxicokinetic) rats and mice (including transgenic strains), ethical use regarding the 3Rs, and other potential cost savings. Challenges and inconsistencies have been previously noted while processing serum and plasma samples using glass capillary-based techniques. Therefore, in this study, a novel microsample technique was used to obtain and process samples. Female rats [Crl:CD(SD)] and mice [2 strains were used, Crl:CD1(ICR) and RasH2 (Hemizygous)] were administered a single gavage dose (150 mg/kg for rats and 100 mg/kg for mice) of acetaminophen. Rats were bled at approximately 0.5, 2, 4, 8, and 24 hour post dose from a tail vein for the microsample (50 µL). Additionally, for comparison, blood was also collected via jugular vein for standard sample volume collections (0.5 mL). Mice were serially bled at approximately 0.5, 4, and 24 hours post dose from a saphenous vein (50 µL). Blood microsamples (for serum and plasma) were collected from rats and mice using a 50uL plastic blood tube via capillary action. Blood was then dispensed using the internal piston of this plastic blood tube into a trumpet shaped tube with a push cap. The trumpet shaped tube was placed in a small carrier tube for ease of centrifugation. The serum and plasma (target volume of 10uL) was harvested using a pipette. Samples were processed and evaluated for acetaminophen. Results indicate exposure to acetaminophen in female rats was similar between serum and plasma matrices and between jugular vein and tail vein collection methods. This indicates the collection site does not impact the results. Exposure to acetaminophen in female mice was similar between serum and plasma matrices and between Crl:CD1(ICR) and RasH2 (Hemizygous) strains. In conclusion, these results indicate this microsample blood collection method allows for sufficient volumes of serum and plasma to evaluate toxicokinetic exposure.

P212 Establishment of Hematological Reference Intervals and Hemocytological Characteristics in a Colony of Wild-Caught Southern Giant Pouched Rats (Cricetomys ansorgei)

DA Jeffery^*1, L Brandt², P Baneux¹, BS Blank¹, E Silvela¹, BS Singh¹

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²College of Veterinary Medicine, Cornell University, Ithaca, NY

Due to their highly sensitive sense of smell and trainability, giant pouched rats (genus Cricetomys) are garnering increasing interest from the scientific community for applications such as landmine and tuberculosis detection. Since 1997, organizations such as APOPO in Morogoro, Tanzania have operated training and captive breeding programs to use the giant pouched rats’ talents. However, despite their increasing use in multiple African countries and their natural ubiquity in this region, there remains a paucity of publications and normative data regarding these animals. With the development of research colonies likely to increase, so too will the need for baseline normal values from healthy individuals and populations against which clinical cases may be compared. The present study sought to establish reference intervals (RI) for complete blood counts (CBC) and serum biochemistry and to characterize hemocytological characteristics in the southern giant pouched rat (Cricetomys ansorgei). Seventy-three clinically healthy animals (35 male, 38 female, 3 juveniles) were wild-caught in Tanzania, and transferred to standard laboratory rodent housing conditions at our institution. General physical exams, body condition scoring, and phlebotomy via ventral tail artery were performed under isoflurane anesthesia. Two milliliters of blood were collected via 23 g needle into microtainers containing EDTA or lithium heparin, stored on cold packs, and processed by the Animal Health Diagnostic Center Clinical Pathology Laboratory within four hours of collection. Automated CBCs were compared to manual differentials (erythrocytes, leukocytes, platelets) performed by a DACVP-boarded pathologist, with absolute leukocyte counts calculated from the manual differentials. Additionally, cytochemical staining was performed to identify the specific staining characteristics of various leukocyte types. Leukocyte staining characteristics were relatively uniform among male and female individuals. The reference interval values for CBCs and serum biochemistry in the Southern giant pouched rat will be presented.

P213 Establishment of a Home-Cage, In Vivo Method of Rabbit Urine Collection

DL McEntee^*, K Borg, V Markiewicz

Drug Safety Research and Development, Pfizer, Inc., Groton, CT

In preparation for a rabbit study in which stress biomarkers were to be measured, a method to collect urine was developed through a collaborative and innovative effort to ensure an appropriate urine sample could be obtained from multiple rabbits at a time in their home cages. Study design required serial urine sample collections at 8-hour or 16-hour intervals over a 48-hour period. Samples were required to be maintained chilled and protected from light during the collection intervals, while minimizing contamination by food, feces, and drinking water. Several variations were tested and modified, resulting in a reliable and repeatable method of collection. This method primarily consisted of installing a grommet in the home-cage tray, pitching the tray towards the grommet, and running a plastic tube from the grommet to a collection flask enclosed in a cooler. As a result, samples were collected from every animal at sufficient volume, quality, and storage conditions (chilled and protected from light). Causing minimal stress to naturally sensitive rabbits while effectively collecting quality urine samples allows for greater opportunities and for research to be conducted while at the same time ensuring animal welfare.

P214 Administration of an Angiotensin II Receptor Inhibitor, Collagen Expression, and Breast Cancer Progression in an Orthotopic Murine Model

D Dréau^*

Biologic Sciences, University of North Carolina, Charlotte, NC

There are nearly 140,000 cases of breast cancer diagnosed each year with 40,000 deaths caused by the disease. Although treatment regimens have greatly improved the patients’ outcome, no treatment is currently available for triple-negative breast cancer tumors. The tumor microenvironment, in particular through the promotion of a sustained local inflammation, angiogenesis, and extracellular matrix remodeling, has been shown to play a critical role in breast tumor progression toward metastasis. Here we investigated the potential of the angiotensin II receptor inhibitor losartan in preventing breast cancer progression in an immunocompetent orthotopic murine model of breast cancer. Following the orthotopic implantation of the triple-negative mammary tumor 4T1-RFP cells in the mammary fat pads of Balb/c female mice, the tumor growth was monitored overtime using caliper and fluorescent imaging. Mice were dosed orally with either losartan (12-15 mg/kg/day) or vehicle. After 30 days, primary tumors were analyzed for alterations in the matrix and especially the presence of collagen. Differences between groups were tested by repeated measure ANOVA and post-hoc tests with p < 0.05 a priori. Additionally, changes in extracellular matrix composition, especially collagen content were determined. The daily treatment with oral low-dose losartan was not associated with any loss of weight or noticeable behaviors changes. However, primary tumor mass were significantly smaller as determined by caliper measurements and by fluorescence evaluation (P < 0.05). Of note, the fluorescence decreased as the tumor size increased especially in the treated group. Furthermore, losartan was associated with a significant decrease in collagen concentration within the extracellular matrix of the primary tumor (P < 0.05). In summary, low-dose losartan per oral prevented collagen deposition within 4T1 tumor mass and limited the growth of the triple negative 4T1 mammary cells in an orthotopic murine mammary tumor model.

P215 An Analysis of Growth Factor Concentrations in Rabbit Platelet Rich Plasma Intended for Accelerated Ligament Healing

E Milchling^*1,2, N Phillips³, L Goodrich³

¹Atlantic Veterinary College, Charlottetown, Canada; ²Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ³Orthopaedic Research Center, Colorado State University, Fort Collins, CO

Platelet rich plasma (PRP) is an excellent source of growth factors that have therapeutic indications for many soft tissues, including ligament healing. The concentration and type of growth factors in PRP, however, are variable between samples and species. By associating the quantity of growth factors to various PRP samples, we should provide key information for selecting the preferred PRP preparation to promote ligament healing. Since platelets release growth factors via their granules, we hypothesize that PRP samples containing greater concentrations of platelets will have higher concentrations of growth factors. To test our hypothesis, growth factors were quantified by ELISA analysis in 4 New Zealand White rabbit samples (blood, platelet poor plasma (PPP), PRP containing 2 times the concentration of baseline platelets (2XPRP), and PRP containing 4 times the concentration of baseline platelets (4XPRP). This study quantified the levels of 4 prominent growth factors including fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), transforming growth factor 2β (TGF-2β), and platelet derived growth factor (PDGF). Growth factors FGF2, VEGF, and TGF-2β increased as the concentration of platelets increased between PPP, 2XPRP, and 4XPRP groups. There was no change in PDGF between the groups. The clinical significance of this remains unknown. Subsequent studies will explore the possible correlation between rabbit ligament healing and the growth factor quantities injected into the ligament. Histology will be performed to asses the degree of ligament healing. This work aims to improve the reliability of PRP preparations so more consistencies can be seen in accelerated ligament healing across all species.

P216 Identifying Antibodies that Cross-React with White-Tailed Deer (Odocoileus virginianus) Blood Bells for Use In Flow Cytometry

ES Lee^*, C Mathiason

Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO

Chronic wasting disease (CWD) is an endemic and highly transmissible disease of deer, moose, and elk of North America. It is caused by propagation of specific misfolded proteins called prions, which cause fatal neurodegeneration. CWD may be transmitted in various blood cells, saliva, urine, and feces, contributing to its increasing spread across North America. B cells and platelets have been found to harbor prions in CWD infection; in Scrapie, a prion disease of sheep, monocytes and T-lymphocytes are associated with infective prions. Prions are typically classified as self-proteins, and are therefore not considered to stimulate an immune response. However, data collected by our group indicate that immediately postinfection, there is a sharp spike in prion concentration, followed by a slow increase throughout disease progression. Thus we hypothesize that certain types of blood cells attempt to combat CWD infection, and that this enables prion-infected cells to be partially cleared/sequestered by the immune system acutely. Flow cytometry will be used to tease apart the role the different blood cells play in CWD infection. Flow cytometry enables the use of fluorophore-stained cell surface antibodies to identify blood cell types, and detect the presence of prion surface antigen. There is no white-tailed deer hybridoma; therefore, ovine or bovine antibodies that consistently cross-react with cervid cells were identified. Blood was collected from four infected white-tail deer (Odocoileus virginianus) in our research herd. The blood was processed to isolate platelets and peripheral blood mononuclear cells. These cells were stained with various anti-ovine and anti-bovine antibodies (CD4, CD8, CD14, and CD41) that were conjugated to a fluorophore. Cell populations were then analyzed with flow cytometry. We concluded that the selected anti-ovine antibodies cross-reacted reliably with the cervid blood cells. These antibodies, and others yet to be identified, will be used in future flow cytometry studies to determine how various blood cells impact the pathogenesis of CWD in white-tailed deer.

P217 Presence of G-Protein Coupled Receptor Gprc6a in the Human, Mouse, and Sheep Placenta

ES McWhorter^*, R West, Q Winger, G Bouma

Biomedical Sciences/Laboratory Animal Resources, Colorado State University, Fort Collins , CO

Impaired placental development and function can lead to intrauterine growth restriction (IUGR), and negatively impact fetal development and can lead to preterm birth. The placenta is critical in embryo and fetal development and survival and is recognized as a major endocrine organ during pregnancy. Metabolic or environmentally induced hormone changes also affect ruminant pregnancies, and can lead to pregnancy failure, placental dysfunction, and embryo mortality. A number of pregnancy disorders associated with placental abnormalities and dysfunction are associated with increased circulating serum androgens and increased androgen receptor (AR) levels in affected placental tissue The classic genomic effects of androgens involve translocation of the androgen-AR complex to the nucleus, leading to regulation of gene transcription. However, androgens also have rapid nongenomic effects. Recently, GPRC6A, a G protein-coupled receptor, was found to bind testosterone and dihydrotestosterone. GPRC6A is known to bind Osteocalcin (Ocn), an osteoblast-specific hormone, and plays a role in bone calcification and calcium ion homeostasis. Ocn also binds GPRC6A in the pancreas, leading to beta cell proliferation and insulin release, as well as stimulating testosterone production by binding Leydig cells in the testes. Furthermore, Ocn levels increase during pregnancy, crossing the placenta to prevent neuronal apoptosis in the growing fetus. We hypothesize that GPRC6A plays a role in mediating the non-genomic effects of androgens in the placenta. To test this hypothesis, we first describe the presence of GPRC6A as well as AR in placental cells. Studies conducted in our laboratory show the presence of both GPRC6A and AR in the human, mouse, and sheep. Using the telomerase-immortalized (h-TERT) first trimester human trophoblast cell line Swan-71, mouse d12.5 placental tissue, as well as ovine d55 placental tissue, we identified the presence of both GPRC6A and AR protein by Western blot. These findings suggest that androgen signaling may occur through both genomic and nongenomic pathways in the placenta. Ongoing experiments include treatment ovine embryos with lentivirus that will allow us to knock-out GPRC6A in the placenta to determine a possible phenotype.

P218 Filobacterium rodentium, a New Name for CAR Bacillus Rodent Isolates

F Ike^*1, A Kajita¹, A Yoshiki¹, M Sakamoto¹, T Kokubo²

¹RIKEN, Tsukuba, Japan; ²National Institute of Radiologic Sciences, Chiba, Japan

The gram-stain negative filamentous bacterium isolated from rodent pneumonia, formerly known as the cilia-associated respiratory bacillus (CAR bacillus), induced chronic respiratory disease in mice and rats. CAR bacillus was initially named in 1985 according to the finding of its infection sites and morphology, and recently given a scientific name, Filobacterium rodentium gen. nov., sp. nov. in Filobacteriaceae fam. nov., on the basis of biochemical, phenotypic, and 16S rRNA analysis. Here we look at the fundamental nature required to give a name to CAR bacillus. Strain SMR-C^T of CAR bacillus was originally isolated from the rat and cultured in Vero E6 cells culture supernatants. All cultures were performed at 37°C in 5% CO₂ 95% air humidified chamber. Morphologic changes were monitored under phase contrast microscopy. Biochemical reactions were determined with the Rapid ID 32A anaerobic identification kit. Fatty acid methyl esters were extracted and processed to specify fatty acid profiles. Previously obtained full genome data of SMR-C^T were used to deduce 16S rRNA sequence and DNA G+C content. Phylogenetic analyses were carried out by using the neighbor-joining method and the maximum likelihood method with Bootstrap resampling. SMR-C^T showed microaerobic, nonspore-forming, motile (gliding) without flagella, gram-stain-negative, argentophilic, filamentous rods. The doubling time was 20–24 hours. When cultured in ultralow-attachment flasks, cells were 0.8–0.9 x 8.3–10.0 µm in size and presented singly in the planktonic state. Cells in conditioned medium on glassware grew in sessile state and made a netlike structure on glass surface. The dominant cellular fatty acids were iso-C15:0 and anteiso-C15:0. The DNA G+C content was 47.7 mol%. SMR-C^T and closely related strains of CAR bacillus rodent isolates formed a novel family-level clade in the phylum Bacteroidetes with high bootstrap support (98–100 %). From these results, a new scientific name,Filobacterium rodentium, was proposed for strain SMR-C^T of CAR bacillus. The type strain is SMR-C^T (=JCM 19453^T, =DSM 100392^T).

P219 The Effect of Blood Glucose Telemeter Implantation on Oral Glucose Tolerance Testing and Glucose Monitoring in Diet-Induced Obese Mouse

GJ DeMarco^*1, X Chen¹, C Salatto¹, S Tiesma²

¹Pfizer Inc., Cambridge, MA; ²Data Science International, St. Paul, MN

Oral glucose tolerance testing (OGTT) and serial blood glucose (SBG) monitoring are important tools in basic diabetes research and the preclinical development of new treatments for diabetes. Although mouse models are critical to diabetes research, their small blood volume and marked stress response to restraint presents significant challenges to generating reliable and consistent OGTT and SBG data. Since telemetric technology can often mitigate the challenges of physiologic data collection in mouse, the purpose of this study was to evaluate the effects of an implantable telemetric glucose sensor designed for mouse on OGTT and SBG in the diet-induced obese mouse model (DIO) of diabetes. Male C57BL/6J mice fed a high-fat diet to induce and maintain diabetes (DIO), divided into 3 groups were used. Groups consisted of animals implanted with a glucose sensing telemeter (HD-XG, DSI™) placed in the carotid artery, sham surgery, and nonsurgical controls. Excepting weight loss, no significant postoperative complications were noted in telemeterized and sham surgery animals. Telemeterized animals lost 15.6 percent and sham surgery animals 5.2 percent of pre-surgical body weight postoperatively which returned to baseline values at 42 and 18 days respectively. Results from OGTT tests, including the effects of a sodium-glucose co-transporter 2 (SGLT-2) inhibitor, were similar between all groups and data captured by telemetry was not different from that obtained with a glucometer. Data from 11 out of 12 telemeterized animals was well correlated (R² > 0.75), and 1 animal poorly correlated (R²= 0.34) with glucometer results. Bland-Altman plots suggested individual variation in agreement between glucometer and telemeter blood glucose values. Continuous blood glucose measurements from the telemeters were possible for 70 days postdevice implantation. These data suggest the HD-XG telemeter can be used in mouse and that implantation of the device does not significantly impact the DIO phenotype. Data from the telemeter correlates well with glucometer results although significant individual variation can occur.

P220 Physiologic, Behavioral, and Histologic Responses to Different Euthanasia Methods in C57BL/6NTac Male Mice

GP Boivin^*1, M Bottomley², PA Schiml³, L Goss⁴, N Grobe⁵

¹Laboratory Animal Resources, Wright State University, Dayton, OH; ²Mathematical and Microbiological Sciences, Wright State University, Dayton, OH; ³Psychology, Wright State University, Dayton, OH; ⁴Neuroscience, Wright State University, Dayton, OH; ⁵Pharmacology and Toxicology, Wright State University, Dayton, OH

Rodent euthanasia using exposure to increasing concentrations of CO₂ has come under scrutiny due to concerns of potential pain during the euthanasia process. Alternatives to CO₂ such as isoflurane and barbiturates have been proposed as more humane methods of euthanasia. We examined 3 commonly used euthanasia methods in mice: a commercially available, nonsterile solution containing pentobarbital sodium and phenytoin sodium administered by intraperitoneal injection (150mg/kg), CO₂ inhalation (15%, 30%, 50%, and 100% chamber replacement rate), and isoflurane anesthesia (5% with oxygen flow rate at 1L/min) followed by CO₂ inhalation (50% chamber replacement rate). Mice were implanted with telemetry units to measure heart rate, blood pressure, and movement. Mice were videorecorded during all euthanasia procedures. Videos were analyzed for activity (hopping, walking/running, sedentary, standing/rearing), breathing pattern (normal, cessation of breathing), ataxia, face wiping, grooming, recumbency/cessation of walking, and loss of muscle tone/nose resting on the bedding. Plasma was collected for ACTH analysis and lungs for histologic analysis. The nonsterile solution containing pentobarbital sodium and phenytoin sodium led to significantly lower elevations in ACTH (P = 0.0006) than isoflurane and CO₂ euthanasia. There were no significant consistent differences between euthanasia techniques in the telemetry recorded cardiovascular or activity changes. Behavioral differences were limited to time until ataxia and recumbency and elevated frequency of face wipes in the mice euthanized with the solution containing pentobarbital sodium and phenytoin sodium (P < 0.001). Euthanasia with 50% or 100% CO₂ chamber replacement rates was the fastest method of the 3 techniques. Histologic damage characterized by mild to moderate perivascular and peribronchiolar edema was significantly more severe in CO₂ euthanized mice. We conclude that use of CO₂ with or without isoflurane is an acceptable euthanasia method. Isoflurane or euthanasia by the solution containing pentobarbital sodium and phenytoin sodium are better suited when studies rely on analysis of lung histology.

P221 Characterization of Transdermal Pharmacokinetics of Ketoprofen in Sinclair and Gottingen Minipigs

A Stricker-Krongrad, GF Bouchard^*, D Brocksmith, M Zhong, J Liu

Sinclair BioResources, LLC, Auxvasse, MO

Our objective was to characterize the transdermal pharmacokinetics of a ketoprofen cream formulation in the Sinclair and Göttingen breed of minipigs and compared it to published human data. Minipigs have a fixed skin tightly attached to the subcutaneous tissues similar to that in humans, which make them a preferable model for dermal studies. Three minipigs per gender per strain (9.1-9.9 months old) were used in the study. Four mL of 2.5% ketoprofen cream, (100 mg Ketoprofen, a human therapeutic dose), were applied on an area on the back of each minipig that was approximately 3% of the total body surface area. No adverse reactions were observed in the animals. Blood samples were collected at 4, 8, 12, 24, 28, 32, and 36 hours post dose. The plasma samples were analyzed with API 4000 LC/MS/MS system with d³-labeled ketoprofen as a labelled internal standard. The linear range of quantitation for ketoprofen was 50 to 25600 ng/mL. The PK parameters were calculated with PKSolver. Individual animal data were analyzed with the non-compartmental analysis. Transdermal pharmacokinetic parameters of ketoprofen were (N = 6; mean +/- SD; Sinclair versus Gottingen): t1/2 (h): 12.2 +/- 2.0 versus 11.7 +/- 3.9; Tmax (h): 8.0 +/- 2.5 versus 9.3 +/- 2.1 and Cmax (ng/m): 94.3 +/- 65.6 versus 173.1 +/- 71.8. Although ketoprofen was shown to be absorbed better in Göttingen than in Sinclair minipigs in the average plasma concentration time curves, no statistically significant difference was observed between all PK parameters. These data suggested that ketoprofen has a similar ADME process in both minipig lineages.

P222 Troubleshooting Cre Activity in Off-Target Tissues: Lessons Learned from a Villin- cre Mouse Model

HR Holcombe^*1, D Puglisi¹, G Gong¹, E Bryant¹, B Horwitz², JG Fox¹

¹Massachusetts Institute of Technology, Cambridge, MA; ²Brigham and Women’s Hospital, Boston, MA

The Cre-loxP recombination system is a common method for generating conditional knock out mice, with gene deletion being dependent on the tissue specificity of the promoter driving Cre expression. However, many Cre driver lines show aberrant excision activity that leads to deletion of target genes in unexpected tissues, resulting in mosaic and even germline deletion. Here we describe inconsistent excisional activity in Villin-cre transgenic mice driving conditional deletion of exon 2 of the IL22 receptor (IL22R) alpha chain in gut epithelia (Vil-Cre;Il-22r1^flox/flox). A brother-sister breeding pair consisting of a Cre+ female and Cre- male were mated, and their offspring genotyped for Cre and floxed IL22R. Genotyping of the first 2 litters confirmed that all pups were positive for floxed IL22R and negative for the WT allele; however, the third and fourth litters had 1 of 2 pups and 3 of 9 pups, respectively, that were negative for the floxed IL22R allele. Our hypotheses were that both parents harbored one il22r allele that had undergone germline recombination (il22r^fl/D) and that the pups that were negative for the floxed IL22R had undergone germline recombination of both alleles (il22r^D/D). These hypotheses were supported by regenotyping the mice using primers that differentiated between floxed and recombined IL22R. Sequencing the PCR product from il22r^D/D mice confirmed that exon 2 of the IL22R was deleted. Subsequent breeding pairs were genotyped and confirmed to be il22r1^flox/flox. Genotyping litters born to the new breeders demonstrated that spontaneous generation of il22r^flox/D pups occurs in some, but not all, litters, with a frequency of 14-30% within the litter. The colony is currently being expanded, and additional genotyping and immunofluorescence staining for IL22R will be performed to further characterize Cre recombinase activity in this line. Effect of ectopic deletion on study design and expected outcomes will be highlighted.

P223 New TK-NOG Mice as an Optimized Platform for Liver Humanization

H Suemizu^*1, K Kawai², Y Ando¹, Y Higuchi¹, H Hashimoto¹, H Nabekawa³, M Nishiwaki^1,4, M Okazaki^1,4, M Yamamoto⁵, M Goto⁶, R Takahashi⁶

¹Laboratory Animal Research Department, Central Institute for Experimental Animals, Kawasaki, Japan; ²Pathology Analysis Center, Central Institute for Experimental Animals, Kawasaki, Japan; ³In-Vivo Science Inc., Kawasaki, Japan; ⁴CLEA Japan, Inc., Fujinomiya, Japan; ⁵ICLAS Monitoring Center, Central Institute for Experimental Animals, Kawasaki, Japan; ⁶Animal Resources and Technical Research Center, Central Institute for Experimental Animals, Kawasaki, Japan

To address issues related to interspecific differences between animal models, we developed an animal model with a reconstituted liver containing human hepatocytes (humanized-liver TK model). However, the disadvantages associated with this model were male sterility and low transgene expression in females. To overcome these disadvantages, we developed a new humanized-liver model based on immunodeficient NOD/Shi-scid/IL-2Rγ^null (NOG) mice that specifically expresses herpes simplex virus type 1 thymidine kinase gene mutant clone30 in the liver using a transthyretin gene promoter (Ttr-HSVtk mutant30 transgenic NOG; NOG-TKmut30). We established 3 transgenic lines (#4, 5, and 10). Male NOG-TKmut30 transgenic mice in each line were fertile and reproduced normally, transmitting the transgene in expected Mendelian ratios. Furthermore, male and female NOG-TKmut30 mice aged 5 weeks showed severe liver injury by ganciclovir (GCV) injection. One million viable human hepatocytes were transplanted intrasplenically into liver-injured NOG-TKmut30 mice, and serum levels of human albumin (hAlb) were monitored every 4 weeks. Engraftment of human hepatocytes in all transgenic lines was confirmed by detection of hAlb in serum. The recipients produced more than 10 mg/mL hAlb eight weeks after transplantation. In these mice, almost 95% of the hepatocytes were positive for anti-HLA staining, suggesting that the recipient liver was replaced by the transplanted human hepatocytes. The NOG-TKmut30 model has several advantages over the traditional TK model. Firstly, overcoming male sterility will facilitate large-scale production of recipient transgenic mice. Secondly, the absence of differences in GCV-induced liver injury between sexes will enable us to produce twice as many humanized-liver mice. We anticipate that the NOG-TKmut30 mice, in which production efficiency of humanized-liver mouse is significantly improved, will become an optimized platform for generating a humanized-liver model for studying drug metabolism, toxicology, and liver regeneration.

P224 The Plant Flavonol Rutin Suppresses Human-Amylin Misfolding in Vitro and Increases the Lifespan of Diabetic Human-Amylin Transgenic Mice

J Aitken^*1, K Loomes^1,2, i Riba-Garcia³, R Urwin³, G Prijic¹, A Phillips⁴, A Phillips^1,2, P Barran⁴, A Dowsey^3,5, G Cooper^1,3

¹School of Biologic Sciences, University of Auckland, Auckland, New Zealand; ²The Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand; ³Centre for Advanced Discovery and Experimental Therapeutics, University of Manchester, Manchester, United Kingdom; ⁴Michael Barber Centre for Collaborative Mass Spectrometry, University of Manchester, Manchester, United Kingdom; ⁵Department of Electrical Engineering and Electronics, University of Liverpool, Liverpool, United Kingdom

Diabetes exerts an enormous toll on patients, their families and caregivers, and health-care delivery systems globally. Type 2 diabetes (T2D) is the most prevalent form of the disease, accounting for 90% or more of all diabetes globally. Currently, available therapeutic approaches have not stemmed the alarming increase in numbers affected and there is a need for more effective therapeutics that can suppress the causative disease mechanisms. Human amylin (hA) is the main component of islet amyloid in diabetic patients. It is a small protein that can spontaneously misfold and aggregate to form structures that cause β-cell death and onset of diabetes. Amylin-mediated cytotoxicity has been identified as a potential target for developing new anti-diabetic molecules that might act by suppressing β-cell death. The aim of this study was to identify natural compounds that suppress human amylin misfolding and to test their efficacy in vivo in a human amylin transgenic mouse line, which overexpresses human amylin in the pancreatic β-cells. These transgenic mice spontaneously develop diabetes and mirror many aspects of the human disease. We identified rutin, a small, orally active compound present in the human diet which is also used as a dietary supplement, as an inhibitor of hA misfolding. In vitro, rutin bound to hA, suppressed its misfolding, and reverted misfolded hA back towards the physiologic form. Rutin was administered from weaning in the drinking water to hA-transgenic mice (n = 11) versus water-treated hA-transgenic mice (n = 13). In rutin-treated hA-transgenic mice, measurements of glucose, fluid-intake, and body-weight showed that oral rutin-treatment slowed diabetes progression by lowering the rates of elevation in blood glucose (P = 0.030), slowing deterioration of diabetes symptoms to death (P = 0.014) and stabilizing body-weight (P < 0.0001). Glucose tolerance, as measured by ipGTT at 60 days post weaning, was improved in rutin-treated compared with water-treated hA-transgenic mice (P = 0.042). In conclusion, the dietary flavonol rutin, which suppresses hA-aggregation in vitro, doubled the lifespan of diabetic mice (P = 0.011) by a median of 69 days compared with water-treated diabetic mice.

P225 Overexpression of Human Amylin in Transgenic Mice Raises Core Body Temperature and Increases Energy Expenditure which Can Be Reversed by Leptin Replacement

J Aitken^*1, C Walker¹, G Prijic¹, S Johnson¹, G Cooper^1,2

¹School of Biologic Sciences, University of Auckland, Auckland, New Zealand; ²The Centre for Advanced Discovery and Experimental Therapeutics, University of Manchester, Manchester, United Kingdom

Diabetes is now pandemic. Globally, more than 380 million people have diabetes, of whom 90% have type 2. If no action is taken to stop the ongoing increase in prevalence then, in 20 years, there will be 592 million people living with diabetes (1 in 10 of all adults). Effective interventions are required that can prevent the progression of the disease. In order to do this, it is first necessary to understand the molecular basis of diabetes. Human amylin (hA) is the main component of islet amyloid in diabetic patients. It is a small protein that can spontaneously misfold and aggregate to form structures that cause β-cell death and onset of diabetes. Amylin also functions as a satiation signal and has long-term anorectic and weight reducing actions. We have developed hA-transgenic mice which selectively express human amylin in their β-cells. These mice undergo progressive islet damage and develop spontaneous diabetes. The aim of this study was to investigate the effect of over-expression of human amylin in hA-transgenic mice on physiologic parameters such as weight, blood glucose, core body temperature, energy expenditure, and endocrine hormones. Blood glucose, weight, and core body temperature were measured weekly and indirect calorimetry was performed in approxately 100-day-old hA-transgenic and control mice. Food intake, activity, O2 consumption, CO2 production were measured and respiratory quotient and energy expenditure were calculated. hA-transgenic mice (n = 24) showed elevated blood glucose from weaning (P < 0.001) leading to diabetes at 100-150 days of age. They weighed significantly less (P < 0.001), and had significantly higher core body temperatures (P < 0.001) and energy expenditure (P < 0.05) compared to their control littermates (n = 15). Amylin and insulin serum levels were significantly higher (P < 0.01; P < 0.05) while leptin and adiponectin levels were significantly lower (P < 0.05; P < 0.01) in the hA-transgenic mice compared to their controls. Leptin plays a central role in appetite regulation and energy expenditure. Acute leptin replacement in hA-transgenic mice reduced energy expenditure to that of their controls. In conclusion, human amylin regulates body weight in hA-transgenic mice by increasing core body temperature and elevating energy expenditure.

P226 Baseline Cardiac Troponin I in Gottingen Minipigs

JA Scotto^*1, S Hulet², N Vincelli², D Miller²

¹Veterinary Services, Edgewood Chemical and Biologic Center, APG, MD; ²Operational Toxicology, US Army Edgewood Chemical and Biologic Center (ECBC), APG, MD

The use of cardiac troponin I (CTnI) as an emergency medicine biomarker indicative of acute myocardial infarction is generally accepted, with the threshold typically set at the 99^thpercentile value for the population. Over several studies we were able to collect blood samples from a large number of young adult male Gottingen minipigs to assess baseline CTnI levels. We noticed a difference in results of cardiac troponin I between K2EDTA and Lithium heparin tubes. We collected samples in both tubes to compare. All of the animals used in these studies had a cannula surgically implanted into the right external jugular vein which was used for whole blood collection. A subset of these animals had DSI telemetry transmitters surgically implanted. All baseline blood samples were collected after the animals recovered from surgery. For the first analysis, an aliquot of whole blood from each pig was split equally into lithium heparin (LiHep) tubes and K2 EDTA and analyzed for CTnI using i-Stat cartridges. A significant difference (P = 0.032) was identified in the CTnI levels in samples taken from the same animals (n = 48) when stored in LiHep tubes vs. K2 EDTA tubes, with the portion stored in LiHep tube reading significantly higher than the portion stored in the K2 EDTA tube. Blood samples from an additional 61 minipigs were collected solely into LiHep (total n = 109) tubes and blood samples from another 20 minipigs were collected solely into K2 EDTA (total n = 68) tubes and assessed for CTnI levels in the same manner. A significant difference (P = 0.019) was again identified when comparing the 2 larger datasets. No significant difference was identified in baseline CTnI levels (all in LiHep tubes) in animals that had surgically implanted telemetry transmitters (n = 54) when compared to animals without transmitters (n = 55). Mean CTnI in the large LiHep dataset (n = 109) was 0.0233 ng/ml ±0.00642 SEM (Range 0.00-0.67 ng/ml). To calculate the 99^th percentile for the dataset a nonparametric analysis of the population was performed. In this analysis the 25^th (Q1) and 75^th (Q3) percentiles were calculated. The interquartile range (IQR) was calculated as Q3-Q1. The exclusion limit was calculated as Q3 + 3(IQR) with a result of 0.12 ng/ml. This analysis resulted in the exclusion of 2 data points from the data set as statistical outliers.There was a significant difference of the CTnl values from blood collected from K2EDTA and lithium heparin tubes. There was no difference in the animals that had surgically implanted telemetry transmitters. The 99^th percentile of the resultant dataset (n = 107) was calculated as 0.0792 ng/ml. For comparison, the recommended 99^th percentile for marker biologic significance in humans for the same i-stat cartridges is 0.08 ng/ml.

P227 Lactose Dehydrogenase Isozymes as Biomarkers for the Progression of Tuberculosis Infection in Guinea Pigs (Cavia porcellus)

S Murray², JH Kopanke^*1, W Williams¹, A Izzo¹, LV Kendall¹

¹Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ²College of Veterinary Medicine, North Carolina State University, Raleigh, NC

Lactate dehydrogenase (LD) is found throughout the body, with some tissue groups producing signature variations in LD isozyme (LD1-5) content. Damaged cells can release LD into the blood resulting in altered serum isozyme values that may serve as diagnostic markers. Previous studies have demonstrated that serum LD3 levels increase in humans infected with Mycobacterium tuberculosis. Guinea pigs (Cavia porcellus) are a useful model for the study of M. tuberculosis; however, there are no known biomarkers that allow an adequate assessment of disease progression. This study investigated whether M. tuberculosis infection in guinea pigs produces a LD isozyme profile that may be used to track infection severity. Blood was sampled from 5 guinea pigs prior to M. tuberculosis infection and 4 weeks postinfection. Electrophoresis was performed on serum using a lactate dehydrogenase specific electrophoresis kit to separate and visualize the 5 LD isozymes. Densitometry data was analyzed from digital scans of the dried gels allowing comparison of isozyme levels. Guinea pig total serum LD averaged 185 IU/L (range: 86–378 IU/L) pre infection and 132 IU/L (range: 67 – 218 IU/L) postinfection. LD constituent isozymes (LD1:2:3:4:5) were found to be 35%:22%:10%:3%:30% in preinfection serum samples and 45%:24%:9%:2%:21% in postinfection serum samples. LD1 was elevated an average of 12.3 IU/L (+/-12.1 IU/L) and LD5 fell and average 16.1 IU/L (+/- 22.3 IU/L). The consistent increases in LD1 and the frequent decreases in LD5 suggest a correlation between disease advancement and serum LD isozymes. This could provide a reliable diagnostic to gauge the progression of M. tuberculosis infections, and provide the information necessary to determine endpoints that minimize animal pain and discomfort.

P228 Scratching Responses in C57BL/6, DBA/2, BALB/c, and CD1 Mice with and without Epidermal Injury

JL Sargent^*, CV Lohr, HE Diggs

Oregon State University, Corvallis, OR

Whereas early investigations into ulcerative dermatitis (UD) focused on the possibility of a primary dermatopathology, several recent studies have advocated scratching behavior as a primary driver for UD. The aim of this study was to assess whether B6 mice exhibit excessive scratching under resting conditions or when provoked by epidermal barrier disruption. We hypothesized that B6 mice would exhibit more spontaneous scratching behavior and that B6 mice would be more pruritic after mild epidermal barrier injury compared with the other strains and stock tested. The behavior of the retired breeder female C57BL/6J, DBA/2J, BALB/cByJ, and Crl:CD1 mice was videotaped over several 60-minute sessions. Behavior filming occurred at 17:15 and at 07:00 the next morning, prior to (baseline) and after tape-stripping to initiate epidermal barrier disruption. Scratching duration was recorded as brief (less than 3 seconds) or prolonged (3 seconds or longer), on the basis of observations during a pilot study. In contrast to the hypothesis, B6 mice did not scratch significantly more frequently or for longer durations than the other types of mice tested under both resting conditions and when challenged by tape stripping. In fact, B6 mice showed the lowest average scratching frequency at baseline and following epidermal injury. B6 mice demonstrated increased scratching behavior after epidermal barrier disruption, but the increased scratching did not surpass the rate or duration of scratching in the other types of mice tested. These findings do not support the idea that a strain-related tendency toward exaggerated scratching behavior under resting or epidermal barrier disruption conditions predisposes B6 mice to UD.

P229 Glial Fibrillary Acidic Protein: A Potential Biomarker in a Rat Model of Amyotrophic Lateral Sclerosis

J Jeffrey^*, I Smit-Oistad, J Van Dyke, M Suzuki

Comparative Biosciences, University of Wisconsin—Madison, Madison, WI

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by specific loss of motor neurons in the spinal cord and brain stem. For the last decade, our group has used a rat model of ALS, extensively, to understand the complexity of this devastating disease. The specific rat model we used is the NTac:SD-Tg(SOD1G93A)L26H from Taconic Biosciences. Currently, there are limited options for treating ALS and further investigation of the disease etiology and ALS disease progression needs to be completed. Identifying biomarkers is a powerful strategy to detect and study disease progression in ALS. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is expressed by a number of cells related to the central nervous system including glial cells and ependymal cells. We hypothesized that GFAP represents a biomarker of disease progression in ALS rats. We first determined whether GFAP expression was increased in the skeletal muscle of symptomatic and end-stage SOD1-G93A rats. Immunohistochemical staining and Western blotting analyses revealed a significant increase in GFAP expression throughout disease progression in the hindlimb muscle of SOD1-G93A rats. Next, we asked whether serum GFAP levels were elevated in end-stage ALS rats. We measured GFAP concentrations by ELISA and found a correlation between increased GFAP and disease progression in SOD1-G93A rats. Our results implicate GFAP as a potential biomarker for ALS and will allow for further investigation into the specific roles of GFAP during ALS disease progression.

P230 Leptospira Infection of Mice via the Conjunctiva

JP Sullivan^*1, L Richer², H Potula², N Nair², M Gomes-Solecki^2,3

¹Comparative Medicine, University Tennessee Health Science Center, Memphis, TN; ²Microbiology Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN; ³Immuno Technologies Inc., Memphis, TN

Leptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals, such as mice. These reservoir animals harbor pathogenic leptospires in their renal tubules and shed the bacteria in urine continuously or periodically for a period of a few months up to several years. We analyzed the outcome of infection of C3H-HeJ mice with Leptospira interrogansserovar Copenhageni via the conjunctival route and tracked a number of readouts for 15 days postinfection (dpi). Approximately 60 ul of a 10⁸/mL culture of Leptospira placed onto each eye of the mouse led to bloodstream dissemination of the spirochete on days 5 to 11 which was followed by urinary shedding on days 7 to 15 postinfection. In contrast to the controls, significant body weight was lost in mice infected with Leptospira from days 12 to 15 postinfection with an average loss of 10% (dpi0 v dpi15). The acquired humoral response to conjunctival infection led to the production of Leptospira-specific IgM and IgG. Leptospira dissemination led to colonization of the kidney (∼10⁴ Leptospires/mg tissue) and triggered inflammation as determined by increased levels of mRNA transcripts of KC, RANTES, MIP-2, TNFa IL1b, IL6, and IL13 as well as reduced levels of IL17a mRNA in kidney tissue. mRNA levels of IFNg, IL4, IL5, IL17, IL23, IL10, and iNOS were not different from controls. In this study, we show that pathogenic L. interrogans serovar Copenhageni disseminates and colonizes the kidney of mice after inoculation via the conjunctival route. Replication of one of the three natural modes of transmission of human leptospirosis in mice allows this species to be used for studying all natural modes of transmission.

P231 Effect of Housing and Colony of Origin on Immune Phenotype of Rhesus Macaques

J Kramer^*1,2, J Lane^3,2, J Whitney⁴

¹Leidos Biomedical Research, Bethesda, MD; ²New England Primate Research Center, Southborough, MA; ³University of Washington, Seattle, WA; ⁴Beth Israel Deaconess Medical Center, Boston, MA

Nonhuman primates remain an important laboratory animal species and are commonly used in biomedical research. Despite physiologic similarities to humans that may make them favorable to other animals in certain biomedical applications, there are significant scientific challenges to their use. Unlike many other laboratory animal species, there is considerable variation not only in their genetics, but also origin, husbandry background, clinical history, and other traits. These factors may not be considered by scientists or veterinarians when animals are assigned to research studies, which may result in unexplained results and increased animal usage. To evaluate the effect of housing and basic husbandry practices on the rhesus immune system, we obtained blood samples from group-housed SPF rhesus macaques in 3 facilities that, among other factors, raised their animals in different environments. In facility 1 (n = 18), animals were free to roam a large space and had direct contact with the natural environment. In facility 2 (n = 10), animals were also raised outdoors, but in a confined corral on a clean substrate. In facility 3 (n = 16), animals were raised indoors on bedding. Animals were of similar age, sex, and SPF status, but we did not control for other variables such as long-term clinical history. We performed flow cytometry to evaluate inflammatory cell subsets and a serum cytokine analysis. Animals raised indoors had increased percentages of total, central memory, and effector memory CD4+ lymphocytes compared to both groups of animals raised outdoors. In contrast, the frequency of activated (CD38+/DR+) CD4+ and CD8+ lymphocytes were significantly increased in both groups of outdoor animals compared with animals raised indoors. Both groups of outdoor-housed animals also had increased inhibitory cytokines including IL-10 and IL-1Ra compared to animals housed indoors. Together, these findings indicate that colony of origin can significantly alter the immune phenotype of rhesus macaques and that husbandry practices may contribute to this effect. Researchers should consider colony of origin when choosing animals and assigning them to study groups.

