2016 AALAS National Meeting Charlotte, North Carolina

Abstracts of Scientific Presentations 2016 AALAS National Meeting Charlotte, North Carolina

Poster Sessions blood collection method called the “one man.” The rat is manually restrained and bled by one person. In preparation for this technique, P1 A Novel Vascular Button Connection Using Combined Tech- rats are handled daily. The increase in handling reduces the amount nologies while Allowing for Social Enrichment by Pair Housing 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, AJ Hehman*1, A Zuvich1, KA Adams2, D Shuey1 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 1Toxicology, Incyte Corporation, Wilmington, DE; 2Laboratory thoracic portion of the rat’s chest is shaved and wiped with an Animal Resources, Incyte Corporation, Wilmington, DE 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 Traditional practice has been to single-house vessel-cannulated the appropriate amount of blood is collected, the needle is removed rodents post surgically to protect exposed exteriorized catheters. A and digital pressure is applied. A sample size of up to 2mLs can be port/protective cap model is available that allows for pair and group obtained. The restraint of the animal (tightness of the skin and housing of cannulated animals and is compatible with a harness/ alignment of the neck) is critical when learning this technique; swivel automated blood sampling system. Our goal was to develop a however, it can be learned easily and because this technique does not vessel-cannulated surgical model that could be socially housed and require any specialized equipment it can easily be implemented. would be compatible with response movement caging. To do this, we Hematomas have been seen on occasion with this method. One other modified the automated blood sample connection to be compatible risk of this procedure is death. This is rarely seen but can occur if the with the available port/protective cap system. A sterile custom vessel is not occluded properly. While both methods produce the connector was designed in collaboration with an industry vendor. same quality of sample; the refinement made to the commonly used Animals were dual jugular cannulated using the available port/ jugular vein blood collection method reduces cost in supplies, protective metal cap to allow for pair or group housing. The newly personnel needed, and the amount of stress on the animal. designed custom connector was primed and attached to the port after removal of the protective metal cap. The modified connector allows P3 Table Restraint Method for Working with Awake Nonhuman the standard extension line from the response movement caging Primates system to easily connect for blood sample withdraws. Studies were * conducted using our standard inhouse study design for pharmacoki- AM Storm , A Vinar, H Eric netic studies with multilple blood sample collections out to 24 hours post dose. All dosing and blood sample collections were performed NIH Division of Veterinary Resources, SoBran, Inc, Bethesda, MD in accordance with an IACUC-approved animal use protocol. The end result is a connector that allows for dosing and timed collections The need to use physical restraint for hands-on procedures involving of high-quality blood samples using a response movement caging nonhuman primates (NHPs) is common throughout the laboratory automated blood sampling system. The modified connector further animal community. Historically, our organization has used chemical allows for enhancement to the welfare of the animals by providing restraint for the vast majority of these procedures. After experiencing social enrichment through pair and group housing without compro- issues related to drug tolerance, anesthesia-related physiologic mising the integrity of the surgical model. complications, and the initiation of protocols requiring multiple procedures per day or multiple procedures over a period of P2 A Unique Blood Collection Method in a Conscious Rat successive days, our organization began a search for a new method of restraint which could increase efficiency, reduce or eliminate the need AM Walters*1, M Hirashima1, K Tsuyama1, S Wakamatsu2 for anesthesia, and improve overall animal welfare. There is an abundance of information related to the various restraint devices and 1SNBL USA Ltd, Everett, WA; 2Shin Nippon Biomedical Laboratories, protocols in the literature. Some of the more common methods LTD (SNBL) Japan, Kagoshima , Japan include chair restraint with pole and collar and the use of squeeze cages. Our organization adopted the use of a lesser known restraint Pharmakokinetics and toxicokinetics are important components of device, the primate restraint table, in combination with cooperative drug discovery and development process. The removal of blood at behavioral training for a variety of hands-on procedures including multiple time points from an animal is required for these evaluations. blood collection and veterinary evaluations. Animals were trained to Blood sampling can be stressful for animals because of the handling, use this device by applying positive reinforcement and desensitiza- restraint, anaesthesia, or discomfort associated with a particular tion techniques allowing the NHPs to participate in research technique. The jugular vein is one of a few collection sites which can procedures while fully awake. This was accomplished by acclimat- be used for multiple blood collections in a conscious rat. Blood ing the NHP to the table, then progressing through a series of steps parameters, supplies, and number of personal required, along with to shape the desired behavior of cooperative restraint and limb stress of the animal were evaluated using 2 different jugular vein extension. Training success was measured by the number of fully puncture methods. The first method requires the use of a jugular acclimated animals to the restraint table prior to the initiation of board for the blood draw and requires minimally 2 people (bleeder medical and research procedures. The implementation of this and holder). The rat is restrained in an unnatural position, which can technique has reduced the labor involved with these procedures by cause stress. The ties used to hold the forepaws can cause damage to eliminating the time needed for anesthesia induction, monitoring the limbs and nerves in this area, causing lameness. Tilting the head and recovery, and the complications associated with the use of of the rats in the head cap can cause haemorrhage to the ears. The rat anesthesia. We recommend this restraint device for laboratories can also lose consciousness. This method is commonly used for serial interested in alternative restraint methods in their research and blood collections in rats. The second method evaluated is a unique medical procedures.