P232 In Vitro and In Vivo Flutamide Effects on Cell Proliferation and Tumor Growth Associated with Steroid Hormone Secretion in Human and Canine Inflammatory Breast Cancer Cell Lines

S Caceres Ramos¹, L Peña¹, MJ Illera¹, B Monsalve¹, W Woodwar², J Reuben³, JC Illera^*1

¹Fisiologia Animal, Universidad Complutense de Madrid, Madrid, Spain; ²Radiation Oncology, MD Anderson Cancer Center, Houston, TX; ³Hematopathology, MD Anderson Cancer Center, Houston, TX

Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. The investigation on the role of androgens in breast cancer is dramatically increasing in recent years in an attempt to find new anti-androgen therapeutic strategies. We studied the effects of the anti-androgen drug flutamide on cell proliferation, in vivo tumor growth and steroid production in canine and human IBC triple negative cell lines. Canine cell line, IPC-366, was cultured in free steroid DMEM/F12 media. Human cell line SUM149 was maintained in free steroid Ham›s F-12 media. Cell cultures were exposed to flutamide treatment. Flutamide concentrations added to the culture media were: 5 µM, 10 µM, and 15 µM for 72 hours and proliferation rates were assayed daily. Additionally, IPC-366 and SUM149 xenotrasplanted mice were used for in vivo assays. Experimental groups of 5 female mice each were divided in a control group and 3 treatment groups (administrated orally with a solution of: 5 µM, 10 µM, and 15 µM of flutamide 3 times a week for 2 weeks). Both experiments were done at the same time. Steroid hormones determination in culture media and tumor homogenates (pregnenolone (P5), progesterone (P4), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 17β-estradiol (E2), and estrone sulphate (SO4E1)) were assayed by EIA previously validated. In vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. In vivo flutamide provoked a tumor size reduction in around 55-65% (IPC-366) and 50-60% (SUM149) of mice treated. In IPC-366 xenotrasplanted mice, metastasis rates decreased compared with control group. P5, P4, SO4E1, A4, T, and DHT hormonal secretion to culture media of cell lines was increased with flutamide addition in contrast to E2 levels that decreased in the treated groups. In tumor homogenates of treated mice, high levels of P5, A4, T, and SO4E1 were found. DHT, P4, and E2 levels were lower compared to control group. Flutamide reduced cell proliferation in vitro, in vivo tumor growth and caused significant changes in steroid hormone secretion by increasing T production leading to lower E2 levels. These results support future therapy approaches of human and canine IBC triple negative breast cancer with anti-androgen drugs.

P233 The Effects of Isoflavones on Estrogens Levles during the Onset of Puberty of Male Wistar Rats

S Caceres Ramos¹, L Peña², P Millan¹, C Perez Garcia³, I Diez Prieto³, JC Illera^*1

¹Fisiologia Animal, Universidad Complutense de Madrid, Madrid, Spain; ²Medicina y Cirugia Animal, Universidad Complutense de Madrid, Madrid, Spain; ³Medicina, Cirugia y Anatomia Veterinaria, Universidad De Leon, Leon, Spain

Isoflavones are phytoestrogens that have a similar structure to the estrogen 17β estradiol. Therefore, they can affect hormonal modulation and some studies have described the association between exposure to phytoestrogens and hormonal-related disorders in children. There are commercially available isoflavone-containing products that have estrogen-like effects such as milk with added genistein. It has been shown that a continuous administration of isoflavones to prepuberal Wistar male rats can delay the onset of puberty. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study is to determine how the 2 most important isoflavones, genistein and daidzein, affect estrogen levels and the onset of puberty on male prepuberal rats, by analyzing the 17β-estradiol and estrone sulphate levels in serum with a competitive enzyme immunoassay (EIA), previously validated for this species. One-hundred twenty five male prepuberal Wistar rats divided in 7 groups, 1 control group and 6 experimental groups, were orally daily administered a high and low dose (HD and LD respectively) of genistein, daidzein, and a mixture of both isoflavones, over 5 weeks. Higher estrogen levels were detected in all experimental groups compared with the control group from the third week of experiment. In control group at the third week there was a significant decrease of estrogen levels associated to the onset of puberty. However, in LD groups, a similar decrease was found at the fourth week of the experiment indicating a delay in the onset of puberty in these groups. Moreover, HD groups did not show this decrease, making undetectable the onset of puberty in these animals. We can conclude that oral administration of isoflavones in male rats affect to the gonadal axis increasing estrogen levels and causing the delay of the onset of puberty, and consequently, could cause physiologic and developmental problems.

P234 Disruption of Folliculin Interacting Protein-1 Prolongs Survival and Improves Response to Therapy in a Mouse B Cell Lymphoma Model

JA Ramirez^*1, M Tsang¹, H Park¹, D Margineantu², D Hockenbery², H Gu³, D Raftery³, B Iritani¹

¹Comparative Medicine, University of Washington, Seattle, WA; ²Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, WA; ³Department of Anesthesiology and Pain Medicine, Mitochondrial and Metabolism Center, University of Washington, Seattle, WA

Folliculin interacting protein-1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with Folliculin (Flcn), a tumor suppressor mutated in human Birt-Hogg Dubé syndrome. Fnip1 and Flcn interact directly with the master metabolic regulator AMP kinase (AMPK), an enzyme that stimulates energy/nutrient production and inhibits energy/nutrient consumption in response to low ATP. Using Fnip1-deficient mice, we previously determined that Fnip1 is required for the development of B lymphocytes, and for B lymphocyte transformation induced by the Myc oncogene in a murine model of human Burkitt’s lymphoma. We hypothesize that inducing Fnip1 deficiency in the tumor cells will prolong survival of the host and improve the response to treatment. In this study, we used the Cre-LoxP system to conditionally disrupt Fnip1 in primary murine B cell lymphomas to determine the potential clinical efficacy of inhibiting Fnip1 in cancer. Groups of 8-9 C57BL/6J syngeneic recipient mice were injected intravenously with spontaneously occurring B cell lymphomas fromFnip1floxedEµ-MycMx1Cre mice. The floxed Fnip1 gene was inducibly deleted in tumor cells from these mice by intraperitoneal injection of polyinosinic-polycytidylic acid (PolyIC; 5-10 mg/kg) to induce Cre expression from the Mx1-Cre (interferon-inducible Cre) transgene. We found that conditional knockdown of Fnip1 significantly delayed lymphoma onset and prolonged survival following chemotherapy (cyclophosphamide, 150 mg/kg IP). Mass spectrometric analysis revealed that inhibition of Fnip1 significantly altered the representation of metabolites involved in glutaminolysis, nucleic acid metabolism, antioxidant, and serine biosynthesis pathways. Using flow cytometry, we found that disruption of Fnip1 increased apoptosis in primary Eμ-Myc lymphoma cells in vivo and also increased metabolic demand by significantly increasing both mitochondrial number and membrane potential. These results suggest that inhibition of Fnip1 may provide a novel strategy to improve response to chemotherapy in lymphoma patients, in part by sensitizing tumor cells to cytotoxic and metabolic stress.

P235 Joint Angles and Limb Alignment of the Humerus and Radius in Nonchondrodysplastic and Chondrodysplastic Feline Breeds

KL Chesney^*1, LA Lyons²

¹Veterinary Pathobiology, University of Missouri, Columbia, MO; ²Veterinary Medicine and Surgery, University of Missouri, Columbia, MO

Orthopedic disease, once thought to be a rarity amongst felines, has recently been shown to affect a large portion of their population. Most senior-age felines have widely variable osteoarthritis, and the incidence of angular limb deformity and chondrodysplasia is on the rise, the latter characteristic of the Munchkin breed and its variants. Treating these conditions in felines is difficult due to the lack of published normal joint angle and limb alignment values. One cadaveric study has examined normal values for the feline pelvic limb, and no data exists for the forelimb. The goal of this study was to develop a range of values for the normal joint angles and limb alignment of the radius and humerus in nonchondrodysplastic mixed-breed felines to characterize the abnormal limb orientation of the chondrodysplastic feline breed, the Munchkin. We hypothesized chondrodysplastic cats would have significantly different limb values than nonchondrodysplastic cats. The entirety of both forelimbs, inclusive of the shoulder joint and carpus, were radiographed in the frontal and sagittal planes in 12 adult, mixed-breed, nonchondrodysplastic cats under sedation. Radiographs were analyzed to determine normal ranges of frontal and sagittal plane anatomic axes and joint angles using a methodology for alignment quantification and preoperative surgical planning described in human surgery, the Center of Rotation of Angulation (CORA). The same procedure was performed for chondrodysplasic cats. Nonchondrodysplastic cats show narrow, normally distributed ranges for each limb value. Chondrodysplastic cats show significant differences (P < 0.05) for 9 of 12 mean limb values, the most significant of which is the CORA of the radius at 10.04 ± 1.30; and 29.56 ± 6.27 (P = 0.0002) for normal and chondrodysplastic radii, respectively. Ranges for normal feline radial and humeral joint angles and limb alignment can be used to characterize newly developed chondrodysplastic breeds and for angular limb and traumatic injury correction when there is bilateral deformity. Many treatment modalities, such as dome osteotomy, are heavily reliant on appropriate preoperative planning, and verified normal values for treatment will be vital for the future of feline orthopedic treatments.

P236 Gender and Microbiota Effects on Gastric Mucosa Tight Junctions in Gnotobiotic INS-GAS Mice

KE Scott^*1, TJ Caron¹, MT Whary¹, JG Fox¹, SJ Hagen²

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Beth Israel Deaconess Medical Center, Boston, MA

During active secretion, the gastric epithelium generates ion gradients that depend on limited paracellular transport of ions across the mucosa. This paracellular “barrier” to permeability is created by tight junctions (TJs), which are regulated, in part, by the gut microbiota. TJs are disrupted and contribute to mucosal pathogenesis in Helicobacter pylori infection. Infected male insulin-gastrin (INS-GAS) transgenic mice have greater pathology than infected female INS-GAS mice. Development of pathology is delayed in H. pylori-monoassociated INS-GAS mice compared to H. pylori-infected gnotobiotic INS-GAS mice co-colonized with select species of Altered Schaedler Flora (ASF). In ASF colonized INS-GAS mice, bacteria that would normally colonize the large intestine also colonize the stomach. We sought determine whether the paracellular barrier in male mice is more permeable than in female mice and to determine the role of gut microbiota in this process. For this, transepithelial resistance (TER), potential difference (PD), and the rate of dextran flux was measured in 4-month-old male and female germfree (GF) and ASF-colonized INS-GAS mice (n = 3-4 per group). A high TER suggests a tight barrier and a high negative PD suggests cation (Cl-) secretion into the stomach lumen. All studies were done with acid blocking agents in the buffer. The results showed GF female INS-GAS mice had a significantly higher TER and negative PD compared to GF male INS-GAS mice, and ASF colonized male and female INS-GAS mice (P ≤ 0.01, P ≤ 0.0001, P ≤ 0.001, respectively). These results suggest that female GF INS-GAS mice maintain a tighter gastric barrier and maintain more Cl- in the gastric lumen than do all other groups. Generally, GF INS-GAS mice have a higher TER and negative PD than ASF colonized INS-GAS mice, and female INS-GAS mice in both groups have a higher TER and negative PD than males of their respective groups. These results support that there is a difference in the composition of TJs in male versus female mice and also support that the microbiota, modeled here using ASF, make gastric epithelia less tight. Future research will evaluate specific differences and regulation of gastric TJ function in male and female mice.

P237 Comparison of Pharmaceutical versus Chemical Grade Streptozotocin and Bolus versus Multiple Low-Dose Regimens in Induction of Diabetes in C57bl/6 Mice

KP Storves^*1, L Lacefield², K Buac¹, J Hutcheson¹, SB Harvey², S Sanchez², U Blas-machado², N Chronos¹, S Chavan¹

¹ViCapsys, Athens, GA; ²University of Georgia, Athens, GA

Streptozotocin (STZ) is an alkylating agent that destroys insulin producing cells and is used in research for diabetes induction. Animal welfare regulations stipulate that pharmaceutical grade agents be used instead of chemical grade unless scientifically justified and IACUC approved. Models using STZ often do not result in 100% induction of diabetes and result in toxicity. Dosing regimens in the literature range from 1 bolus dose to multiple low doses. Previously our collaborators have used 200mg/kg once IP and achieve roughly 80% induction of diabetes with varying amounts of mortality. Chemical grade STZ requires reconstitution with sodium citrate tightly pH buffered to 4.5 and use within 30 minutes of reconstitution. Pharmaceutical grade STZ has an extended shelf life (12 hours) and is more stable suggesting that it may be a safer product for use. We hypothesized that pharmaceutical grade STZ would provide a greater induction percentage with less adverse effects than chemical grade STZ. Our second hypothesis was that multiple low doses would result in less toxicity than a single bolus dose. We used 50 male C57BL/6 mice. Groups 1 and 3 received 3 doses of pharmaceutical and chemical grade STZ at 83mg/kg intraperitoneally (IP), respectively. Groups 2 and 4 received 1 dose of 250 mg/kg IP pharmaceutical and chemical grade STZ, respectively. Groups 5 and 6 were controls. Animals were evaluated over 6 weeks by body weight and glucose readings. All animals in both the high-dose pharmaceutical and chemical-grade STZ groups required early euthanasia due to poor clinical conditions and severe weight loss. All animals in the low-dose pharmaceutical and low-dose chemical groups survived for the duration of the study without clinical signs or weight loss. All animals in both groups achieved and maintained blood glucose levels above 200mg/dL for the extent of the study. The animals in the low-dose chemical STZ group maintained higher blood glucose levels as a group than the pharmaceutical grade animals maintained. Histopathology revealed hydronephrosis, hepatocellular necrosis, and changes in the pancreas in all animals treated with STZ. Changes were similar regardless of grade of STZ or dosing regimen. These results indicate that there is no significant difference in toxicities between chemical and pharmaceutical grade STZ and administration of either form at a high bolus dose results in severe clinical signs requiring euthanasia. Based on the consistently elevated glucose levels and lack of clinical signs in the multiple low dose chemical grade STZ group (group 3), we have adopted this regimen as our method for diabetes induction of C57BL/6 mice for future work.

P238 The Role of Platelets in the Pathogenesis of Cytomegalovirus Infection in Mice

JK Brockhurst^2,1, K Najarro¹, CG Cryer^2,1, S Guerrero-Martin¹, Y Su³, R Boger³, KA Metcalf Pate^*1

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, MD; ²School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; ³Division of Pediatric Infectious Disease, Johns Hopkins University, Baltimore, MD

Cytomegalovirus (CMV) a herpesvirus that is excluded from many specific pathogen-free animal facilities. It is difficult to eradicate without rederivation because it forms a latent state in tissue myeloid cells and the mechanisms underlying the development of this latency are incompletely understood. Platelet decline is characteristic of the acute stage of many viral infections, including CMV, and can be partly driven by platelet sequestration in associations with monocytes and endothelial cells. Such associations affect the characteristics of the myeloid monocyte and the endothelial-organ border, and may change the ability of the monocyte to become infected and invade into tissues by causing the monocyte to adopt an activated Ly-6C^lo phenotype. We hypothesized that platelet decline and platelet-monocyte aggregate (PMA) formation would occur in CMV-infected mice, and that platelet depletion would decrease viral load in the salivary gland and spleen. Male BALB/c mice were infected with 3 X 10⁶ plaque forming units of murine CMV on day 0, and bled for complete blood count every other day to longitudinally track platelet numbers over 21 days. The lowest platelet counts were observed on day 3 of infection, and were followed by a rebound thrombocytosis on day 8 (N = 5, P = 0.04). To determine whether sequestration in PMAs contributed to this decline, additional mice were infected and euthanized on days 0, 3, 8, or 14 for blood and tissue harvest to quantify PMA formation by flow cytometry (N = 5 per timepoint). PMA formation, especially Ly-6C^lo PMA formation, decreased rather than increased at all timepoints following infection compared to baseline (P = 0.05), indicating that PMA formation was not contributing to platelet decline during acute infection. A subset of mice were treated with platelet depleting antibody at the time of infection to determine the effect of platelets on viral entry into tissues at days 0, 3, 8, and 14 (N = 5 depleted and 5 undepleted per timepoint); organ viral load was quantified by plaque assay and compared to undepleted controls. Three of 4 undepleted mice demonstrated higher levels of CMV in the spleen than the depleted mice. Platelets therefore may enhance the ability of CMV to enter into tissues such as the spleen through associations with cells other than monocytes.

P239 Detection and Pathogenesis of Bordetella Hinzii in 4 Commonly Used Mouse Strains

KS Henderson^*1, KR Pritchett-Corning², P Momtsios¹, VS Dole¹

¹Research Animal Diagnostic Services, Charles River, Wilmington, MA; ²Office of Animal Resources, Faculty of Arts and Sciences, Harvard University, Cambridge, MA

Bordetella hinzii is a gram-negative rod-shaped bacterium. Consistent with other members of the genus Bordetella, it resides in respiratory pathways and has been reported to contribute to respiratory inflammation. Up until 2008, our laboratory occasionally isolated an organism from laboratory mice which was consistently identified as B. avium because of the similar biochemical profile. There are reports of isolates consistent with B. avium biochemically, but which also metabolized alkali to malonate and had marginally distinct 16s sequence, both which were consistent with B. hinzii. As a result of this information, our laboratory identified multiple isolates that were consistent with the B. avium biochemical profile, but had a 16s sequence similar to B. hinzii. We further sequenced a portion of the of bacterial RNA polymerase β subunit which contained a larger set of unique nucleotides compared to closely relatedB. spp. which accommodated the development of a highly specific fluorogenic PCR. This assay was added to our routine PCR screening panels to determine the prevalence of B. avium. To further understand the pathogenesis and detection by PCR and bacteriology, we intranasally inoculated 4 each of 3-4 week-old CD-1 NU/NU, SCID, and C57BL/6N, and CD-1 mice with (2 of each isolated as negative controls). Minimum to mild bronchitis was observed in inoculated mice of all strains, with the interesting exception of bronchitis being absent in the Nu/Nu. Tracheitis and rhinitis was minimal in CD-1, min-mild in B6 and NU/NU, and mild-moderate in SCID mice. Nasal aspirate, bronchial lavage fluid, and fecal pellets were positive by PCR, and oral swabs, lung tissue, external genitalia, and cecum contents were mostly negative by PCR. Approximately 2-3% of field samples received for PCR quarantine and routine screening panels received 2013-2016 were positive for B. hinzii with the majority being detected in transgenic mouse lines. In conclusion, our investigation identified B. hinzii as a prevalent primary pathogen contributing to respiratory inflammation in both immunocompetent and immunodeficient strains.

P240 Functional Validation of Tensin2 SH2-PTB Domain by CRISPR/Cas9-Mediated Genome Editing

K Marusugi^*1, K Nakano^1,2, R Kaneda¹, H Sasaki¹, J Kimura⁴, R Yanobu^2,3, T Okamura^2,3, N Sasaki¹

¹Laboratory of Laboratory Animal Science and Medicine, Faculty of Veterinary Medicine, Kitasato University, Towada, Japan; ²Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; ³Department of Infectious Diseases, Section of Animal Models, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; ⁴Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Podocytes are terminally differentiated and highly specialized cells in the glomerulus that form a crucial component of the glomerular filtration barrier. The ICGN mouse is a model of glomerular dysfunction that shows gross morphologic changes in the podocyte foot process accompanying proteinuria. Previously, we demonstrated that proteinuria in ICGN mice might be caused by the deletion mutation in the tensin2 (Tns2) gene (designated Tns2^nph). To test whether this mutation might cause the mutant phenotype, we created knockout (KO) mice carrying Tns2 protein deleted of its the C-terminal Src homology domain and phosphotyrosine binding (SH2-PTB) domain (designated Tns2^ΔC) via CRISPR/Cas9-mediated genome editing. Tns2^nph/Tns2^ΔC compound heterozygotes and Tns2^ΔC/Tns2^ΔC homozygous KO mice displayed the podocyte abnormality and massive proteinuria similar to those seen in ICGN mice, indicating that these 2 mutations are allelic. Further, this result suggests that SH2-PTB domain of Tns2 is required for podocyte integrity. Tns2 knockdown in mouse podocyte cell line significantly enhanced actin stress fiber formation and the cell migration. Thus, this study provides evidence that alteration of actin remodeling by deficiency of Tns2 causes the morphologic changes in podocytes, and subsequent proteinuria.

P241 Photoreceptor Proliferation and Dysregulation of Cell Cycle Genes in Early Onset Inherited Retinal Degenerations

KL Gardiner^*1,2, L Downs², A Berta-Antalics^2,3, E Santana², G Aguirre², S Genini²

¹Pathobiology, University of Pennsylvania, Philadelphia, PA; ²Section of Ophthalmology, Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, University of Pennsylvania, Philadelphia, PA; ³Augenklinik Uniklinik Erlangen, Erlangen, Germany

Mitotic terminally differentiated photoreceptors (PRs) are observed in early retinal degeneration (ERD), an inherited canine retinal disease caused by mutation in the NDR kinaseSTK38L (NDR2). In this study, we examined whether PR proliferation may also occur in other early onset inherited retinal diseases to determine if common molecular pathways were involved. Retinas and retinal pigmented epithelium (RPE)/choroid samples were obtained from dogs that were either normal, xlpra2, rcd1, or ERD-affected at timepoints before cell death peak (induction: 3 weeks), at cell death peak (execution: 5–10 weeks), and during sustained but reduced cell death rate (chronic cell death: ≥ 14 weeks). Samples were analyzed by immunohistochemistry and Western blot analysis, and by real-time PCR (qRT-PCR; n = 3 per timepoint per condition) for evidence of PR proliferation, as well as differential expression of genes involved in cell cycle regulation, PR and eye development regulation, and HIPPO/LATS1 pathway. We show that a proliferative response similar to that observed in ERD, but lower in magnitude, occurs in 2 other early onset disease models, xlpra2 and rcd1 by immunolabeling with PCNA and phospho-histone H3 antibodies. Proliferating cells were determined to be rods. Statistical significance of differentially expressed retinal genes (P < 0.05; fold change FC>+/-2) was assessed with an unpaired t-test. Expression of the cell cycle-related genes RB1 and E2F1, as well as CDK2,4,6 was up-regulated, but changes were mutation-specific. Changes in cyclin expression differed across all genes, diseases and time points analyzed, although CCNA1 and CCNE1 expression increased with age in the 3 models, suggesting that there is a dysregulation of cell cycle gene expression in the 3 diseases. Unique to ERD, however, are mutation-specific changes in the expression of NDR kinases and Hippo signaling members with increased expression of MOB1 and LATS1 in the newly generated hybrid rod/S-cones using Western blot analysis. Our data raise the intriguing possibility that terminally differentiated normal PRs are kept from dividing by NDR2-MOB1 interaction. Furthermore, they provide the framework for the selection of candidate genes for further investigation as potential targets of therapy.

P242 Prolonged Therapeutic Levels of Sustained Release Buprenorphine with Repeat Dosing in Rats

LE Neidig^*1, M Haenisch¹, SM Meeker¹, AM Martinson², C Murry², TL Brabb¹

¹Comparative Medicine, University of Washington, Seattle, WA; ²Pathology, University of Washington, Seattle, WA

Sustained release formulation of buprenorphine (SR Bup) has become widely used in laboratory animal medicine, particularly in rodents, where reduced handling likely decreases stress while providing extended analgesic coverage. Current literature has documented blood plasma levels at or above 1 ng/ml for 72 hours following a single subcutaneous (SQ) dose in rats; however, there is a lack of published data on repeated dosing for animals undergoing multiple survival surgeries. Side effects associated with higher levels of buprenorphine in rats such as rebound hyperalgesia, respiratory depression, gastrointestinal distress, and pica highlight the need for SR Bup to be administered at appropriate dosages and time intervals. In this study, the pharmacokinetics of SR Bup were examined when a second dose was administered 4 days after the initial dose. Adult athymic nude male rats (Hsd:RH-Foxn1rnu) received a dose of SR Bup, 1mg/kg SQ, on day 0 and again approximately 90 hours later. Half of the animals (n = 3) underwent ischemia reperfusion cardiac injury via thoracotomy (ketamine/xylazine anesthesia) followed by a repeat thoracotomy (isoflurane anesthesia) for stem cell therapy 4 days later. The control group (n = 3) underwent the same anesthetic protocol, but no surgery. Serum buprenorphine levels were determined at 12-14 time points (0 to 8-10 days) after the first dose. Serum chemistry screens, before the first procedure and preceding the second dose of SR Bup, indicated no underlying renal or hepatic impairment. Rats in the control group had serum buprenorphine levels consistently above 1 ng/ml (1.12 ± 0.07 ng/ml) >100 hours (>4 days) following the repeat dose, and above 0.5ng/ml (0.78 ± 0.13) for 150 hours (>6 days) after the second dose. In rats that received surgery, serum buprenorphine levels remained above 0.5 ng/ml for 100 hours (> 4 days) following the second dose of SR Bup. Area under the curve analysis revealed elevated serum buprenorphine levels in the three days following the second dose compared to the 3 days after the first dose in the control group (P = 0.001). These results suggest that protocols with multiple survival surgeries within close time proximity may need to consider altering the dose and timing of repeat SR Bup administration.

P243 A Novel Model of Regional Hepatic Arterial Infusion in Hepatitis-Infected Woodchucks (Marmota monax) with Hepatocellular Carcinoma

LI Curtin^*1, M Kim², C Powers², S Sexton¹, A Gudkov³, A Purmal⁴, R Iyer⁵

¹Laboratory Animal Shared Resource, Roswell Park Cancer Institute, Buffalo, NY; ²Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY; ³Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY; ⁴Incuron LLC, Buffalo, NY; ⁵Department of Medical Oncology, Roswell Park Cancer Institute, Buffalo, NY

The woodchuck (Marmota monax) is the only large animal model of spontaneous hepatocellular carcinoma (HCC) in the context of chronic hepatitis infection. This makes it ideally suited for preclinical studies of antineoplastic therapies. Intravenous chemotherapy for surgically unresectable tumors that are limited to the liver is often mired by undesirable side effects and low response rates. Regional cancer therapy through a hepatic artery infusion pump (HAIP) takes advantage of the first-pass effect owing to the liver’s unique blood supply and the fact that liver tumors are fed exclusively by arterial flow. HAI has been used extensively in primary and metastatic human liver tumors, albeit, with mixed clinical results. The purpose of this study was to develop an animal model of HAI in a woodchuck with a preexisting HCC tumor. Four woodchucks bearing 3-5 cm tumors underwent prophylactic cholecystectomy, hepatic artery collateral vessel ligation, and placement of a 2Fr. vascular access port (VAP) into the gastroduodenal artery under a surgical microscope. The port head was secured subcutaneously to the rectus muscle. Correct placement of the VAP catheter end was verified during surgery by direct inspection using a surgical microscope and palpation using microforceps, and was successful in all 4 animals. All 4 ports supported an experimental drug infusion of 3 mL/kg over 30 minutes without complications and were functional at the time of euthanasia in all animals (4-28 days post implantation). One of the four woodchucks suffered postoperative bleeding at the port head site, which was attributed to the animal’s degree of liver dysfunction, and 4/4 woodchucks were partially anorexic for up to 7 days post op. In conclusion, we describe a new model of regional HAI chemotherapy using a VAP that provides a feasible and practical treatment modality for evaluating novel regional cancer chemotherapy agents in a large animal model of HCC.

P244 Vertical Sleeve Gastrectomy in Mice: Validating a Mouse Model for the Surgical Intervention Of Obesity

LA Stewart^*1,2, TA Knotts^1,2, DA Kieffer¹, K Lloyd^1,2, KD Evans^1,2, K Grimsrud^1,2

¹Mouse Biology Program, University of California—Davis, Davis, CA; ²Mouse Metabolic Phenotyping Center, Davis, CA

Obesity is rapidly becoming a global health epidemic. Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is currently the most effective long-term treatment for obesity and has shown to markedly improve glucose homeostasis. The VSG surgical procedure removes approximately 75% of the stomach creating a small remnant tube that limits food intake. In order to investigate the metabolic effects of these surgeries, it is critical to have appropriate translational, clinically relevant animal models. We developed and validated the VSG surgical procedure in diet-induced obese (DIO) C57BL/6J mice (n = 160). During the developmental phase, we modified an existing procedure to improve survival rates and reduce the occurrence of surgical site leakage, ischemia, and abscess formation. Our modified technique improved the survival rate from approximately 50% to 90%. The validation was comprised of mice fed a 60% high-fat diet for 9 weeks prior to surgery. VSGs, pair-fed shams (PFS), ad-lib shams (ALS), and control mice (C) were measured for body weight, body composition, feed intake, and glucose tolerance over a 3-month period. Significant differences in body composition of VSG mice were observed. VSG male and female mice gained significantly less body weight post surgery compared to the ALS mice; no difference between VSG and PFS mice was observed. Male VSG mice had improved glucose tolerance compared to ALS mice. No difference was observed among female mice. Our validation demonstrated that the pattern of body weight change and the effect on food intake and body composition in our VSG mouse model paralleled the human prognosis following VSG surgery. In summary, the surgical mouse model we created and validated is translational and can be used to further investigate the effects of VSG as a surgical intervention of obesity.

P245 Marble Burying Test as a Practical Pain Assessment in Rats

NN Lee, LV Kendall^*

Laboratory Animal Resources, Colorado State University, Fort Collins, CO

Behavioral assessments using ethograms are frequently used as a means to assess pain in rats. These measurements are often subjective and prone to inter-observer variability. As a prey species, rats may hide behavioral signs during observations, which further complicates pain assessment. Rats typically bury novel objects as a natural defense mechanism to cope with stress or anxiety caused by novel objects. Marble burying test are frequently used to assess anxieolytic drugs and obsessive compulsive disorders. We evaluated the marble burying test in an effort to identify a more objective measurement of pain in rats. Six male Sprague–Dawley rats were anesthetized with isoflurane and underwent a surgical vasectomy. Six rats received anesthesia only, no surgery. Three vasectomized rats received postoperative analgesia treatment with meloxicam (1 mg/kg s.c. bid) and 3 received saline (0.1 ml s.c. bid). Three rats in the anesthesia-only group received meloxicam and 3 received saline. Pain was assessed at 1, 3, 6, 12, 24, and 48 hours post operatively. Rats were observed for 5 minutes to evaluate behavioral indicators of pain including rearing, grooming, wound licking, posture, orbital tightening, and general activity. Following the 5-minute observation period, rats were placed in a new cage with 12 marbles. Rats were removed after 30 minutes and the number of marbles that were buried 2/3 of their diameter were counted. Vasectomized rats had reduced rearing, increased wound licking, and increased orbital tightening scores compared to unsurgerized control rats. Wound licking was greatest in the saline treated group. Marble burying in saline treated vasectomized rats was reduced for the first 12 hours post-operatively; whereas, meloxicam treatment increased the marble burying behavior 3-6 hours post-operatively. This was similar to the unsurgerized, saline treated rats. Meloxicam treatment in unsurgerized rats had the greatest marble burying activity. Compared to the ethogram observations, marble burying behavior showed the most apparent differences between the 2 surgical groups. Although there were no statistically significant differences, this pilot study suggest that marble burying behavior may be a reliable and objective way to assess pain in rats.

P246 Continuous Assessment of Marble Burying in Rats Is Inadequate for Assessing Postoperative Pain

W McGee, LV Kendall^*

Laboratory Animal Resources, Colorado State, Fort Collins, CO

Behavioral assessments of post-operative pain in rats can be subjective and prone to interobserver variability. We demonstrated that marble burying behavior could be used as an objective means to assess post-operative pain in a pilot study using vasectomized rats. This study evaluated the use of marble burying behavior to assess post-operative pain in female rats following an ovariectomy. Twelve Long Evans rats were anesthetized with isoflurane and underwent a surgical ovariectomy, and 12 rats received anesthesia only. Six ovariectomized rats received post-operative analgesia treatment with meloxicam (2 mg/kg s.c. bid) and six received saline (0.1 ml s.c. bid). Six rats in the anesthesia only group received meloxicam and 6 received saline as controls. Pain was assessed at 1, 3, 6, 12, 24, and 48 hours post operatively. Rats were observed for 2 minutes to evaluate behavioral indicators of pain including rearing, grooming, wound licking, posture, orbital tightening, and general activity. Marbles (1.5 cm diameter) were placed in the recovery cage and the number of marbles that were buried 2/3 of their diameter were counted at the time of pain assessment. Ovariectomized rats had increased orbital tightening and wound licking, and reduced rearing compared to unsurgerized rats. There was no significant difference in the number of marbles buried over time between the 4 groups. This study demonstrates that marble burying behavior may not be an objective means to assess post-operative pain if the marbles are left continuously in the home cage.

P247 The Cynomolgus Macaque (Macaca fascicularis) Model of Pneumonic Tularemia

LL Lanning^*1, P Sanz¹, L Wolfraim¹, C Houchens², K Omland³, M Williams¹, J Hewitt¹, T Guina¹

¹NIH/NIAID/DMID, Bethesda, MD; ²BARDA/HHS, Washington; ³Mergus Analytics, Jerricho, VT

The National Institute of Allergy and Infectious Diseases (NIAID) and the Biomedical Advanced Research and Development Authority (BARDA) formed a Working Group for qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia as a Drug Development Tool with the Food and Drug Administration. Qualification of an animal model through FDA’s Animal Model Qualification Program is limited to those proposed for use in product approval under the Animal Rule. This is a regulatory mechanism for approval based on evaluation of the efficacy of new-generation therapeutics and vaccines in suitable animal models [21 CFR 314.600 for drugs; 21 CFR 601 Subpart H for biologic products]. The adequacy of an animal model for qualification is based on data from natural history studies and from the human disease or condition showing that the specific animal species given a specific challenge agent by a specific route, produces a disease that corresponds to the human disease or condition of interest in multiple important aspects. We intend to qualify the natural history model of pneumonic tularemia in the cynomolgus macaque based on results of 2 LD50 and 5 natural history studies that have been completed. Mortality, average time to death, average time to fever onset, average interval between fever and death, initial level of bacteremia post-challenge, timing of bacteremia onset, clinical signs, necropsy findings, and histopathology from the studies were analyzed and form the basis for the proposed cynomolgus macaque model parameters. Data collected to date support the conclusion that symptoms and signs of pneumonic tularemia in cynomolgus macaques exposed to a target dose of 1,000 cfu (range 300 to 3000 cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar.

P248 Safety of Epidural Morphine with Preservative in Domestic Goats (Capra aegagrus hircus)

MG Lin^*1, JJ Elliott¹, M Jones², M Wight-Carter³, G Carroll²

¹Comparative Medicine Program, Texas A&M, College Station, TX; ²College of Veterinary Medicine, Texas A&M, College Station, TX; ³ARC Diagnostic Lab, University of Texas Southwestern Medical Center, Dallas, TX

The management of perioperative pain in livestock species using opioid epidurals has become increasingly popular. Preservative-free morphine sulfate (PFM) is currently the standard for epidural use, as phenol and formaldehyde preservatives found in morphine with preservative (MWP) have been shown to be neurotoxic. However, due to costs and national shortages of PFM, many veterinarians are being forced to use MWP instead. The objective of this study was to determine if epidurally (L6-S1) administered MWP produced behavioral or spinal cord changes that were different from those seen with PFM, normal saline, or no epidural administered. One hundred young adult goats were randomized into 7 groups, and necropsied 2-4 weeks following no epidural (2 weeks, n = 15), saline epidural (2 weeks, n = 15; 3 weeks, n = 11), PFM epidural (2 weeks, n = 15; 4 weeks, n = 14), or MWP epidural (2 weeks, n = 15; 4 weeks, n = 15). Behavior and clinical signs of toxicity were evaluated. At necropsy, spinal cords were removed from the lumbar region to the cauda equina and histology performed. All slides were examined by a veterinary pathologist (blinded to treatment groups) for evidence of neurotoxicity by the presence of gliosis, central chromatolysis of neurons, white blood cell infiltrates, thickening of dura mater, and fibrosis. Histologic lesions of neurotoxicity were not found in any treatment group. Behavior did not vary among treatment groups, and no clinical signs of toxicity were noted. Thus, a single dose of MWP administered epidurally in goats does not cause histologic evidence of neurotoxicity and may be considered a safe practice.

P249 Nutritional Supplement and Its Effect on Tumor Growth and Body Weight in NSG Mice Undergoing Chemotherapy

M Tewodros^*1, L Yao¹, M Cheng¹, M Creamer²

¹In Vivo, The Jackson Laboratory, Sacramento, CA; ²Comparative Medicine and Quality, The Jackson Laboratory, Sacramento, CA

Chemotherapeutics are effective in the treatment of cancer; however body weight loss and subsequent malnutrition are potential complications during therapy. The use of supplemental nutrition in cancer patients remains controversial. This study seeks to investigate whether nutritional supplement can alleviate body weight loss during chemotherapy and its effects of tumor growth using NSG mice. Female NSG mice (n = 60) were subcutaneously engrafted with human lung patient derived xenograft (PDX) fragments. A subset of mice (n = 30) were used to evaluate tumor growth response with or without nutritional supplement and the remaining engrafted mice (n = 30) received the chemotherapeutic Docetaxel with or without nutritional supplement. Tumor growth and body weight were measured twice weekly for all groups. Nutritional supplement consisted of ad libitum access to 2oz cups of a commercially available nutrient-fortified water gel placed within the cage. Cups were replaced twice weekly. The study found that mice receiving Docetaxel had an equal amount of body weight loss regardless of access to nutritional supplement. However, mice that received nutritional supplement saw a significantly faster (P < 0.05) increase in body weight once chemotherapy decreased or ended and were able to regain their body weight 5-7 days sooner than mice which did not receive supplementation. There was no significant difference in tumor growth of chemotherapeutic treated mice irrespective of nutritional supplementation. Interestingly, tumor growth in mice not undergoing chemotherapy was significantly greater (P < 0.05) in groups that received nutritional supplement compared to mice that did not receive nutritional supplement. The results of this study suggest an altered response to tumor growth in mice that receive nutritional supplement prior to but not during chemotherapy. The improvement on body weight recovery for mice receiving supplement during chemotherapy may improve the overall health of the animal subjects and extend the life of the research study. If nutritional support is used during chemotherapy studies with mice we recommend providing supplement to all cages during dosing to avoid variability in tumor growth or body weight loss between cages.

P250 Urine Cortisol:Creatinine Ratios as an Assessment of Stress and HPA-Axis Function in Rhesus Macaques (Macaca mulatta)

SC Adams^*1, C Guyot¹, K Berry¹, A Loar², M Leblanc¹

¹ARD, Salk Institute, La Jolla, CA; ²STAT Veterinary Lab, San Diego, CA

Monitoring biomarkers of stress in laboratory animals is critical to evaluate the potential impact of research activities on animal welfare. Cortisol is a sensitive marker of stress in many species, including macaques. Cortisol levels may be evaluated in several samples, including serum, saliva, feces, hair, and urine. While serum and saliva best reflect real-time cortisol levels, they require stressful restraint that may alter results. Fecal cortisol is non stressful, but results may be confounded by urine contamination, diet, and intestinal transit. Hair cortisol is also noninvasive but provides retrospective values over weeks to months. Urine cortisol:creatinine ratios (UCCR) are commonly used in small animal and human medicine and reflect hypothalamic-pituitary-adrenal (HPA) activity over a period of a few hours to a day; however, information is lacking regarding its use in nonhuman primates (NHPs). We therefore evaluated the use of UCCR in macaques to assess animal welfare and HPA axis. We hypothesized that UCCR values would be elevated in working cranially implanted macaques and would be responsive to dexamethasone suppression. Urine was collected from singly housed animals that were separated due to veterinary or scientific exemptions or paired-housed macaques separated at the time of feeding or during work. We first compared UCCR in working macaques with cranial implants (n = 4) and naïve non-working monkeys (n = 8). UCCR values were significantly higher (P < 0.001) in working monkeys (58.2 ± 23.4) as compared to nonworking animals (12.5 ± 12.7) suggesting higher levels of stress associated with research activities. We also evaluated the use of UCCR to assess HPA function by performing a Low Dose Dexamethasone Suppression (LDDS) test. Oral dexamethasone (DEX) (0.01 mg/kg) was given at 6 am, 12 pm, and 6 pm on the same day and urine samples were collected before and at 6, 12, 24, 32, and 56, and 102 hours post first DEX dose. UCCR levels decreased by 67% in the first 12 hours and reached nadir at 32 hours before gradually returning to normal at 102 hours, thus suggesting that cortisol is predictably suppressed by low doses of DEX and can be used to assess near-real time cortisol levels and HPA function in macaques. Overall, these results suggest that UCCR is a noninvasive method that can be used to evaluate near-real time cortisol levels, stress, HPA axis, and welfare in research macaques.

P251 Antigen, Antibody, and Echocardiographic Results for Cats Experimentally Infected with L3 of Dirofilaria Immitis and Implications for Future Studies

M Krecic^*1, J Lizer², T McTier²

¹Zoetis, Florham Park, NJ; ²Zoetis VMRD, Kalamazoo, MI

Feline heartworm disease caused by Dirofilaria immitis remains difficult to diagnose. Serological tests do not always correlate well with infection and little controlled data is available on the use of echocardiography in determining infection and infection levels. The objective here, part of a wider feline heartworm diseases program, was therefore twofold: to determine serological heartworm status through 8 months post experimental infection and to correlate serological heartworm test results and echocardiographic findings to the known numbers of adult heartworms identified at necropsy 238 days postinfection. Twenty cats experimentally infected with 100 third stage larvae (L3) of D. immitis (day 0) were tested for antigen on and between days 28 through 238 and antibody on and between days 56 through 238, and examined echocardiographically on days 177 and 231. Sixteen of 20 (80%) cats developed adult heartworm infections; 13 of 16 (81%) cats had antigen on day 238 and 16/16 (100%) cats had antibodies on days 182 and 238. Four of 20 (20%) cats did not develop adult heartworm infections. These cats were also negative for antigen and antibody on days 182 and 238 and also negative for adult heartworms with echocardiography on days 177 and 231. Results suggest that combined diagnostics are useful in a research setting for accurately identifying adult heartworm infections in cats at 8 months post experimental infection with 100 L3 of D. immitis without the need for necropsy. Veterinarians examining cats suspected of adult heartworm-induced disease may equally be able to exclude infection with negative serological test and echocardiographic results as early as 7 months post natural infection.

P252 Isolation of Lytic Bacteriophages against Multidrug-Resistant Enterococcus faecalis ST55 Isolated from Research Macaques

MT Lieberman^*1, RJ Citorik², TK Lu³, JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Department of Biology, Massachusetts Institute of Technology, Cambridge, MA;³Department of Biologic Engineering, Massachusetts Institute of Technology, Cambridge, MA

Multidrug-resistant Enterococcus faecalis (MDR-EF) is a serious cause of human nosocomial infections. We have identified 14 MDR-EF isolates of sequence type (ST) 55 (n = 7) and ST4 (n = 7) from cephalic recording chambers of macaques used in neuroscience research. Whole genome sequencing identified genes encoding resistance to aminoglycosides, tetracycline, erythromycin, and macrolide antibiotics. Due to the difficulties in treating infections caused by multidrug-resistant bacteria, new antimicrobial strategies are needed. Lytic bacteriophage therapy has historically been used to treat bacterial infections in Eastern Europe since the early 1900s. We hypothesized that we could isolate lytic bacteriophages with activity against MDR-EF. Bacteriophage enrichments were prepared by adding sterile-filtered sewage samples to double-strength phage broth and individual inoculation with single MDR-EF isolates. Following overnight incubation, enriched samples were centrifuged and supernatants were spot tested on each MDR-EF isolate using a modified double-agar overlay method. Individual phages were purified by 3 serial passages of isolated plaques, then concentrated from lysed broth cultures via polyethylene glycol precipitation and stained with uranyl actetate for transmission electron microscopy (TEM). Lytic activity was observed against all 7 ST55 isolates with 3 isolates showing the strongest lysis as indicated by plaque size. Initial TEM imaging identified the 3 phages as likely Siphoviruses of 2 distinct morphologies featuring long, flexible, apparently noncontractile tails. Capsid morphologies included icosahedral (50.7 ± 2.4 nm in diameter) and 2 sizes of prolate capsids (104.5 ± 5.7 nm x 37.64 ± 1.1 nm and 92.8 ± 4.6 nm x 36.5 ± 1.3 nm). Lytic phage therapy offers an alternative to traditional antimicrobials and may be especially valuable against highly antimicrobial-resistant bacteria. Future studies include continued efforts to isolate lytic phage with activity against ST4 isolates, as well as phage sequencing and characterization of anti-biofilm activity by ST55 lytic bacteriophages.

P253 Pharmacologic Inhibition of Rho-Associated Protein Kinase Pathways as a Therapeutic Strategy for Attenuating Corneal Fibrosis and Neovascularization in Vivo

MK Fink^*1,5, S Gupta^2,5, M Possin^3,5, PR Sinha^2,5, EA Giuliano², RR Mohan^4,5

¹Veterinary Pathobiology and Comparative Medicine Program, University of Missouri, Columbia, MO; ²Veterinary Medicine and Surgery, University of Missouri, Columbia, MO; ³Mason Eye Institute, University of Missouri, Columbia, MO; ⁴Veterinary Medicine and Surgery, Veterinary Pathobiology, and Mason Eye Institute, University of Missouri, Columbia, MO; ⁵Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO

Corneal scarring (fibrosis) is a leading cause of blindness worldwide as a result of the normal corneal wound healing response. Rho-associated protein kinase (ROCK) pathways regulate cellular proliferation, migration, adhesion, wound healing, angiogenesis, and fibrosis in vivo. We tested the postulate that topical application of ROCK inhibitor HA1077 to the eye following corneal insult will attenuate corneal fibrosis and corneal neovascularization (CNV) in vivo by mitigating exuberant wound healing. The study was approved by the IACUC. Corneal fibrosis and CNV were produced in 12 New Zealand White rabbits by a single topical alkali (1N NaOH) application for 1 minute to the central cornea. Following corneal wounding, animals were divided into 2 groups: Group 1 served as controls and received 50μL balanced salt solution (BSS) topically twice daily for 3 days. Group 2 served as treatment cohort and received 50μL HA1077 (3nM) topically twice daily for 3 days. Contralateral untreated eyes served as negative controls. Serial slit and stereomicroscopy evaluated the degree of ocular inflammation, corneal edema, and corneal opacity. Corneas were harvested on day 14 with H&E and immunofluorescence staining employed to characterize levels of fibrosis, CNV, inflammation, and apoptosis. Biomicroscopy detected a significant decrease (∼2.8 fold; P < 0.01) in corneal fibrosis and CNV in eyes treated with HA1077 compared to BSS-treated controls. Further, HA1077-treated corneas showed significant decreases in fibrosis markers (smooth muscle actin, fibronectin, and F-actin; 55-60%; P < 0.0001), and did not exhibit significantly increased numbers of CD11b+ (2-11%) or TUNEL+ (0-5%) cells compared to BSS-treated controls during immunofluorescence analyses. A substantial decrease in CNV was also noted in HA1077-treated eyes as compared to BSS-treated controls (quantification pending). Clinical ocular examinations and histologic evaluation did not reveal evidence of acute toxicity from topical application of HA1077. Topical application of ROCK inhibitor HA1077 is a viable option for treating corneal fibrosis and neovascularization resulting from corneal insult. Further in vivo analysis is warranted.

P254 Noonan Syndrome with Multiple Lentigines Associated Hypertrophic Cardiomyopathy Treatment with Angiotensin II Receptor Blocker

MA Bellrichard^*1, M Krenz^2,3

¹Veterinary Pathobiology, University of Missouri, Jonesburg, MO; ²Medical Pharmacology and Physiology, University of Missouri, Columbia, MO; ³Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO

Noonan syndrome with multiple lentigines (NSML) is a rare genetic disease that leads to a variety of clinical manifestations. These include skin, skeletal, and cardiovascular abnormalities. Importantly, 80% of NSML patients have hypertrophic cardiomyopathy (HCM). Genetic studies have shown that 90% of NSML cases are due to mutations in the nonreceptor protein tyrosine phosphatase Shp2. Mechanistically, animal studies have shown that mutations in Shp2 result in hyperactivation of downstream signaling through Akt and mammalian target of rapamycin, both of which are potent stimulators of cardiac growth. Myocardial hypertrophy and fibrosis are a major part of the pathology of HCM and both of these processes can be stimulated by angiotensin II. In addition, another study has revealed that angiotensin II directly activates Akt through angiotensin II receptor type 1. This information lead to the hypothesis that blocking angiotensin II via losartan, an angiotensin II receptor antagonist, will improve cardiac morphology and function in NSML. We chose the Q510E-Shp2 mutation for our studies since this mutation is associated with a particularly severe form of NSML in both humans and mice. Expression of Q510E-Shp2 in mice causes severe early onset HCM with interstitial fibrosis, thickened ventricular walls, and depressed contractile function. In our study, 8-week-old Q510E mutant mice and nontransgenic controls were treated with losartan or a control for 8 weeks. The mice were evaluated with echocardiography at both the onset and conclusion of the treatment trial. Cardiac function, as measured by fractional shortening, was decreased by 20% in the untreated NSML mice. However, the NSML mice treated with losartan showed no decrease fractional shortening in comparison to their nontransgenic counterparts. At the conclusion of the study, heart to body weight ratios were assessed as a measure of cardiac hypertrophy. Our data show a trend with NSML mice having 20% heavier hearts than the nontransgenic mice and the losartan treatment decreased this hypertrophy to only 10%. Taken together, this information shows losartan has promise as a novel treatment modality for maintaining cardiac function in NSML patients, but would have only minor effects on the degree of cardiac hypertrophy.

P255 Characterization of the Fecal Microbiota of Mink (Neovison vison): A Representative Carnivore

NR Compo^*1, B Tapscott², D Gomez¹, J Weese¹, PV Turner¹

¹Pathobiology, University of Guelph, Guelph, Canada; ²Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Canada

The mammalian gut microbiota has received increasing attention because of its role in health and disease. In particular, reports have emerged from mice, rabbits, and other species suggesting that differences in gut microbiota can influence research outcomes. To date, most microbiota work has been conducted in herbivores and omnivores, although some work has also been conducted in companion cats. The objective of this study was to characterize and compare the fecal microbiota of mink (Neovison vison), to contribute to the understanding of comparative microbiota differences in carnivore models. In addition, we sought to determine whether temporal or age differences exist in the mink fecal microbiota, and whether dietary changes, as occur in female mink prior to breeding, affect the gut microbiota. Pooled fecal samples (n = 344) were collected from healthy females and weaned kits from mink farms during 2 consecutive summers and from healthy females (n = 36) before winter breeding. Bacterial DNA was extracted and characterized using 16S rRNA gene PCR and next generation sequencing. Following quality control filtering, approximately 7.2 million sequences have been identified. The predominant phyla in the feces of females and kits are Firmicutes, with mean relative abundances of 54.7 and 57.3%, respectively, and Proteobacteria, with means of 42% and 39.7%, respectively. The latter is greater than the that seen in other species, such as mice, rabbits, and humans. Other phyla were identified but all were uncommon accounting for <2% of total sequences each. There were no significant differences in phyla identified between females and kits. At the genus level, >900 genera were identified, only 3 of which represented >4% of the total sequences: Atopostipes (∼5%), previously only isolated from an underground swine manure storage pit; Lactobacillus (∼9%), involved in lactic acid production; and Ignatzschineria (∼21%), the predominant genus in the gut of spotted flesh fly larvae. It is unclear whether this last finding represents a fecal contaminant or true gut colonization in mink, which receive a significant portion of their diet from livestock byproducts. Limited data are available on the microbiota of carnivores, so this data will be valuable for understanding comparative modeling.

P256 Antibiotic Drinking Water for Immunocompromised Strains: Does the Type of Water Make a Difference?

PH Myers^*, TE Whiteside, J Locklear, TL Blankenship

National Institutes of Health, Research Triangle Park, NC

Immunocompromised mice are often placed on acidified water to reduce the spread of bacterial disease among animals through the drinking water. It is questionable whether this is an effective protocol in minimizing clinical disease in strains that are susceptible to specific bacterial agents. In these instances, supplementation with antibiotics may be recommended. Normally antibiotics are provided to the mice via the drinking water; however, acidification of the drinking water may interfere with antibiotic efficacy. In this project, we evaluatedStaphylococcus xylosus (S.xylosus) cultured from immunocompromised mice being treated with either acidified water only or neomycin prepared with acidified water. Thirty female B6.129S-Cybb^tm1Din/J (Cybb) mice were received and housed 2/cage and placed on either acidified water (Group 1-5A) for the duration of the study or neomycin acidified water (Group 1-5B and 6-10). Animals in Group 1-5B alternated between acidified and neomycin acidified every 2 months. Group 6-10 were treated with neomycin acidified water for 5 months. At the end of the study both groups were removed from antibiotic treatment and placed back on acidified water. Oropharangeal (OP) cultures were performed weekly until S. xylosus was recovered and antibiotic susceptibility testing could be performed. Sensitivity disks were prepared with 0.25 inch blank disks saturated with 20 ul of each treated water neomycin acidified or acidified as well as neomycin in water deionized by reverse osmosis (RO-DI) to confirm the reduction in microbial inhibition was indeed due to the acidified water. Sensitivity disks were placed on 5% Sheep Blood Agar plates streaked with S. xylosus and incubated at 37 degrees for 24 hours. Zones of inhibition were recorded. S. xylosus could not be cultured while on antibiotic water; however, S. xylosus was recovered 3 weeks after animals returned to acidified water. Microbial sensitivity testing revealed neomycin had a significant reduction in microbial inhibition when prepared with acidified water compared to neomycin prepared with RO-DI water. Therefore the type of water used when preparing antibiotic treated water should be considered.

P257 Reference Growth Curves for Long-Tailed Macaques (Macaca fascicularis) from Mauritius

S Naiken, P Honess^*

Animal Welfare, Bioculture Group, Riviere des Anguilles, Mauritius

The long–tailed macaque (Macaca fascicularis) of Mauritian origin is widely used in biomedical research. However, there is little information on its growth pattern. The purpose of this study is to provide weight for age and sex reference standards for this important animal model. Reference values were derived from retrospective data over a 10-year period for all live births of captive-bred monkeys from birth to a maximum of 84 months. The animals were weaned at between 12 and 24 months into peer groups housed outside under ambient conditions. Housing and care meets or exceeds standards set in the NRC Guide (2011) and the EU Directive (2010/63/EU). The smooth curves of weight for age and sex were created using Generalised Additive Models for Location Scale and Shape (GAMLSS) in R statistical software (v.3.1.2) and included 244,809 weight observations from 37,105 males (mean = 6.6 observations/animal, SD=8.53) and 223,432 from 35,455 females (mean: 6.3 observations /animal, SD=8.13). It was found that males not only grow faster than females (regression coefficient, 0-84 months: male=0.09, P = 0.000; female=0.065, P = 0.000), but they also continue to grow well after females have attained their peak body mass at around the age of 48 months. Males appear to go through an adolescent growth spurt from around 30 months, whereas females continue to grow at a constant, but lower rate (regression coefficient, 30-84 months: male= 0.094, P = 0.000; female=0.048, P = 0.000). The results from this study establish age-weight reference ranges which can be useful for assessing growth in long-tailed macaques of Mauritian origin.

P258 Evaluation of Anthelmintic Resistance and Exhaust Air Duct PCR as a Diagnostic Tool in Mice Endemically Infected with Aspiculuris tetraptera

P Kapoor^*2, Y Hayes², L Jerrell¹, DA Bellinger², R Thomas³, C Fletcher², J Nielsen²

¹Sobran Inc, Dayton, OH; ²Division of Laboratory Animal Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; ³Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill, Chapel Hill, NC

The influx of infectious agents in rodent colonies occurs despite a robust sentinel monitoring program, strict quarantine measures, and superior biosecurity practices. Due to several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for the early detection ofAspiculuris infection. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude strains of mice were experimentally inoculated with embryonated Aspiculuris ova harvested from endemically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/ml piperazine in water for 4 or 8 weeks. Regardless of the strain and treatment, none of the mice demonstrated anthelmintic resistance as indicated by the absence of worms at necropsy. In order to evaluate the effectiveness of EAD PCR, 69 cages of breeder mice endemically infected with Aspiculuris were housed on a sterilized IVC rack. Pooled fecal pellets and fur swabs were analyzed by PCR and fecal centrifugation floatation (FCF). At day 0, 56-58% of the cages on this rack tested positive for Aspiculuris by PCR and FCF. PCR from EAD swabs became positive forAspiculuris within 1 week of placing the cages on the rack. Following treatment with 150 ppm fenbendazole in feed, the EAD PCR reverted to negative within 1 month of treatment and remained negative for the duration of the study (8 weeks). Additionally, no Aspiculuris worms were observed in the colons of culled mice at necropsy. The sensitivity of EAD PCR was investigated by housing 6 Aspiculuris-infected mice on another sterilized IVC rack. The EAD PCR from this rack was positive for Aspiculuris within 1 week of placement. Aspiculurisinfection was confirmed by direct visualization from cecum/colon samples. The IVC racks and air handling units were sanitized prior to each study and confirmed negative by pinworm PCR, as a rack control. These results demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive, and reliable assay that can be an important diagnostic tool for antemortem detection of Aspiculuris infection in conjunction with fecal PCR and FCF.

P259 Development of a New Adenovirus Antigen for Serological Detection of Adenovirus Infections in Guinea Pig Colonies

RK Dhawan^*, ML Wunderlich, R Brouillette

BioAssay Services, Charles River Laboratories, Wilmington, MA

Adenovirus has a moderate prevalence in lab guinea pigs and therefore it’s important to have a high throughput serological screening assay for detection of antibodies against it. Failure to make conventional guinea pig adenovirus (GPAV) antigen in the past was due to difficulty in virus propagation and/or in making a recombinant antigen. Historically, highly purified conventional and recombinant mouse adenovirus (MAV) antigens, both type-1 and type-2, failed to demonstrate significant cross-reactivity with GPAV antibodies. We hypothesized that a new GPAV cross-reactive conventional MAV-2 antigen (nMAV-2) could be prepared from CMT-93 mammalian cells infected with mouse adenovirus type-2 (MAV-2, K87) virus. Reactive viral proteins were isolated from infected cells by using a combination of techniques including low speed centrifugation, ultra-centrifugation, and detergent extraction/washing. Analysis by ELISA and MFIA^® confirmed that purified antigen was cross-reactive with GPAV seropositive antisera collected from naturally and experimentally infected animals. Sensitivity of this nMAV-2 antigen was evaluated by screening 20 GPAV antibody positive field sera previously confirmed positive by IFA. All of these samples, 20/20 (100%) were found positive by both nMAV-2 ELISA and MFIA^®. Early detection capability of nMAV-2 antigen was compared to previously purified conventional and recombinant MAV-2 antigens. Antisera collected from experimentally MAV-1 and MAV-2 infected mice and rats at various time points post inoculations (day 0-35) were tested by ELISA and MFIA assays. Assays developed using nMAV-2 antigen detected early seroconversion at the same time points (7 DPI) as seen by both conventional and recombinant MAV-2 antigens. Antigen specificity was evaluated by testing sera from historically known negative guinea pig colonies. None of the 300 tested sera showed positive results thus demonstrating a 100% specificity of both nMAV-2 ELISA and MFIA assays. Purity of the nMAV-2 antigen was tested by screening heterologous sera positive for other infectious agents (i.e. LCMV, MPV, MTLV). Results showed no cross-reactivity with non-MAV antibodies. In summary, ELISA and MFIA assays developed using nMAV-2 antigen demonstrated species cross-reactivity for detection of GPAV sera antibodies and can be used for routine screening of GP colonies. IFA test can continue to be used for confirmation of suspect ELISA and MFIA positive GPAV sera samples.

P260 Development and Confirmation of a Novel Technique for Orthotopic Engraftment of Colon Organoids in Mice

R Garcia-Gonzalez^*1, EP Chua¹, J Yamada¹, C Sohn¹, P Manzanillo², J Lesch², K Leong²

¹Laboratory Animal Resources, Genentech, Inc., South San Francisco, CA; ²Research, Genentech, Inc., South San Francisco, CA

Inflammatory bowel diseases (IBD), including both Crohn’s disease and ulcerative colitis, are disorders of chronic inflammation of the gastrointestinal tract marked by episodes of relapse and remission. These chronic, lifelong conditions can be treated symptomatically but not cured. The incidence and prevalence of IBD has been increasing worldwide across pediatric and adult populations. IBD can significantly affect a patient’s quality of life and may have a high financial burden. Researchers at our institution developed a new animal model for studying IBD and other gastrointestinal diseases. We aimed to verify a recently published approach to transplant GFP-labeled colon organoids into a mouse’s intestinal tract via enema infusion. Organoids are 3D structures that are generated from stem cells or tissue progenitor cells. These small systems organize themselves to recreate the organ’s architecture and demonstrate organ-specific functionality. We hypothesized that colon organoids could easily engraft or integrate into a DSS-damaged intestinal epithelium by infusing the intestinal tract via enema with these organoids in suspension. Developing this model required several refinements to the original approach. We sought to ensure organoids remain in the mouse colon for several hours to settle and implant themselves into the damaged epithelium. The original published reference used tissue glue around the delicate skin and mucus membranes of the perianal region to allow for the infusion of organoids to remain in the GI tract for up to 6 hours. We determined we could refine the technique with a more consistent and less invasive approach by applying common veterinary methods to treat rectal prolapses and administering suppositories or enemas. The major areas of refinement that was added was the introduction of sterile petrolatum/mineral oil-based ointment after intrarectal injection of the labeled organoid infusion, followed by placement of a grain of Israeli couscous to function as an intestinal plug, coupled with a purse string suture around the perianal region. Shortening the time for intestinal infusion to 4-5 hours, suture was removed and animals were allowed to normally defecate. The animals were constantly monitored, and only 1 animal that presented hunched posture before the final timepoint had the purse string suture removed. Ten days post transplantation, proper engraftment of organoids was a success, as determined by microscopic examination of the intestinal epithelium and viewing GFP-labeled gastrointestinal stem cells. The potential for the ability of intestinal stem cells to repair damaged epithelium brings great hope for all inflicted with IBD.

P261 Assessment of Sensitization and Pain Medication Treatment in the Guinea Pig Maximization Test

RM Morales^*, PC Modine, T Smith, D Maillett

Quality Biology Laboratory, Edwards Lifesciences, Irvine, CA

The Guinea Pig Maximization Test (GPMT) is the most sensitive commonly employed skin sensitization assay used in preclinical testing of medical devices. The assay requires use of complete Freund’s adjuvant, which raises concern for the associated pain and distress that animals may experience throughout the test. As a potential refinement, 2 pain medications were evaluated for effect on GPMT in correctly identifying known sensitizers and alleviation of test-related discomfort. Testing followed ISO 10993-10 guidelines. Pain medication was given subcutaneously twice daily on the initial induction phase, and on bandage removal of the topical induction and challenge phases. The pain medications were Buprenorphine (0.05 mg/kg), an opioid, and Flunixin Meglumine (2.5 mg/kg), a nonsteroidal antiinflammatory drug. Test materials included polar and nonpolar non-sensitizers (0.9% sodium chloride (NaCl) and sesame oil (S.Oil)), an organic strong sensitizer (0.2% dintrochlorobenzene (DNCB)), and an inorganic weak sensitizer (1-3% nickel sulfate (NiSO₄)). Each test material group had 36 guinea pigs divided into 2 test groups (test material + pain medication) and 1 control group (test material only, no sham injection). Additionally, 2 groups of 6 guinea pigs were included to control for test material vehicles. Guinea pigs were weighed and clinically observed periodically. Observations included behavioral signs such as movement, vocalization, and pruritus. Throughout all treatment groups, DNCB had sensitization responses of intense erythema and edema while NiSO₄ had only mild erythema. DNCB showed a higher proportion (83% - 100%) and amplitude (scores 0-3) of sensitization than NiSO₄ (33% - 67%, scores 0-1). No sensitization (0%) was detected for NaCl, S.Oil, and the two negative control groups. Buprenorphine groups were generally characterized by diminished vocalization, resistance, movement, interactivity, and weight gain as compared to the no pain medication group. In contrast, Flunixin Meglumine groups had similar weight gain and clinical signs as the no pain medication group. In all cases, the GPMT correctly distinguished nonsensitizing and sensitizing materials, demonstrating noninterference of Buprenorphine and Flunixin Meglumine with the GPMT.

P262 Modulation of Kidney Development and Polycystic Kidney Disease by Folliculin Interacting Protein 1

R Centini^*, M Tsang, J Ramirez, H Gu, D Raftery, BM Iritani

Department of Comparative Medicine, University of Washington, Seattle, WA

Folliculin Interacting Protein 1 (Fnip1) is cytoplasmic protein which interacts with Folliculin, Fnip2, and the master metabolic regulator AMP kinase (AMPK). In response to low energy, AMPK stimulates mitochondrial biogenesis to produce more ATP, while inhibiting energy consuming biosynthetic and growth pathways regulated by mechanistic target of rapamycin (mTOR). Whereas the cellular and molecular functions of Fnip1 are unclear, the Fnip1 gene is amplified in up to 16% of renal cell carcinomas, suggesting an important role for Fnip1 in kidney development and/or function. In addition, Folliculin (encoded by the Bhd gene) is mutated in Birt-Hogg Dube’ Syndrome, an autosome recessive disorder in humans characterized by renal tumors, skin hamartomas, and lung cysts. We hypothesized that Fnip1 has a normal role in the development and function of the mammalian kidney, in part by regulating AMPK and/or mTOR activation. For this study, we used mice lacking Fnip1 (Fnip1^-/-C57BL6J), which we previously generated using ENU chemical mutagenesis, and wildtype controls (n = 4-8 mice per group), to define how loss of Fnip1 alters AMPK and mTOR activation, kidney metabolism, gene expression, and renal function. By comparing kidney/brain weight ratios to assess kidney size, we found that Fnip1^-/- kidneys are significantly larger than wildtype kidneys (P = 0.018). Hematoxylin and Eosin stained histologic sections showed increased numbers of small cysts in the renal cortex of the Fnip1^-/- relative to WT mice (P < 0.01). Metabolomics using mass spectrometry revealed increased levels creatinine and metabolites associated with amino acid metabolism and the Krebs cycle. Transcriptomics using RNAseq showed significant changes in the expression of genes involved in immunity, metabolism, and ion transport. Altogether, our data suggests important roles for Fnip1 in renal development, function, metabolism, and that alterations in Fnip1 expression may predispose to polycystic kidney disease and renal cancers by altering amino acid metabolism and ion transport.

P263 Species Variation in Pinworm (Aspiculuris tetraptera) Susceptibility in a Wild-Derived Colony of Mice of the genus Mus

RC Curtis^*1, J Murray¹, P Campbell², Y Nagamori³, A Molnar⁴, TA Jackson¹

¹Animal Resources, Oklahoma State University, Stillwater, OK; ²Integrative Biology, Oklahoma State University, Stillwater, OK; ³Veterinary Pathobiology, Oklahoma State University, Stillwater, OK; ⁴Statistics, Oklahoma State University, Stillwater, OK

Pinworms are common parasites found in wild and laboratory rodents. Despite being relatively nonpathogenic in immunocompetent models, pinworm infections add an unwanted variable and may play a role in compromising certain types of research. For this reason, health monitoring programs and biosecurity measures aim to minimize the spread of pinworm infections into colonies free from disease. Wild-derived and laboratory strains of mice infected with Aspiculuris tetraptera have shown varied susceptibility in the literature. Work with laboratory and wild-derived strains of Mus musculus has illustrated higher susceptibility in wild-derived mice, in younger animals, and in males. Routine surveillance at our institution revealed pinworm infection (Aspiculuris tetraptera only) within a colony of multiple, wild-derived species of Mus, although only certain species showed positive results during initial sampling. It was our hypothesis that there were differences in susceptibility between various species within the genus Mus. To examine this further, fecal samples from every cage of the colony were analyzed (using the Wisconsin egg-counting test) to assess fecal egg counts of Aspiculuris tetraptera. Our results revealed significant differences in susceptibility between various species/subspecies of Mus. Egg counts in M. spicilegus were found to be significantly higher than counts in M. m. domesticus (WSB/EiJ) (p < 0.001) and M. macedonicus (p = 0.001). M. spretus was found to have significantly higher egg counts than M. m. domesticus (WSB/EiJ) (p < 0.001), M. m. musculus (PWK/PhJ) (p = 0.003), and M. macedonicus (p < 0.001). We did not find significant differences in regard to age, sex, and number of mice per cage. As wild-derived mouse models continue to compliment research largely based on laboratory strains, it will be important to understand host-parasite interactions and their effect on research.

P264 Comparison of Cross-Foster Rederivation and Antibiotic Administration in the Drinking Water to Eradicate Bordetella pseudohinzii

SE Clark^*1, JE Purcell¹, X Bi², JD Fortman¹

¹Biologic Resources Laboratory, University of Illinois at Chicago, Chicago, IL; ²University of Illinois Urbana—Champaign, Champaign, IL

Bordetella pseudohinzii is a microbial agent of potential importance in mice and may confound pulmonary research. At our institution there is an association between infection with B. pseudohinzii and increased neutrophils (> 4%) in bronchoalveolar lavage fluid. We sought to evaluate cross-foster rederivation and antibiotic administration in drinking water as methods to eradicate B. pseudohinzii. To evaluate cross-foster rederivation 29 litters representing 16 strains of mice were cross-fostered from B. pseudohinzii positive cages to B. pseudohinziinegative Crl:CD1-Elite surrogate dams. To evaluate antibiotic administration, sulfamethoxazole at a concentration of 0.66 mg/ml and trimethoprim at a concentration of 0.13 mg/ml (TMS), and tetracycline at a concentration of 4.5 mg/ml were administered in drinking water. There were 3 antibiotic treatment groups with twelve B. pseudohinzii positive cages per group (6 cages of CD1 and 6 cages of C57BL/6 mice), TMS for 4 weeks, TMS for 6 weeks, and tetracycline for 6 weeks. Twenty-four of the 29 litters that underwent cross-foster rederivation were negative for B. pseudohinzii. Five of 12 cages treated with TMS for 4 weeks were negative for B. pseudohinzii 2 weeks post treatment, and 1 of 12 cages treated for 6 weeks with TMS was negative 2 weeks post treatment. Three of 12 cages treated with tetracycline were negative for B. pseudohinzii 2 weeks post treatment. Pearson chi square test analysis revealed a significant association (p < .0001) between method of eradication (cross-foster rederivation or antibiotic administration) and B. pseudohinzii infection, and an odds ratio estimate from a logistic regression demonstrated that cross-foster rederivation was more successful. Antibiotic administration in drinking water failed to eradicate B. pseudohinzii, however cross-foster rederivation was successful and has been used to establish a B. pseudohinzii negative barrier that has remained negative for 11 months.

P265 APE2 Couples SSB End Resection to Checkpoint Signaling in Oxidative Stress Response in Xenopus laevis Egg Extracts

S Yan^*

Biologic Sciences, University of North Carolina—Charlotte, Charlotte, NC

Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defenses. ROS include hydrogen peroxide and hydroxyl radicals, and are generated endogenously from normal cellular metabolism and exogenously from chemotherapeutic and environmental agents. Oxidative stress leads to DNA damage in genome, such as DNA single-stand breaks (SSBs) and AP (apurinic/apyrimidinic) sites. Oxidative stress is often the underlying pathology in a variety of diseases including cancer and neurodegenerative disorders. To eliminate oxidative DNA damage, base excision repair (BER) has evolved as a major DNA damage repair mechanism. However, it remains unknown how unrepaired oxidative DNA damage is sensed and recognized by cells to coordinate the cell cycle progression and DNA repair. APE2 (AP endonuclease 2) has weak AP endonuclease activity and strong 3’-phosphodiesterase and 3’-5’ exonuclease activities, playing essential role in PCNA-dependent repair of hydrogen peroxide-induced oxidative DNA damage. We hypothesize that APE2 may play an essential role in the DNA damage response (DDR) pathway. Xenopus laevis egg extract system is a cell-free biochemical system from extract derived from the eggs of African clawed frogs. Xenopus egg extract is supplemented with sperm chromatin (4000/μl) and hydrogen peroxide (100mM). After a 45-day incubation, total extracts were examined via immunoblotting analysis. We found that hydrogen peroxide triggers Chk1 phosphorylation at the Serine 344 residue, indicating the activation of the ATR-Chk1 DDR pathway. Notably, the hydrogen peroxide-induced Chk1 phosphorylation was compromised when endogenous APE2 was depleted in Xenopus egg extracts via immunodepletion with anti-APE2 antibodies. This observation suggests that APE2 plays a vital role in ATR-Chk1 checkpoint signaling in response t oxidative stress. We then added back wild type APE2 recombinant protein or mutant APE2 recombinant protein deficient for its exonuclease activity to the APE2-depleted egg extracts supplemented with sperm chromatin and hydrogen peroxide. Samples were further analyzed via immunoblotting analysis after a 45-min incubation. We found that wild type APE2, but not mutant APE2, rescued the hydrogen peroxide-induced Chk1 phosphorylation in APE2-depleted egg extracts, suggesting that APE2 resects the SSB in the 3’ to 5’ direction via its exonuclease activity to generate single-strand DNA for checkpoint activation. We have demonstrated distinct mechanisms of how APE2 couples SSB end resection with checkpoint signaling following oxidative stress. Our findings from Xenopus egg extracts system will help to better understand how cells trigger oxidative stress response to prevent genomic instability, a hallmark of cancer.

P266 Bone Marrow Transplantation with Autologous Lenti-GFP Transduced Cells in Nonhuman Primates for Assessment of Cell Mobilization in Tissue Regeneration

SS Lankford^*, A Dean, JK Williams

Regenerative Medicine, Wake Forest University, Winston-Salem, NC

A major question remaining in the development of tissue engineering medicine approaches to tissue and organ replacement is the role of injected/implanted versus native cells in tissue regeneration. The goal of this study was to address this gap in knowledge by developing a method to pullulate the bone marrow with autologous cells carrying a lentivirus promotor for green fluorescent protein (GFP). Bone marrow samples were collected from the humerus of 10 female cynomolgus monkeys (Macaca fascicularis). The buffy coat was isolated using gradient centrifugation and the sample was placed on a fibronectin coated plate and cultured in EGM-2. Adherent stromal cells were expanded in culture and labeled with a lentivirus GFP promoter. Five million autologous cells (30±5% of those exhibiting positive fluorescence for GFP) were injected intravenously 4 days following the monkeys receiving 3 intravenous injections (on 3 consecutive days) of busulfan (1 mg/kg/day). The recipient monkeys had surgically induced urinary sphincter deficiency produced 6 weeks post injection of the labeled cells. Tissue content of the labeled cells was assessed in the urinary sphincter complex. We analyzed the BMCs by immunohistochemistry. We will present data of successful transduction of BMCs by lenti-GFP (30% efficiency), including the presence of lenti-GFP+ cells in the bone marrow (30% positive) and the presence of lenti-GFP+ cells in the lamina propria of the urinary sphincter complex. Labeled cells strongly expressed markers for endothelial and stromal progenitor cells (CD133, CD34, CD117, and CD44) both in vitro and in vivo. It is concluded that this is a safe and effective method to efficiently label bone marrow cells for assessment of their migratory/mobilization behavior. Safety was assessed by examining the urinary sphincter tissue histologically for evidence of infection, inflammation, atypical cells, and necrosis. None were observed, nor were there any effects of treatment on plasma white cell counts, red cell counts, kidney, or liver function markers.

P267 Targeting tMUC1 for Treatment of Pancreatic Cancer Using Monoclonal Antibody TAB004

S Wu^*1, A Fowler², C Ogle², P Mukherjee¹

¹Biological Sciences, University of North Carolina—Charlotte, Charlotte, NC; ²Chemistry, University of North Carolina—Charlotte, Charlotte, NC

Pancreatic cancer (PC) is a lethal disease with a 5-year survival rate of 7% and an annual death rate of ∼41,000. Poor prognosis is the result of being diagnosed late in the progression. More than 80% of PC cases are locally advanced or metastatic at diagnosis and less than 20% of patients eligible for surgical resection. Pancreatic ductal adenocarcinoma (PDA) accounts for more than 85% of PCs and over 80% of PDAs over-express tumor-associated Mucin-1 (tMUC1), a membrane-tethered glycoprotein protein. Tumor-associated MUC1 exhibits changes to its glycosylation pattern that expose its protein core, thus making it identifiable to the TAB004, a specific antibody we developed and have shown to only recognize this form of MUC1. There is a need to diagnose pancreatic cancer efficiently and to target the delivery of anti-cancer treatments to specific areas to avoid adverse side effects in patients. Thus, we hypothesize that TAB004 antibody can be used as a targeting agent to accurately diagnose PDA and to increase the accumulation and duration of anti-cancer treatments, which will increase their overall therapeutic index. Confocal microscopy and high-resolution fluorescence microscopy were used to determine TAB004 specificity and internalization and nanoparticle (NP) internalization in several MUC1-expressing PDA cell lines. Cells were played into 4-well chamber slides and incubated with TAB004 conjugated to a fluorophore or fluorophore containing nanoparticles for increasing amounts of time, starting at 0. After the incubation process, the cells were fixed and imaged. NuLink kit was used in TAB004-NP conjugation (T-NP). Nanoparticles were suspended in sterile, ultrapure water and treated with the NuLink linking reagent and TAB004 antibody. Cell viability was determined using the Dojindo CCK -8 Cell and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The cells were plated and treated with different treatments, ranging from controls, blank nanoparticles, drug loaded nanoparticles, and TAB004-conjugated nanoparticles. After 1.5 hours of treatments, the cells were washed and allowed to incubate for 48 hours before results were read with a plate reader. An IVIS Spectrum was used to image mice injected with TAB004 conjugated with ICG, NPs with indocyanine green (ICG), and T-NPs with ICG. Mice were injected with TAB004-ICG, NP-ICG, and T-NP-ICG retroorbitally and intraperitoneally and imaged at increasing time points starting at 0. Results show that TAB004 was specific for and internalized significantly more in tMUC1 expressing cell lines, able to target tMUC1 expressing orthotopic tumors, able to increase the duration, and accumulation of NPs in tMUC1 expressing cells in vitro and in vivo upon conjugation. TAB004 is a promising targeting agent for diagnosing and treating tMUC1 expressing PDA.

P268 Postoperative Analgesia of Liposomal Bupivacaine in a Model of Incisional Pain in Rats (Rattus norvegicus)

S Kang^*1, K Jampachaisri², TL Seymour¹, S Felt¹, C Pacharinsak¹

¹Comparative Medicine, Stanford University, Stanford, CA; ²Naresuan University, Phitsanulok, Thailand

Bupivacaine is a local anesthetic that is valuable for multimodal analgesia. Here, we evaluated the application of liposomal bupivacaine for postoperative analgesia. The aim of this study was to assess whether liposomal bupivacaine effectively attenuates mechanical and thermal hypersensitivity in the postoperative period in an incisional pain model in rats. Rats (Rattus norvegicus) (n = 36) were randomly assigned to 5 treatment groups: 1) saline 1 ml/kg SC BID for 2 days (C); 2) buprenorphine HCl 0.05 mg/kg SC BID for 2 days (Bup HCl); 3) 0.5% bupivacaine 2 mg/kg SC local infiltration once (Bupi); 4) liposomal bupivacaine 1 mg/kg SC local infiltration once (Exp-1); 5) liposomal bupivacaine 6 mg/kg SC local infiltration once (Exp-6). Mechanical and thermal hypersensitivity were evaluated daily on days 1, 0, 1, 2, 3, and 4 post surgery. Bup HCl attenuated mechanical hypersensitivity for 2 days and thermal hypersensitivity for 1 day post surgery. Bupi attenuated only thermal hypersensitivity for 4 days. Exp-1 attenuated both mechanical and thermal hypersensitivity for 4 days post surgery. Exp-6 attenuated mechanical hypersensitivity on day 0 and thermal hypersensitivity for 4 days post surgery. These data suggest that Exp-1 effectively attenuates both mechanical and thermal hypersensitivity for up to 4 days in a rat incisional pain model.

P269 Use of Goat Antisera in Virus Identification for Global Influenza Surveillance

T Rowe^*, E Blanchard, X Xu, JL Weed, G Langham

Center for Disease Control and Prevention, Atlanta, GA

Can goats be used in virus identification for global influenza surveillance? Seasonal influenza outbreaks have the potential to affect 15-60 million people in the US each year. The majority of the influenza A (H3N2) viruses that circulated in 2014-15 influenza season were antigenic drift variants. This season was characterized by increased hospitalizations rates and higher mortality among affected populations. Constantly evolving viruses make it difficult to select the ideal strains of influenza viruses that will best protect against multiple cocirculating drift variants in the upcoming influenza season. Researchers are developing ways to rapidly deploy counter measures or test isolates to track these emerging pathogens. One approach is the use of the WHO Influenza Reagent Kit for Identification of Influenza viruses. These kits, sent worldwide each year, represent a simple and effective way to identify circulating influenza viruses with relative ease. Our question is whether the goat is a suitable model to use for reagents for virus identification for global influenza surveillance. The CDC is continuing to optimize methodologies to increase the precision of these kits in detecting emerging influenza viruses. Previously, sheep or ferret antisera were included in the kits. To improve sensitivity and specificity of the WHO kits’ antisera, goats were used in this study. Animals were primed (SubQ with 100 µg) and boosted (SubQ with 15µg) 2 to 4 weeks with bromelain purified hemagglutinins from representative seasonal influenza viruses, then apheresed at intervals following boosting. The methods chosen demonstrated that immunized goats provided larger volume of antisera (compared to ferrets) and demonstrated less nonspecific cross-reactivity than that obtained from sheep. Therefore, goat antisera appears to be a valuable reagent in addressing global influenza virus identification. In addition, reduction in time required to generate large quantities of quality antisera allows for refinement of antisera production. Furthermore, experimental animals are maintained and boosted with new variants each year to broaden the coverage of responses to current influenza viruses. These findings assure that high quality reagents are provided each year, reducing the number of animals used and refining the length of time they are on study.

P270 Anesthesia Does Not Affect Development of Diabetes in ZDF Rats

V Karicheti^*, Y Luo, R Velez, L Williams, GB Mulder, A Williams, JA Cornicelli

Charles River Laboratories, Wilmington, MA

The obese ZDF rat develops diabetes over time and is an excellent model of late stage type 2 diabetes. The development of diabetes in that strain is diet dependent and can be influenced by many environmental factors. Anesthesia has been associated with transient changes in blood glucose in rodents. However, long-term effects of anesthesia on regulation of blood glucose levels and development of the diabetic phenotype is not known in rodents. We sought to determine whether or not administration of anesthetic agents would alter the development of the diabetic phenotype in these animals. Thirty, 8-week-old male ZDF obese rats were distributed to 3 groups of 10 animals (ketamine/xylazine, isoflurane, and control). Diet, bedding, and water were provided ad libitum for this IACUC-approved study. Blood glucose (BG) was measured from a tail nick blood sample using a handheld glucometer. Anesthesia was induced by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (6 mg/kg), or inhalation of isoflurane using a chamber (2 to 4%) and followed by a 15-minute maintenance period (0.5 to 2%). Control animals experienced identical cage changes as the other groups. Immediately post anesthesia, food was returned to half the animals in each group, while the remaining animals were fasted through the acute sampling period. Acute BG was measured every 30 minutes for 4 hours. Chronic BG was measured once every week for 11 weeks (until 20 weeks of age). Fasted glucose was estimated 16 hours following collection of the nonfasting sample. All the animals tolerated the study well and showed similar weight gains post anesthesia. Ketamine/xylazine increased BG immediately following anesthesia in both fasted and non fasted animals, followed by a decrease compared to controls. BG in isoflurane animals was lower in fasted animals only relative to the control group. Long-term assessment of both fasting and nonfasting BG demonstrated no effect on the conversion to the diabetic phenotype in the animals. Based on these data, we conclude that ketamine/xylazine or isoflurane acute exposure does not affect the onset and development of diabetes in male ZDF obese rats.