606 Abstracts of scientific papers 2016 AALAS National Meeting

P4 Strategies to Increase Time Usage and Accessibility of Environ- P6 Optimization of in Vivo Blood Sampling for Pharmacokinetic mental Enrichment Provided to Socially Housed Primates to Studies Improve Animal Welfare and Wellbeing ST Ulufatu, CK McCaughey* CA Carrier* Safety Assessment, Genentech, San Francisco, CA Covance Research Products, Alice, TX Currently blood collection for preclinical mouse pharmacokinetic Group housing is the standard method for housing nonhuman studies has been refined from rotated retro-orbital (RO) eye bleeds to primates at our facility. Delivering enrichment to all social group a microsampling technique via the “whole blood” or “tail prick” members can be difficult due to the social hierarchies. Often the method. This technique in conjunction with an automated, miniatur- dominant animal will guard and chase off other group members when ized immunoassay platform is now primarily employed such that we enrichment is thrown in. While training can be done to combat some of may obtain a full serum-concentration time profile from each individ- this dominant behavior, we looked at other changes that could be ual mouse. By using this combination, we were able to produce a made to improve our environmental enrichment delivery methods to more robust data set than using the RO method and ELISA. The tail large groups of primates. Simple refinements in our methods were prick method involves warming the tail of a mouse and then using a tried to improve animals’ access to offered enrichment. The novel 27g needle which is injected into the tail vein and quickly taken out. strategies discussed here are piñatas, game feeders on timers, and Approximately 20ul of whole blood is collected from the tail vein via larger frozen forage blocks. Experience with primates reveals a hematocrit glass tube and spun down after 30 minutes of clotting. ingestible items encourage foraging and destructible items prove most The serum is collected by cutting the glass hematocrit and pipetting interesting. As a result, selected new strategies targeted these “likes.” at least 10ul of serum. The serum is then processed with an automat- The most successful enrichment strategies provided the group a longer ed, miniaturized immunoassay. Previous methods used staggered time of use, gave them something to do, and provided foraging RO blood collection which combines data across animals to produce opportunities. The longer it took to empty the item, the sooner the a full pharmacokinetic profile. The limiting factor with RO bleeding dominant animal moved on. This allowed other animals in the group a is the amount (~150ul) and frequency blood is able to be collected. chance to forage. The new methods also provided more accessibility to The microsampling technique offers us the opportunity to take a group-housed primates especially the lower ranking members who smaller amount of blood (~20ul) at every time-point from the same typically only had access to leftovers. These simple and practical to mouse. In the large molecule space, the need began to arise for employ refinements, increased duration of use, and allowed for more researching data to make direct comparisons between molecules and animals to access the environmental enrichment being offered in the mouse strains, based on the need for a more predictive preclinical group setting. As such, these methods should increase the welfare and pharmacokinetic screening tool. Limited data existed from molecules wellbeing for low ranking animals in a group setting. 