P271 Prevalence of Rotavirus, Astrovirus, and Hepatitis E Viruses in Domestic Rabbits

W Xie^*1, J Bil², E Shantz², J Hammermueller¹, PV Turner¹

¹Pathobiology, University of Guelph, Guelph, Canada; ²University of Guelph, Guelph, Canada

Gastrointestinal viruses acting alone or in concert with other gut flora play a significant role in causing animal disease. Often, diseases caused by viral infections are difficult to characterize and differentiate from other infections, and there are few validated diagnostic tools for rabbits. Astrovirus, rotavirus, and hepatitis E virus infections are common in many species, and have been associated with enteric disease in both humans and rabbits, and rabbit hepatitis E virus is potentially zoonotic. None of these viruses are currently reported for rabbits in vendor quality assurance surveillance reports. We hypothesized that virus prevalence would differ between Canadian domestic rabbit populations, and further, that subclinical astrovirus infection would be more common in commercial meat rabbits, based on reduced biosecurity and hygiene practices. The prevalence of these 3 viruses was evaluated in pooled fecal samples collected from clinically healthy rabbits from 28 commercial rabbit farms (n = 101), individual companion and shelter rabbits (n = 76), and pooled rabbit fecal samples from 7 research facilities (n = 13). Viral RNA was extracted, reverse-transcribed into cDNA, and PCR amplified using rabbit-specific primers. Agarose gels run using the PCR products confirmed viral infections. Astrovirus was found in 50% of commercial rabbit samples, with one rotavirus positive and no rabbit hepatitis E virus. Rabbit hepatitis E virus was isolated from 3 companion rabbit samples. None of the 3 viruses were detected in pooled fecal samples from multiple specific pathogen-free research rabbits or the vendor from which these rabbits were sourced. The presence of astrovirus and rotavirus in farmed rabbits is of concern, as conventional research rabbits may be purchased from these sources. Although the research rabbits used in this study were negative for the tested viruses, continued monitoring of viral infections is important to prevent confounding factors in research, and improve understanding of clinical disease in research animals.

P272 A Study of Investigating High-Fat Diet with Coronary Balloon Compression Technique Modeling of Resting Electrocardiograph Effect

X Gao^*1, G Yang², Z Zhe¹, N Yin¹, J Pan¹, S Wang², Y Feng², Y Gao², H Zhou²

¹Department of Scientific Research, Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang City, China; ²Liaoning University of Traditional Chinese Medicine, Shenyang, China

This study investigates the effect of high-fat diet on pigs treated with coronary balloon compression to induce myocardial ischemic injury. The study proceeded for 48 weeks, with the effect of the diet being measured by resting electrocardiograph (ECG). Fiftt-five Bama minipigs were randomly divided into a control group of 5 and treatment group of 50. The 5 in control group received basic feed without coronary vessel injury, whereas the treatment group was given a high-calorie diet for 24 weeks. At the second week, coronary balloon compression was conducted at the left anterior descending coronary artery to induce vascular injury in the treatment group. ECG was recorded during the operation to generate a baseline measurement of cardiac function. At 0 week, 4th week, 12th week, 24th week, 36th week, and 48th week, 8-channel electrocardiogram was conducted and RR interval(s), heart rate (BPM), PR interval(s), P duration(s), QRS interval(s), QT interval(s), QTc(s), JT interval(s), Tpeak Tend Interval(s), P amplitude (mV), QRS amplitude (mV), ST height (mV), and T Amplitude (mV) was recorded. At the 0, 4th, and, 12th week, there was no statistical significance between the treatment group and the control group in ST height (P > 0.05), but at the 24th week, 36th week, and 48th week, ST height was significantly lower in the treatment group. Difference in T Amplitude between groups reached statistical significance by the 12th week (P < 0.05), with the T wave appearing flat and two-way or inverted in the minipigs that had received the high fat diet. In animals given a high-fat diet with coronary balloon injury, ST segment changes relatively slowly and T wave changes relatively early. Eight-channel electrocardiogram measurement, together with coronary artery angiography was one of the criteria for the evaluation of this animal model of chronic myocardial ischemia.

P273 Alfaxalone as an Adjunct Constant Rate Infusion Provides Safe and Stable General Anesthesia in Rats (Rattus norvegicus)

K Heng^*1, C Pacharinsak¹, K Jampachaisri²

¹Comparative Medicine, Stanford University, Menlo Park, CA; ²Naresuan University, Phitsanulok, Thailand

Because ketamine/xylazine provides an unreliable surgical plane of general anesthesia in rats, a constant rate infusion (CRI) of alfaxalone was added as an adjunct, and the combination was evaluated. The aim of this pilot study was to investigate whether alfaxalone CRI in combination with ketamine/xylazine safely and effectively extends a stable surgical plane of general anesthesia in rats. Rats (n = 8) were randomly assigned to 2 treatment groups: 1) KX - ketamine/xylazine (80/8 mg/kg, SC), and 2) KXA - ketamine/xylazine (80/8 mg/kg, SC) and alfaxalone (10 mg/kg/hr, IV). In both groups, time 0 was assigned when the rats lost righting reflex (RF) after KX injection. In the KXA group, tail vein catheter placement was initiated at time 0, and alfaxalone CRI was started at time 5. All rats were monitored every 5 minutes for 45 minutes and provided 100% supplemental oxygen throughout. The surgical plane was evaluated by a surgical stimulation (ST, 0.5 cm incision on right lateral thigh) after loss of jaw tone (JT) or paw withdrawal reflex (PWR). Measured anesthetic parameters included oxygen saturation (%SpO₂), heart rate (HR), respiratory rate (RR), apnea, and body temperature (T). Recovery time was evident by the presence of RF after subcutaneous atipamezole at 45 minutes to reverse xylazine’s effects. After full recovery, animals were monitored for 3 days. KX provided up to 15 minutes of a surgical plane with some animals responsive to JT or PWR. KXA provided up to 45 minutes of a stable surgical plane with unresponsiveness to JT, PWR, and ST. There were no significant differences in the measured anesthetic parameters (%SpO₂, HR, RR, or T). Apnea was not observed in either group. Animals receiving KX recovered faster than the KXA group after atipamezole. These findings suggest that alfaxalone CRI in combination with ketamine/xylazine safely and effectively extends the stable surgical plane of general anesthesia in rats.

Platform Sessions

PS1 Comparison of Per Diem Rates in Europe and the USA

J Hau^*, O Kalliokoski

Experimental Medicine, University of Copenhagen, Copenhagen, Denmark

Mice are used in large numbers in present day’s high-profile biomedical research, and housing large numbers of mice for a long time is expensive. Per diem rates for mice vary between universities and are a subject of much discussion between scientists. Institutions generally subsidize their laboratory animal care and use program with researchers paying per diem rates that only reflect a certain proportion of the real costs. It is often claimed by scientists that high per diem rates are inhibitory to their research, and render institutions noncompetitive compared to institutions offering low per diem rates. This is a statement with interesting far-reaching implications, which is why we decided to sample per diem rates at different institutions and to benchmark our own rates. We were particularly interested in investigating whether scientifically high-ranking institutions deliberately kept their per diem rates low to attract top scientists and stimulate scientific production. We analysed per diem rates at 37 research institutions (95 different programs), 28 universities, and 9 other nonuniversity research institutions. All per diem rates were calculated as median costs per mouse and day. The data are associated with some uncertainty because of the different caging types used at the various institutions and differing pricing models (whether researchers are charged by day or week and by animal or cage). As expected per diem rates were significantly lower in US institutions compared with European institutions, which may be due to overall higher costs in Europe than in the US. The per diem rates varied from 2 cents to $3 per animal, and the European Max Planck institutes were unique in not charging their scientists for animal accommodation. When plotting the per diem rates against the institutions’ ranking in the Times Higher Education World University Rankings no correlation could be found. This suggests that the institutions do not use per diem rates and subsidization of their animal care and use program as a tool to attract scientists reliant on mouse models in their research, or to stimulate the quality and quantity of their research performance.

PS2 Creation of a Risk Assessment and Operational Procedures Matrix for the Use of Hazardous Chemical in Animal Studies

M Zelivyanskaya^*, A Hall, M Blayney

Office for Research Safety, Northwestern University, Chicago, IL

A vast amount of health science research is based on the use of hazardous chemical compounds in animal studies. There are known and unknown hazards and risks associated with this research. Not surprisingly, there are potential hazards and risks to scientists and vivarium staff from these chemicals, as well. Despite decades of animal-based science, there is still no standardized risk assessment process for commonly used hazardous chemicals in the animal care facility. Of greater concern is that institutionally approved study precautions may not be practical or achievable within the operational limits of the vivarium. In cooperation with the veterinary staff, we developed and implemented a risk assessment and operational procedures matrix. For simplicity, we focused on the most commonly encountered hazardous chemicals in our animal studies. The risk assessment process included expected criteria such as animal species, dose, route of administration, and excretion. The information was further organized, not by individual chemical, but as generalized groups of chemicals with the same approximate degree of health hazard. The risk assessment information was then distilled into a table consisting of 9 operational procedures (and combinations of procedures) matched to the day-to-day operational standards of the vivarium. Each of the 9 procedures contains safety guidelines for husbandry (disposal of bedding, animal housing, and cage changing locations, etc.), personal protective equipment, and warning signs. The table was designed as a tool for investigators to complete the occupational health and safety section of the animal study proposal. Faculty report that they find this table helpful in preparing their proposals. The long-term effectiveness of this approach will be evaluated within the vivarium by comparing the approved work practices of the study with the operational outcomes and understanding of the animal care staff.

PS3 Out with the Old, In with the New: How We Replaced a Tunnel Washer in a Fully Functioning Research Facility

JR Chipps^*

Animal Resources Core, Nationwide Children’s Hospital, Columbus, OH

Typically, a tunnel washer is one of the largest and most expensive pieces of equipment in the vivarium. Like many large research facilities that are 20+ years old, our tunnel washer was becoming unreliable and in dire need of replacement. Size, cost, and sheer importance in day-to-day operation make the thought of replacing it a very intimidating situation. Roughly 80% of our caging needs were being washed in that specific machine. In addition to cost concerns, there were numerous operational concerns. We used a systematic approach to our machine replacement that required interaction with a number of different departments, including budgeting, finance, strategic planning, business process improvement, engineering, and epidemiology. Focus groups containing people from many different areas of expertise were constructed to determine machine needs, what functions were important, removal details, and installation concerns, among others. Cage wash functions were shifted to a secondary vivarium through use of a leased box truck and cage wash staff conducted mock exercises on driving the truck, as well as the process of large scale material handling. A failure mode analysis was also conducted to think through scenarios that could disrupt this process. A complete overhaul of our typical push/pull cage wash approach was needed during the project. Once the new machine was installed and functional, widespread testing and validation were completed while new wash techniques were found to increase operational efficiencies. The new machine has allowed us to streamline our push/pull approach while also being far more environmentally friendly (less water) and employee friendly (far less noise, steam, and better ergonomics). By using a thorough and systemic approach based on business improvement practices, our downtime and impact for such a large scale project was decreased significantly.

PS4 Location-, Equipment-, and Task-Associated Assessment of Lab Animal Allergen Exposure in Animal Care and Use Spaces

P Preisig^*1, JD Macy³, C King², D Dicks², D Ormrod², D Scavone², J Merk³, R Kline⁴

¹Medicine/Nephrology, Yale University, New Haven, CT; ²Environmental Health and Safety Office, Yale University, New Haven, CT; ³Comparative Medicine, Yale University, New Haven, CT; ⁴Coastal Safety, Clinton, CT

Laboratory animal allergen (LAA) exposure is a well-established occupational hazard of animal care staff and researchers, and an increasing concern for incidental exposure to others. Protecting staff is critical, but has cost implications. Thus, evidenced-based performance standards are needed to ensure appropriate protection while minimizing cost and regulatory burden. A systematic approach was taken using standard industrial hygiene exposure monitoring methods to collect data across animal care and use locations, equipment, tasks, and procedures to establish performance-based personal protection equipment (PPE) requirements. Using >5 ng/m3 mouse urinary protein (MUP) as a threshold for requiring respiratory protection, the data show that there is high exposure variability for tasks with the potential to generate larger amounts of allergens, e.g., cage dumping and cage changing without use of a clean air device (CAD). This variability is likely related to a combination of staff technique and ventilation control efficiency. However, MUP levels were consistently <5 ng/m3 in housing rooms, regardless of cage type or manufacturer, on the clean side of washroom, in animal facility hallways, and in staff breakrooms. Importantly, MUP levels were near or below detection in spaces outside the vivaria, e.g., in labs while surgical or other procedures were being performed and where mice were held for >24 hrs. These data suggest that: 1) respiratory protection is likely required when dumping cages, even when a CAD is used because of variability in staff technique and equipment, and when changing cages without use of a CAD, 2) LAA exposure is insignificant when entering vivaria hallways, animal housing, or procedure spaces and 3) the risk of incidental exposure is negligible when entering spaces where live animals are taken outside of the vivaria. A gown is required for handling live animals and/or soiled/used caging in any location, given the potential for direct exposure with clothing and the potential for subsequently re-exposing oneself or exposing others (second-hand allergens) to low levels of MUP until clothes are removed. Removing PPE requirements for vivarium hallways and animal room entries (no animal handling) should realize a substantial cost savings.

PS5 Smartphone or Smartphomite?: Evaluation of 6 Bacterial Sanitization Methods

MT Lieberman^*, CM Madden, JG Fox

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Smartphone usage has increased in popularity, with both smartphones and tablets used frequently in laboratory animal facilities. Portable devices present a risk for fomite transmission when devices are transferred between facilities. The goal was to evaluate different bacterial sanitization methods for smartphones. We hypothesized that ultraviolet light (UV) would be an effective nonliquid method to decrease aerobic bacterial colonization of phones. Sterile swabs were used to sample the smartphone face (PF), the phone-case junction (PJ), and the back and sides of the smartphone case (PC) before and after different sanitization methods. Phones were sanitized using 2 commercially available UV devices lasting either 30 seconds (UV-30) or 5 minutes (UV-300) according to manufacturer instructions, a bleachwipe (BW), quaternary ammonium disinfectant spray (QA), 70% ethanol spray (ET), or by cleaning with a sterile cleanroom wiper (KW). Swabs were plated on tryptic soy agar with 5% sheep blood and incubated aerobically overnight at 37°C. Following incubation, the number of colony-forming units (CFU) was enumerated for plates pre and post disinfection and percent reduction of bacterial colonies calculated. The mean and standard deviation for CFU on PF, PJ, and PC prior to sanitization were 17.5±24.1, 29.7±30.1, and 25.6±26.0, respectively. All sanitization methods were effective at decreasing bacterial colonization levels and there was no significant difference in percent reduction of bacterial colonization between UV-300, BW, QA, ET, or KW (Wilcoxon Signed-Rank Test). Percent reduction of bacterial colonies was significantly increased using UV-300 compared to UV-30 sanitization (P < 0.001, Mann-Whitney Test) and UV-300 was significantly more likely to reduce aerobic bacterial colonization to zero as compared to UV-30 (P < 0.001, Fisher’s Exact Test). Overall, “dry” sanitization methods using UV light or cleaning with a sterile cleanroom wiper were as effective in percent reduction of bacterial colonization as “wet” disinfection methods using bleach, quaternary ammonium disinfectants, or 70% ethanol. Further investigation is needed to evaluate optimal UV exposure times to reliably minimize aerobic bacterial colonization of smartphones.

PS6 Doing More with Less: Consoldiation of Small, Inefficient Facilities

J Percifield, MP Mottet^*

HDR Architecture, Atlanta, GA

Many Institutions have seen small to significant reductions in their research funding and have been forced to operate existing facilities and programs with less money. These instituions are looking for ways to improve the efficiency of their programs with various strategies, including higher-density caging systems, faster throughput washing equipment, and other means of making their animal facilities more efficient. Several Institutions have or have begun to assess their existing vivarium facilities inventory to determine if and what opportunities exist to consoldiate older, inefficient facilities into fewer, larger, and more efficient facilities. The first step in the approach to the consolidation of a group of facilities is to assess their condition and their functionality and to develop and document the baseline conditions. Older, inefficient facilities are highlighted, and the animal census and research programs are incorporated into a spreadsheet to track this data. Meetings with institutional leadership are used to discuss and document areas of anticipated growth so that the long-term needs of the institution can be charted against existing capacities. Discussions with vivarium operations leadership will uncover operational inefficiencies and individual facility challenges that will also impact consolidation opportunities. Finally, a thorough review of the animal progam’s financial status is required to understand the total operating costs and the sources and amount of income. The results of applying the outlined apppoach at several institutions vary, but many similar outcomes have evolved. First, most institutions do not have spare vivarium capacity, which makes the reduction of existing facilities a challange and often leads to the recommendation for a need for a new facility. Consolidations through the implementation of new caging systems and cage washing equipment have benefitted many institutions and allowed some smaller facility closures. However, major vivarium facility consolidations continue to be a challange. The review of existing animal facilities, their operational efficiencies, and the animal program financial structure can lead to lower operating costs for their institution.

PS7 Starting an AALAS Certification Mentorship Program through Your Local AALAS Branch

LJ Hughes^*

AHCS/NINDS, National Institutes of Health, Bethesda, MD

The AALAS National Capital Area Branch (NCAB) Education/Workshops Committee launched the NCAB AALAS Certification Mentorship Program (NACMP) for NCAB members seeking to obtain AALAS certification The goal of this somewhat unorthodox program was to foster AALAS certification among NCAB members. This program partnered a senior laboratory animal professional (manager/supervisor, trainer, senior technician with LATG certification, or a veterinarian) with a laboratory animal professional desiring to obtain AALAS certification. Since this program was newly launched, we solely focused on the Laboratory Animal Technologist (LATG) certification exam. This type of collaboration provided NCAB members the benefit of one-on-one training sessions with a senior lab animal professional, attentiveness to different learning styles, and scheduling flexibility. This also gave the mentee a personal cheerleader. So far, all of those who successfully completed the program and took the exam have successfully obtained LATG certification. This presentation will share the behind-the-scenes development and coordination of the program, the pros and cons of the program, selection process of mentors, mentees, and their pairings, as well as the use of technology to aid in the delivery of material and flexibility of one-on-one meetings.

PS8 Measuring Intracage Ammonia Concentrations: What’s All the Stink About?

R Morrow^*, R Wiler

TgT, Genentech, Davis, CA

The measurement of intracage ammonia is often used as a surrogate for quality of husbandry. The literature documents a variety of sensors and methods to measure intracage ammonia. However, attempts to duplicate results are a challenge due to poorly defined methods, incomplete reporting of methods, and differences in facility operations. Adopting a standardized methodology for ammonia measurement will benefit the lab animal research community by delivering reproducible results. Selecting the appropriate gas sensor, while understanding the limitations of a measurement device, is critical to understand how these factors affect the microenvironment and the generation of ammonia. We sought to develop defined methodology to measure intracage ammonia. A side-by-side comparison of 3 types of ammonia gas sensors: electrochemical (EC), photoionization (PID), and colorimetric was performed. The EC and PID sensors have continuous real-time measurements, while the colorimetric sensor provides a single snapshot of the intracage ammonia levels. Similar concentration results were achieved using the EC and PID sensors, but reproducible results were unachievable using the colorimetric sensor, a device that is commonly used for assessing intracage ammonia in the lab animal setting. Furthermore, ammonia concentrations should reflect a core data set that includes both macro- and microenvironmental ammonia, temperature, relative humidity, air changes per hour, bedding material and volume, animal occupancy, and latrine locations. By adopting a standardized reproducible measurement methodology, the lab animal research community will improve the reporting of intracage ammonia. Moreover, it will enable discussion and comparison between institutions.

PS9 An Economical Solution to Whole Room Scavenging of Chlorine Dioxide Gas

J Ludwig^*, JM Criley

University of Illinois, Urbana, IL

Chlorine dioxide gas generated with a portable gas generator is an effective method of sterilizing many pieces of equipment and animal rooms. The use of the generator requires multiple steps: sealing off a room or chamber, increasing the humidity in the room, introduction and exposure of room/chamber to the gas to set parameters, and removal of the gas. Room exhaust or active scavenging can both be used to remove gas from the room. The majority of animal rooms have dedicated exhaust without recirculation of air so room exhaust is typically used in our animal facilities. We have recently had requests to sterilize areas outside of the animal facility and without a portable active scavenging unit our decontamination services were limited to rooms with nonrecirculating room exhaust. The largest obstacle in obtaining a portable whole room scavenging system was financial. A commercial scavenging unit can cost between $1,800 and $2,000. With an understanding of the mechanics of a scavenger we were able to build a unit ourselves with materials available from local home improvement stores for less than $650. By adding a whole room portable scavenger we were able to increase our decontamination services to accommodate more areas of our institution and provide service to our larger campus community.

PS10 Fine Tuning Steam Sterilization: One Cycle Does Not Fit All

K Lux^*, K New, J Vollmer, R Santos

The Jackson Laboratory, Sacramento, CA

The use of steam sterilization is common practice in laboratory animal operations to prevent the introduction of pathogenic or opportunistic microorganisms. Controlling sterilization variables is critical to maintaining biosecurity. Although traditionally steam autoclaves are regularly evaluated and monitored to ensure their effectiveness via steam tape, biologic indicators (BIs), and/or chemical integrators, we suggest that additional variables are taken into account. As part of a steam autoclave optimization project, the following were evaluated for the 16 autoclaves that we have between our 2 sites: cycle parameters, load contents, load configuration, indicators, documentation, operator training, and machine differences. We first performed temperature distribution studies in an empty chamber to identify cold spots and create a baseline for autoclave operation. Materials were classified into families and material(s) within each family identified as the most challenging to sterilize within a cycle. The biologic indicator selection (product and concentration) was made after evaluating the bioburden of certain materials, ensuring that the BIs were more difficult to “kill” than our materials. BIs were placed in the center of the materials and strategically placed throughout the load during fractional studies (a process of reducing exposure time until the failure point is determined). Differences in load configuration were noted to impact sterility of materials, which lead us to establish minimum/maximum loads and load configuration guides for each cycle and autoclave. Several surprising lessons were learned regarding challenging materials and mechanical components that impact cycle effectiveness. Each unit must be evaluated with special consideration to the contents and their configuration, and it is critical to ensure that your quality control check is more challenging than your most challenging material, paying careful attention to the selection and placement of BIs. With the current trends towards maximum barrier and gnotobiotic housing, sterility is key to our research results.

PS11 It’s Not Where You Live but with Whom You Live: Housing Effects on Tumorigenesis in Murine Colon Cancer

J Zaias^*1,2, JK Lang³, R Dheer³, R Santaolalla³, M Phillips³, J Grant⁴, M Abreu³

¹Division of Veterinary Resources, University of Miami, Miami, FL; ²Pathology and Laboratory Medicine, University of Miami, Miami, FL; ³Gastroenterology, University of Miami, Miami, FL; ⁴Physiology and Biophysics, University of Miami, Miami, FL

Colitis-associated cancer is a widespread disease that affects 130,000 people annually in the US alone. Colitis-associated cancer can be modeled in mice by treating them with a combination of the genotoxic agent azoxymethane (AOM) and the colitis inducer dextran sodium sulfate (DSS). We aimed to see if environmental enrichment, social housing, and sex have an effect on intestinal tumorigenesis using the AOM-DSS murine model. We used aged-matched C57BL/6J (n = 64) mice that were randomly assigned to groups, each having an equal number of males and females. Mice were housed either singly or in groups (4 mice/cage) as a model for social housing. Nesting substrates were provided to half of the mice as environmental enrichment. All mice were given AOM (14.6mg/kg, i.p.) on day 1 of the study. On day 14, mice were administered a cycle (7 days) of 3% DSS. Following this, mice were given 2 weeks of recovery time and subsequently treated with a second cycle of 3% DSS for an additional 7 days. On day 56, we measured tumor number, tumor size, calculated tumor load, and characterized the inflammatory and neoplastic changes in the colon by histopathology. We also examined the welfare of the animals by recording body weights and nest scores over the course of the treatment. We found that singly housed animals (n = 24) get higher numbers of tumors (P < 0.05), and had an increased tumor load (P < 0.05) compared to their group housed counterparts (n = 32). Tumor number and load were also significantly higher in female than in male mice, especially when singly housed (P < 0.05). Environmental enrichment had no effect on tumorigenesis. Although environmental enrichment had no effect in tumorigenesis, sociality and sex both had significant impacts. These results provide performance-based data that can be used when providing recommendations for the use of social housing, environmental enrichment, and when selecting sex of study subjects. Ultimately, this study has shown that both social housing and sex can influence rodent welfare, revealing the importance of selecting proper environmental and housing conditions, specifically in murine models of colitis-associated neoplasia.

PS12 Animal Room Light Phase LED Light Enhances Nighttime Circadian Melatonin Inhibition of Rodent Hepatoma Metabolism and Growth

MA Wren-Dail^*1, RT Dauchy¹, AE Hoffman², JP Hanifin³, B Warfield³, GC Brainard³, SM Hill¹, VP Belancio¹, EM Dauchy¹, DE Blask¹

¹School of Medicine, Department of Structural and Cellular Biology, Tulane University, New Orleans, LA; ²Epidemiology, Tulane University School of Medicine, New Orleans, LA; ³Neurology, Thomas Jefferson University, Philadelphia, PA

Melatonin, produced by the pineal gland in mammals primarily at night, regulates linoleic acid (LA)-dependent metabolism and proliferation in human xenograft tumors in vivo.Melatonin inhibits LA-uptake and conversion to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE), a lipoxygenase product that enhances epidermal growth factor and insulin-like growth factor-I-induced mitogenesis. Previously, our laboratory demonstrated how transmission of light in the blue-appearing portion of the visible spectrum (465-485 nm) through standard rodent caging during light phase results in amplification of the nighttime melatonin signal in rats and leads to suppression of human prostate tumor metabolism and growth. Here we tested whether light phase exposure of hepatoma-bearing rats to white light-emitting diode (LED) lighting, enriched in blue emissions, results in amplified nighttime melatonin levels and influences tumor metabolism and growth. Male Buffalo rats (BUF/CrCrl; 250 g; n = 12/group) bearing “tissue-isolated” Morris hepatoma 7288ctc xenografts were maintained on a lighting regimen 12L(172.0 ± 12.3 lx; 70.5 ± 5.0 mW/cm² [inside cage]; lights on 0600):12D under control broad spectrum cool white fluorescent (CWF) or experimental LED lighting. Tissue-isolated hepatoma latency-to-onset of growth and growth rates were significantly delayed (P < 0.001) in the LED group (21 days; 0.36 ± 03 g/d) versus CWF group (11 days; 0.71 ± 0.03 g/d), respectively. Arteriovenous measurements were taken when tumors reached an estimated weight of 6-8g. Results showed that arterial plasma melatonin levels (mean ± 1 SD) were low in the light phase (1200 h) in both groups (1.0 ± 0.2 pg/mL), but significantly higher (P < 0.001) in LED (1065.0 ± 38.0 pg/mL) compared to CWF (154.0 ± 16.5 pg/mL) at peak, mid-dark phase (2400 h). Hepatoma xenograft cAMP levels, uptake-metabolism of LA to 13-HODE, aerobic glycolysis (Warburg effect) and growth signaling activities were significantly reduced in rats maintained in LED versus CWF lighting environments (P < 0.001). These findings are the first to show in vivo that amplification of nighttime melatonin levels by exposing rats to daytime blue-enriched LED light significantly reduces rodent hepatoma metabolic and proliferative activities.

PS13 Ophthalmologic Assessment of the Common Marmoset (Callithrix Jacchus) Involved in Visual Neuroscience

C Boisvert², J Mitchell³, SC Adams¹, J Reynolds⁴, M Leblanc^*1

¹ARD, Salk Institute, La Jolla, CA; ²UC Irvine Gavin Herbert Eye Institute, Irvine, CA; ³Brain and Cognitive Sciences, University of Rochester, Rochester, NY; ⁴Systems Neurobiology Laboratories, The Salk Institute for Biologic Studies, La Jolla, CA

The common marmoset (Callithrix jacchus) is gaining popularity as a leading nonhuman primate (NHP) model for visual neuroscience. As with the macaque, marmosets can easily be trained to perform complex behavioral tasks, have natural gaze behavior, and highly developed oculomotor systems that enable smooth-pursuit and saccades, as well as brains with organizational features very similar to humans. Marmosets have the added advantage of a nearly lissencephalic cortex that facilitates area mapping, laminar electrode recording, planar array recording, and 2-photon and optical imaging. In addition, marmosets are prolific breeders with a short generation time and are not carriers of serious zoonotic agents. The recent development of transgenic marmosets is also a major scientific breakthrough that will allow the exploration of complex brain functions in NHP using genome-editing tools. As is true in humans, marmosets are susceptible to refractive errors that may affect research outcome. Here, we report ophthalmology and visual acuity examinations in a colony of marmosets used in visual neuroscience. Across the colony, the mean refractive error was -0.35±1.48D for the right eye and -0.20±1.55D for the left eye. A high correlation coefficient (R²= 0.93, P<0.001) was found between the spherical equivalents of both eyes in individual animals. Refraction was correlated with age with mild hyperopia to emmetropia at a younger age evolving to myopia after 3 years of age. Myopia (as defined as a spherical equivalent refraction less than or equal to −0.50D) was found in 13 (36.1%) eyes, with 2 adult animals being severely nearsighted, with refractions exceeding -3.25D in both eyes. In those animals, vision would be blurry over a 30 cm working distance and would greatly limit performance on standard behavioral and visual tasks unless corrected. There was also a tendency for adult females (-1.06±0.84D) to be more nearsighted than males (-0.28±0.63D) (NS). There were no other pathologies observed on ocular examination. This study stresses the importance of screening marmosets involved in visual science for refractive errors and ocular pathologies on a regular basis throughout their experimental life and especially after 3 years of age to ensure the quality of research.

PS14 Characterization of Immunodeficient Mouse Models

J McClellan^*1, R Macasocal², C Buckner², T Hare², MJ Horn¹

¹Veterinary Science, Research and Support, Envigo, Indianapolis, IN; ²Envigo, Indianapolis, IN

Leakiness in SCID mouse models refers to the occasionally productive VDJ rearrangement leading to clonal expansion of these limited B and T cell clones. The rate and extent of leakiness in immunodeficient models is a factor of age, as well as housing environment and the background strain of the model. Here, serum IgG was used to determine the extent of leakiness in various immunodeficient mouse models; survival and lymphoma incidence was also analyzed. Male and female (n = 23/26) C.B-17-SCID, NOD SCID, SCID Beige, SHrN^®, Beige Nude XID, and Nude mice were housed in flexible film isolators. Blood was collected and analyzed using dried blood spot technology at 12, 16, 20, and 24 weeks of age for IgG concentration analysis using multiplex fluorescent immunoassay. The leakiness threshold was set at 5 µg/mL of IgG for analysis. At 24 weeks of age, necropsies were performed to determine incidence of lymphoma. At 24 weeks of age, the SHrN^® model had the smallest percentage of leaky animals followed closely by the NOD SCID. Beige Nude XID mice display an age associated increase in serum IgG levels, as well as an increasing percentage of animals above the 5 µg/mL threshold from 12-24 weeks of age. Nude mice also show increases in IgG from 12 to 24 weeks of age. The NOD SCID and SHrN^® mice had the lowest survival rate at 24 weeks; however, survivability in all models was greater than or equal to 79%. There is a positive correlation between the percentage of leaky animals and survival in males and females across all models. The C.B17-SCID shows low incidence of lymphoma at 24 weeks of age whereas the NOD SCID model has the highest incidence of lymphoma. But when the hairless mutation is crossed onto the NOD SCID mouse to create the SHrN^®, incidence of lymphoma is decreased. This is the first study reporting IgG concentration in multiple immunodeficient models to examine leakiness as well as 24-week survival and lymphoma incidence. Additional studies measuring immune cell activity and function will be useful to further characterize immunodeficient models; however, these data will be valuable information in determining the best model for xenograft research.

PS15 Daytime Blue-Enriched LED Light Enhances Nighttime Circadian Melatonin Regulation of Rodent Metabolism and Physiology

RT Dauchy^*1, MA Wren-Dail¹, AE Hoffman¹, JP Hanifin², B Warfield², GC Brainard², SM Hill¹, VP Belancio¹, DE Blask¹

¹Tulane University School of Medicine, New Orleans, LA; ²Department of Neurology, Thomas Jefferson University, Philadelphia, PA

Light entrains the master biologic clock within the suprachiasmatic nucleus (SCN), which tightly controls the nocturnal pineal melatonin signal, a critical contributor to the temporal coordination of circadian rhythms in metabolism and physiology of all mammals. Previous studies from our laboratory demonstrated that the spectral transmittance (color) of light passing through standard laboratory rodent caging impacts these responses in rats. Here, we examined the hypothesis that white light-emitting diode lighting (LED) during daytime, high in emission of light in the blue-appearing portion (460-480 nm) of the visible spectrum, compared to standard cool white fluorescent lighting (CWF), alters the nocturnal circadian melatonin signal, thereby altering normal metabolic and physiologic functions in laboratory rodents. Male Buffalo rats (BUF/CrCrl); n = 12/group) were maintained on a common lighting regimen 12L(172 ± 12.3 lx; 70.5 ± 5.0 mW/cm²[within cage]; lights on 0600):12D in translucent clear polycarbonate rodent cages in an AAALAC-accredited facility. After 1 week, animals were subjected to a series of six low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, 2000, and 2400) over a 4-week period to assess arterial blood melatonin, total fatty acid (TFA), glucose, lactic acid, pO₂, pCO₂, insulin, leptin and corticosterone concentrations. Results showed lower dietary and water intake, and body growth rates in LED versus CWF (P < 0.05). Plasma melatonin levels in pg/mL (mean ± 1 SD) were low (>2.0 ± 0.3) in the light phase (1200 h) in both groups, whereas peak, mid-dark phase levels (2400 h) in LED (1064.9 ± 37.7) were significantly higher compared to CWF (153.8 ± 16.5) (P < 0.001). Arterial blood diurnal rhythms of TFA, glucose, lactic acid, pO₂, pCO₂, leptin, insulin, and corticosterone levels were significantly altered in LED, compared to CWF (P < 0.05). Together with our previous results, the present findings suggest that daytime broad spectrum LED light with high blue emissions, compared to CWF light, has a profound positive impact on the circadian regulation of neuroendocrine, metabolic, and physiologic parameters that influence laboratory animal health and wellbeing, and ultimately the outcome of scientific investigations.

PS16 A Nonhuman Primate Model for Prediction of Clinical Outcomes in Regenerative Medicine Approaches to Tissue and Organ Repair

JK Williams^*1, A Dean¹, SS Lankford¹, K Andersson^1,2

¹Regenerative Medicine, Wake Forest University, Winston-Salem, NC; ²Urology, Aarhus University, Aarhus, Denmark

It is not unusual for animal models of disease to inaccurately predict clinical outcome of clinical studies. One such example is stem cell therapy for stress urinary incontinence (SUI) where preclinical studies report almost complete remission of symptoms, whereas clinical studies report only around 50% remission in 50% of patients. The answer is most likely because animal models (which create acute SUI in relatively young animals) do not represent the most common clinical scenario where SUI is most common as a chronic disease in peri/post-menopausal women with coexisting risk factors such as obesity and Type 2 diabetes. To better predict the effects of cell therapy for UI, we developed a cynomolgus monkey model of urinary incontinence (surgical nerve and muscle damage to the urinary sphincter complex) that reproduces the functional and structural changes in the urinary sphincter complex seen in women with clinical SUI. In these studies, we modeled both acute and chronic SUI in younger and older female NHPs with varying degrees of estrogen deficiencies and impaired glucose/insulin metabolism. With an n = 6 experimental group, autologous skeletal muscle precursor cells (skMPCs) were isolated from a muscle biopsy, expanded to 5 million cells, and injected directly into the urinary sphincter complex of NHPs with SUI. skMPCs almost completely restored sphincter muscle content and urethral pressures in younger (5-8 years) NHPs (P < 0.05 vs SUI/no treatment), but not older (15-28 years) NHPs (P > 0.05 versus SUI). This same pattern of efficacy was observed in NHPs with acute versus chronic SUI, in intact versus ovariectomized NHPs, in normal cycling dominant NHPs versus dysmenorrheic subordinate NHPs, and in normal weight/normal glucose metabolism versus heavier impaired glucose/insulin ratio NHPs. Thus, there are multiple determinants of cell therapy efficacy that can be modeled in NHPs and are critical to translational applicability of regenerative medicine approaches to tissue repair.

PS17 A Mouse Model to Study Zika Virus Pathogenesis

CM Nagamine^*1, K Majzoub², Y Ooi², R Mateo³, KA Kirkegaard⁴, J Carette²

¹Comparative Medicine, Stanford University, Stanford, CA; ²Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA; ⁴Genetics, Stanford University School of Medicine, Stanford, CA

Zika virus (ZIKV), a mosquito-borne virus in the family Flaviviridae, is an emerging pathogen that is associated with congenital microcephaly and Guillain-Barré syndrome, an autoimmune disorder where the immune system attacks the peripheral nervous system, leading in severe cases to life-threating paralysis. Well-characterized animal models are critical to generating insights into ZIKV pathogenesis and to test antiviral compounds and vaccines. The AG 129 mouse strain (129/SvEv-Ifnar1^tm1AgtIfngr1^tm1Agt) has knockouts for the receptors for both Type I (IFN-a/b) and Type II (IFN-g) interferons on the 129 genetic background. We placed the double KO on the C57BL/6 genetic background generating the AG B6 strain (C57BL/6J.129-Ifnar1^tm1AgtIfngr1^tm1Agt). We compared the DKO strains, AG 129 (8-9 weeks of age, n = 14) and AG B6 (8-14 weeks, n = 20), and the single KO strains, A B6 (C57BL/6J.129S2-Ifnar1^tm1Agt; 8 weeks, n = 22) and G B6 (C57BL/6J.129S7- Ifngr1^tm1Agt, 4-8 weeks, n = 14), to challenge from ZIKV (PF/13 strain). AG 129, AG B6, and A B6 were inoculated at 10³ and 10² pfu/mouse in a volume of 0.1 ml in Leibovitz (L-15) medium by either the retro-orbital (RO) or subcutaneous routes. Age-matched controls (AG 129, n = 3; AG B6, n = 5; A B6, n = 5) were inoculated with L-15 alone (0.1 ml, RO). G B6 mice were inoculated with ZIKV only (10³ pfu/mouse, RO). AG 129, AG B6, and A B6 strains were susceptible to ZIKV infections resulting in death or euthanasia, the latter based on the criteria of paralysis or loss of > 20% of initial body weight, between 8 and 13 days post inoculation (dpi). G B6 mice were resistant to ZIKV. The first indication of infection was weight loss that started at 4-5 dpi. While there was 100% mortality of the AG 129 and AG B6 mice, 4/22 (18%) of A B6 mice, inoculated SC with either 10³ or 10² pfu/mouse, survived. By characterizing this ZIKV model in adult and pregnant mice, this research will elucidate how ZIKV affects the developing fetus and the peripheral nervous system and will transform how we treat ZIKV infections by generating a desperately needed small animal model to test antiviral compounds and vaccines.

PS18 Murine Norovirus Alters B Cell Development in the Bone Marrow of Stat1 Knockout Mice

CC Hsu^*, SM Meeker, JA Ramirez, S Escobar, B Iritani, TL Brabb, L Maggio-Price

Comparative Medicine, University of Washington, Seattle, WA

Murine norovirus (MNV) is known to infect macrophages and dendritic cells. Recently, it was reported that MNV also infects B cells. Therefore, we hypothesized that MNV would alter the proportions of B cell subsets during development in the bone marrow of infected mice, and that this effect would be more pronounced in Stat1 knockout mice that have impaired responses to viral infections. Female, 7- to 8-week-old wild-type 129 mice (129S6/SvEvTac) and Stat1 knockout mice (129S6/SvEv-Stat1^tm1Rds) were infected with MNV-4 by oral gavage and B cells from the bone marrow were stained with markers to identify subsets of developing B cells including B220, CD19, CD43, IgM, and IgD, and stained cells were evaluated by flow cytometric analysis. Stat1 knockout mice infected with MNV had significantly (P < 0.05) decreased developing B cell populations in the bone marrow including pro-B/pre-B, immature, and mature B cells compared to mock-infected control mice. This effect was observed as early as 7 days postinfection (PI) as well as at 3 weeks PI. No differences in any B cell subsets were noted in MNV infected 129 mice versus. mock-infected control mice at 3 weeks PI. MNV was detected by RT-PCR in the bone marrow of all infected mice. Additionally, splenic B cells isolated from Stat1 knockout mice and then infected with MNV-4 in vitro showed a 1.9-fold and 3.0-fold increase in MNV-4 genome copies at 24 and 48 hours PI, respectively, when compared to MNV-4 genome copies at 2 hours PI. This increase in viral genome copies was not seen in similarly infected splenic B cells isolated from wild-type 129 mice. These results suggest that MNV-4 infection can alter B cell development in the bone marrow and can replicate in splenic B cells, and these changes are dependent on Stat1 deficiency.

PS19 Drug-Induced Neurotoxicity Assessments: Validation of a Neurologic and Functional Observation Battery in the Sinclair Miniature Swine

A Stricker-Krongrad^*, D Brocksmith, M Zhong, GF Bouchard, J Liu

Sinclair BioResources, LLC, Auxvasse, MO

The nervous system is one of the most complex organs in the body, and has its unique vulnerabilities to toxic compounds. The central nervous system, along with cardiovascular and respiratory systems, has been listed in the safety pharmacology core battery of the harmonized test guidelines for preclinical testing of human pharmaceuticals. A functional observational battery (FOB) has been recommended in the guideline as the first tier neurotoxicity screening that encompasses motor activity, behavioral changes, coordination, sensor/motor reflex responses, and body temperature. The miniature swine is an appropriate species for neurologic and behavioral studies. Its central nervous system and specifically the brain are similar to those of human with respect to tissue composition, gyrencephalic structure, and developmental growth and myelination patterns. In addition, behavioral studies, as well as modeling of neurologic human diseases, have been increasingly performed in minipigs. Because the miniature swine has increasingly been used as an alternative to dog or nonhuman primate in regulatory toxicology studies, we validated a FOB for the safety pharmacology assessment of pharmaceutical products intended for human use. A swine FOB protocol was developed with multiple observation parameters to monitor effects on 6 majors categories related to sensory and motor functions, autonomic and voluntary functions, and behavior. Known CNS acting agents were administered to the Sinclair minipigs via intramuscular route. Amphetamine, ketamine, and diazepam were demonstrated to positively or negatively dose-dependently affect different parameters related to autonomic functions, excitability, behavior, motor activities, gait, and reflexes.

PS20 Successful Group Formation of Mature Male Long-tailed Macaques (Macaca fascicularis): Can Minimizing Coalitions Help Reduce Aggression?

T Andrianjazalahatra, P Honess^*

Animal Welfare, Bioculture Group, Riviere des Anguilles, Mauritius

Forming groups of mature male macaques in captivity without potentially fatal aggression has long been a challenge. With roughly equal birth sex ratios in breeding facilities where breeding groups are single-/two-male, bachelor groups are a necessity for efficient use of caging. We aimed to reduce aggression and improve welfare when creating groups of long-tailed macaque males over 4 years old, by reducing the number of potential existing coalitions (cooperative relationships based on prior acquaintance) between introduced males through minimizing previous experience of each other. It was hypothesised that minimizing coalitions would reduce aggression recorded in animal records as fight injuries requiring veterinary attention. In a retrospective analysis of colony records (January 2010–November 2015) we compared aggression levels for 30 days following mergers using the new reduced coalition system (43 groups) and the old system (36 groups) (overall mean = 11.03/group). Study animals were group-housed outside under ambient conditions. Housing and care meets or exceeds standards set in the NRC Guide for the Care and Use of Laboratory Animals and the EU Directive on the Protection of Animals used for Scientific Purposes (2010/63/EU).There was no difference between methods in aggression (events/merger) (P = 0.771). However further analyses showed the new system produced 47% lower aggression (P = 0.048) among wild-caught males and 86% higher aggression (P = 0.031) in captive-bred males. In conclusion, success of the refined system depends on the animals’ origin highlights the importance of tailoring the management system to the characteristics of the animals. These findings have relevance for those working in a range of captive contexts including breeding facilities, laboratories, zoos, and sanctuaries with potential significant improvement in animal welfare during mergers.

PS21 Murine Astrovirus Infection and Detection

SR Compton^*, JD Macy

Yale University School of Medicine, New Haven, CT

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice, yet little is known about its pathogenesis. Our goal was to determine the duration of MuAstV infection, susceptibility of pups, and the efficacy of soiled bedding sentinels and environmental monitoring. Eight CD1 dams with litters of 3-day-old mice and 8 CD1 dams with litters of 13-day-old mice were inoculated orally with filtered colon homogenates containing MuAstV. No clinical signs were observed in the dams or pups and MuAstV had no effect on weight gain in pups. MuAstV RNA was detected in feces from 15/16 dams for 18 days postinfection (dpi) and 9 dams were shedding MuAstV at dpi 29 or 39. MuAstV RNA was highest in intestines of mice infected at postpartum day (ppd) 3 at 12 dpi and highest in intestines of mice infected at ppd 13 at 4 dpi. Low levels of MuAstV were sporadically detected in spleen, liver, and kidney of mice infected at 13 dpi. MuAstV was detected in the feces of all 3 to 6 week-old weaned mice and in 94% of feces from 7-9 week-old mice. Many of the dams became pregnant immediately after delivering their first litters and intestines from their second litter pups were MuAstV negative at ppd 0-5, indicating that fostering may be feasible for generating MuAstV negative mice. Weekly swabs of cages housing MuAstV infected mice were MuAstV positive through 6 weeks. Swabs of the rear plenum of the ventilated rack were MuAstV negative at 6 and 13 dpi and positive at 20 dpi and beyond. Ninety-seven percent of sentinels that received soiled bedding from cages at 7-18 dpi and 80% of sentinels that received soiled bedding from cages of 4-6 week old mice were MuAstV positive. Feces from sentinels exposed to soiled bedding stored for 1, 2, or 3 weeks prior to addition of the sentinels were MuAstV positive indicating that MuAstV is stable in the environment for several weeks. Collectively, these results show that MuAstV is shed for extended periods, from 3 weeks to more than 5.5 weeks in dams and at least 8 weeks when the mice are infected as pups; detection of MuAstV on plenums was effective at 2 wpi; soiled bedding sentinels and cage swabs were effective from 1 wpi; and MuAstV remained infective in soiled bedding for at least 2 weeks.

PS22 Laboratory Mice Are Colonized with Colibactin-Encoding Escherichia coli

A Garcia^*1,2, A Mannion², CM Madden², V Bakthavatchalu², Y Feng², Z Shen², Z Ge², JG Fox²

¹Molecular Sciences Research Center, University of Puerto Rico, San Juan; ²Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Colibactin (Clb) is a genotoxin encoded by Escherichia coli strains belonging to phylogenetic group B2 that have been isolated from humans with intestinal diseases, including cancer as well as from urogenital disease, meningitis, and bacteremia cases. E. coli NC101 is a prototype murine Clb-encoding E. coli that has been used to experimentally infect mouse models and investigate cancer pathogenesis. However, the relative prevalence of naturally occurring Clb-encoding E. coli in laboratory mice is unknown. Furthermore, E. coli, historically considered a commensal, has not been excluded from laboratory rodent colonies. We obtained rectal swabs or feces, and/or extraintestinal samples from surveillance (sentinel) mice [Crl:CD1(ICR)], a breeding colony (which supplies mice for our surveillance program), 2 commercial vendors (1 vendor supplied mice for our breeding colony), and nonsurveillance (nonsentinel) mice (various strains). The extraintestinal samples included nare/lung wash samples from surveillance mice (collected as part of our health monitoring program) and uterine samples from clinically affected genetically modified mice (collected as part of necropsy procedures). Samples were cultured and E. coli isolates were characterized biochemically, molecularly, and by serotyping and cytotoxicity assay. Fifty-one E. coli isolates were obtained from 45 surveillance and nonsurveillance mice. Forty-five of 51 (88%) isolates were PCR positive for Clb genes (clbA and clbQ). Clb-encoding E. coli belonged to phylogenetic group B2. Selected E. coli isolates were serotyped as O2:H6, OM:H+, O22:H-, and O-:H-. Clb-encoding E. coli induced megalocytosis, a cytotoxicity phenotype, in HeLa cells. A Clb-encoding E. coli was isolated from a uterine sample of a clinically affected genetically modified mouse diagnosed with cystic endometrial hyperplasia. Our study suggests that laboratory mice are colonized with Clb-encoding E. coli that may be associated with disease and potentially confound research studies. This is the first report documenting the presence of Clb-encoding E. coli in laboratory mice and suggests the monitoring and exclusion of these Clb-encoding E. coli strains from rodent colonies.

PS33 Reduce, Replace, and Refine the Due Diligence Process for Vendors, Contract Research Organizations, and Academic Research Organizations

CM Charlebois^*

MRL QA Animal Welfare, Merck, West Point, PA

Site visits to vendors, contract research organizations (CROs), and academic research organizations (AROs) are a necessary part of the due diligence process. Universities, government, and pharma spend significant funds sending staff to service providers to evaluate their quality and compliance. If resources are not available to conduct site visits, compliance may be at risk. Service providers are also impacted due to the commitment of multiple staff members for visits that may last several days. Site visits can be inconsistent due to the auditors’ qualifications and experience, questions posed, time allotted, and available access to the facility. Additionally, these site visits are duplicative as each university, government agency, and pharma conducts a site visit to each service provider. Alternatively, the lab animal industry can use the 3Rs to decrease the resources needed to ensure the animal care and use program at any service provider meets the same high standards of your own institution. Options may include sharing site visit reports, using third party auditors, and standardizing the site visit process. We will initiate a conversation about these options and the way they can reduce the industry’s resources for site visits while still ensuring animal welfare compliance at CROs, AROs, and vendors.

PS34 The New R: How an Animal Welfare and Ethical Review Body in the UK Is Tackling Reproducibility

JC Gebbie^*, B Whitelaw

The Roslin Institute, Easter Bush, United Kingdom

The remit of an Animal Welfare and Ethical Review Body (AWERB) in the UK includes encouraging implementation of the classic 3Rs of humane animal science. Of late, there have been several new Rs appearing in science publications and media, lead by “reproducibility.” Our AWERB recognised that reproducibility of scientific studies is fundamentally an issue of science quality and is critically important in animal studies. Studies that cannot be reproduced because of poor quality work represent use of animals that has not achieved a useful objective and therefore the animals’ adverse experiences and lives have been wasted. Also public trust in animal science will be eroded if it is not perceived to meet high standards.We established a reproducibility subgroup to consider issues related to reproducibility and identify means to improve the reproducibility of animal work conducted at the institute. To achieve this aim we have shared and discussed relevant publications and guidelines, introduced the subject to academic development seminars, and incorporated reproducibility issues into a new “ethics day” for PhD students and postdoctorate staff. We have also trialled a process of following the paper trail of a study from planning to publication to see how reproducible the study has been and what we can learn about practices in our Institute which affect reproducibility. The AWERB also has a statistician member who raises general issues regarding experimental design and statistical analysis and is also involved in the local review of detailed study request forms.The number of publications on reproducibility is growing and our scientists are increasingly aware of these issues. The AWERB has taken the lead in our Institute because of the importance of reproducibility in animal studies, but it is a wider issue for all our science. Our conclusion is that this is an issue that is not going to go away. The AWERB needs to be actively involved in identifying relevant issues in our Institute, gathering sources of advice and tools, and promoting communication and education through creative means.

PS35 A Robust and Mandatory Training Program Promotes High-Quality Science

NA Rogers^*, E Horrigan, JG Fox

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

In casual conversations with training coordinators at other academic institutions, there often seems to be a concern to not burden researchers with mandatory training, instead presenting a minimal slate of required introductory training and offering further training as optional or merely recommended. In our experience in a large and diverse academic research program (with over 1,500 active animal users), we have found that a comprehensive mandatory training program for the investigative community (students, postdocs, staff, and faculty) is not only achievable, but has reaped rewards in compliance, competency, and fostering a spirit of cooperation and collaboration with the research community. Over the course of the past decade, we have dramatically expanded our training program while simultaneously streamlining and simplifying the onboarding process for investigators. Mandatory introductory training for all animal users now includes online e-courses, hands-on species-specific training, and animal facility orientations. Supplemental training in surgery, hazmats, imaging, irradiation, and food/water regulation is required, as necessary. Continuing education for all mouse and rat users comes in the form of an annual “rodent refresher”; an online continuing education program for nonhuman primate users is to be launched in the fall of 2016. Compliance is achieved by gating certification to work with animals, protocol approval, and access to animal facilities through completion of the requisite training. Efforts have been made to minimize growing pains; new mandatory training is phased in where feasible and, when warranted, can be waived for long-standing investigators. All training can now be completed or scheduled online. While the requirements can appear formidable on first glance, feedback from researchers has been positive; comments commending our staff both for our efforts and the scope of our training program are common. The rewards of instituting a robust training program extend beyond our own animal care program to the broader scientific community—more thorough training fosters better informed and skilled researchers, well cared for animals, and, ultimately, higher-quality research.

PS36 Spreading the Word: Creating a Veterinary Technician Student Outreach Program

TT Mufford^1,2, JA Reisinger^*2

¹Office of Animal Resources, University of Colorado—Boulder, Boulder, CO; ²Division of Medical Oncology, University of Colorado, Aurora, CO

Veterinary technicians play a unique and vital role on the laboratory animal professional team. They are an essential bridge for various species, specialties, and procedures for which traditional clinical veterinary technicians generally are not prepared. Most vet tech degree programs offer little more than basic introduction to laboratory animal medicine, as required for AVMA for accreditation. This amounts to the equivalent of 3 credit hours of lecture, minimal hands-on experience in technical procedures, and virtually no exposure to research vivaria. Oftentimes this leads to misunderstandings of the mission of the lab animal field, and fosters most to seek positions in companion animal clinics. We sought to change the negative bias towards the field by starting an outreach program geared towards veterinary technician students. Through networking with instructors in local technician programs, we integrated standard lab animal classes to generate exposure and interest, as well as to educate students on the wide variety of related topics. Lectures were geared towards the importance of the veterinary technician in research, relevance to their lives, and educational and advancement opportunities with employment. A central theme focused on how a background in animal science is precisely what the field needs for healthy animals and valid science. Instructors surveyed the students pre and post lecture with questions regarding student feelings towards lab animal medicine. The overwhelmingly positive results from these lectures was used to tailor future discussions to answer the most pressing concerns about the field, and help guide expansion of the outreach program. Over the last 4 years, our lectures have been presented to 32 classes and hundreds of students at 5 different vet tech schools. Although discussing a career in laboratory animal research can be a sensitive subject, this program has demonstrated that with the proper information, enthusiasm, and personalization on how animal research has affected their lives, outreach programs should help persuade this essential cohort to embrace their role in supporting animal research.

PS37 Real versus Perceived Costs of Self-Imposed Regulatory Burden in Different Administrative Models of Animal Welfare Assurance

JM Wallace^*1, VK Bergdall⁶, JF Bradfield¹⁰, LA Conour⁴, LJ DeTolla⁷, AW Grady¹¹, DL Hickman⁹, SM Niemi⁵, JN Norton³, JD Thulin², BJ Weigler⁸

¹Pathology, Microbiology and Immunology/Comparative Medicine, Vanderbilt University Medical Center, Nashville, TN; ²Biomedical Resource Center, Medical College of Wisconsin, Milwaukee, WI; ³Division of Laboratory Animal Resources, Duke University, Durham, NC; ⁴Laboratory Animal Resources, Princeton University, Princeton, NJ; ⁵Office of Animal Resources, Harvard University, Cambridge, MA; ⁶University Laboratory Animal Resources, The Ohio State University, Columbus, OH; ⁷Program of Comparative Medicine, University of Maryland, Baltimore, MD; ⁸Office of the Vice President for Research and Innovation, University of Nevada, Reno, Reno, NV; ⁹Laboratory Animal Resource Center, Indiana University School of Medicine, Indianapolis, IN; ¹⁰AAALAC International, Frederick, MD; ¹¹Laboratory Animal Facilities, University of Mississippi Medical Center, Jackson, MS

A growing number of biomedical research institutions, particularly in academia, have organizationally separated administrative support for their IACUC from their animal resources program (ARP), with each entity reporting separately to their institutional official (IO). There are a variety of valid management rationales for adopting this particular organizational structure, including the limited veterinary management resources in small programs. However, a common driver has been the fear of a purported conflict of interest for the attending veterinarian (AV), who typically directs the ARP. The reasoning is that in also directing the IACUC administrative staff, the AV could unduly influence IACUC activities and decisions, especially as they pertain the the IACUC’s regulatory responsibility for oversight of the ARP. Yet by the same reasoning, any individual charged with directing the IACUC administrative support could be similarly conflicted because each could inappropriately influence IACUC activities and decisions for personal advantage unrelated to welfare concerns. In spite of this largely theoretical conflict, the specific separation of IACUC administrative support from ARP management is repeatedly portrayed as a best practice, a characterization too often misinterpreted as favored or required rather than merely one of several acceptable options. In addition to claimed advantages, separation of IACUC and ARP administrative support has drawbacks, including duplication of effort, inefficiencies, and unnecessary discord. These outcomes in turn can result in increased costs and regulatory burden without appreciable benefit to animal welfare or research quality. We considers the merits of an alternative organizational arrangement in which both ARP and IACUC support staff report to the AV, preserving the tripartite approach for trustworthy oversight involving the IO, IACUC, and AV. Effective communication, full transparency, constructive conflict resolution, and mutual respect are essential and ensure a healthy system of checks and balances.

PS38 Making Matters Worse: A Snapshot how Outside Influences Can Impact Model Endpoints and Animal Welfare

JK Lang^*1,2, J Zaias³

¹Department of Medicine/Gastroenterology, University of Miami, Miami, FL; ²Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA; ³Divison of Veterinary Resources, University of Miami, Miami, FL

Suggestions for study design often come from outside sources. For example, IACUC directives can change animal protocols, especially when related to welfare matters. The Guide has been updated with recommendations for enrichment and social housing. Recently, the NIH has strongly suggested using both sexes in animal experiments. It is difficult to understand how these potentially positive directives can impact longstanding predictable model endpoints. We demonstrate how outside influences suggesting alterations to study variables can impact model outcomes and animal welfare. Our lab studies colitis using DSS and colitis-associated cancer (CAC) using AOM-DSS. Both are widely accepted models in the GI field. Using 2 separate study designs, we tested the effects of several variables which were recently addressed by the Guide, NHI, and our IACUC. These variable were environmental enrichment, social housing, using both sexes, and fluid therapy as a medical refinement. We performed a CAC study using various housing variables, enrichment, grouping, and sex to determine how strongly these might affect our model outcome. Our results demonstrate these variables had an effect on the tumorigenicity of our model, as nonenriched single-housed females were prone to higher tumor numbers and loads than the other groups.Our IACUC suggested we hydrate our DSS colitis mice. We provided fluid hydration and examined the mice at several time points during the study. Interestingly, mice had dramatically lower inflammation when provided with fluids. We were no longer able to use our protocol as a robust model of colitis. Therefore, the outside proposal from IACUC, which was suggested to refine our model, ultimately would have caused an increase in sample size, as many of the animals were not inflamed enough for our studies.Both study results highlight the need for strong standardization of experimental design. Seemingly minor deviations in the environment can cause significant differences in experimental outcomes. This can be difficult and limiting due to animal availability, cost factors and convenience. Hence prior to accepting a suggestion from an outside source, it is important to consider what sort of impact minor changes could have on your model.

PS39 Engaging Investigators Using a Diversified Approach to Postapproval Monitoring (PAM) Process

E Koncza^*, E Kapsalis, K Sklavounos, J Zaias

Office of the Vice Provost, University of Miami, Miami, FL

The purpose of our postapproval Mmnitoring (PAM) processi is to engage with animal users in constructive exchange, to assist researchers in identifying and correcting any deviations, and to verify research is being conducted as approved. Our initial PAM process included semiannual inspections, monitoring by veterinary personnel, and limited one-on-one meetings with the PI and IACUC Director. In 2011, a new PAM process was launched revealing that of those investigators that underwent the process, a significant number had medical recordingkeeping deficiencies. It soon became evident that a typical PAM process took much longer than we originally expected and that we needed to develop a more systemic and time-efficient approach. In 2012, a process called self-PAM was initiated in which investigators perform their own internal protocol monitoring and report their outcomes to the IACUC. In 2014, a pre-initiation monitoring (PIM) process was piloted. The PIM process consists of additional protocol review and discussion with individual investigators, who are either new to animal work at UM or their protocols are at high risk for noncompliance. The PIM process occurs prior to any animal work by new investigators on newly approved protocols. During the first year, of 22 investigators that participated in PIM, 19 opted to revise their protocols subsequent to the feedback they received during the PIM process. In 2015, a new cross-PAM process was piloted. In this process, we focus on a particular compliance concern such as rodent enrichment or tumor burden and review its implementation across several investigators’ protocols. In first 5 months, we were able to work with 69 investigators and 115 protocols. Diversifying our approach to PAM increased our efficiency in reaching animal users and provided us with an expanded platform for positive educational and preventive interaction with investigators.

PS40 Rodent Survival Surgery: Where Are Your Investigators Learning Aseptic Technique?

JL Haupt^*, JG Fox

Massachusetts Institute of Technology, Cambridge, MA

Aseptic surgical technique for rodent survival surgery is mandated by the Guide, as well as being required in many institutional standards. Proper training of sterile surgical techniques and specific surgical procedures is vital for institutional educational programs and for the welfare of laboratory animal species. With advances in web-based training, many investigators are seeking outside sources for procedure specific training. Concerns regarding these resources include the demonstration of inappropriate sterile surgical technique, as well as inadequate anesthetic and analgesic regimens. To investigate potential deficits in these areas, 100 randomly selected video-based articles on the topic of rodent survival surgery were assessed for aseptic surgical principles, appropriate tissue handling and wound closure, analgesic and anesthetic use, and postoperative monitoring. All videos were viewed and scored by a boarded veterinary surgeon. Assessment of the video articles revealed that only 44% of the articles described or demonstrated appropriate preoperative skin disinfection. In addition, surgical asepsis was deemed to be maintained in only 10% of the videos with 51% and 35% having minor and major breaks in aseptic technique, respectively. Appropriate tissue handling and wound closure were present in only 67% of the videos and 13% of videos had no mention or use of analgesics. This study highlights many of the potential deficits in video-based articles for rodent surgery and alerts the laboratory animal community regarding the use of these video articles by investigators for purposes of surgical training.

PS43 Comparative Review of Key Issues for Biocontainment and Gnotobiology IVC System Use versus Glove-Box Isolators in Rodent-Based Research

PR Hardy^*1, PC Willan²

¹Veterinary & Professional Services, Allentown Inc., Bussy Saint Georges, France; ²Allentown Europe, Reading, United Kingdom

Biosafety requirements include the use of biosafety cabinets or other physical containment devices (primary containment) and suitable working practices. They must respond to a risk assessment and a related mitigation plan, be validated accordingly, properly maintained, and used by experienced and qualified personnel. Both for animal care and use, practical constraints and risk assessment outcome related to the use of BSC-III/glove-box isolators (sensitive procedures, space requirements, ergonomics, use of sharp items, etc.) led to the development of innovative biocontainment IVC systems, associated to the use of suitable biosafety cabinets. Such alternatives must respond to the specific and integrated needs of scientists and technicians carrying rodent-based research, as well as to biocontainment regulations and best practices and biosafety officers expectations. A comparative review of the design, validation, and use of IVC-based systems and associated working cabinets allows comparing their benefits and drawbacks when compared to isolator use. A similar reverse approach was used to develop, validate, and use bioexclusion IVC systems designed for gnotobiology study requirements. Provided that the solution is selected according to the specific features of each option, to the outcome of a comprehensive risk analysis, and that, as illustrated by examples, appropriate training and practices are used, IVC-based solution represent a reliable and practical alternative for biocontainment and gnotobiology.

PS44 Combined Use of Isolators and IVC System for Production, Maintenance, and Experimentation with Axenic Animals

J Bom^*1, K Berman², A Ribeiro¹, M Pereira¹, AS Leocádio¹, M Rebelo¹, J Demengeot¹

¹Animal House Facility, Instituto Gulbenkian de Ciência, Lisboa, Portugal; ²Baylor College of Medicine, Houston, TX

In 2005 we started with rigid walls isolators for axenization and maintenance of mutant and wild-type mouse strains of different genetic backgrounds. Gnotobiology techniques allow maintaining animals in axenic environment, with the possibility to restore specific components of the microflora. In 2012, the need to implement gnoto experiments led us to reorganize space, optimizing human and financial resources. The IsoCage P system was designed to achieve the safety and the protection features of an isolator, with the advantages in terms of ergonomics, flexibility, and density of an IVC. Each cage in an IsoCage P system is a microisolation cage, allowing multiple studies on the same rack, providing full bioexclusion for maximum protection, excluding cage-to-cage contamination. This IVC system has been in use since 2013 for experiments with GF animals, in combination with the IsoCage Biosafety Station (IBS). The IBS was specifically conceived to enable the manipulation of cages and animals in a fully sterilized environment. The use of the IsoCage P system for gnoto experiments allowed freeing space in isolators and expanding the number of GF animals produced. From 2013 to 2015, 44 different experiments were performed using this system. Standard operational procedures have been implemented and improved, registering a decrease of contaminated experiments from 45% to 5-10%. To validate our working system, GF sentinel animals were implemented in the IsoCage P system. These animals are manipulated exactly the same way experimental animals are, and their health status checked every month for quality control. The ongoing results demonstrate that sentinels have been maintaining their axenic status for more than 1 year, indicating that our system is working efficiently. In summary, the combined use of isolators for production and maintenance of GF animals stocks and IsoCage P system for experimentation with axenic animals has been running for the last 3 years. Based on the low fraction of contaminated experiments and our sentinel program we conclude that this system was successful, optimizing human, financial, and space resources.

PS45 Effects of Bedding Material and Water Treatment on the Gut Microbiota

WA Bidot^*1, A Ericsson^2,3, CL Franklin^2,3

¹Veterinary Pathobiology and Comparative Medicine Program, University of Missouri, Columbia, MO; ²Metagenomics Center, University of Missouri, Columbia, MO; ³Mutant Mouse Resource and Research Center, University of Missouri, Columbia, MO

Rodent models are invaluable to understanding health and disease in many areas of biomedical research. Unfortunately, many models suffer from lack of phenotype reproducibility. Our laboratory has recently shown that differences in gut microbiota (GM) can modulate phenotypes of models of colon cancer and inflammatory bowel disease. We and others have also shown that a number of factors associated with rodent research, including vendor, housing ventilation, and bedding can alter GM. The objective of this study was to expand the latter studies to examine the effect of additional bedding materials and methods of water decontamination on GM diversity and composition. To this end, Crl:CD1 (ICR) mice were housed on corncob or compressed paper chip bedding and provided water that was decontaminated by reverse osmosis, autoclaving, sulfuric acid treatment, or hydrochloric acid treatment. Feces was collected at days 0, 7, and 14. At day 28, animals were euthanized and both fecal and cecal material samples were collected. DNA was extracted from samples, amplified by PCR using conserved bacterial primer sets and subjected to next generation sequencing. Raw sequence data was analyzed using Qiime and resulting metagenomics data compared among groups using principal component analysis and multivariate ANOVA. Principal component analysis of data revealed differences in microbiota composition in the following comparisons: corncob versus paperchip in mice given either sulfuric acid-treated or autoclaved water, and sulfuric acid-treated water versus autoclaved water in mice on corncob bedding. These findings expand the list of husbandry factors that can modulate GM to water decontamination methods and bedding/water interactions and further emphasize the need to consider microbiota changes as possible sources of phenotype variability.

PS46 Static and Individually Ventilated Caging Acceptable for Mouse Housing Density Exceeding Guide Recommendations

E Helman^*, D Duvall, JM Cadillac, E Dohm, T Schoeb, SC Cartner

Animal Resources Program, University of Alabama—Birmingham, Birmingham, AL

Many references have determined that the mouse housing density recommendations of the Guide can be exceeded when IVC is used. However, having different requirements for animals housed in IVC or under static conditions is not practical for the mouse housing density policy at our institution. Therefore, a study was undertaken to both validate published IVC housing density recommendations and to confirm that those same higher housing densities are also acceptable for cages kept under static conditions. The experiment consisted of a breeding and a nonbreeding group housed in standard cages of approximately 70 inches² of floor space with hardwood chip bedding. Each group contained 24 cages of inhouse bred C57BL/6 mice equally divided into control and test densities and housed on static or IVC racks. The breeder control density consisted of 8-week old mice set up in a trio breeding scheme, 1 male and 2 females, with no more than 9 pups total per cage, strict weaning at 21 days, and no more than 14 days separation in age of the pups. The breeder test density was also a trio breeding scheme, but with no limit on pups and no restriction in litter age difference. The nonbreeder groups included a control density of 5 and test density of 7 weaning age males. Measurements of intracage conditions included ammonia at cage change, which occurred once weekly for IVC and twice weekly for static cages. Evaluation of the soiled bedding, water, food, nest material, pups born, and health assessment were recorded daily. At weaning the litter weight was recorded, and number of pups surviving from birth through weaning was calculated. Initially, ATP luciferase test swabs were used at cage change as an objective assessment of cage hygiene, but it was quickly determined that food dust confounded results, so testing was changed to CONTACT PLATES cultures. Observations continued for at least 2 months. There were no significant differences between any of the groups for ammonia, CONTACT PLATES growth, soiled bedding, nest evaluations, pup birth rate, and survival. Housing of mice at densities exceeding those recommended in the Guide is acceptable at our institution regardless of static or IVC status and in agreement with other published data on the subject.

PS47 Quick and Easy Determination of Rat Housing Density by Husbandry Staff Using Tail Diameter as a Surrogate for Body Weight

K Browning^*, JM Cadillac, E Dohm, SC Cartner

Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL

Determining the appropriate housing density for rats is difficult because of the large difference in weight as they grow and lack of any visible markers for various stages of adult life. Current cage space recommendations are based on weight. Considering the potential for cross-contamination of different colonies, it is not advisable to use a single scale from room to room, and it is cost prohibitive to have a dedicated scale for each rat colony. An alternate technique to determine the proper housing density of rats is needed. Skeletal size has been shown to be proportional to body weight in previous studies. However, the studies were not done on live animals and were not correlated with size of the tail, therefore this study explored the feasibility of using tail diameter instead of weight as a surrogate marker of weight. A digital caliper was fitted with polyoxymethylene blocks, 25 x 38mm, to create a standardized surface for the measurement. Rats were then weighed and their tails measured at the boundary of fur to no fur near the tail base. A series of 3 measurements were collected and averaged. Average data from no less than 50 adult male and 50 adult female animals from several different colonies resulted in a positive correlation of 0.85 between tail diameter and body weight. Using that information, it was calculated that the diameter of the tail at critical weights for housing density is approximately 7.7mm for a 200g animal, 8.6mm for a 300g animal, and 9.5mm for a 400g animal. We will use these measurements to create a standardized rat tail gauge for verification of proper housing density. It was determined that the diameter of a rat’s tail at the fur to no fur boundary has a high correlation to its body weight, and can be used to determine the appropriate housing density without the use of a scale, which will save money and time of the husbandry staff.

PS48 Cross-Fostering within the Same Animal Housing Room to Eliminate Murine Norovirus and Helicobacter spp. in Mice

CA Stevens^*

Central Michigan University, Mount Pleasant, MI

In efforts to preserve the SPF status of our facility with mice arriving from other institutions, cross-fostering to eliminate pathogens in the space housing room was initiated. Our goal was to minimize potential unexpected changes in transgenic mice which had previously occurred with rederivation. A cost and time effective protocol was implemented to cross-foster incoming mice with known MNV and Helicobacter spp. status. The procedure was put in place involving cross-fostering litters in the same housing room due to limited housing space. The pathogen-positive colony and the surrogate cross-foster CD1-Elite females were Mouse Surveillance Plus Pria tested prior to being housed on separate ventilated racks in quarantine. Clear-cut, extensive steps were implemented to prevent breaches in cross-contamination. Mouse Surveillance Plus Pria testing was repeated several times up to 12 weeks of age to ensure negative MNV and Helicobacter spp. status of cross-fostered pups and surrogate mothers prior to transferring the offspring from quarantine into regular housing. Quarterly health monitoring has continued to confirm the efficacy of our cross-fostering protocol. Our results indicated that our protocol was 100% successful in eliminating both MNV and H. ganmani,H. hepaticus, H. mastomyrinus, and H. typhlonius. This protocol is recommended to eliminate pathogens in cross-foster programs within the same housing room.

PS49 An Assessment of Recycling Crinkled Paper Nesting Material for 2 Cage Changes in Individually Ventilated Cages

ML Kain^*1, TL Martin², P Velleman¹, C Hedrick², N Jurcak², S Lewis²

¹Cornell University, Ithaca, NY; ²The Ohio State University, Columbus, OH

Nesting material as environmental enrichment has been shown to reduce stress, anxiety, pup mortality, and aggression. Recent studies indicate that mice prefer to build nests with naturalistic bedding, such as commercially available brown crinkled paper. Literature has also demonstrated that mice prefer dirtier cages to clean ones, with an increase in adverse behaviors such as aggression and stereotypies observed following cage changes. We hypothesized that recycling crinkled paper for 2 cage changes (28 days total) would result in reduced adverse behaviors in mice without affecting nest quality. We proposed that the crinkled paper would preserve its strength and cleanliness throughout the 28-day period. To test this, we gave 20 individually ventilated cages of mice 8g of crinkled paper and monitored behavior, nest scores (as a measure of paper strength), and intracage ammonia levels (as a measure of paper cleanliness) for 28 days. Half of the cages (n = 10) received new crinkled paper at cage change (day 14), while half (n = 10) retained the original crinkled paper. Strength testing and visual assessments of paper cleanliness were also conducted at each cage change (days 14 and 28). We observed no significant behavioral changes in mice receiving clean versus recycled paper. We found no significant difference in intracage ammonia levels in cages receiving clean versus recycled crinkled paper, although the recycled paper did subjectively appear dirtier upon visual examination. We found reduced paper strength via strength testing, and significantly lower nesting scores in cages receiving recycled paper (p = .0049). We did observe strain differences in nest score and structure, indicating that uniform scoring systems in the literature should be tailored to the strain being examined to be most useful. Overall, we concluded that the crinkled paper did not retain sufficient strength and visual cleanliness to be recycled for 2 cage changes.

PS50 Prevalence of Murine Adventitial Infections in Biomedical Research Facilities

JO Marx^*, AL Smith, D Gaertner

Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Adventitious infections in mice used in biomedical research may have a profound impact on study outcomes. Effects are not limited to deaths and clinically apparent illness in mice, since undetected infections may have subtle effects on the immune system, hormone production, and other physiologic systems that can unknowingly alter collected data. Many immunosuppressed mouse strains are highly vulnerable to these normally benign infections and any viral, bacterial, or parasitic infection can profoundly impact study results from mice used in immunology research. Additionally, genetically modified mice may have subtle, undetected alterations in immune function. Because of this, research institutions spend a great deal of time and resources to control these infections in their colonies. We recently conducted a survey querying 62 of the top 100 NIH-funded biomedical research institutions regarding the prevalence of all common infections in their colonies during the previous 2-year period. These results were compared with those from 2 previous published surveys conducted in 1996 and 2006. We found that the frequency of most viral, bacterial, and parasitic infections decreased over time, despite improved sensitivity of diagnostic techniques. The most commonly identified agents included fur mites (56% of institutions reporting infections during the 2-year period), pinworms (44%), and mouse parvovirus (37%). The prevalence of mouse hepatitis virus (13%), epizootic diarrhea of mice virus (15%) and minute virus of mice (3%) were dramatically decreased compared to the previous surveys. Twenty-two percent of institutions completely excluded norovirus, while 48% of institutions had select suites or facilities which were maintained free of norovirus. In both situations, 21% of institutions experienced norovirus outbreaks. Management of viral and fur mite outbreaks varied widely among institutions. Ultimately, our ability to completely eradicate these infections will depend on the use of strict barrier housing practices, the prevalence of the organisms in wild mouse populations, the biology of the agent, and the perceived impact of the agents on research.

PS51 Preferential Treatment: Enrichment Options for Mice and Rats

J McClellan^*1, M Strine², M Gallimore², J Bowman², L Figueroa², MJ Horn¹

¹Veterinary Science, Research, and Support, Envigo, Indianapolis, IN; ²Envigo, Indianapolis, IN

Enrichment is important for laboratory rodent models as it can reduce stress and abnormal/aggressive behaviors. Proper enrichment can lead to a reduction in variability in research outcomes and reducing the number of animals needed for a study. Two studies were performed to evaluate the use of several enrichment types in various rat and mouse models. In the initial study, a cotton fiber pad (DN) was evaluated. Usage of this enrichment was examined in breeding pairs of the Brown Norway rat, C57BL/6N, ICR, Athymic Nude, SHrN^®, and FVB/N mouse models for 6 weeks starting at 8-10 weeks of age. DN usage was also examined in C57BL/6N, ICR, Athymic Nude, SHrN^®, and FVB mouse models for from weaning to 6 weeks of age. The number and size of pups per litter was recorded in the breeding cages. Nests were scored weekly according to the following system: 0-5 where 0=undisturbed, 1=disturbed, 2=flat nest, 3=cup, 4=incomplete dome, and 5=complete dome. The second study evaluated usage and preference of various enrichment types in breeding pairs: DN, a compressed cotton pad (IB), rolled tissue paper (DT), or crinkle paper (SN) for 13 weeks in Athymic Nude, BALB/c, DBA/1, C57BL/6N mice, and Sprague–Dawley^® rats. Production efficiency index, aberrant findings, and enrichment usage were recorded weekly. There were no aberrant findings in either study with the use of enrichment. All models built nests with the DN enrichment. The number of pups per litter was significantly higher in ICR mice with DN compared to no enrichment. The percent of pups weaned was also significantly higher in the Athymic Nude mice with DN compared to those with no enrichment. The SHrN^® breeders built better nests with SN compared to DN. BALB/c mice built nests more readily with DN but didn’t build nests with IB until 6 weeks after initial exposure. C57BL/6N mice also preferred DN to IB; only 50% of cages nested with the IB compared to 100% of cages with DN. Brown Norway breeding pairs built nests with DN and the Sprague–Dawley^® rats efficiently used the DT. Studies such as these will enable us to determine best practices for nesting enrichment in laboratory rodent models to better care for our animals and promote the 3Rs.

PS52 Methods Development for Cohousing Male Crl:CD1(ICR) Mice

RR Hukkanen, L McFadden, J Murray*, J Reinart, J Wachner, J Lacher

Dow Chemical Company, Midland, MI

Mice readily seek conspecific contact and are capable of forming social relationships. However, males have strain-dependent territorial tendencies that often prohibit pairing of adults. Group housing of mice is advantageous and encouraged as enrichment to promote species-specific behavior and deter stereotypy. Nonetheless, individual housing is the current practice on toxicological studies here due to the potential for male aggression and the need to normalize experimental environments across sexes/subjects. Despite historical challenges, we hypothesized that male Crl:CD1(ICR) mice could be successfully paired through reducing factors associated with aggression. We explored the effect of pheromone continuity, age at first pairing, partner permanence, and delayed intervention within a 28-day repeat-dose toxicity test study design. We further explored the presence of novel enrichment within new and sustained-pairings over a 1-5 month period. Four groups of 10 mice aged 5 or 6 weeks were paired once or twice using husbandry techniques designed to transfer pheromones at cage change-out (marking clean cages with urine-soiled gauze, transfer gauze from dirty to clean cages, and transfer feed on separate days from cages). A control group of 10 mice was paired using standard husbandry techniques. Under veterinary supervision, animal care staff did not separate pairs exhibiting nontraumatic interactions including vocalization and fighting. After 4 months, the first 4 groups of mice were transitioned to standard husbandry and offered novel enrichment. All pairings were successfully maintained throughout the study. A single injury (minor laceration) was observed within the first 28 days of pairing; however, the pair was maintained and the laceration resolved. These data indicate that pheromone continuity, age at first pairing, and partner permanence do not significantly impact pairing success. We therefore conclude that male Crl:CD1(ICR) mice may be successfully paired on repeat-dose toxicity studies up to 90 days of duration.

PS53 The Placental Estrogen, Estriol, Protects Female Mice against Influenza A Virus by Reducing Pulmonary Inflammation

MS Vermillion^*1,2, SL Klein²

¹Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, Baltimore, MD

Estriol (E3) is a pregnancy-specific estrogen in humans and mice that is produced by the placenta and found in highest concentrations during late pregnancy. In humans and mice, E3 has potent immunomodulatory functions and can ameliorate diseases caused by inflammation, including multiple sclerosis (MS). Like MS, pulmonary disease associated with influenza A virus (IAV) infection is predominantly caused by aberrant inflammation and immunopathology, rather than virus replication. We hypothesized that exogenous E3 may protect female mice against IAV-associated disease by reducing pulmonary inflammation and tissue damage. Adult gonadally intact female C57BL/6 mice received a subcutaneous implant of either 21-day release 5mg E3 or placebo (n = 20 animals/treatment). This dose of E3 increased concentrations to levels found in pregnant female mice as measured by mass spectrometry. Females were intranasally infected with a sublethal dose of mouse-adapted 2009 H1N1 IAV or vehicle, and body mass was recorded daily as a measure of morbidity. Pulmonary inflammation and immune cell populations were evaluated by histology and flow cytometry at 9 and 14 days postinfection (dpi), corresponding to peak disease and recovery, respectively. Following infection with IAV, E3-treated females lost significantly less body mass compared with placebo-treated females. Clinical protection against IAV was associated with significantly less pulmonary inflammation and tissue damage, which was characterized by reduced perivascular and peribronchiolar inflammation at 9 dpi. Characterization of the T cell response by flow cytometry showed that E3-treated females had a significantly greater ratio of CD4+ to CD8+ T cells at both 9 and 14 dpi, and skewing of CD4+ T cell responses toward Th2 and away from Th1 and Th17 responses at 9 dpi. These data suggest that pregnancy levels of E3 are protective against IAV infection and indicate that the estrogen-driven immunomodulation during pregnancy is not the cause of pregnancy-associated IAV disease severity. These data also provide evidence that E3 has broad immunomodulatory effects and may be beneficial in the context of inflammation-driven disease.

PS54 In Vivo Methods for Monitoring Disease Progression in Mouse Models of Alzheimer’s Disease

NM Tataryn^*1,2, H Ahn², S Strickland²

¹Veterinary Services, Memorial Sloan Kettering Cancer Center, New York, NY; ²The Rockefeller University, New York, NY

Several mouse models of Alzheimer’s disease (AD) employ the integration of human transgenes into the mouse’s genome that upregulate the production of amyloid-β peptides leading to the deposition of amyloid plaques in the brain. Traditionally, methods to monitor disease progression in these models have included behavioral testing and postmortem histologic evaluation. These methods are often insensitive and preclude longitudinal analysis of individual mice. We evaluated multiple in vivo imaging modalities to monitor disease progression and to equate these findings with innovative 3-dimensional immunohistochemical techniques in 2 mouse models of AD. The MRI method of arterial spin labeling (ASL) was used to measure cerebral blood flow. Additionally, positron emission tomography (PET) was performed using 2 different radiotracers: ¹⁸Fluordeoxyglucose (FDG) to measure cerebral metabolism, and Pittsburgh compound-β (PiB) to measure amyloid plaque accumulation. MRI revealed decreased cerebral blood flow and PET revealed decreased cerebral metabolism and increased levels of amyloid plaques. These findings correlated with the histologic findings, which employed novel 3-dimensional immunofluorescence imaging. We propose these techniques as sensitive methods to evaluate the progression of AD in mice, allowing longitudinal evaluation in lieu of postmortem analysis at specific time points.