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 P5 Use of Hydrophobic Sand as a Refinement for Urine Collection was hypothesized that strain differences combined with limitations in Mice and Rats of a staggered PK approach confounded our historical data. By using an automated, miniaturized immunoassay in conjunction with micro- CA Pinkus*1, A Pereira1, A Nguyen2, MC Debrue1, L Garcia Menendez1 sampling for our in vivo work, we are able to drastically reduce the number of animals used on study which speaks directly to the 3Rs, 1Compartive Medicine, Pfizer Inc, Cambridge, MA; 2CVMET, Pfizer and the added benefit of providing improved quality of our data by Inc, Cambridge, MA producing a more robust data set all while helping us to better understand intrastrain variability. Metabolic cages used to collect urine are a barren, nonenriched environment known to increase stress in mice and rats. This study P7 Training Rhesus Monkeys to Walk on a Treadmill looked at using hydrophobic sand in a regular disposable cage as an alternative way to collect urine from mice and rats. Water bottles DH Sedlak*1,2, A Evans1,2, M Szarowicz1,2, E Tilmont1, K Vaughan1,2, J were a concern due to potential leakage, so water bottles and Mattison1 hydrating gel were also compared in both settings. Thirty-two male C57BL/6NCrl mice and 32 Sprague–Dawley rats were chosen for the 1NIA, NIH, Dickerson, MD; 2SoBran, Bethesda, MD study. Each species was divided into 4 groups of 8, comparing hydrophobic sand versus metabolic cages, and water bottles versus There has been recent scientific interest in the positive influence of hydrating gel in each setting. Urine was collected and volume record- exercise on health and wellbeing, including its effect on various ed at 3, 6, and 24 hours for mice and 2, 4, and 6 hours for rats. In disease states. As such, our lab has recently begun a collaborative mice, after the 6-hour time point there was no significant difference project aimed at characterizing the progression of peripheral artery in urine volumes collected from hydrophobic sand and metabolic disease (PAD) and evaluating exercise as a potential intervention. cages. However, at the 24 hour-time point the volume of urine This involved developing a monkey model to measure lower limb collected from the sand was significantly lower than the volume ischemia, while also implementing a novel exercise program for collected from the metabolic cages. In rats, significantly lower adult rhesus monkeys. After a thorough review of the literature and volumes of urine were collected in hydrophobic sand than in consultation with another lab, we elected to employ a treadmill to metabolic cages at all time points. A significant decrease in urine achieve exercise. Having worked with monkeys for many years, this volume was found in the 2 rat groups that received hydrating gel seemed a daunting task. However, with some suggestions from the compared to the groups that had water bottles. In mice, the provision behavior staff in our facility we were able to successfully train adult of gel versus water bottle did not impact the urine volume. Using rhesus monkeys to walk at varying speeds for 30 minutes per day. hydrophobic sand with mice was not as effective for maximum This was a slow process beginning with the animal sitting on the volume collection at the 24 hour-time point but it might be an option treadmill completely stopped for multiple days for short periods of when shorter collection time points are required and maximum urine time getting many treats and novel rewards for positive reinforce- volume is not required. Hydrophobic sand was also not as effective ment. We currently have 2 study groups of stable treadmill walkers for maximum volume of urine collection in rats compared to that are helping us proceed with our PAD study. Here, the pertinent metabolic cages. Providing hydrating gel impacted rat urine volume steps and considerations necessary for developing a successful but was found to be unnecessary since water from water bottles was nonhuman primate exercise model are presented. 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.