PS55 Host Genetic Drift and Gut Microbiota Variability between C57BL/6- Apc^MinMouse Colonies Affects the Intestinal Tumor Phenotype

JE Moskowitz^*, S Busi, ML Hart, CL Franklin, J Amos-Landgraf

Comparative Medicine Program, University of Missouri, Columbia, MO

Reproducibility in animal studies has emerged as a significant concern across a breadth of research fields, yet the underlying causes of phenotypic variability among animal models often remains unclear. Host changes such as genetic drift between different colonies are likely to influence these phenotypes, but other poorly characterized factors such as the gut microbiota (GM) may have a considerable impact as well. The Apc^Min mouse model of colorectal cancer is a highly used model that has demonstrated phenotypic variability among colonies. Consistent with these reports, our laboratory has found significant differences in both small intestinal (SI) and colonic tumor numbers between two Apc^Min mouse colonies; the C57BL/6J-Apc^Min (B6-Min/J) from the Jackson Laboratory, and the C57BL/6JD-Apc^Min (B6-Min/D) closed colony that has been maintained at the University of Wisconsin. To determine if phenotypic variability between these 2 colonies is due to host genetic changes or the environmental impact of the GM, we used complex microbiota targeted rederivation (CMTR) to rederive embryos of the 2 Apc mutant colonies onto CD1 surrogate dams that possessed GM from either The Jackson Laboratory (Crl:CD1_GMJAX) or Envigo (Hsd:CD1_GMHSD). We generated a total of 4 Apc^Min groups: B6-Min/J_GMJAX, B6-Min/J_GMHSD, B6-Min/D_GMJAX, and B6-Min/D_GMHSD. Tumor counts revealed significant increases in both SI (P = 0.03) and colonic tumors (P = 0.006) in the B6-Min/J group with a GM from HSD compared to the B6-Min/J group with a GM from JAX, suggesting that the GM is critical in the SI and colonic tumor phenotype. Additionally, B6-Min/D animals had more SI tumors than B6-Min/J animals within each GM group. However, there were no differences between these groups in colonic tumor numbers, suggesting that host genetic differences play a role in the SI phenotype, but not the colonic phenotype. We conclude that both the GM and host-genetic factors within the C57BL/6J-Apc^Min population contribute to the SI tumor phenotype, while the GM primarily defines the colonic tumor phenotype. These results demonstrate how interactions between host and environment can shape a given phenotype within our animal models, and thus significantly impacts reproducibility in animal studies.

PS56 Comparison of the Vaginal Environment in Rhesus Macaques (Macaca mulatta) and Cynomologus Macaques (Macaca fascicularis)

GJ Daggett^*1, C Zhao³, F Connor-Stroud¹, F Villinger²

¹Department of Animal Resources, Emory University, Atlanta, GA; ²New Iberia Research Center, New Iberia, LA; ³Centers for Disease Control and Prevention, Atlanta, GA

Rhesus (Macaca mulatta) and cynomologus macaques (Macaca fascicularis) are valuable animal models for the development and study of Human Immunodeficiency Virus (HIV) prevention strategies. Prevention of vaginal acquisition of HIV is highly relevant as it represents a major portal of viral entry that is heavily influenced by local infections, inflammation, hormonal changes, and the resident microbiota. To this end we tested the vaginal microbiota and pH of rhesus and cynomolgus macaques to characterize the normal microflora present in these models of HIV infection. In women, Lactobacillus spp dominate a healthy vaginal microbiota, leading to the production of lactic acid, a lower pH, and the inhibition of other bacterial species, as well as viruses. Higher Nugent scores, referred to as bacterial vaginosis, and higher vaginal pH have been shown to increase susceptibility to mucosal transmission of HIV in humans. In this study we compared the Nugent scores and vaginal pH of rhesus and cynomologus macaques to determine if a significant species difference exists, which could suggest that the vaginal environment of one is a closer analogue to humans than the other. This experiment has shown that both parameters are significantly different between the 2 species. The natural vaginal environment in both species was equivalent to bacterial vaginosis in humans. We therefore attempted to colonize the macaque vagina with Lactobacillusrhamnosus to create an induced model of a healthy vaginal environment. Successful colonization indicated that both macaque species are able to host Lactobacillus spp vaginally. The colonization was sustained for a longer period of time in rhesus macaques with significant decreases in Nugent scores. The Nugent scores in cynomologus macaques did not change significantly. Vaginal pH following colonization with L. rhamnosus decreased significantly in both species. These results suggest that normal rhesus and cynomologus macaque vaginal environments more closely represent humans with bacterial vaginosis. Future studies of vaginal HIV transmission should consider the normal vaginal environment when selecting an animal model. Induction of a healthy vaginal environment via colonization with Lactobacillus spp should also play an important role in future investigations.

PS57 Diluted Buprenorphine Degrades over Time in Several Common Storage Conditions

JM DenHerder^*1,4, RL Reed^2,3, JL Sargent^1,4, BT Bowers^6,5, JF Stevens^2,3, HE Diggs^1,4

¹Department of Biomedical Sciences, Oregon State University, Corvallis, OR; ²Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR; ³Linus Pauling Institute, Oregon State University, Corvallis, OR; ⁴Laboratory Animal Resources Center, Oregon State University, Corvallis, OR; ⁵College of Veterinary Medicine, Oregon State University, Corvallis, OR; ⁶College of Pharmacy, Oregon State University, Corvallis, OR

Buprenorphine is a partial mu opiate agonist used for analgesia. Due to the small size of rodents, buprenorphine HCl is typically diluted 10- or 20-fold with a sterile diluent such as saline to aid in accurate dosing. Protocols for preparing and storing diluted buprenorphine vary by institution, and there is little information in the literature regarding beyond-use dating of specific preparations. Buprenorphine HCl is packaged without a preservative in 1 ml glass ampules, vials, or syringes intended for immediate use as a single human dose. Inadvertent microbial contamination when diluting buprenorphine poses a significant risk to the patient and degradation of the compound as a result of dilution and storage could decrease efficacy. The purpose of this study was to determine the chemical and microbiological stability of diluted buprenorphine stored for up to 21 days. Our hypothesis was that the concentration of buprenorphine would decrease over time and that the rate of degradation would be affected by container type and storage temperature. Buprenorphine was diluted 1:10 with sterile saline using aseptic technique in a Class II, type A2 biologic safety cabinet. Diluted samples were stored in glass vials or plastic syringes protected from light at refrigerated or room temperature. Samples were stored for several time points from 0 to 21 days and then analyzed with high performance liquid chromatography (HPLC) to determine relative concentration of buprenorphine. Samples were also submitted to the Oregon Veterinary Diagnostic Laboratory for sterility testing. All samples stored for 0 to 21 days were negative for bacterial and fungal growth. HPLC results suggest that buprenorphine concentrations decreased under all storage conditions. In addition, there was another peak on the chromatograms of all samples stored in plastic syringes which increased over time, suggesting a chemical was eluting from some component of the plastic syringe.

PS58 Characterization of Multi-Drug Resistant Enterococcus faecalis Isolated from Research Macaques

MT Lieberman^*1, SE Woods¹, F Lebreton², J Dzink-Fox¹, MS Gilmore², JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA

Multi-drug resistant (MDR) Enterococcus faecalis is a common and serious cause of nosocomial infections. Previous characterization of 14 E. faecalis isolates from cephalic recording chambers of macaques used in neuroscience research revealed two lineages with marked multi-drug resistance. Lineage A (LA) isolates (n = 7) showed differing susceptibilities to gentamicin, with 4/7 isolates displaying high-level gentamicin resistance while Lineage B (LB) isolates (n = 7) displayed resistance to neomycin. All isolates from Lineages A and B displayed marked streptomycin resistance. Resistance to tetracycline, chloramphenicol, erythromycin and trimethoprim-sulfamethoxazole was also noted. DNA was extracted from 2 LA and 1 LB isolate and sequenced on a single SMRT cell on a Pacific Biosciences RS2 sequencer. Genomes were assembled and FASTA sequences were analyzed by PubMLST, ResFinder and VirulenceFinder to confirm sequence type (ST) and identify genes of interest. Two isolates from LA were confirmed to be ST4 and the isolate from LB was confirmed to be ST55. All isolates had a unique antimicrobial resistance gene profile with the lsa(A) gene encoding intrinsic resistance to lincosamides and streptogramins A as the only common gene between all 3 isolates. Additional macrolide resistance encoded by erm(B) was identified in 2 isolates. Four genes encoding aminoglycoside resistance were identified: str and 3 aminoglycoside-modifying enzymes: aph(3’)-III, aac(6’)-aph(2”) and ant(6)-Ia. Genes encoding tetracycline resistance included mechanisms for both efflux pumps (tetL) and ribosomal protection (tetSand tetM). Phenicol resistance was conferred by the cat gene and was present in ST4 isolates, but not the ST55 isolate. Trimethoprim resistance encoded by dfrG was noted in 1 ST4 isolate. Other virulence factors identified included cytolysin, enterococcal surface protein, aggregation substance, gelatinase, collagen adhesion precursor and endocarditis antigen. E. faecalis isolates from cephalically-implanted macaques display genetic similarities to isolates associated with human nosocomial infections. Macaques represent a unique research model to study nosocomial infections due to their long-term residence following cephalic implantation, and intermittent antimicrobial exposure.

PS59 Intraperitoneal Administration of Ethanol as a Means of Euthanasia in Neonatal Mice (Mus musculus)

CE de Souza-Dyer^*, JO Marx

Unit for Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA

The euthanasia of animals in research is of utmost importance to our field. Regrettably, euthanasia of neonatal mice can be challenging because of differences in physiology compared to adults. In 2013, the AVMA’s Guidelines for the Euthanasia of Animals included intraperitoneal (IP) injection of ethanol as “acceptable with conditions,” and a recently published study confirmed that this method is adequate for euthanasia of adult mice; however, neonatal mice have not been tested. This study examined whether IP ethanol results in humane euthanasia in neonatal mice. Mouse pups (C57BL/6 and CD1 (n = 162)) were injected with 100% ethanol, a pentobarbital/phenytoin combination, or saline at 7, 14, 21, 28, or 35 days of age. Electrocardiogram, respiratory rate, and times to loss of righting reflex and death were recorded. No mice recovered after injection with pentobarbital or ethanol. The average time to death (TTD) was not significantly different between strains so results were pooled. However, there was a significant difference in TTD between ethanol and pentobarbital at 7, 14, and 21 days, as well as between ethanol groups at 7, 14 and 21 days compared to 35 days. The average TTD (time±SD) in minutes at 7, 14, 21, 28 and 35 days old using ethanol was 70.3±39.8, 51.7±30.5, 32.3±20.8, 13.1±14.1, and 4.9±1.4 respectively, and 2.8±0.4, 2.9±0.5, 3.9±1.2, 3.9±0.7, and 4.4±0.5 for pentobarbital, respectively. Although there was no statistically significant difference in TTD between ethanol and pentobarbital at 28 days of age, the TTD in 2 of 12 mice was greater than 30 minutes following ethanol administration. Therefore, 100% ethanol IP should not be used as a method of euthanasia in mice younger than 35 days, as the criteria for humane euthanasia (rapid and minimally distressful) were only met in mice 35 days or older.

PS60 Effects of Low-Level Brodifacoum Exposure on the Feline Immune Response

JH Kopanke^*1, K Horak², E Musselman¹, K Bennett¹, S VandeWoude¹, S Bevins²

¹Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ²USDA APHIS, Fort Collins, CO

Anticoagulant rodenticides have recently been implicated as a potential inciting factor in the development of notoedric mange in bobcats and other wild felids that may regularly consume rodents. To date, however, these studies have failed to provide direct evidence of a causative association between anticoagulant rodenticide exposure and immune suppression in non-target species. Therefore, the present study sought to determine whether chronic, low-level exposure to brodifacoum resulted in alterations in the direct and recall immune response in felines. Age-matched, specific pathogen-free (SPF) domestic cats were exposed to either 0.05 mg/kg brodifacoum or sham bait on a weekly basis for 6 weeks (days 0, 7, 14, 21, 28, 35) to model environmentally realistic exposure scenarios. Complete blood counts (CBC) and prothrombin time (PT) were monitored biweekly as measures of immune function and coagulation. Cats were vaccinated and boosted with 50 μg of two irrelevant antigens (ovalbumin and keyhole limpet hemocyanin (KLH)) at different points during the course of the experiment to assess the recall and direct immune responses, respectively. Measures of immune response included delayed-type hypersensitivity (DTH) tests and cell proliferation assays in the presence of ovalbumin and KLH. Ovalbumin- and KLH-specific antibodies were detected and quantified via ELISA assays. Cytokine induction following exposure to vaccine antigens was also analyzed. The mean blood brodifacoum level in exposed cats was 4.76 ± 0.577 ng/mL on Day 70, and the mean liver brodifacoum level was 1806 ± 106 ng/g on Day 77. Remarkably, no coagulopathies developed during the course of the study, and there was only one time point (Day 42) where the KLH DTH response and KLH-specific antibody titers were lower in the brodifacoum-treated group than the control group. This study indicates that cats maybe more resistant to clinical effects of brodifacoum exposure than other species and suggests that gross impacts of environmentally realistic brodifacoum exposure on humoral and cell mediated immunity against foreign antigen exposures in domestic cats are minimal.

PS61 Staphylococcus aureus Strains Isolated from an Outbreak of Preputial Gland Abscesses in C57BL/6 Mice under Exclusion Barrier

UM Jonnalagadda^*1, L Ward², K Zhang², W Pan², C Pantigoso², C Fielding², C Geary-Joo², D Morck², M Ayroud²

¹Laboratory Animal Medical Services, University of Cincinnati, Cincinnati, OH; ²Cummings School of Medicine University of Calgary, Calgary, Canada

Colonization of laboratory mice maintained in barrier conditions by Staphylococcus aureus is relatively common. We describe an outbreak of preputial gland abscesses (PGA) in C57BL/6 mice in an exclusion barrier room in a transgenic mouse facility, housing only commercially sourced mice or mice generated within the facility. The PGA in the index case, a founder mouse, was lanced and treated with antibiotics. Within the next 14 days, 22 additional mice developed PGA in the same room. Males had been rotated through breeding cages, which may have contributed to the additional cases. Offspring subsequently born to the infected sires also developed PGA at 6-8 weeks of age, despite having euthanized the clinically apparent sires. While identifying the source of this PGA problem, further cases of PGA occurred in commercially sourced mice in a separate mouse room within 1-2 weeks of arriving from the commercial vendor. The only observable sign of infection in all affected mice was nodular swelling at the base of the penis. Histologically, lesions consisted of extensive preputial gland suppurative inflammation with large numbers of intralesional coccoid bacteria. Methicillin-susceptible S. aureus (MSSA) was isolated in pure culture from samples of affected mice. Molecular characterization including pulsed-field gel electrophoresis, Staphylococcus protein A typing and multilocus sequence typing were further performed on the isolates involved in the outbreak. Of the 6 isolates genotyped, 3 were identified as ST1-t127-MSSA, 2 as ST88-t2649-MSSA and 1 as ST88-t7464-MSSA. ST88 has been previously reported to be associated with PGA. To avoid potential spread to other parts of the barrier, we salvaged valuable strains through embryo rederivation and decontaminated the room and equipment. For the past 15 months, no further PGA cases have been observed. In cases of persistent PGA, we recommend the causative agents be identified and typed as some bacterial strains may be commonly involved. Control strategies may require depopulation of affected animals.

PS62 Estimated Prevalence of Demodex spp. in a 1600-Cage Mouse Colony and in Imported Mice from Other Academic Institutions

RJ Ricart Arbona^*1, MA Nashat², NS Lipman¹, FR Wolf¹

¹Center of Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College, New York City, NY; ²Tri-Insitutional Training Program in Laboratory Animal Medicine and Science, New York, NY

We attempted to estimate the prevalence of Demodex spp. in a colony of mice (∼ 1600 cages) of various strains used by 14 laboratories, which were imported en masse, using a PCR assay offered by a commercial diagnostic laboratory. Two rounds of testing were performed 3 weeks apart. For each round, cages and mice were randomly selected. Fur swabs from 1 mouse per cage from 10 cages were pooled into a single sample. Twenty four samples (representing 240 cages) were submitted. Thirteen of 24 samples were positive for Demodex spp.In the second round of testing, 5 additional samples of 27 (representing 270 cages) were found to be positive. All cages tested in the second round were different than those tested in the first round. This data suggests the prevalence of this ectoparasite in this group may be between 3.5% and 35%. Additionally, from February to May 2016, the same PCR assay was used to test smaller imported groups of mice (1- 10 cages) from different academic institutions while in quarantine. Five of 38 groups (point prevalence = 13.2%) tested positive for Demodex spp. Skin testing (fur pluck and deep skin scrape evaluated by microscopy) was also performed on these groups simultaneously. Only one group was confirmed positive by microscopy. All groups tested negative by PCR after a 4-week antiparasitic treatment protocol. These data may indicate that the Demodex spp. PCR is significantly more sensitive than fur pluck and deep skin scrape and the prevalence of Demodex spp. may be greater than suspected. Additional characterization and comparison of the PCR assay is warranted.

PS63 Pharmacokinetics and Pharmacodynamics Fail to Support the Use of 3 Commonly Considered Delivery Methods for Postoperative Analgesia Using Meloxicam in Mice

JA Herrod^*1, AM Mexas¹, C Doane²

¹Veterinary Medicine, Midwestern University, Glendale, AZ; ²University of Arizona, Phoenix, AZ

Meloxicam, a commonly used NSAID, is routinely given to mice following minor surgical procedures. Researchers have explored the use of NSAID’s such as Meloxicam administered in sustained-released formulations, solid feed pellets, or gel as a stress-free way to deliver analgesia postoperatively. These delivery methods allow for less frequent handling following a surgical procedure and therefore should reduce pain and stress. Anecdotal evidence supports the use of these delivery methods, but objective data comparing the different formulations is lacking in the literature. In this study the pharmacokinetics and pharmacodynamics of Meloxicam-containing feed (dosed at 3.3mg/kg), Meloxicam-containing gel (dosed at 4mg/kg), and injectable sustained-release Meloxicam (dosed at 5mg/kg) were compared. An osmotic pump was placed in the subcutaneous region between scapulae of each mouse to demonstrate a routine, minor surgical procedure. We measured Meloxicam drug plasma levels using liquid chromatography/mass spectrometry and evaluated common appearance parameters such as the mouse grimace scale (MGS), hair coat, nest building, activity, and subjective pain scoring to evaluate pre and postoperative pain. All mice displayed signs of pain postoperatively; mice showed a decrease in activity and grooming and an increase in pain based on the MGS on postoperative days 0, 1, and 2. Meloxicam blood plasma levels in all groups were below therapeutic plasma concentrations. Although the highest concentration of meloxicam in the blood plasma was noted in mice that received subcutaneous Meloxicam SR, these values were below reported therapeutic levels, consistent with the subjective pain scores. We concluded based on low Meloxicam blood plasma levels and observations of pain postoperatively in all groups, that these three approaches are not as effective in treating postoperative pain as desired at the given doses. Higher doses of Meloxicam may need to be considered when formulating medicated pellets or gel in order to achieve therapeutic concentrations.

PS64 Hypercortisolemia and Depression in a Cranially Implanted Rhesus Macaque (Macaca mulatta)

SC Adams^*1, C Guyot¹, K Berry¹, S Wallack², A Loar³, M Leblanc¹

¹ARD, Salk Institute, La Jolla, CA; ²Veterinary Imaging Center of San Diego, San Diego, CA; ³STAT Veterinary Lab, San Diego, CA

A 10-year-old cranially implanted rhesus macaque (Macaca mulatta) with a history of weight loss and alopecia was presented for change in mentation described as dull in the cage. Initial physical examination was normal while bloodwork revealed marked hypercortisolemia (23 μg/dL). Other CBC and biochemistry values including fT4 and ACTH were normal. Differential diagnosis for hypercortisolemia, weight loss, and alopecia included Cushing’s and Pseudo-Cushing’s Syndrome. As an initial screening, we compared Urine Cortisol:Creatinine Ratio (UCCR) in the principal animal and naïve animals. UCCR was 10x higher in the presenting animal suggesting a possible Cushing. We then performed a low-dose dexamethasone suppression (LDDS) test by administering oral dexamethasone (DEX) to the presenting animal and 3 clinically normal nonworking animals for comparison. Oral DEX was given the same day at 6 am, 12 pm, and 6 pm and urine samples were collected before and at 6, 12, 24, 32, 56, and 102 hours post first dose of DEX. All animals showed suppression and release of suppression at a similar time, which was interpreted as a nonpathologic response. Abdominal ultrasound and radiographs of the presenting animal revealed marked bilateral adrenal mineralization, which is a common finding in macaques, but no overt adrenal tumor. Overall, these results excluded Endogenous Cushing’s Syndrome and prompted us to evaluate different causes of Pseudo-Cushing. Obvious differentials such as obesity or diabetes were immediately excluded based on previous results. Chronic stress and depression were then evaluated by recording video and quantifying behaviors in the principal animal and naïve nonworking colony animals. A presumptive diagnosis of depression was made based on published criteria, which includes time spent in huddling posture (>5 min/hr) with concomitant hypercortisolemia. The animal was placed on Fluoxetine and provided increased environmental enrichment. To evaluate the effect of treatment, we performed serial UCCR and behavioral measurements and observed a return of UCCR values within normal range and improvement of behavioral scores after 6 weeks of treatment. This case highlights the importance of evaluating chronically implanted animals for depression and the utility UCCR at baseline and following LDDS for screening behavioral and HPA abnormalities.

PS65 Use of a Murine Sepsis Scoring System (MSS) for a Mouse Model of Hemolytic Uremic Syndrome

PA Castro, LV Gigliello^*

Research, VA Palo Alto, Palo Alto, CA

Seven C57BL/6 (WT) and 7 Carboxypeptidase N knockout (CPN -/- “KO”) adult male mice were each given a single intraperitoneal injection of Shiga toxin 2 (6ug/kg) and E. coli LPS (1.5mg/kg) mixed in 500uL of saline to induce Shiga toxin associated kidney disease as a mouse model of Hemolytic Uremic Syndrome (HUS). These mice were monitored and scored using the Murine Sepsis Scoring (MSS) system at 6 hours intervals for up to 132 hours. The MSS evaluated mice based on 7 clinical variables: appearance, level of consciousness, activity, response to stimuli, eyes, respiration rate, and respiration quality. Each of the 7 variables were scored from 0 (normal) to 4 (significant clinical abnormalities) with a composite score range of 0 to 28. Mice were additionally given subcutaneous injections of sustained release buprenorphine (1mg/kg) every 72 hrs. The mean survival time for all experimental mice was 106.3 hours with a MSS mean composite score of 20.7 measured 6 hours prior to being found dead. The mean survival time for WT mice was 116.6 hours with a MSS mean composite score of 22.9 measured 6 hours prior to death while comparatively the mean survival time for KOs was 96.0 hours with a mean composite score of 18.6. Prior to death, 85.7% of all mice reached a MSS composite score of at least 18. Six hours prior to being found dead, 13 of the study mice (92.8%) had a score of at least 12 when combining MSS variables appearance, level of consciousness, activity, and response to stimuli. An activity score of at least 3 was measured in the majority of all mice (92.8%) prior to death and all experimental mice had a score of at least 3 or more in appearance measured 6 hours prior to death. Based on this study we conclude that an MSS score of 18 indicates that a mouse will likely die within the next 6 to 24 hrs. The data from this study also highlights the possible need for different endpoint criteria for the different mouse strains used. Furthermore, this work demonstrates the ability to measure clinical conditions based on a clinical observational scoring system (e.g. Murine Sepsis Score) that can be used with other disease models, such as HUS in this study, to more precisely determine suitable criteria for study endpoints.

PS66 Use of PCR for Posttreatment Testing of Murine Fur Mites

DM Molk^*, S Shainker

Massachusetts General Hospital, Charlestown, MA

Pooled PCR testing is used to identify the extent of a fur mite outbreak at our institution; fur pluck testing is the standard post-Ivermectin treatment follow-up. We wanted to determine if PCR testing could be used as an alternative to fur plucks for follow-up testing. However, dead mites can lead to false positive PCR findings. Based on the work of other institutions that have shown that live mites are readily passed to offspring and that young mice are likely to have larger, detectable mite loads, we devised a new follow-up PCR-based testing plan focused on the testing of weanling mice to minimize the likelihood of false positive findings. PCR was compared to fur pluck testing. We had 2 outbreaks of fur mites: the first consisted of 185 cages consisting of multiple transgenic lines on various backgrounds quarantined for Radfordia. Breeding continued, and at the end of the treatment period, 8 cages contained mice of weanling age; PCR and fur plucks were performed on dams and weanlings, and all results were negative. Outcome from this sample group suggested that PCR was at least as accurate as fur pluck testing. The second outbreak consisted of 256 cages positive for Myobia. Seven cages contained mice of weanling age at the end of the treatment period, and PCR and fur plucks were performed on the weanlings and dams. One dam was PCR positive, while all offspring were negative. This suggests that the breeder had been infected, treatment was effective, and that live mites did not transfer to the offspring. Fur plucks were negative on this same dam. Sentinel testing resulted in positive PCR results but negative results on direct animal testing. These results support the claim that PCR is more sensitive for fur mite detection than fur plucks, and supports the possibility that targeting offspring of weanling age for testing via PCR after treatment avoids false positive findings. These scenarios foster consideration of how PCR can be used for follow-up testing in outbreak situations.

PS67 Novel Cageside Methods for Recognizing Unalleviated Postoperative Pain in Mice

JL Lofgren^*1, Y Kang¹, C Luke², D Myers²

¹Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MA; ²Conrad Jobst Vascular Research Laboratory, University of Michigan, Ann Arbor, MI

Here we describe 2 novel cageside methods to quickly and objectively identify unalleviated postoperative pain in mice. With normal grooming, a nontoxic material that glows under black light is transferred from the top of the head to the paws, face, body, nest, and cage environment in a reproducible pattern. Additionally, mice readily consolidate nesting material, which can be quantified in any location of the home cage. Adult C57BL/6 male mice were evaluated by a blinded-observer postisoflurane anesthesia +/- analgesia and postlaparotomy with inferior vena cava (IVC) ligation +/- analgesia. Buprenorphine (0.05mg/kg) (n = 9) or saline (n = 9) were provided preemptively and then twice daily for 48 hours. To assess grooming, a glow in the dark material was applied to the top of the mouse’s head; a black light was then used to identify transfer to body and cage areas according to a novel scoring system ranging from 1 (original application site only) to 10 (completely groomed away). To assess nesting consolidation behavior, one quarter of a cotton nesting square was placed in each corner of the home cage. Consolidation was scored from 0 (no pieces) to 4 (all pieces) consolidated within a 2.5 x 3.5 inch area of one another. Nest consolidation was significantly depressed after surgery compared to anesthesia for the first 24 hours post surgery. Grooming behavior was significantly depressed after surgery compared to anesthesia throughout the 48 hour post operative period. Taken together, laparotomy with IVC ligation resulted in postoperative pain for at least 48 hours. At 1-hour postsurgery, buprenorphine treated mice had significantly fewer differences in nest consolidation behavior and grooming behavior between the postanesthesia and postsurgery conditions compared to saline treated mice, indicating buprenorphine likely provided some analgesia. However, by 8 hours postsurgery buprenorphine-treated mice did not significantly differ from saline controls, indicating a higher dose or additional analgesia was needed to effectively alleviate postoperative pain. This pilot study provided proof of concept that the Nest Consolidation Test and Grooming Transfer Test are effective, objective, titratable cageside methods for assessing unalleviated postlaparotomy pain in mice.

PS68 Baboon (Papio hamadryas) as Animal Model For Uterus Transplantation

K Rivas-Wagner^*

The Mannheimer Foundation Inc, Homestead, FL

There are several key factors and recent developments that shed new interest on uterus transplantation (UTx) as a medical treatment for women with absolute uterine factor infertility who hope to achieve full-term gestation. In Sweden, this quite anticipated option has now resulted in several full-term pregnancies with successful births. As in any field of ongoing research, the beginning and continuity are never definitive and almost invariably the work needs to improve over what has begun. Therefore, continued research seeks to make the surgical approach safe and effective for donors (live donor) and recipients alike. Since 2011, we been a part of these investigations where our Hamadryas baboons (Papio hamadryas) have played an invaluable role and have taken part in moving toward the first uterus transplantation in the United States. For most of the phases of this project, nulliparous females in reproductive age were used as recipient subjects whereas multiparous females in reproductive age were used as donors. A critical component of the study was the use of immunosuppressive drugs such as Tacrolimus, Mycophenolate Mofetil, Anti-thymocyte globulin, Solu-Medrol, Ganciclovir, and a few more, which presented a unique challenge to administer and balance when compare to other laboratory animals. Our investigators keep on working arduously to refine their surgical technique and reduce the operation time. An ambitious project nowadays is to retrieve the uterus from living donors through laparoscopy. This involves dissecting and re-connecting the main blood vessels that irrigate this important organ once is transplanted. Based on preliminary results, and the experience gained thus far, we can conclude that baboons may represent an ideal model for this type of transplantation work.

PS69 Comparison of 2 Saliva Collection Methods and Saliva-Serum Cortisol Correlation in Rhesus Macaques, Cynomolgus Macaques, and African Green Monkeys

K Rapp-Santos^*1, CC Hofer¹, L Altamura², L Lugo-Roman¹

¹Veterinary Medicine Division, The United States Army Medical Research Institute for Infectious Diseases, Fort Detrick, MD; ²CTR Diagnostic Systems Division, The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD

Serum cortisol measurement has been widely used as a stress indicator due to activation of the hypothalamic-pituitary-adrenal axis in nonhuman primates. Salivary cortisol measurement may be superior because it allows for repeated sampling in a relatively short period, represents relative acute stress levels, and is less subject to degradation or contamination than other biosamples. The purpose of this study was to evaluate a human clinical saliva collection device for measurement of salivary cortisol in 3 nonhuman primate species. Serum and saliva samples were collected from 20 nonhuman primates of each species. Saliva was collected using the human clinical collection device and compared to passive drool. Both serum and saliva samples were analyzed using a commercial ELISA kit to determine cortisol levels. Mean serum cortisol was 370.2 ± 23.8 ng/mL in African green monkeys, 374.4 ± 17.9 ng/ml in cynomolgus macaques, and 492.5 ± 25.3 ng/mL in rhesus macaques. Mean saliva cortisol was 33.4 ±4.3 ng/mL in African green monkeys, 24.37 ± 4.2 ng/mL in cynomolgus macaques, and 34.3 ± 3.3 ng/mL in rhesus macaques. The mean saliva cortisol was greater in both African green monkeys (P = 0.03) and cynomolgus macaques (P < 0.001) when collected by passive drool compared to the clinical collection device. In the rhesus macaque group, the mean saliva cortisol was independent of collection technique. In both the African green and the rhesus macaque group, the clinical saliva collection technique yielded greater volume than the passive drool technique (P < 0.001, P = 0.02). Serum and saliva cortisol demonstrated adequate correlation in all species. This study represents the first evaluation of this human clinical collection device for salivary cortisol determination in nonhuman primates. It also provides the first known published ranges of salivary free cortisol in cynomolgus macaques and African green monkeys, as well as serum total cortisol in African green monkeys. Saliva cortisol demonstrated adequate correlation with serum cortisol, and the clinical saliva collection device was validated for use in 3 nonhuman primate species.

PS70 Albumin Is an Overlooked but Highly Relevant Allergen Potentially Affecting Laboratory Animal Professionals

O Kalliokoski^*, TM Kousted, J Koch, J Hau

Experimental Medicine, University of Copenhagen, Copenhagen, Denmark

Although long recognized as a prevalent allergen, mouse serum albumin has taken the back seat to the urinary protein mus m1 in investigations of mouse-derived allergen levels in laboratory animal facilities. Two implicit assumptions have been made: that albumin functions mainly as a contact allergen, which is less of a workplace health concern, and that quantification of the more volatile mus m1 gives a more accurate indication of the presence of airborne (inhalable) allergens in a laboratory animal facility. To assess the prevalence of airborne allergens in an under-ventilated animal unit, we collected air samples using an IOM sampler and quantified mus m1 and albumin collected on the filters using ELISAs (a commercial and an inhouse assay, respectively). We found, somewhat surprisingly, that albumin was present in quantities comparable to mus m1, but that the levels of the 2 allergens in the same sample were not correlated. The findings likely reflect the allergens’ different means of spread in a laboratory animal facility. Mus m1 is a smaller protein, thus an easily dispersed carrier of pheromones, whereas albumin is likely to spread adsorbed onto larger particulates like dust and dander. Consistent with this theory, the room-level ventilation was able to reduce the levels of airborne mus m1 following cage changing, whereas the levels of airborne albumin were stable over time in the animal rooms. Even though improved cage-level ventilation and aerated cage-changing stations are becoming the norm in laboratory animal facilities, higher stocking densities and old buildings with inefficacious room-level ventilation may outbalance the benefits of the last few decades’ technical innovations. Continuously monitoring and addressing sources of airborne allergens in animal facilities is important, in particular to reduce the sensitization risk for those who visit the facilities infrequently. In doing so, we suggest that controlling exposure to the albumin family of proteins is as important as the much-focused-on lipocalin family (e.g., mus m1 and its rat counterpart rat n1). Assessing only lipocalin levels when determining the airborne allergen status of a laboratory animal environment paints, in our experience, an incomplete picture.

PS71 Husbandry on a Budgett: Caring for Carnivorous Frogs (Lepidobratrachus laevis) and the Challenges of a Novel Model Organism

I Bukovnik^*, A Huffine

Laboratory Animal Resources, North Carolina State University College of Veterinary Medicine, Raleigh, NC

Lepidobatrachus laevis, commonly known as the Budgett’s Frog, is a carnivorous aquatic anuran from the Chaco region of South America. This animal has historically been kept as an exotic pet, but has also recently begun to gain popularity as a developmental research model due to its unusually large embryo size (over twice the size of X. laevis) and rapid speed of development. Current research efforts span the fields of development, evolution, morphogenesis, and tissue regeneration. Our research facility maintains over 50 L. laevis of various ages and origins, and we are currently working to establish an F3 generation as a known research strain. Because ours is the only known L. laevis research colony in the United States, husbandry protocols have been developed from the ground up and demand constant innovation. L. laevis husbandry differs from normal aquatic amphibian husbandry in several key ways, most noticeably in feeding habits and tank design requirements. As a pioneer in L. laevis husbandry, we have made many improvements to our colony, including designing an individualized feeding schedule, additional enrichment in the form of aquatic plants, and implementing a regular weighing schedule to monitor health and development. In the future, we plan to make further improvements by building an integrated tank system and developing further enrichment ideas to more closely mimic this species’ wild habitat. We predict these measures will improve frog health and breeding success by lowering stress, increasing appetite, and improving water quality.

PS72 Litter Box Training of Rabbits Housed in Pens

C Merrill^*, JA Ordile

Comparative Medicine, Pfizer, Pearl River, NY

The Guide has recommended social housing of social animals as the default housing system. AAALAC also supports this initiative. With these standards in place, the industry has begun to social house rabbits. Group housing rabbits in a laboratory facility has shown to be an excellent source of enrichment and a way to improve animal welfare. Here, rabbits are socially housed in pairs or triples in dog runs that have been converted into rabbit pens. This group-housing program resulted in increased social contact and improved animal welfare. The daily husbandry and maintenance of these pens, however, proved to be laborious and time consuming. Therefore, it was decided to attempt to litter box train rabbits with the expectation of reducing manual labor, reducing chemical usage, increasing time efficiency, and improving animal welfare. Two litter boxes containing irradiated corncob bedding were introduced in the corners of the pens where rabbits were housed and a sample of their excrement was added to each box. Over time, the less-used box was removed from the pen. Boxes were emptied a minimum of every other day, and a sample of soiled bedding was taken from the dirty box and placed into the clean box. This helped the rabbits identify the area they marked previously for consistent use of the box. Used boxes were replaced with clean boxes a minimum of twice a week to prevent urine scale build up. Litter box training of pen-housed rabbits was successful despite encountering several challenges that allowed for the opportunity for brainstorming and innovation. Overall, the success of this program has led to increased husbandry efficiency, overall cleaner pens, and improved animal welfare.

PS73 Evaluation of 4 Commercially Available Applications as Environmental Enrichment for Domestic Cats (Felis catus) in a Laboratory Animal Setting

RG Hairston^*

Unit for Laboratory Animal Resources, The Ohio State University , Columbus, OH

Institutions seek to find creative and innovative ways to encourage species-specific behavior via their enrichment program in order to promote higher-quality welfare for the animals in their care. The institution must meet this goal while also dealing with the reality of budget constraints, necessitating creative and cost-effective methods to create enrichment whenever possible. Most programs have tablet and/or smart phones available through the institution. These technologies offer a myriad of quick and useful tools that can be used for a multitude of species. This purpose of this study was to determine if smart technology applications (apps) can be used as an enrichment device for domestic cats (Felis catus) housed in a laboratory setting. Using a smart tablet, 4 commercially available cat applications were chosen to be evaluated. All consisted of different animations that were developed to entice cats into interacting with the screen through visual engagement and/or touch. The applications evaluated consisted of 1 of the following: a laser light simulator, coy fish, insects, or mice in order to elicit the cats natural prey drive. A colony of 5 gang-housed cats were provided access to each app twice a day for 10-minute periods for 4 days. During each 10-minute time frame, ethogram data for each cat was collected at 1-minute scans by the same individual observing the animals through a window. It was observed that the cats were most engaged in the applications that featured single animations that moved in quick, random patterns. These are features present in both the laser and the mouse animation applications. Variation in individual interest was also observed, with some cats consistently engaged while others demonstrated little to no interest in any of the applications. Most cats showed decreased interest in the apps within the first 2 days. While not a substitution for human interaction, smart tablet applications are a relatively simple tool that can be easily used as a component of an enrichment program.

PS74 Implementation of a Supplemental Feeding Program Improves Survival in Neonatal Common Marmosets (Callithrix jacchus)

MA Burns^*1, M Siddalls¹, J Hyman², S Sancricca¹, RP Marini¹, L Wachtman¹, JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Somerville, MA; ²Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA

The common marmoset (Callithrix jacchus) is a nonhuman primate species used in research as an attractive alternative to larger NHP species due to their small size, high fecundity, and relative lack of zoonoses. Marmoset mothers typically give birth to twins in the wild, but often produce triplets in captivity. A number of studies have evaluated factors that are predictive for infant survival, including birth weight, maternal weight, and number of infants per birth, among others. Triplet births have been associated with higher infant mortality. There is considerable variation in how institutions address the problem of failing infants, ranging from providing no support to daily supplemental feeding. While studies describing supplemental feeding guidelines have been published, there remains a need to validate and document increased survival after instituting supplemental feeding programs. Initially, no infant support was provided to neonates born in our colony. During 2015, 44 infants were born, with 28 born to triplet (n = 24) or quad (n = 4) litters. Only 24 survived the neonatal period (50% infant mortality). In an effort to improve infant survival, a supplemental feeding plan was instituted. Feeding began 24 hours after birth, starting with frequent feedings of a dilute dextrose/infant formula mixture that gradually changed to infrequent feedings of infant formula mixed with rice cereal by 28 days post birth. Supplemental feeding was rotated among all infants in the litter. Each infant was fed no more than once every 3 days unless weight loss was noted. Early on in supplementation, infants were kept in an incubator for approximately 8 hours per day, and this was decreased to <1hr by the end of the 28-day feeding period. A total of 32 infants have been born in our colony this year, and 28 infants have undergone the supplemental feeding and incubator support program. Of these 28 infants, 23 have survived the neonatal period (18% infant mortality). Institution of a supplemental infant feeding program significantly reduced infant mortality (P < 0.01; Fisher exact test). This daily feeding program also allowed for establishment of neonatal growth and diet consumption volume curves, which aid in assessment of neonates.

PS75 Effects of Pair Housing on Sleep Parameters Evaluated with Actigraphy in a Rhesus Monkey (Macaca mulatta)

LF Berro^*1,2, ML Andersen^1,2, LL Howell²

¹Psychobiology, Universidade Federal de São Paulo, São Paulo, Brazil; ²Yerkes National Primate Research Center, Emory University, Atlanta, GA

According to the Animal Welfare Act, institutions that house nonhuman primates must develop a plan to provide for the animals’ psychologic wellbeing and address their social needs. Rhesus monkeys (Macaca mulatta) are known to live in social groups in nature and behavioral management strategies have been focusing on promoting pair-housing in order to decrease distress. Although social housing shows countless benefits, pair-housing may have effects on research outcomes. In humans, it is well known that co-sleeping can have a major impact on bed partner’s sleep. In the present study we investigated if pair-housing would influence home-cage partner’s sleep in rhesus monkeys and if separation through socialization panels would disrupt this pattern. Sleep parameters of 5 naïve adult rhesus monkeys (1 single-housed socialized monkey and 2 pairs of pair-housed monkeys) were evaluated for 15 consecutive days using Actiwatch monitors. Sleep measures of pair-housed monkeys were significantly inferior compared to the single-housed subject, with the single-housed monkey showing higher sleep efficiency and lower sleep latency and fragmentation. Correlational analysis indicated a positive correlation between sleep measures of 1 paired monkey and its home-cage partner, suggesting that pair-housing influences sleep quality. Paired animals were then separated through socialization panels and sleep-like measures were evaluated for 15 days. After separation, sleep patterns of previously paired monkeys did not differ from that of the single-housed monkey. Further correlational analysis indicated a lack of correlation for all sleep measures between previously paired monkeys, suggesting that when separated the home-cage partner’s sleep does not influence the other monkeys’ sleep. In conclusion, our results suggest that social housing has a strong impact on home-cage partner’s sleep and that this pattern can be disrupted by separation through socialization panels. Studies evaluating sleep in pair-housed monkeys should take into consideration the effects that the partner’s sleep may have on the subjects’ sleep parameters.