607 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P8 Outreach Outside of the Box ing pathogenesis of HF and screening novel therapeutics.

DJ Disselhorst*1, P Crowder2 P10 Refining Urine and Fecal Collection Methods Using Nonstress- ful Acupressure during Normal Routine Handling 1Texas A&M, Ft Stockton, TX; 2Iraan High School, Iraan, TX EP Chua*, R Garcia-Gonzalez, J Yamada, R Scott, C Sohn Typical biomedical research outreach programs involve research personnel going out to schools and giving short, impromptu Laboratory Animal Resources, Genentech, Inc., South San Francisco, presentations to science classes on what biomedical research is. CA Science teachers have a general understanding of the necessity of animals in research and are commonly receptive to these sessions. Collecting urine and fecal samples from laboratory mice is often Although useful in getting the message out, what if the biomedical performed in support of various metabolic, biochemical, nutritional, research community were to actively pursue alternative methods for behavioral, and physiologic studies, as well as routine diagnostic getting our message out? Animal rights groups start programs in the testing for animal health status. Although multiple collection education system through a formalized PETA cirriculum, which techniques are available, these methods may be stressful to the provides online lesson plans and the National Compassionate animal (manual restraint), require additional sample handling Classroom Report Card. Animal rights groups also target 4-H shows (collection of urine/feces on plastic or metabolism cage), and may and high school agriculture programs such as FFA. High School FFA yield samples that are heavily contaminated. Our method was and 4-H youth programs are excellent starting points for biomedical developed as an alternative nonstressful collection method of urine research to spread the message of applicable and responsible and/or fecal material. Use of this method has many benefits research. Commonly thought of as livestock programs, genetics, including minimal animal handling, eliminating the need of a breeding records, husbandry, and basic research concepts can be metabolism cage, speed of sample collection, and ability to collect presented by incorporating biomedical research concepts into samples at specific time points. Using this method, the mouse existing programs. Adjunct faculty with a background in biomedical remains in a lordosis-like posture. In this posture, the pelvis rises up research can be enlisted to incorporate these same concepts through with front of animal angling downward. Based on our experience, junior and senior high school agriculture and 4-H programs. By within a short duration of time of assuming this posture, the animal requiring college-focused students to complete a research project will defecate for a fecal sample. For urine collection, the bladder can before graduation, a sense of accomplishment is achieved, while be expressed by gently massaging the area over the bladder while showing how research is applicable in their projects. As most of the keeping the mouse relaxed in the lordosis-like posture. modern day medical discoveries are extrapolated to veterinary science, this concept can enrich an agriculture/4-H program. By P11 A Novel Way to Reduce the Number of Swine Required to displaying the posters in the school or 4-H meeting rooms, other Support Trauma and Resuscitation-Focused Research nonscience-specific disciplines can be exposed to the responsible nature of biomedical research. We explore this approach in a FFA EL Dean*, T Brown, P Barnicott, L Harris program and in a 4-H program. 59th Clinical Research Division, San Antonio, TX P9 Minimally Invasive Tachypacing-Induced Heart Failure Model in Beagle Military surgeons and researchers seek novel approaches to mitigate morbidity and mortality of battle-field injuries and hemorrhage. We DK Hirenallur Shanthappa*, W Sutherland, I Brun del Re, SM Davis, are often asked to supply large quantities of swine fresh whole blood S Barry, M Escamilla, J Viray Jr., H Vargas, J Eswaraka (FWH) or blood components for transfusions to support this research. To meet the growing demand for FWH and blood compo- Amgen, Thousand Oaks, CA nents but reduce overall animal usage we developed the Nonhuman Research Subject Blood Donor Initiative (NHRSBDI). Until this The canine tachypacing-induced heart failure (TPHF) model has been development, the method for blood collection involved exsanguina- widely used to study heart failure (HF) mechanisms and the tion of donor animals under general anesthesia with an average discovery of new therapeutics for HF in drug development. The most collection of 6 units of blood per animal. Of these 6 units only the common model of TPHF involves invasive surgical instrumentation first 3 bags are optimal for transfusion due to unpublished findings. of large deep-chested dogs (mongrel) to enable placement of a This resulted in a high cost-benefit ratio and we saw a need to refine pacemaker, electrodes, and pressure-sensing catheters. This model is the blood collection process and reduce the numbers of animals valuable for the acquisition of a variety of cardiovascular parameters, needed to support trauma research. We began housing a pool of but significant disadvantages include prolonged postoperative blood donor swine (>70 kg) that have blood (3 bags of 450 ml/unit) recovery time, low study throughput, hardware-associated complica- collected every 2-4 weeks depending on health status and blood tions, extensive training of canines, and intensive veterinary care. parameters (suggested values: WBC 9-23k, Hg ≥ 9 g/dL, HCT ≥ 30%,