PS76 Artificially Intelligent Weight Management System for Nonhuman Primate Research

GD Lawrence^*1, TR Hobbs²

¹Director’s Office, Oregon National Primate Research Center, Portland, OR; ²Surgery Unit, Oregon National Primate Research Center, Beaverton, OR

Excessive variation in weight can in uncertain ways adversely effect the suitability of an animal for its assigned research purpose, in addition to impacting on the animal’s overall health and wellbeing. We are challenged with managing the weight of nearly 600 nonhuman primates (NHPs). To do so we have developed a software application that allows us to review each case thoroughly yet quickly, modifying diet regimens and feeding methods when necessary. Weight is automatically tracked with respect to targets, with alerts being issued when the difference between target and actual weight exceeds a predefined threshold, or when body condition score deviates from ideals. The system puts the key components of the animal record in easy reach of the clinician, and a structured case-entry interface ensures that the weight management (WM) record is machine readable. This is essential to a rigorous determination of efficacy for individual NHP weight-management regimens, as well as evaluating performance of the WM program as a whole. In particular, we wanted to know whether particular dietary interventions, changes in feed, ration, and/or feeding methods, were actually effective in producing the desired weight change (gain, loss, or stability). Having established this relationship, we would be better placed to automate additional aspects of the WM program, including diet adjustment, allowing the system to identify cases where a modified regimen might be called for, and what sort of ration change would be appropriate. This would further optimize time allocation, presenting clinicians with recommendations for review and approval, or to be adjusted as appropriate. We proceeded by introducing new functionality in the WM software application that allowed us to graphically visualize the sequence of WM interventions (feed, ration, feeding methods) superposed on animal weight plots. We were able to assess time required for an intervention to have effect, and identify instances in which interventions were not timed well. By embedding clinical expertise in our tools, making them more “intelligent,” we found that we are able to both refine that expertise and deliver a better weight management outcome.

PS77 Challenges and Successes of Implementing Automated Feeders in an Outdoor Nonhuman Primate Facility

B Hughes^*, MT Sharpless, J Moran, T Meeker, R Stavisky, J Cohen, KF Ethun

Division of Animal Resources, Yerkes National Primate Research Center, Emory University, Lawrenceville, GA

Over the past 3 years, 32 commercially available automated feeding stations have successfully been deployed in 8 outdoor compounds, housing approximately 42% of the rhesus macaque breeding colony. As animals obtain food pellets, the computer-controlled system records grams obtained in real-time by detecting unique radio frequency identification (RFID) microchips implanted subcutaneously in each hand of individual monkeys. Because this novel system is a significant departure from standard husbandry practices, the animal care staff has been faced with various challenges related to network and infrastructure installation, equipment maintenance, feeder sanitation, diet storage and dispensement, database management, record keeping, and animal care technician staffing and training. We took steps to address each of these challenges including the establishment of internal written procedures, validation of an automated feeder sanitation protocol, formulation of new pelleted diets, employment of an operating system manager, development of effective staffing strategies, and establishment of an effective animal care technician training program. To ensure the successful implementation of these automated feeding stations on multiple outdoor compounds, a team of animal care, colony management, and veterinary medicine personnel met routinely to review and evaluate new automated feeder operating procedures. Information from this report will be of interest to the management staff of captive nonhuman primate colonies considering the utilization of commercially available automated feeding stations in either their indoor or outdoor facilities. Successful implementation of automated feeding stations has led to enhanced husbandry practices of socially housed rhesus macaques.

PS78 Physiologic and Pharmacokinetic Effects of Multilevel Caging on Sprague–Dawley Rats under Ketamine-Xylazine Anesthesia

A Dodelet-Devillers^*1,2, C Zullian¹, F Beaudry¹, J Gourdon², J Chevrette², P Helie¹, PJ Vachon¹

¹Medecine Veterinaire, Universite de Montreal, Montreal, Canada; ²Comparative Medecine and Animal Ressources Centre, McGill University, Montreal, Canada

While cage refinement is a necessary step towards improving the welfare of research rats, increasing the complexity and surface area of the living space of an animal may have physiologic impacts that need to be taken into consideration. In this study, ketamine (80 mg/kg) and xylazine (10 mg/kg) caused a short duration anesthesia that was significantly decreased in Sprague–Dawley rats housed in multilevel cages (MLC; n = 6), compared to rats housed in standard cages (SDC; n = 6). The withdrawal reflex, the palpebral reflexes, and the time-to-sternal all occurred earlier in MLC-housed rats (P < 0.02), suggesting an effect of housing on the physiology of the rats. In addition, during anesthesia, cardiac frequencies were increased in animals housed in the smaller SDC (P < 0.05). Respiratory frequencies, the blood oxygen saturation, and rectal temperatures during anesthesia did not vary between conditions during the anesthesia. While xylazine pharmacokinetics were unchanged with caging conditions, the clearance and half-lives of ketamine and its metabolite, norketamine, were altered in the rats housed in MLC. Finally, while no difference was ultimately seen in rat body weights, isolated liver and adrenal gland weights were significantly lighter in rats housed in the MLC (P < 0.05). Increasing cage sizes, while having a positive impact on wellbeing in rats, can alter anesthetic drug metabolism and thus modify anesthesia parameters and associated physiologic processes.

PS129 Using Trends to Understand Animal Resource Center Challenges

P Preisig^*1, R McDonald², JD Macy²

¹Medicine/Nephrology, Yale University, New Haven, CT; ²Comparative Medicine, Yale University, New Haven, CT

IACUCs, attending veterinarians, animal resource centers (ARCs), and investigators are facing financial, operational, and regulatory challenges that can impact the sustainability of animal-based research. Such challenges likely require reengineering and/or reprioritization of current practices to provide the bandwidth and resources to resolve the challenges and implement sustainable solutions. Analysis of discrete and longitudinal data from archived and recent Yale Animal Resource Cost and Benchmarking survey data (reflecting ARCs in US academic Institutions) quantifies, qualifies, and/or contextualizes contemporary issues facing animal programs. The analysis profiles the highest-ranked challenges in the context of trends in the funding environment, consumer price index (CPI), and federal regulatory requirements. The highest-ranked financial challenges had the theme of reducing operating costs and managing the budget, consistent with expectations as a result of the plateauing of the NIH budget, steady increase in the CPI, and finding that 40-70% of participating institutions report the inability to fully recover mouse care costs, the latter possibly due to limits on per diem rate affordability. The highest-ranked operational challenges were managing human resources, increased compliance requirements, and inefficient facility size and layout. The highest-ranked technical challenges were tracking census and service charges. Despite these challenges, participants felt that the noncompliance-related program changes increased researcher productivity, but also increased operational costs, possibly contributing to the cost under-recovery of ARCs. Overall, compliance-related program changes more negatively effected researcher productivity. The most effective ARC program improvements focused on animal care and welfare, outcomes that require financial support but can be incorporated into the per diem rates; ∼60% of participants reported per diem rate increases associated with these improvements. In contrast, the most effective IACUC improvements focused on making the lives of the Investigators easier. While these improvements also require financial support, they are often not included in the per diem rate.

PS79 Eradication Of Demodex musculi Using a Topical Parasiticide in an Infested Transgenic Mouse Strain with Defective Adaptive Immunity

MA Nashat^*1,2, NS Lipman³, FR Wolf³, RJ Ricart Arbona³, SF Santagostino³

¹Comparative Bioscience Center, The Rockefeller University, New York, NY; ² Center for Comparative Medicine and Science, Tri-Institutional Training Program of Laboratory Animal Medicine and Science, New York, NY; ³Center for Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center and Weill Cornell Medical Center, New York, NY

A unique transgenic mouse strain (B6.Cg-Rag1^tm1MomTyrp1^B-w Tg(Tcra,Tcrb)9Rest/M, TRP-1/TCR mice) with defective adaptive immunity presented with ulcerative dermatitis and suppurative ocular and skin infections. Corynebacterium bovis and Pasteurella pneumotropica were isolated from the lesions and Demodex musculi mites were observed in skin sections from affected regions. As rederivation would be time consuming and costly, we assessed whether a parasiticide could be used to eradicate D. musculi from the colony. Thirty mice were confirmed mite-positive by fur plucks and deep skin scrapes. Infested mice were randomly assigned to 3 groups: untreated control, ivermectin (12 ppm) diet ad libitum for 8 weeks, or moxidectin 3.3 mg/kg and imidacloprid 13 mg/kg weekly for 8 weeks. Following treatment, half of the mice from each group were euthanized and necropsied, or they were euthanized 3 months after cessation of treatment. Fur plucks, deep skin scrapes, and histologic skin sections revealed large numbers of mites in untreated controls while ivermectin-treated mice had a reduced mite population and moxidectin/imidacloprid-treated animals were mite free. Subsequently, a shorter 4-week treatment trial revealed that 3/3 (100%) control and 1/7 (14.3%) moxidectin/imidacloprid-treated mice remained infested. These results indicate that an 8-week treatment regimen of moxidectin/imidacloprid is necessary to eradicate D. musculi mites in an infested immune-compromised mouse strain and that ivermectin at 12 ppm in feed is not sufficient. Additional studies evaluating other drug combinations, routes of administration, and newer parasiticides are warranted.

PS80 Exhaust Air Particle PCR: A Highly Reliable Alternative Microbiological Monitoring Method for the Detection of Excluded Pathogens in IVC-Housed Rodent Colonies

M Brielmeier^*

Research Unit Comparative Medicine, Helmholtz Zentrum München, Neuherberg, Germany

Recently, alternative health monitoring strategies for IVC-housed rodent colonies have been proposed to improve the detection of excluded pathogens. One novel method uses exhaust air particles polymerase chain reaction (EAP-PCR) to detect pathogen nucleic acids associated with particles in the exhaust air of IVC cage rack systems. Although the idea of environmental samples for microbiological monitoring is not new, only recently, suppliers of IVC systems have presented alterations to their equipment that provide user-friendly technical solutions. Yet, there is only limited peer-reviewed information on the reliability and sensitivity of EAP-PCR. Intensive research was conducted to provide evidence that the method of EAP-PCR is superior to soiled bedding sentinel serology, a method flawed by serious drawbacks. EAP real-time PCR was systematically tested for Mouse Norovirus (MNV),Pasteurella pneumotropica, and Helicobacter hepaticus with multiple repetitions and at different pathogen prevalence. Sentinels were exposed to soiled bedding during multiple 3-month monitoring periods and gauze pieces pinned on the prefilter of an air handling unit of an IVC system were exposed to particles in the IVC racks exhaust air. Under field conditions, in animal rooms with medium to high prevalence, EAP PCR detected MNV in 57/59, SBS serology in only 28/59 3-month periods, respectively. EAP-PCR detected Pasteurella pneumotropica in 26/26 quarterly monitoring periods whereas SBS serology failed completely (0/26). At low prevalence, EAP-PCR detected MNV- and Pasteurella pneumotropica-infected animals in 6/6 monitoring periods, while SBS detected MNV in only 1/6 periods and failed to detect Pasteurella pneumotropica in all instances (0/6). EAP PCR proved highly sensitive and the minimum prevalence of MNV, Pasteurella pneumotropica, and Helicobacter hepaticus for a positive PCR result was repeatedly 1 cage with 5 infected mice in a 63-cage IVC system. In addition, EAP-PCR was able to detect infections within 1 to 2 weeks after onset. The use of EAP-PCR is highly recommended as an additional method to exclude unwanted organisms.

PS81 Detection of Passalurus ambiguus in a Laboratory Rabbit Colony

CL Perkins^*1, KS Henderson¹, LA Burlingame², P Lester²

¹Charles River, Wilmington, MA; ²University of Michigan, Ann Arbor, MI

Passalurus ambiguus is an oxyurid pinworm of rabbits (Oryctolagus cuniculus) which has typically been reported in domestic and wild rabbits, but has historically thought to have been uncommon in laboratory rabbits. In this study, we investigated a colony of laboratory rabbits from an academic institution as a result of repeated low-positive findings in fecal samples by qPCR pinworm screening over a 1-year period. An investigative transition to using environmental swab samples and perianal hair swabs for PCR testing provided substantially higher and reproducible detection of a pinworm infestation. To confirm the presence of P. ambiguus, a 750bp segment of the 28S rRNA gene was amplified and sequenced. Obtained sequence was consistent with P. ambiguus sequence previously obtained from individually isolated rabbit pinworms. There is no P. ambiguus sequence currently available in GenBank, but BLAST analysis indicates that the target sequence is ∼90% homologous to Aspiculuris spp. To further investigate detection methods, a diagnostic comparison of the colony was performed using PCR targeting the 28s rRNA gene, fecal float, and tape test methods. Ten male and 10 female New Zealand White rabbits were evaluated at each of 3 age ranges; <6 months, 6-12 months, and >12 months of age. Tape test and fecal float evaluation was performed at the colony facility and perianal hair swabs were collected for qPCR screening and submitted to a diagnostic laboratory. Pinworm detection rates were 68% via PCR, 38% for tape test, and 8% for fecal float. Although overall PCR was more sensitive, there were some instances where eggs were detected by tape test and not by PCR. Infestation rates, and the estimated copy number detected by qPCR, are both higher in younger animals and decrease with age. There does not appear to be a notable gender bias in infestation rate. Environmental swab samples were also collected from the ceiling exhaust vents and floor of a newly populated room and were tested using the qPCR screening assay. P. ambiguus was detected in the environment swabs after 6 weeks of exposure. As most rabbit colonies are housed in open cages, environmental sampling provides a simple way to monitor for P. ambiguus by PCR testing.

PS82 Development and Qualification of a Probe-Based Fluorogenic Rt-qpcr for the Detection of Zika Virus In Nonhuman Primates

C Wang^*, P Momtsios, KS Henderson

Charles River Laboratories, Wilmington, MA

Zika virus (ZIKV) is a single-stranded RNA virus that is an emerging agent related to other Flaviviruses such as West Nile virus, yellow fever virus, and dengue virus. ZIKV was initially isolated from a rhesus macaque (Macaca mulatta) in 1947 in Uganda. Like other Flaviviruses, it can infect multiple host species and can be transmitted by mosquitoes (Aedes spp.) which is the major vector for transmission to humans. In humans, infection of ZIKV often have influenza-like signs and symptoms and could be misdiagnosed during the acute phase. Recently, outbreaks of ZIKV infection have occurred in Brazil and distributed to USA and several other areas in South America. There is no valid vaccine available to protect hosts from ZIKV infection. Because nonhuman primates (NHPs) in southern United States are sometimes housed in facilities with access to outdoor air, ZIKV-infected mosquitos could pose a risk for infection of NHPs and to caretakers. To date, rapid laboratory detection of the presence of ZIKV in nonhuman primates and other research animals, particularly for those housed outside in research facilities, has not been reported. Based on ∼90 ZIKV sequences in the NCBI database, we developed a fluorogenic real-time RT-qPCR assay targeting a conserved region of the nonstructure protein gene. The RT-qPCR assay limit of detection was determined to be in the 1-10 copy range, and there was no cross-reactivity to genomic DNA of multiple host species and other pathogens. Inactivated ZIKV was acquired and tested to confirm that the process from RNA isolation to PCR testing was functional. We obtained 45 NHP blood samples from 3 different institutions in the southern USA. These NHPs were housed outdoor and may be exposed to ZIKV infected mosquitos. When compared with the inactivated ZIKV template, the results from all 45 samples were determined to be negative. ZIKV serology assay may not distinguish between different flavivirus NHP seroconversion antibodies, such as antibodies from Dengue virus (DENV) and West Nile virus (WNV). Therefore, individual flavivirus infections can only be confirmed by agent specific PCR assays. Samples from ZIKV infected animal or infected colony will be further validated by this RT-qPCR assay. In summary, we developed a ZIKV RT-qPCR assay that may be used to routinely monitor for active infection of ZIKV in NHP research colonies, and may also be considered as a tool in ZIKV research studies.

PS83 Detection and DNA Sequencing of Pinworms and Novel Fur Mites in a Laboratory Vole Colony (Microtus ochrogaster)

CL Perkins^*1, WR Shek¹, SM Sullivan², SE Perkins³

¹Charles River, Wilmington, MA; ²Northeastern University, Boston, MA; ³Tufts University, Boston, MA

Voles (Microtus spp.) are occasionally used in laboratory research for neurobiology, genetics, and social behavior studies. In this study, we compared methods of detection for pinworms and fur mites infesting an academic colony of prairie voles (Microtus ochrogaster). In addition, we speciated the parasites by DNA sequencing. Comparative testing was performed on 8 male and 12 female voles of various ages. Using a dissecting microscope, fur was examined for fur mites and macerated GI tract for adult pinworms. Also, qPCR assays targeting conserved regions of the 18S rRNA gene in the fur mite genera Myobia and Radfordia, and the 28S rRNA gene of the pinworm genera Aspicularis and Syphacia were performed on body swab and fecal pellet pools. Direct examinations detected fur mites in 100% and pinworms in 55% of the animals. PCR detected fur mites in 80%, and pinworms in 50% of the animals. The lower rate of detection of fur mites by PCR may be due to sampling limitations. The estimated pinworm copy number detected was higher in younger versus older animals whereas the fur mite DNA copy number remained consistent. There were no apparent differences observed based on gender. DNA sequencing was performed on a 572bp segment of the fur mite 18S rRNA gene and an 719bp segment of the pinworm 28S rRNA gene amplified from PCR positive samples. Using Vector NTI, the consensus sequence for the vole fur mite 18S rRNA gene was compared to sequences generated in our laboratory for Myobia musculi, Radfordia affinis, Radfordia ensifera, and Myocoptes musculinus; it was found to be 99% homologous to both M. musculi and R. affinis, but with base pair differences suggesting a novel fur mite species. It is unclear whether this mite species may infect mice, rats or other rodent species. Using BLAST analysis, the consensus pinworm 28S rRNA sequences was compared to pinworm sequences in GenBank; it was found to 99% homologous with Syphacia montana. It is unknown whether S. montana may cross-contaminate other rodent species, although literature categorizes this species as specific to the subfamily Microtinae.

PS84 Using One-Time Fetal Surgery Measurements to Predict Fetal Health and Growth

JS Streeter^*1,2, S Louey², S Jonker²

¹Comparative Medicine, Oregon Health and Science University, Portland, OR; ²Knight Cardiovascular Institute, Oregon Health and Science University, Portland, OR

Pregnant sheep are the gold standard model for studying cardiovascular and pulmonary development because of close parallels to human development. Large animal studies are labor intensive and expensive, making selection of healthy, normally growing fetuses essential for acquisition of quality data. Surgical indicators of fetal wellbeing would be a critical refinement, reduce wasted cost and effort invested in suboptimal fetuses, improve data quality, and reduce unnecessary use of animals in research. It is likely measurable deviations from the expected range of arterial blood gases and related parameters (e.g. arterial pH, pCO2, pO2, hematocrit) at the time of fetal surgery that can be used to establish whether a fetus is within the normal, or suboptimal ranges for further study. To date, no systemic studies have used measures at the time of fetal surgery to assess fetal wellbeing, growth trajectories, and suitability to continue to experimental protocols. To test whether a single blood sample predicts fetal outcomes, we obtained fetal arterial samples at surgery and 3-7 days post surgery; blood gases were analyzed on a Radiometer ABL825 and hematocrit was determined by microcapillary tube centrifugation. Maternal samples were collected for measurement of cortisol by ovine-validated ELISA. Data were compared by Pearson Correlation and Student’s T Test. Surgical and postsurgical fetal hematocrit were correlated (P < .0001, R=0.6120). Given known relationships between hematocrit with long-term trends in fetal arterial oxygen pressure (PaO₂), postsurgical PaO₂ values were compared for fetuses with a surgical hematocrit <40% or ≥40%; this relationship is confirmed in our dataset (P = 0.0551). Post-surgical maternal cortisol and post-surgical fetal hematocrit were significantly correlated (P = 0.0424, R=0.6836). Interestingly, fetal surgical pH was not correlated with post-surgical hematocrit or PaO₂. These findings suggest that fetal surgical hematocrit, which reflects the physiologic status over days or weeks (versus pH, which changes rapidly), and maternal postsurgical cortisol values, reflective of maternal stress, may be useful to predict postsurgical fetal health.

PS85 Cardiovascular Phenotyping in Mice through the Measurement of Pulsed Doppler Ultrasound Blood Flow Velocity

AK Reddy^*1,2, S Madala^1,2

¹Indus Instruments, Webster, TX; ²Medicine, Baylor College of Medicine, Houston, TX

Evaluation of cardiovascular (CV) function in genetically altered mouse models that mimic human CV diseases requires reliable measurements with minimal effects from experimental conditions. Invasive measurement of blood pressure or flow are reliable but can be traumatic and/or terminal. Therefore, a noninvasive method to evaluate CV function in mice and one that meets “reduction” and “refine” aspects of the 3Rs of animal welfare is desirable. Pulsed Doppler ultrasound is such a method which allows for single or serial noninvasive measurements in living animals. Noninvasive blood velocity measurements can be made from the heart and the peripheral vessels in a less cumbersome and relatively shorter time using pulsed Doppler ultrasound versus echocardiography. Blood flow velocity measurements, along with electrocardiogram, are made in isoflurane anesthetized mice. Measurement times for a given site can range from 5-15 min. The following measurements can be made: (1) left ventricular filling and ejection velocities to determine diastolic and systolic function; (2) coronary velocity to determine coronary flow reserve; (3) pulse transit time in an arterial segment to determine pulse-wave velocity (arterial stiffness index); (4) flow velocity waveforms from peripheral vessels to determine indices of arterial compliance, resistance, and wave reflections; and (5) stenotic velocities for estimation of pressure gradient; and 6. tail artery velocity for estimating blood pressure using a tail-cuff. This convenient and easy method can be used by investigators to evaluate cardiovascular function in various genetically, surgical, and/or pharmacologically altered mouse or other small animal models. Because flow velocity is independent of body size due to the scaling of lengths and time constants of vascular systems, the magnitude and shape of velocity waveforms in mice are similar to the waveforms obtained in humans. Thus, much of what we learn from mice is readily translatable to humans for the diagnosis, evaluation, and treatment of cardiovascular diseases.

PS86 Effects of Buprenorphine, Methylnaltrexone, and Their Combination on Gastrointestinal Transit in Rabbits (Oryctolagus cuniculus)

M Martin-Flores^*1, BS Singh², CA Walsh³, EP Brooks³, LC Taylor³, LM Mitchell¹

¹Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY; ²Center for Animal Resources and Education, College of Veterinary Medicine, Cornell University, Ithaca, NY; ³College of Agriculture and Life Sciences, Cornell University, Ithica, NY

Buprenorphine (BUP) is likely the most commonly used opioid analgesic in rabbits (Oryctolagus cuniculus). However, like most opioids, it can decrease gastrointestinal (GI) motility and contribute to ileus. The peripheral opioid antagonist methylnaltrexone (MTX) might be useful in relieving the GI effects of BUP without affecting analgesia. We evaluated the effect of buprenorphine and methylnaltrexone on GI transit time in 8 healthy male rabbits. The rabbits were administered 4 treatments using a randomized crossover design (at least 1 week apart): BUP 0.05 mg/kg, MTX 1 mg/kg, both agents combined (B/M), or normal saline (control). All treatments were administered subcutaneously every 12 hours for 2 days. Prior to each treatment, 20 barium-filled plastic spheres were administered via orogastric tube to be later identified by radiography of feces. Feces were collected every 6 hours, and body weight, food, and water consumption were measured once daily, for 5 days. The time to first detection of spheres in feces was compared between the groups using Kruskall-Wallis test. Changes in body weight, daily consumption of food and water, and daily fecal weight were compared between the groups with mixed effects models. Time to first passage of spheres was significantly longer for BUP [median (minimum - maximum)] [30 (24-48) hours] than for control [18 (18-18)] and for MTX [18 (18-24)]. The weight of collected feces per day was significantly lower for BUP and BM than for the other 2 groups. Water and food consumption was lowest for groups BUP and B/M than for control and MTX, however, there was no difference between the groups in daily change in body weight. BUP delayed GI transit time, and decreased daily fecal output and food and water consumption. MTX (1 mg/kg) administered concurrently was insufficient to prevent these effects.

PS87 Quantification of Induced Hypothermia from Aseptic Scrub during Rodent Surgery Preparation

FC Hankenson^*3, A Skorupski², D Ferguson³, J Zhang¹, F Lawrence¹

¹Center for Statistical Training and Consulting, Michigan State University, East Lansing, MI; ²College of Veterinary Medicine, Michigan State University, East Lansing, MI; ³Campus Animal Resources, Michigan State University, East Lansing, MI

Laboratory mice, Mus musculus, are common models in biomedical research, often which involve surgical procedures as part of approved biomedical research objectives. Mice may be prone to develop undesirable levels of hypothermia while under anesthesia for surgery; hypothermia may impede anesthetic recovery, wound healing, and future health. Previously, whole body temperatures of mice were shown to be influenced by a variety of supplemental heat sources while under isoflurane anesthesia. This related study was designed to quantify the whole body cooling effect, particularly by ethanol (EtOH) with its evaporative properties, that scrub applications may have on the progression of hypothermia in anesthetized mice (n = 47) and to determine if a certain scrub application should be preferentially recommended. EtOH, room-temperature saline, and warmed saline (37°C), coupled with the iodophore betadine, were assessed as to their impact on both core temperature, recorded by rectal probe, and surface temperature, recorded by infrared scanner. Scrub options were applied in triplicate to a 2cm x 2cm shaved abdominal area on mice that were maintained on a warm-water recirculating blanket (at 38°C) in a surgical suite (20-23°C room temp average) and given isoflurane anesthesia (1.5-2.0% @ 0.6 L/min) for a 30-minute period. Interestingly, all applications that included betadine resulted in animals with the coolest body temperatures, significantly different from control animals (P < 0.05) at the time of application and for the remainder of the surveillance phase of 30 minutes. Betadine plus saline options resulted in body temperatures that lowered throughout the 30-minute period, with no differences observed despite the fluid warming. EtOH alone demonstrated the most rapid cooling after application, both for surface and core temp readings, followed by an unanticipated warming (rebound) phase that equilibrated with the control animals’ baseline temperature within 12 minutes of application. These findings demonstrate that EtOH can be part of an acceptable scrub protocol to prepare an incision site, as it resulted in near normothermia in mice that were maintained on an appropriate heat source while under isoflurane anesthesia.

PS88 Pharmacokinetics of a Novel, Transdermal Fentanyl Solution in Rhesus Macaques (Macaca mulatta)

GW Salyards^*1, HK Knych², AE Hill^3,4, KL Christe^1,4

¹Department of Primate Medicine, California National Primate Research Center, University of California—Davis, Davis, CA; ²K.L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California—Davis, Davis, CA; ³California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California—Davis, Davis, CA; ⁴Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California—Davis, Davis, CA

Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate biomedical model, consisting of both research and clinical procedures requiring analgesia. Providing analgesia is imperative and opioids are a mainstay of analgesic therapy. A novel, transdermal fentanyl solution (TFS) was developed to be a long-acting, single-administration, topical opioid that delivers ≥4 d of therapeutic plasma concentrations as previously published in beagles (Canis familiaris). The TFS dose was applied to clipped dorsal skin under ketamine (10 mg/kg IM) sedation. We describe the pharmacokinetic profile of TFS in healthy, adult rhesus macaques (n = 6; 3M, 3F) in this 2-period, 2-treatment crossover study of a single topical administration of 1.3 (25) and 2.6 mg/kg (50 μL/kg) TFS. We hypothesized TFS in rhesus macaques would provide ≥4 d of therapeutic plasma concentrations (≥0.2 ng/mL) as compared to beagles. Plasma fentanyl concentrations were determined by tandem liquid chromatography-mass spectrometry prior to drug administration and for up to 21 d post administration (0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, 240, 336, 408, and 504 hours). Noncompartmental pharmacokinetic analysis was performed. For each dose (1.3 and 2.6 mg/kg), respectively, maximum plasma concentration was 1.95 ± 0.40 and 4.17 ± 0.70 ng/mL, occurring at 21.33 ± 4.09 and 30.67 ± 8.68 hours. The area under the curve was 227.25 ± 31.67 and 446.97 ± 49.08 h/ng/mL and the terminal elimination half life was 93.65 ± 7.06 and 98.75 ± 5.40 h. No adverse effects were noted at either dose. Macaques maintained plasma fentanyl concentrations ≥0.2 ng/mL as compared to beagles for at least 7 days after 1.3 mg/kg and at least 10 days after 2.6 mg/kg topical administration of TFS. A single TFS dose may provide efficacious therapy with less stress, discomfort, and risk to animals and personnel.

PS89 The Use of Ear Pressure Pain Analysis to Assess Pain and Analgesic Efficacy in the Laboratory Rabbit (Oryctolagus cuniculus)

K LaVallee^*1, A Yorsh¹, A Hess², LV Kendall¹

¹Laboratory Animal Resources, Colorado State, Fort Collins, CO; ²Statistics, Colorado State University, Fort Collins, CO

The ability to assess pain in laboratory animals is critical in order to provide appropriate analgesia, while ensuring optimal animal care and welfare. New Zealand White rabbits are common animal models for surgical implant biomedical research due to their large size, which enables relatively easy surgical operations. These procedures are predictably painful, but methods to quantitatively assess analgesic efficacy in rabbits are poorly described. We evaluated ear pressure pain analysis (EPP) using the Randall-Siletto anlgesiometer as a means to assess analgesic efficacy in rabbits. Ear pressure pain analysis was conducted once per day for 3 days to acquire baseline pain tolerance for 3 rabbits. Rabbits were treated with either topical EMLA cream (Lidocaine and Prilocaine), Meloxicam (0.5 mg/kg SC), or Buprenorphine (0.05 mg/kg SC) in a cross-over design with a 3-day washout period between treatments. EPP was conducted 30 minutes after EMLA application, and 2 and 6 hours after administration of Meloxicam and Buprenorphine. Baseline EPP measurements were 209 g. Rabbits receiving ELMA had a significant increase in EPP to 550 g (P < 0.001). There was an increase in the EPP of Meloxicam treated rabbits (240 g) and Buprenorphine (277 g) at 2 h (P = 0.9 and 0.4, respectively). There were no differences noted at 6 hours. These results suggest that EPP has the potential to quantitatively assess pain in laboratory rabbits.

PS90 Prophylactic Antibiotics Prevent Corynebacterium bovis Infection Following Acute Exposure

CA Manuel^*1,2, MJ Schurr³, CJ Henry^3,4

¹Office of Laboratory Animal Resources, University of Colorado—Denver, Aurora, CO; ²Department of Pathology, University of Colorado—Denver, Aurora, CO; ³Department of Immunology and Microbiology, University of Colorado—Denver, Aurora, CO; ⁴Department of Biochemistry and Molecular Genetics, University of Colorado—Denver, Aurora, CO

Corynebacterium bovis is sensitive to commonly used antibiotics that are able to reach therapeutic blood concentrations in mice when administered orally. However, it is believed that prophylactic antibiotics are ineffective at preventing C. bovis infections. An experiment was performed to evaluate if prophylactic antibiotics can prevent a C. bovis infection following acute exposure and to investigate the innate immune response to infection in athymic nude mice (n = 32). Amoxicillin and clavulanic acid were administered in the water to naïve mice for 14 days following C. bovis exposure. Treatment was started 3 days prior (-3 d, n = 8), or immediately following (0 d, n = 8), acute exposure to C. bovis. C. bovis positive and negative controls (n = 8 each) and antibiotic treatment groups were serially sampled by oral and dermal swabs on days 0, 5, 10, 15, 21, 35, 49, and 63 for C. bovis DNA by qPCR. Half of mice were euthanized at day 15, with the remaining mice euthanized 63 days post exposure. At harvest, splenocytes were isolated and evaluated for activated macrophages (Mac-1⁺, IFNg⁺) and NK cells (DX5⁺, IFNg⁺) by flow cytometry. Our results show that all mice given prophylactic antibiotics starting on -3 day and 0 day of acute exposure were C. bovis negative by qPCR at both endpoints. At 15 and 63 day post exposure, the number of activated splenic macrophages was significantly lower in both antibiotic treated groups as compared to C. bovisinfected controls (P < 0.05). However, at 15 day post exposure, the number of activated macrophages was significantly higher in the 0 d treatment group as compared to the -3 day treatment group (P < 0.05). There were no significant differences in activated NK cells between positive and negative controls at either time point. Our results demonstrate that prophylactic antibiotics can prevent C. bovis infections in athymic nude mice when started -3 day and 0 day of an acute exposure. In addition, our data also shows that macrophages of the innate immune response are activated when antibiotics are started on the day of C. bovis exposure, but are not activated when antibiotics are started 3 days prior to exposure.

PS91 “Functionally Appropriate Primate Environments” as an Alternative to the Term “Ethologically Appropriate Environments” to Describe Environments for Research Primates

MA Bloomsmith^*1, J Hasenau³, R Bohm²

¹Yerkes National Primate Research Center, Atlanta, GA; ²Tulane National Primate Research Center, Covington, LA; ³Lab Animal Consultants, Reno, NV

The term “ethologically appropriate environments” was first published in the report of the Institute of Medicine Committee on the Use of Chimpanzees in Biomedical and Behavioral Research in 2011. In 2015, the United States Department of Agriculture requested comments on a petition which called for amending the Animal Welfare Act so that all research primates would be housed in “ethologically appropriate physical and social environments.” We are critical of this term because (1) it does not provide clarification beyond that in current regulatory language, (2) it does not provide for balance between animal welfare goals and the reasons why the primates are housed in research facilities, (3) it discounts the adaptability that is inherent in the behavior of primates, (4) it conveys that duplication of features of the natural environment are required for suitable holding environments, (5) objective studies reveal that environments that appear to be more ethologically appropriate do not necessarily better meet the needs of animals, and (6) using the term “ethology” is inherently confusing. We propose that the term “functionally appropriate primate environments” be used instead, as it emphasizes how environments work for nonhuman primates, it better describes current activities underway to improve nonhuman primate welfare, and the balance that is achieved between meeting the needs of the animals and the requirements of the research in which they are involved. We propose a process to further refine this term through literature reviews, interviews with experts, identifying areas with convergence of opinions on major features needed to support welfare, conducting a gap analysis to ascertain needs for future research, and developing a mechanism to integrate research findings into animal care standards. We believe this approach will be fruitful as we work to further refine environments for nonhuman primates in biomedical research.

PS92 Intermittent versus Continuous Pair-Housing in Laboratory Rhesus Macaques (Macaca mulatta) and Activation of the HPA-Axis

D Hannibal^*2,1, L Cassidy⁵, A Barnard¹, J Vandeleest¹, K Chun³, S Semple⁴, B McCowan^2,1

¹California National Primate Research Center, University of California—Davis, Davis, CA; ²Population Health and Reproduction, University of California—Davis, Davis, CA; ³Semel Institute for Neuroscience and Human Behavior, University of California—Los Angeles, Los Angeles, CA; ⁴Life Sciences, Roehampton University, London, United Kingdom; ⁵The German Primate Centre, Göttingen, Germany

A social environment provides optimal captive primate welfare. At indoor research facilities, laboratory rhesus macaques are often housed in pairs and may be temporarily or permanently separated for study procedures, concerns over compatibility, medical surveillance, or other reasons. While long-term separations, as well as introductions, can stimulate the stress response systems to the point of impacting inflammation and immune function, it is unknown whether short-term separations for less than a day can have an effect. In this study, we investigated differences in urinary cortisol among 24 adult female rhesus macaques over a 10-week period. The sample consisted of 12 females in continuous pair-housing prior the start of the study, then switched to intermittent pair-housing half-way through the study (CI subjects), and 12 females in intermittent pair-housing for management reasons prior to the start of the study, then switched to continuous pair-housing half-way through the study (IC subjects). Creatinine and cortisol were assayed from urine samples collected 2 times per week. Behavioral data was collected on each pair using 8-minute focal observations twice each morning and afternoon for measures of affiliative, aggressive, anxious, abnormal, and activity-state behaviors. At the end of the study, subjects were scored for personality and pair-rating attributes. Cortisol data were analyzed using a generalized linear model with gamma distribution and an information theoretical approach to determine the predictors for a best-fit model. The final model (X2=49.98; df=6; P < 0.001) shows subjects that are dominant (beta=-0.55; P < 0.001), paired with a close relative (beta=-0.61; P = 0.004), and in the IC pairing group (beta=-0.65; P < 0.001) have lower cortisol. There is also an interaction of intermittent-housing with “tense” pair rating, showing that subjects in the intermittent condition are likely to have increased urinary cortisol if they are in a “tense” pair (beta=0.24; P = 0.002). This suggests that to avoid chronic activation of the HPA-axis, rhesus macaque females should be housed in continuous contact in compatible pairs rather than maintained through intermittent housing in less compatible pairs.

PS93 Use of Temperament in Behavioral Management Practices for Captive Nonhuman Primates

K Coleman^*

Oregon National Primate Research Center, Beaverton, OR

Effective behavioral management programs are designed to address the behavioral needs of the animals. However, there is often a one size fits all approach, in which techniques that have been found to be beneficial for some are assumed to be good for all. However, individuals vary with respect to a wide range of traits, such as temperament or personality, that may affect how they respond to these practices. Temperament influences how individuals cope with novel or stressful situations. Thus, it stands to reason that it may affect how individuals react to various management procedures, including positive reinforcement training, socialization, and response to environmental enrichment. Over the past 5 years, an increasing number of studies have examined how measuring temperament can help guide behavioral management decisions. For example, knowing the temperament of macaques can assist in social introductions. Further, an individual’s propensity to use environmental enrichment can be influenced by its temperament. Shy, inhibited individuals may not use or get the same benefits from some common environmental enrichment options compared to more exploratory individuals. Temperament can also affect an individual’s trainability. Bold monkeys are easier to train using positive reinforcement techniques than their shy conspecifics. This presentation will examine how individual differences in temperament might affect behavioral management practices for captive nonhuman primates.

PS94 An International Survey of Approaches to Monkey Chair Restraint

JL McMillan^*1, M Prescott², MA Bloomsmith¹

¹Yerkes National Primate Research Center, Atlanta, GA; ²National Centre for the Replacement, Refinement and Reduction of Animals in Research, London, United Kingdom

The use of specifically designed restraint chairs is the preferred method of restraint for studies that require nonhuman primates to sit in place for sustained periods of time. Restraint chairs have been used for decades within the research environment and there is a lot of variation in the equipment employed and the methods used for preparing animals for these procedures. With increasing emphasis on refinement of restraint procedures to promote animal welfare, it’s important to have a better understanding of this potentially stressful procedure. Chair restraint is used all over the world, yet there is little published information available for researchers on the subject. We developed a survey to obtain a better overview of chair restraint equipment, procedures, and plans for improvement. We received 101 responses representing academic, pharmaceutical, and government facilities within the United States, Canada, Europe, and Asia. Findings indicated that the majority of researchers using restraint chairs work with macaque species and conduct a wide range of procedures including cognitive testing, recording neuronal activity, fMRI, intravenous infusion, and blood sampling. Two-thirds of laboratories reported using an enclosed chair, where the animal enters and is trained to extend his/her head through an opening on top of the chair, and 39% use an open chair which typically requires the use of the pole and collar system to transfer and secure the animal into the chair. Researchers reported considering a variety of factors when choosing the type of chair for their studies including comfort for the monkey, ease of use, and ability to adjust fit between different-sized monkeys. We were encouraged with the number of facilities incorporating positive reinforcement training methods into their approaches. The most commonly reported period of training time before research procedures begin was 1-2 weeks (38%) for users of open chairs and 1-2 months (38%) for users of enclosed chairs. Similar proportions of open (69%) and enclosed chair users (71%) felt additional preparation time would be beneficial. As a research community using restraint procedures, there is a need to work together to define methods of best practice and this survey can help movement in that direction.

PS95 Primate Behavior Management Certification Course for Animal Care Staff

JE Perlman, S Hastings, J Cowan-Brown^*, K Neu, MA Bloomsmith

Yerkes National Primate Reseach Center, Emory University, Atlanta, GA

Animal care technician responsibilities include identification of adverse or abnormal behaviors in the species with which they work. The Guide states animal care personnel should receive training in the behavioral biology of the species. Our Behavioral Management Unit developed a certification course for the purpose of teaching the behavioral biology of nonhuman primates and to provide a framework, practical tools, and a system of communication with which to address behavioral issues. Five areas of behavioral management are presented. These topics are primate behavior, environmental enrichment, animal training, social housing, and welfare. The course includes instruction on the behavior of laboratory primate species in the wild and captivity (chimpanzee, macaque, squirrel monkey, and managbeys); environmental enrichment includes applications of the 4 inanimate enrichments; animal training covers terminology, techniques, and application that promote positive reinforcement techniques and how to apply alternative methods; and social housing includes the impact of different housing options on behavior and daily management. Captive wellbeing research is discussed with an emphasis on practical applications such as identifying and reporting behaviors of concern. Information is presented via lecture, discussion, demonstrations, and group exercises. Testing is conducted at the end of each unit to demonstrate information is synthesized and can be applied to everyday care. Students receive certificates for each unit successfully completed and receive full certification in behavioral management after all of the units are passed.

PS96 Size Doesn’t Matter: Behavior Management of a Small Population of Nonhuman Primates in a Research Setting

AN Rehrig^*

University of Rochester, Rochester, NY

Implementing and managing a behavior program for a small population of nonhuman primates may seem like a simple endeavor at first glance; however, program implementation in a close knit research community poses numerous challenges. Regardless of size, successful programs depend on securing funding for behavioral staff and resources, staff training specific to behavior management in a research setting, and fostering program acceptance with the IACUC, principal investigators, and husbandry staff. At our institution, the animal behaviorist oversees the behavior management for the entire research collection, including everything from rodents to primates and is involved in all aspects of the program including hands-on animal work and administrative duties. Prior to start up, an outside consultant was brought in to assess the existing program and the behaviorist visited sister institutions to gain insight on how to improve and grow the program using best practices in the field. Acceptance was achieved through open and effective communication with all involved parties, demonstration of improved animal welfare, and increased compliance and recognition from regulatory institutions (e.g., USDA). Effective behavior management programs, even in small-scale operations, can have a significant and long lasting, positive impact on primate welfare, as well as the culture of the research institution overall.

PS97 Taking Behavioral Management to the Next Level: Providing Chimpanzees with Opportunities to Participate in Their Own Care

SJ Schapiro^*1,2, L Reamer¹, ER Magden¹, S Lambeth¹

¹National Center for Chimpanzee Care, University of Texas MD Anderson Cancer Center, Bastrop, TX; ²Experimental Medicine, University of Copenhagen, Copenhagen, Denmark

Behavioral management programs have advanced considerably during the 5 years since the publication of the most recent version of the Guide. We have focused our efforts on behavioral management techniques that allow the animals to make meaningful choices and to control some aspects of their care. Many of the chimpanzees are greater than 30 years of age, and some are experiencing aging-related maladies, particularly arthritis. In addition to the administration of typical arthritis medications, we provide our chimpanzees with opportunities to voluntarily seek a variety of other treatments for their arthritis symptoms, including acupuncture, low-level laser therapy, and a medication choice procedure. Many chimpanzees voluntarily participate in these treatment regimens, and a number of them have demonstrated behavioral improvements (including increased mobility, decreased inactivity, and increased sociality; some of the changes are statistically significant) as a function of acupuncture, laser therapy, and/or choosing their medication. We are continuing to 1) expand the number of treatment options that chimpanzees can choose to receive and 2) refine our techniques for assessing the benefits of such treatments. The ultimate goal of the behavioral management program at the NCCC is to establish a communication system that allows the chimpanzees to ‘tell’ us how they want to be managed and treated.

PS98 The Critical Role of Animal Care Staff in Executing Behavioral Management Programs

MT Sharpless^*, JE Perlman, J Cowan-Brown

Field Station, Emory University/Yerkes Primate Center, Lawrenceville, GA

Primate behavioral management programs encompass animal enrichment activities, animal training procedures, and socialization efforts. The size of the facility, the number and type of species housed, and the nature of the research being conducted are some of many factors in the design, structure, and staffing of an organization’s behavioral management program. Behavioral management programs, especially in facilities housing nonhuman primates, are often dynamic and can be complex. These programs need to be flexible and adaptable in order to accommodate a range of approved activities and options and to implement any necessary changes or modifications. Animal care technicians are key components to the behavioral management program. They are often responsible for some or all aspects pertaining to the preparation, distribution, implementation, and record keeping associated with enrichment activities. Care technicians are also on the front lines throughout the socialization process during the course of their daily husbandry and care duties and responsibilities. Enrichment and socialization efforts continue to increase as we gain better understanding and as we increase our knowledge of the impact of behavioral management on the physical health and psychologic wellbeing of captive animals as well as the impact on research data and data collection. Laboratory animal care technicians are part of this positive forward movement and work culture. Care technicians enter the field with a compassion for animals and they are seeking guidance, information, and mentorship from supervisors and managers on ways to improve and enhance the lives of the animals for which they provide care. Discussion topics will include challenges to behavioral management programs and possible resolutions, methods used in the development of a cooperative work culture, strategies employed to motivate and engage staff, and ways to provide support and additional educational resources to care technicians.

PS99 Changes in Practices over Time and Variations Associated with Facility Size

KC Baker^*

Veterinary Medicine, Tulane National Primate Research Center, Covington, LA

Surveys of behavioral management programs for nonhuman primates (NHPs) housed in laboratories reveal notable progress between 2003 and 2014, particularly with respect to social housing, positive reinforcement training (PRT), the use of enrichment devices, and personnel resources. The aim herein is to evaluate whether this progress is manifest irrespective of facility size, and to identify how current practices vary with facility size. Among 2014 survey responses, facility size was dichotomized per the 2003 ratio of caged animals to behavioral management technicians (358:1), resulting in 23 large facilities (LFs; mean population 1,952 NHPs) and 18 small facilities (SFs; mean population 82 NHPs). In comparison to the use of social housing for caged NHPs in 2003 (41% of NHPs across all facilities surveyed), it is significant that gains are evident both in LFs (67%) and SFs (66%). Increases in the use of enrichment devices since 2003 are also seen in both. In both LFs and SFs, animal care technicians (ACTs) participate more in 2014 than 2003, particularly in human interaction as enrichment, PRT, and implementation of intervention for abnormal behavior. Currently, SF programs are primarily overseen by veterinarians (67% of facilities) as opposed to behavioral scientists (6%). In contrast, LFs are overseen more often by behavioral scientists (44%) than veterinarians (31%). While 87% of LFs employ behavioral management technicians, a third of SFs do so. This appears to be compensated for by some increase in ACT participation, but to a greater degree by veterinary technician participation in social housing, manipulanda, devices, and new employee training. While long-term program progress is not limited to large facilities with dedicated behavioral management scientists and technicians, SFs report fewer recent program changes as well as fewer future plans to alter programs, underscoring the utility of pertinent literature, conferences, and other mechanisms for cross-facility communication.

PS100 Procedural and Socialization Similarities and Differences between Pair Housing Macaques (Macaca mulatta) and African Greens

J Weed^*1, M Jorgensen²

¹Comparative Medicine Branch, Centers for Disease Control, Atlanta, GA; ²Pathology, Wake Forest School Of Medicine, Winston-Salem, NC

Vervets or African green monkeys are a nonhuman primate species widely used in biomedical research. At present there are relatively few references available on how to effectively social house this species in a captive environment. Social housing is currently the default value promoted in the Guide for captive-housed nonhuman primates unless there is a valid scientific justification for individual housing. Herein we describe the subtle similarities and differences between techniques for social housing African green monkeys versus macaques under laboratory conditions. We describe procedures implemented for social housing African greens in several laboratories and provide an overview of the methods used in one captive population with over 6 years of experience of pair housing. Compatibility was measured at 14, 30, and 60 days post introduction. In 1 cohort (vervet research colony), successful pairing rates at 14 days for males was 100%: females 96% (n = 72 female pairs, n = 52 male pairs). By 30 days post introduction, rates for both male and female pairs remained high, 98% and 94%, respectively. At 60 days post pairing, 75% of males and 82% of female pairs remained intact. Some pairs were separated for practical purposes, such as research needs rather than due to antagonistic behavior or distress. We emphasize that African greens are not macaques and should not necessarily be treated the same way. Gradual introduction techniques may not be beneficial or suitable for this species. Variables such as age, sex, and prior familiarity with new partners influenced successful socialization rates. The discussion will focus on behavioral differences between African green monkeys and macaques as well as future directions for research on pair housing this species.

PS111 Preparing a Research Facility for Work on Emerging Infectious Diseases

BL Kick^*2,1, S Jean¹, M Thompson¹

¹Yerkes National Primate Research Center, Atlanta, GA; ²Division of Animal Resources, Emory University, Atlanta, GA

In early February the WHO declared Zika virus a Public Health Emergency of International Concern. In response, several institutions have begun working with Zika virus, both in vitro and in vivo. We will cover the steps and challenges associated with preparing a research institution for work on Zika virus in both rodents and nonhuman primates. We will also emphasize the collaborative efforts needed between biosafety professionals, occupational health providers, researchers, institutional administrators, animal care resources, and the veterinary care team. The first step to developing a good team and functional facility for work on emerging infectious disease agents will be to develop an agent specific risk assessment. Once the comprehensive risk assessment is completed and the appropriate risk level determined, the core team moves forward with developing a facility plan and work flow. When working with emerging infectious disease agents that may require higher levels of containment several questions have to be addressed. For example, where will this research occur? Who will conduct the animal work with this agent? What quantity of animals are required and what training will be needed (both humans and animals)? How will diagnostic samples be processed and how will occupational health requirements be met? Based on a risk assessment, work on Zika Virus at our facility is managed at an animal biosafety level 2+. We will lay the framework for how we developed our policies, procedures, facility work flow, and the challenges we faced. Hopefully this information will assist other organizations to develop their risk assessment to aid in preparing their facilities for work on emerging infectious disease agents.

PS112 New NIH Requirements for Cell Line Authentication: What You Need to Know

BA Bauer^*1, LK Riley²

¹Genetic Services, IDEXX Bioresearch, Columbia, MO; ²IDEXX Bioresearch, Columbia, MO

The National Center for Biotechnology Information estimates that between 18–36% of cell lines are contaminated contaminated with cells from other cell lines, with the International Cell Line Authentication Committee (ICLAC) listing 475 known cross-contaminated or misidentified cell lines in existence today. There is more attention than ever on this topic with the recent publication of NIH Notification (NOT-OD-16-011) calling for grant applicants to include an authentication plan for key biologic materials with new and competitive renewal grant applications. This presentation will discuss the new requirements for cell line authentication in NIH grants and how to implement cell line authentication monitoring programs. Participants will learn what is involved in cell line authentication and different strategies for implementing monitoring programs. In addition, participants will learn how cell line authentication fits into a more comprehensive biologics monitoring program to ensure materials used in animal studies to improve reproducibility and minimize risk to animal facilities. The target audience includes veterinarians, facility managers, and technical personnel who work with animal models using cell lines or human patient derived xenograft (pdx) models.

PS113 Development of a Flexible Port for Repeated Biopsy of Spontaneous Ovarian Cancer in Domestic Laying Hen (Gallus gallus domesticus)

S Galaviz², AT Richert^*1, BD Smith¹

¹Comparative Medicine, Texas A&M, College Station, TX; ²Texas A&M, College Station, TX

Ovarian cancer is diagnosed in approximately 225,000 women per year accounting for 4% of all cancer cases worldwide on an annual basis. There is an estimated 140,000 deaths per year that are attributed to ovarian cancer. Diagnosis at advanced stages and resistance to treatments contribute to the high 5-year mortality rate. Domestic laying hens (Gallus gallus domesticus) have been identified as a spontaneous model for ovarian cancer with the potential, once fully characterized, to be the first predictive model and model for therapeutic trials. The chicken develops spontaneous and progressive ovarian cancer that have epidemiologic, morphologic, and molecular traits that are similar to humans. The incidence of spontaneous ovarian cancer in chickens ranges somewhere between 40-70% in chickens over 2 years of age. This is a consequence of constant ovulation resulting in DNA damage and repair of ovarian epithelial cells leading to ovarian cancer, similar to postmenopausal women which account for over half the diagnosed cases of ovarian cancer in women. Chickens make an ideal model due to their limited genetic diversity and predictable age of onset of disease. The development of this model will allow for development of predictive assays and allow for new therapeutics to be tested on spontaneously occurring ovarian cancer. Currently, the only way to diagnose chickens with ovarian cancer is to do invasive endoscopy. Development of surgically implanted ports created with a 3D printer allows for a minimally invasive repeated visualization of the ovary, following the progression of disease, repeated sampling, and treatment monitoring. This allows for the endoscopic visualization and biopsy of the ovary to change from requiring a surgical procedure to only being an anesthetic event. The original design was a hard plastic port that was easily implanted but suffered the complication of rejection. The material was then switched to a soft, flexible silicone and the shape was adjusted to accommodate the location. The new material and design has shown no evidence of port rejection, allowed endoscopic visualization of the ovary, and ports have remained implanted for over 9 months.

PS114 The Grooming Sleeve: Encouraging Conspecific-Like Behavior and Increasing the Human-Animal Bond

CM Sands^*, C Evans, CA Buckmaster

CCM, Baylor College of Medicine, Houston, TX

Nonhuman primates are naturally social animals that live in large family groups called troops. In the wild, monkeys forage and build bonds with one another through grooming and other social activities. The act of grooming has been found to greatly decrease stress, heart rate, and cortisol levels in nonhuman primates. This decrease in stress and cortisol is seen in the animal being groomed as well as the groomer. In the research setting, singly housed nonhuman primates do not have the opportunity to groom another individual. In order to offer our singly housed macaques the option of grooming, we designed a grooming sleeve. This device is worn over the protective gear, on the arm of the caregivers, and gives our singly housed macaques the opportunity to groom. We had a high success rate, with 80% of our macaques grooming the device, and 50% presenting to be groomed. Due to the overwhelmingly positive response to the grooming sleeve we recommend that it be used in other facilities housing nonhuman primates.

PS115 Adding Fuel to the Fire: Performance-Based Evaluation of Welfare Interventions in the DSS Murine Model of Colitis

JK Lang^*1,4, J Zaias², R Santaolalla¹, R Dheer¹, M Phillips¹, S Diaz¹, J Grant³, M Abreu¹

¹Department of Medicine/Gastroenterology, University of Miami, Miami, FL; ²Divison of Veterinary Resources, University of Miami, Miami, FL; ³Physiology and Biophysics, University of Miami, Miami, FL; ⁴Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA

Ulcerative colitis (UC) affects a large percentage of the population by causing inflammation mainly in the colonic mucosa. The DSS-induced UC model in mice can cause debilitating consequences (e.g., diarrhea, weight loss, bloody stool, hunched posture, and difficulty walking). These clinical signs are often viewed as painful by the IACUC, thus possibly conflicting with researcher goals, as adding medical treatments to mitigate symptoms may interfere with experimental endpoints. To directly assess this, we examined the effect of fluid supplementation on animal welfare and experimental endpoints in DSS-treated mice. Nine mice were assigned to 4 treatment groups, with or without fluid supplementation and at 2 endpoints: 1 day (acute) or 6 days (recovery) after DSS cessation. 2.5% DSS was provided ad lib in water for 5 consecutive days. Fluid supplementation was started on day 3 of DSS treatment and continued for 4 days. Welfare associated behaviors were measured (nest building and time to nest integration (TNT)). Clinical parameters included body weight/condition, stool characterization, and intestinal inflammation. We found that acutely, there were no differences in clinical signs or parameters measured; however, TNT and nest scores acutely were greater in fluid supplemented mice. During recovery after DSS cessation, mice who received fluids regained weight faster, had higher serum protein and albumin, and improved health than those who did not receive fluids. Colonic inflammation did not differ acutely, but was significantly reduced 6 days after DSS cessation in mice given fluid supplementation. Overall, fluids given to DSS-treated mice did not have a significant impact on welfare acutely, but significantly improved their health and welfare during recovery. Fluid supplementation does impact colonic inflammation and serum protein levels during recovery. Therefore, use of fluid supplementation as a welfare tool is not clear cut acutely and it affects (decreases) our model endpoint (induction of inflammation) which could ultimately result in an increase in total numbers of animals used. These data can be applied henceforth by IACUC and investigators to provide performance-based decisionmaking for therapy options and humane endpoints for this model.

PS116 Establishment of Novel IACUC Guidelines for Evaluation of Mild Traumatic Brain Injury Devices for Mice and Rats

KL Cavanaugh^*1, S Pritt¹, R Hammer²

¹IACUC, UT Southwestern, Dallas, TX; ²Biochemistry, UT Southwestern, Dallas, TX

Mild traumatic brain injury (mTBI), such as a concussion, comprises 70-80% of all traumatic brain injuries. To mimic these injuries, investigators create mTBI within an experimental animal model using various methods of impact. These methods, such as weight drop, may use linear or rotational accelerated force to strike an animal’s head at a desired region. These methods produce a mild, noninvasive closed head injury that simulates injuries commonly sustained through sports, motor accidents, or other events. As commercially available devices are not viable for the experimental needs of close head injuries, the published literature references the use of noncommercial, individually created devices. Therefore, investigators wishing to reproduce these methods of injury must create their own devices, which then come under IACUC oversight for approval. As investigators embark on creating their own equipment, it becomes increasingly important for the IACUC to set standards for these mechanisms. To ensure consistent consideration of animal welfare and device evaluation, novel standard criteria for mTBI studies using mice and rats were established by the IACUC at an academic research institution. This standard criteria is encompassed within a developed IACUC policy on traumatic brain injury to communicate to investigators the baseline expectations of the IACUC. Once initial device construction is inspected by an ad hoc of the committee and injury parameters are addressed, PIs are then required to complete pilot studies to evaluate device performance. The IACUC requires that pilot studies be conducted in 3 phases, each of which is immediately followed by gross pathology analysis: (1) carcass trials with veterinary supervision, (2) anesthetized animals that are euthanized immediately after injury, but prior to anesthesia recovery, and (3) live animals allowed to recover from anesthesia after injury for a short survival period (typically ≤ 24 hours) for physical and behavioral evaluation, then euthanized. Results of pilot experiments are submitted to the IACUC and veterinary staff for evaluation. By setting standard expectations for device construction and animal welfare, the IACUC may ensure consistency in the evaluation of devices used in mTBI studies.

PS117 Successful Outcomes of a Behavioral Response Plan (BRP): A Standardized Approach to Respond to Stereotypic Behaviors in Nonhuman Primates

JN Camacho^*, DM Jarrell

Massachusetts General Hospital, Charlestown, MA

Our standardized approach to manage stereotypic behavior is documented using a Behavioral Response Plan (BRP) combined with an Intervention Enrichment Assessment (IEA) and an Individual Behavior Assessment (IBA). The BRP categorizes 4 stereotypic behaviors: repetitions, alopecia, feces Smearing, and self-directed behaviors. Additionally, the plan recognizes 4 other NHP behaviors that indicate concern: fear, aggression, higher needs, and social conflict. Dependent on the frequency and/or severity of each behavior, a grading system is used and a specific enrichment approach prescribed, followed by the use of a quantitative scale to determine the effectiveness of the enrichment to match the animal’s behavioral needs and eliminate the stereotypic behavior. An example of this approach is an NHP that presents as having overgrooming alopecia, categorized as displaying moderate severity (classified by 2-4 patches). The NHP would have an initial intervention of destructible enrichment daily, followed by a quantitative assessment used by husbandry staff. An IEA score of 1 indicates enrichment is completely used, while a score of 3 indicates the enrichment is not being used, and an IBA score of 1 indicates the enrichment poses a benefit while a score of 3 indicates a negative response and could result in worsening the stereotypic behavior. After analyzing a group of 80 NHPs identified to have stereotypic behaviors and were subsequently treated with the BRP, 55 (60%) of the NHPs responded successfully to the BRP with stereotypic behavior resolved (without reoccurrence for 4+ months) within an average of 156 days (15-323 day range). While tracking prior to the use of the BRP plan was not as standard, it would not be ununusual for a NHP identified as having a stereotypic behavior to be receiving some sort of treatment for years with little change in behavior. The BRP plan has been highly successful at treating stereotypic behaviors observed by primates at our institution, using animal care staff making daily observations and responding based on individual animal needs.

PS41 Withholding of Analgesics: When Is Justification Simply Enough?

DA Pellecchia^*

Comparative Medicine, Pfizer, Pearl River, NY

A researcher’s decision to not administer analgesics should always be accompanied by adequate justification in the animal use protocol. Many times though, this rationale could be lacking appropriate scientific evidence. An older article may be cited from a renowned medical journal describing a reliable study which indicated that a particular class of analgesics is “very likely” to interfere with scientific objectives. For example, an analgesic’s mechanism of action may inhibit tumor growth in an oncology study. Or, an analgesic might interfere with an immune-modulated response on a vaccination study. But do we truly verify whether this tried and true “evidence” is still scientifically valid? Has the prior science been disproven with the discovery of other innovative pain relievers that use a different mechanism of action which consequently may not interfere with the study objectives? IACUCs are charged with ensuring that principal investigators have indeed conducted through literature searches to support their stance to withhold analgesics. But, how far do we expect the committee to delve into whether alternative options are available to mitigate potential pain and distress? We aim to provide guidance to IACUC members, regulatory personnel, veterinarians, and researchers regarding how to determine whether the justification to withhold analgesics is scientifically valid and capable of withstanding the scrutiny of regulatory and accreditation agencies. Some methods will be described to help ensure that IACUCs have conducted due diligence when deciding to approve a protocol in which analgesics will be withheld.

PS42 IACUC Meeting Minutes: Defining the Balance between Adequate and Too Much Information

DA Pellecchia^*

Comparative Medicine, Pfizer, Pearl River, NY

IACUC meeting minutes are frequently a bone of contention among the regulatory community in regards to the level of detail they should be. Ideally, minutes would be an accurate reflection of the discussions that transpired during the course of convened meeting. However, some IACUC deliberations can become extremely tangential and spiral into more of a political debate than a productive and efficient dialogue. IACUCs must abide by the regulations governing their institution and industry expectations regarding compilation of meeting minutes. Unfortunately, that particular guidance is not provided anywhere in an enumerated or concise description for the research community to follow. Most institutions tend to convey way too much information about the events that occurred within a 2-hour meeting. What probably could have been captured succinctly in a simple page or 2 ends up instead looking like a multipage transcription. How do you avoid this pitfall? Although there is no straightforward answer, there is a method to tackle the problem. By studying several years’ worth of our IACUC’s meeting minutes along with queries from our regulatory authorities and accreditation organizations, our institution decided to pare down our meeting records to just enough to cover the basics while avoiding the minutiae and not burying all of the details. It definitely helps to have folks on your IACUC who have an affinity toward concise editing. This session will help to provide guidance on how to trim the fat without losing too much of the core of your IACUC deliberations. Your meeting minutes can fulfill regulatory requirements without being overly verbose.

PS118 Evaluation of Analgesia Efficacy in a Guinea Pig (Cavia porcellus) Surgical Pain Model

V Oliver^*1, K Simon¹, S Athavale¹, LV Kendall², JA Nemzek¹, J Lofgren¹

¹University of Michigan, Ann Arbor, MI; ²Colorado State University, Fort Collins, CO

Approximately 50,000 guinea pigs (Cavia porcellus) are used in research studies in which more than momentary or slight pain is expected. As a prey species, they do not readily display overt signs of pain, thus challenging our ability to accurately assess efficacy of current analgesic recommendations. Our laboratory examined analgesia efficacy for treating postoperative pain. Briefly, 24 female Hartley guinea pigs were placed into analgesic treatment groups: extended-release buprenorphine, carprofen, or multimodal treatment with both analgesics. Assessments included daily body weight measurements, electronic vonFrey testing, and video recording for ethogram analysis at 2, 8, 24, 32, 48, 72, and 96 hours in the following time-matched conditions: baseline, anesthesia and analgesia only, and hysterectomy surgery. An additional 8 guinea pigs were used for pharmacokinetic evaluation of the extended-release buprenorphine. Animals treated with a single analgesic experienced increased mechanical sensitivity for at least 96 hours post surgery. In contrast, animals receiving multimodal analgesia only demonstrated increased mechanical sensitivity at 2 hours post surgery. Ethogram data indicated the extended release buprenorphine had a sedating but analgesic effect during all anesthesia/analgesia and surgery time points, whereas carprofen-treated animals displayed behaviors indicative of insufficient analgesia at 8 hours post surgery. After both anesthesia and surgery conditions, the multimodal and buprenorphine treated animals lost at least 10% of their baseline weights (P < 0.05) and were unable to return to baseline weights within 96 hours. In contrast, carprofen treated animals animals lost significantly less weight (∼3%, P < 0.05) and returned to baseline weights within 72 hours. Plasma levels of extended-release buprenorphine were above 0.9ng/ml from 8 to 96 hours post-injection with peak levels (1.2ng/ml) at 48 hours. Of the 3 analgesia regimens evaluated, multimodal analgesia appeared to provide the greatest analgesia efficacy, however the significant weight loss and mild sedation observed in the extended-release buprenorphine-treated groups may need to be considered when selecting an analgesia regimen.

PS119 Spontaneous Group B Streptococcal (GBS) Septicemia in Gravid Long-Evans Rats

CM Bodi^*1, V Bakthavatchalu¹, M Esmail², Y Feng¹, L Richey², J Dzink-Fox¹, B Beall³, SE Perkins², JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Division of Laboratory Animal Medicine, Tufts University, Boston, MA; ³Streptococcus Laboratory, CDC/NCIRD/DBD/RDB, Atlanta, GA

Group B Streptococcus (S. agalactiae, GBS) is a gram-positive opportunistic pathogen that inhabits the respiratory, urogenital, and gastrointestinal tracts of humans and animals. Maternal colonization of GBS is a risk factor for infectious disease in humans and a principle cause of neonatal meningitis and septicemia. It also is an emerging pathogen in the elderly, immunosuppressed, and pregnant women. We describe the presentation of polymicrobial sepsis including GBS in 3 gravid adult female Long-Evans rats experiencing acute mortality (septicemia, meningitis, endocarditis, nephritis) from a colony of long-term breeding pairs. Fluorescent in situ hybridization was performed to confirm the organism associated with the pathology in the affected tissues. Antibiotic susceptibility testing indicated similar resistance and susceptibility patterns to that of human strains of GBS. Seven GBS isolates obtained from affected and nonaffected animals identified by 16S rRNA sequencing were submitted to the CDC for whole genome sequencing and comparison of the rat GBS genomes to human isolates collected as part of their multi-state GBS surveillance pipeline. The isolates had virulence factors (β-hemolysin/cytolysin, hyaluronate lyase, CAMP factor) known to contribute to pathogenicity, and shared serotypes (Ib, V) with invasive isolates collected from human cases. SNP analysis revealed similar genetic relationships among isolates, strengthening the notion that zoonotic transmission is possible. To our knowledge, this is the first report of GBS associated with spontaneous maternal septicemia in gravid female rats. GBS is zoonotic, but the factors contributing to intraspecies and zoonotic transmission remain unknown. Due to the potential for significant GBS-induced disease, particularly in gravid and neonatal animals, prevention and consideration of elimination of GBS in the colony are advised. Preventative measures including stringent PPE practices and repopulation have decreased the incidence of clinical disease in the rat colony. In the interest of both public health and assisting in ensuring healthy rats, consideration of excluding GBS as a potential pathogen for immunocompromised or stressed animals is recommended.

PS120 Mycoplasma hyorhinis: Resident Flora and Opportunistic Pathogen of Swine Conjunctiva

MP Bradley^*, MJ Hoenerhoff, JA Nemzek

Unit for Laboratory Animal Medicine, The University of Michigan, Ann Arbor, MI

In the fall of 2015, 5 adult pigs (35-55kg), representing approximately 16% (5/32) of those acquired from one SPF agricultural farm over a 3-month period, were reported at intake exam for mild bilateral conjunctivitis. The condition became severe over the next 3 days following arrival. The conjunctivitis was typified by conjunctival hyperemia, chemosis, elevated nictitans, and purulent discharge. Diagnostic workup ncluded conjunctival cytology, biopsy, and aerobic bacterial culture. PCR analysis was performed for several pathogens, including Porcine Reproductive and Respiratory Syndrome virus (serum), Swine Influenza Virus (oropharyngeal fluids), Chlamydia, and Mycoplasma (conjunctival swabs). PCR analyses were negative for viruses and Chlamydia, but positively identified Mycoplasma hyorhinis in all samples. Cytology revealed neutrophils, lymphocytes, and basophilic inclusions within epithelial cells. Biopsy samples revealed widespread submucosal lymphoplasmacytic infiltration with marked lymphoid follicle formation. Aerobic cultures grew various bacteria including Staphylococcus spp., Streptococcus spp., and multiple coliforms. Prophylactic oxytetracycline (2mg/kg IM SID) for 4 days prior to shipment reduced the incidence of severe conjunctivitis to 2% (1/57) over the following 6 months. PCR analysis of conjunctival swabs from 9 clinically normal pigs from the same farm, as well as two other farms revealed M. hyorhinis in all samples analyzed. These findings definitively document that M. hyorhinis, a known component of normal upper respiratory tract flora in pigs, is also a common inhabitant of the conjunctiva. Under certain conditions, M. hyorhinis is an opportunistic pathogen, typically associated with polyserositis, arthritis, and otitis. The diagnostics and response to oxytetracycline in this report strongly suggest M. hyorhinis may also cause severe conjunctivitis. This is the first definitive report of M. hyorhinis as normal flora of the swine conjunctiva, and these data suggest that M. hyorhinis may act as an opportunistic pathogen in the conjunctiva under stressful conditions such as transport and acclimation. Importantly, our data shows that this severe conjunctivitis can be largely prevented with prophylactic antibiotic therapy.

PS121 Evaluation of Firocoxib in Sprague–Dawley Rats Using a Plantar Incision Model of Pain

TE Noe^*, TJ Pavek, WS Schwark, BS Singh

Cornell University, Ithaca, NY

For rodent analgesia, NSAIDs are preferable over opioids due to reduced frequency of required dosing and ease of availability. We sought to evaluate the relative efficacy of firocoxib, a highly COX-2 selective NSAID, versus meloxicam and buprenorphine for reducing allodynia and hyperalgesia in rats in the plantar incision model of surgical pain. Sprague–Dawley rats (n = 12 per group, 6 per group of each gender) were divided into 6 groups: sham (anesthesia, no surgery), control (no analgesia), buprenorphine (0.05 mg/kg SC q8hr), meloxicam (2 mg/kg SC q24hr), firocoxib 10 (10 mg/kg SC q24hr), and firocoxib 20 (20 mg/kg, SC q24hr). An incision was made into the plantar aspect of the right hind foot, the plantaris muscle was elevated, and the incision closed with 6-0 nylon suture. Nociceptive assays were performed using von Frey and Hargreaves methodology to test mechanical allodynia and thermal hyperalgesia, respectively. These assays were performed at 24, 20, 28, 44, 52, 68, and 76 hours after surgery. Data were analyzed using mixed-model ANOVA, and a Bonferroni correction for multiple comparisons was applied. The von Frey assay revealed increased allodynia with no evidence of significant alleviation of nociception after firocoxib or meloxicam injection, and minimal evidence of alleviation of nociception after buprenorphine. The Hargreaves assay revealed no statistically significant difference between any of the analgesic groups and the control group at any timepoint. Additionally, females appeared to experience greater hyperalgesia in the Hargreaves assay than males, though this trend did not reach statistical significance. These findings suggest that commonly recommended dosages of analgesics do not provide sufficient analgesia in rats in the plantar incisional model of pain.

PS122 Spontaneous Mutation with Limb Deformity in a Colony of Swiss Webster Mice

A Dickerson^*1, H Sandy¹, J Blaisdell², CA McGee¹, R Cannon², TL Blankenship¹

¹Comparative Medicine Branch, NIEHS/NIH/DHHS, Research Triangle Park, NC; ²Signal Transduction Laboratory, NIEHS/NIH/DHHS, Research Triangle Park, NC

We report a spontaneous mutation that was observed in a Swiss Webster breeding colony. The colony is used to generate sentinel mice. The mice are bred in an isolator and removed from the isolator at weaning. Although outbred, the colony is closed and inbreeding is likely due to the small numbers of breeding mice. The mice with the mutation demonstrated prominently shortened front limbs with missing digits. The footpads were deformed and found on the dorsal surface of the front limbs (versus the normal ventral location). Radiographs demonstrated absence of an ulna and missing phalangeal bones. Affected males and females were infertile. Breeding experiments were set up with normal siblings to affected animals to determine heritability and heritability pattern. Normal appearing siblings were intercrossed. Once a pair yielded mutant mice, they were identified as potential heterozygotes or carriers of the trait. These were then test mated with other normal siblings. Punnet squares of expected phenotypes were examined; and, assuming basic Mendelian genetics of dominant and recessive phenotypes, the trait appeared to be completely recessive. Tail biopsies were collected from all offspring and identified as either carrier, affected, or wildtype (based on breeding results and phenotype). Literature reviews lead us to believe that the Wnt 7a pathway was involved as this mutation has been reported in outbred ICR mice. A PCR was optimized for the exons of Wnt 7a and the 4 exons were examined. Analyses revealed that there were no exon size differences among the 3 different phenotypes; however, this does not rule out a single nucleotide deletion which has also been reported. When an unexpected phenotype appears in a rodent colony, it is of value to investigate whether it is related to any genetic manipulation the rodent has undergone or whether it is a spontaneous mutation. Spontaneous mutations do occur and can contribute to our understanding of disease processes. They also serve as a valuable educational tool on basic genetics for those in laboratory animal science.

PS123 Focal Naso-Maxillary Swelling and Hemorrhage from an Ear Canal in a Cynomologus Macaque (Macaca fascicularis)

KE Scott^*, V Bakthavatchalu, JG Fox, RP Marini

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

A 15-yr-old, intact, singly housed, male cynomolgus macaque (Macaca fascicularis) with a head-post implant presented with an acute focal swelling infraorbital, overlying the maxillary bone (1 cm diameter), which ulcerated and had serous discharge within 48 hours post presentation. Two and a half years prior to presentation, the animal had been treated for purulent otitis externa and an oral abscess from which Staphylococcus aureus and Streptococcus pyogenes were cultured. Multiple radiographic studies revealed no oral or otic pathology. Biopsies of the facial swelling were nondiagnostic and aerobic, anaerobic, and fungal cultures were negative. An MRI was performed and during the imaging session, the right ear bar was difficult to place due to hyperplastic tissue in the ear canal. Blood drained from the canal after the procedure. MRI revealed that the rostral aspect of the facial swelling had a focal hyperintense linear tract that extended 5mm into the underlying tissues and a less intense soft tissue material extending through a small focal defect in the maxillary bone to the level of the root of the right maxillary canine tooth. MRI also revealed heterogenous T2W hyperintensity filling the right tympanic bulla with extension of strongly T2W hyperintense material (fluid) into the mastoid air cells of the right temporal bone. The bulla wall remained intact and no abnormalities were detected in the cochlea and adjacent brain parenchyma. Based on MRI findings, a diagnosis of otitis media and periodontitis was confirmed. Myringotomy and tympanocentesis was attempted but unsuccessful due to the depth and narrow diameter of the external ear canal. The right maxillary canine was extracted, and histology of associated soft tissue showed an infiltration of a low number of neutrophils, macrophages, lymphocytes, and plasma cells. Due to the small size of the tympanic bulla of cynomolgus macaques, radiographic examination for bulla-associated pathology is not as sensitive as in other species. In this animal, the otitis media remained subclinical, without signs of discomfort or neurologic signs. Otitis media can remain subclinical chronically and is difficult to confirm and culture in cynomolgus primates.

PS124 Urinary System Lesions Associated with a Rat Model of Spinal Cord Injury

RL Munden^*, JL Booth, TK Cooper

Comparative Medicine, Penn State University, Hershey, PA

Each year approximately 12,000 individuals suffer a traumatic spinal cord injury. Rats are the most widely used animal model to study spinal cord injury. Recently at our facility, necropsy evaluation of 5 male Sprague–Dawley rats following experimental traumatic spinal cord injury revealed that all cases had significant disease of the urinary system. Cystitis, pyelonephritis, lower urinary tract obstruction, and urinary bladder rupture were observed. Severe dermatitis and cellulitis secondary to urine scald were observed in one of the cases. Evaluation of the lesions for these 5 rats prompted exploration of treatments and supportive care options for these studies going forward. A literature search revealed treatment and management options such as indwelling (suprapubic) catheters, intermittent urinary catheterization, and the use of a hand-held digital ultrasound imaging system to monitor urinary retention in the bladder. Pharmacologic treatment with prazosin, dantrolene, or light induction with isoflurane may be additional options to facilitate effective and less traumatic emptying of the bladder. Additionally, adjusting the amount of subcutaneous fluids and/or the amount of times the bladder is expressed per day may be other considerations to aid in bladder maintenance for future studies. Awareness of potential complications associated with this model allows better recognition of clinical concerns, guides treatment recommendations and minimizes potential animal distress.

PS125 Adverse Food Reaction to a Novel Diet in Adult Cynomolgus Macaques (Macaca fascicularis)

MS Metzler^*, E Floyd, B Uberseder, T Register, S Appt, K Michalson, A LeGrande, M Gee, C Shively

Pathology/Comparative Medicine, Wake Forest University, Winston-Salem, NC

Five out of 21 socially housed (3-4/group), adult female cynomolgus macaques (Macaca fascicularis) presented with diarrhea and weight loss within 1 year of being switched from commercial monkey chow to a novel diet, designed to mimic healthy human diets with most of the protein and fat from plant sources. Physical exam findings revealed poor body condition, dehydration, and gas-filled intestines. Laboratory findings included hypoalbuminemia (≤ 2.6 g/dL; 5 out of 5 cases), leukocytosis (WBC > 16,000 cells/µL; 4 out of 5 cases), and eosinophilia (> 6%, 4 out of 5 cases). Abdominal ultrasound revealed gas- and fluid-filled intestines with mildly increased mucosal distinction and wall thickening. Clinical and laboratory abnormalities improved with cessation of the novel diet and returned when diet was reinitiated. Clinical improvement persisted after termination of the novel diet. One animal was switched to a Western-like diet (protein from casein and whey), 2 were switched to commercial monkey chow, and 2 were switched to a walnut-free version of the novel diet. Evaluation of laboratory values obtained prior to introduction of novel diet revealed that all cases had serum globulin concentrations in the top 10% (> 3.6 g/dL) of the total study cohort (n = 43) and 4 out of 5 were of subordinate status in their social groups, suggesting a predisposition toward this phenotype. This is the first report of nongluten, adverse food reactions in laboratory macaques. Our findings indicate that social stress (subordinate status) and a preexisting, proinflammatory phenotype (high serum globulins) may have predisposed these animals to an adverse food reaction. Additionally, the findings of elevated serum eosinophils and resolution of clinical symptoms following walnut-elimination diet suggest a potential intolerance of some individuals to tree nuts.

PS126 Review and Enhancement of Rodent Body Condition Monitoring Program

S Shainker^*, DM Molk

Center for Comparative Medicine, Massachusetts General Hospital, Charlestown, MA

We empower our front line husbandry staff, or research animal specialists (RAS), to use our veterinary-derived rodent health poster to perform daily health observations. The poster is composed of a designated treatment plan for 23 common rodent health concerns. Our most commonly reported health concern is poor body condition (PBC) (30-40% of all health concerns) with an average treatment timeline of ∼6 weeks. Monitoring of animals during treatment varied among staff who shared their confusion regarding when to close PBC cases as resolved. Veterinary validation of resolution was common. Using Plan-Do-Check-Act (PDCA), a lean management problem-solving tool, an assessment of 25 current PBC cases in 1 facility was performed. Each cage was followed from initial health alert to resolution. Data on supply cost, staff time, and animal body weight was collected. This assessment revealed that 84 min and $56.60 were spent per case. It also revealed that body weights were stable or improving long before staff stopped performing daily monitoring. Weights were tracked on newly reported cases until they stabilized. Within 2 weeks of daily supplementation, mice maintained, gained, or lost weight; treating mice longer than 2 weeks did not alter the outcome of the case. A new standard approach for treating PBC cases, including a prescribed 2-week monitoring period was created. Cases are treated daily with supplements (Napa Nectar) for 2 weeks, after which the case is closed. This saved 60 mins and $36.40 per case and addressed staff hesitation to close a case. It is important to regularly review standard processes in our facilities to assess if workflow efficiency can be improved and to ensure that staff have clear guidance on veterinary care expectations.

PS127 Putative Virus Identified during Colony Health Monitoring with a Novel MTLV Antigen

RJ Ricart Arbona^*, NS Lipman, FR Wolf

CCMP, Memorial Sloan Kettering Cancer Center, New York City, NY

During routine sentinel health screening, soiled bedding sentinels (SBS) from 2 cages in room A (RA) from a 4-room vivarium (FB) tested positive for Mouse T Lymphotrophic Virus (MTLV) by multiplex fluorescent immunoassay (MFIA) in house (IH) and confirmed at Diagnostic Laboratory A (DLA) by MFIA (using the same antigen [Ag] used IH) and IFA. After discussions with DLA, the positive samples were retested and the IFA results were subsequently classified as nonspecific (TC), and the samples were reclassified as MTLV negative. Concurrently, 7 of the remaining 8 SBS cages, in the other 3 FB rooms, were confirmed MTLV MFIA positive and IFA TC. The only other SBS cage having the same serologic profile on our campus of >360 SBS cages received soiled bedding from mice belonging to Research Group A (RGA), which had most of their mice in FB and routinely transferred animals between these sites. It was determined that DLA had switched to a novel in vitro method (permissive lymphocyte cell line supplemented with several rodent-origin biologics) to generate the Ag used in their MFIA and also the MTLV-infected cells for IFA ∼ 1 year prior. Testing of a select number of the MTLV + samples by Diagnostic Lab B, which uses the classic in vivo produced MTLV Ag, were negative. Further analysis revealed that the MTLV+ sera reacted, although less intensely, to the uninfected cells suggesting that the serologic activity was directed to an unknown virus carried by the cell line. Results from additional tests including mouse antibody production, anatomic pathology, electron microscopy, and genomic sequencing of the cell line will be presented and important issues to consider when interpreting diagnostic assays will be reviewed.

PS128 Gross and Morphologic Characterization of an Unusual Bilobed Placenta in a Sprague–Dawley Rat

EF Tena Betancourt^*2,1, S Jimenez³, G Carrillo-Marhx¹, V Graullera³, AM Zúñiga-Muñoz³, C Jiménez-Gutiérrez¹, P Hernández-Jáuregui⁴

¹Animal Facility Services and Experimental Surgery, Universidad La Salle, A.C., Mexico City, Mexico; ²Ethology, Wildlife and Laboratory Animals, Universidad Nacional Autónonoma de México, Mexico City, Mexico; ³Pathology, INC Ignacio Chávez, Mexico City, Mexico; ⁴Dirección, Cyta Labs, AC, Puebla, Pue, Mexico

Placental development constitutes a key element in pregnancy. Extensive research in eutherian mammals has reported numerous placental types depending on their gross shape and histologic structure of the materno-fetal interface, among other structures. However, no studies have reported an unusual twin placenta form in laboratory rats. This report characterizes a bilobed placenta in a multiparous, 8-month-old Sprague–Dawley rat suffering maternal dystocia. This rarity was accidentally found while the dam was assisted through Cesarean section. Major findings showed abdominal hemorrhage from a ruptured, grossly distended left uterus displaying 1 single fetus having a bilobed placenta. Subsequent obstetric manipulation for extraction resulted in 9 fetuses among 11 pups. It was estimated that the dystocia was the result of the presence of 2 larger, malposed dead fetuses observed through the translucent uterine wall in caudal position blocking the birth canal. All fetuses and their placental remmants were retrieved and weighed and the crown-rump index recorded. Pups numbered 1 to 9 weighed 5.98, 4.84, 5.92, 4.63, 5.98, 5.94, 6.33, 6.39, 6.44 g (5.8278 ± 0.654) while the overdeveloped fetus blocking the birth canal 6.44 g. Total body lengths (TBL) measured 4.88, 4.55, 4.48, 4.32, 4.93, 4.62, 4.83, 4.81, 4.93 cm (4.7056 ± 0.2204), whereas the placentas weighted 0.51, 0.48, 0.49, 0.52, 0.47, 0.49, 0.49, 0.53, 0.48, 0.57 (0.559 ± 0.176) respectively (fetuses 1-9). Microscopically, we studied placentas belonging to animal 7 naming them primary (PP) and secondary (SP). Histologically, both placentas showed remarkable comparative analogies including the labyrinth zone, basal zone, and decidua. Morphologic features of PP demonstrated the labyrinth and basal zones congested with profuse erythrocytes delimitated by 1 hematoma. The SP exhibited a smaller hematoma among the basal and labyrinth zones and scarce trophoblastic giant cells. The communicating umbilical cord showed a typical morphology including 2 arteries and 1 vein buried within Wharton’s jelly. No anomalies were discerned among both placentas. This study highlights a striking reproductive rarity never reported in an extensively studied mammal as the laboratory rat.

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