Additionally, surgical manipulation and instrumentation creates pO2 ≥ 100 mmHg, Lactate ≤ 2.5 mmol/L). Animals are under general inflammation in and around the heart that could affect cardiovascu- anesthesia and on volume control ventilation during the collection

lar function, ultimately resulting in low-quality data. To overcome process with vitals (HR, SpO2, RR, TV, PIP, PEEP, etCO2, %Iso, Temp., some of these challenges, we evaluated a minimally invasive %O2) collected every 15 minutes. Volume replacement with warmed transvenous right ventricular pacemaker in Beagles. In this model, normal saline occurs at the completion of the procedure. Once the the pacemaker was inserted via a right jugular vein cut-down and animals recover they are housed in facility for 24-48 hours and if no secured into the right ventricular endocardial wall. Following the complications are noted they are moved to a paddock where they are postoperative period, HF was induced in 16 dogs by tachypacing the monitored daily until it is time for another donation. Animals remain animals at 240 bpm for 2-4 weeks. HF progression was monitored within the pool until it is deemed that further blood draws could using echocardiography. All dogs recovered from surgery with compromise animal health. We successfully removed approximately minimal complications by day 5. At day 28, dogs had Stage C (AHA/ 20-30% of a swine’s circulating blood volume for every 2-4 weeks. ACC classification) heart failure as evidenced by decreased ejection This amount nearly doubles the single blood draw amount currently

fraction (< 40%), fractional shortening (dP/dtmax), increased left reported in the literature. The implementation of the NHRSBDI ventricular end diastolic volume, and increased left ventricular end resulted in a significant reduction in the number of animals required diastolic pressure compared to normal dogs. Overall, findings to support trauma related protocols. Implementing this initiative on a suggest that the minimally invasive tachypacing-induced heart single trauma focused research study resulted in saving approxi- failure model in beagles is a refinement to the more invasive mongrel mately 420 animals. If this initiative was instituted across other model of TPHF. This model is robust and reproducible for investigat- facilities it could reduce the number of swine required to support

608 Abstracts of scientific papers 2016 AALAS National Meeting

trauma and resuscitation focused research, resulting in a more viable and identification methods were performed and the progeny were research process and enhancing accessibility. assigned to a breeding colony room. To date, this method has produced a 100% success rate with 18 lines rederived. By performing P12 Handling Ease and Behavior Enhancement in Sinclair sterile cesarean rederivations animal care technicians are efficiently Miniature Swine able to eliminate known pathogens from imported colonies and maintain adherence to the bio-exclusion standards while facilitating D Brocksmith, G Rivard*, I Stewart, GF Bouchard, A Stricker-Kro- research Importantly, this was a challenging and rewarding develop- ngrad ment opportunity for technicians to expand skillsets, while eliminating outside contract services and saving investigators Sinclair BioResources, LLC, Auxvasse, MO valuable time.

We have developed a unique behavior enhancement system program P14 Use of a Remote Video Monitoring System as a Refinement in for miniature swine (MS). One of the program’s components consists a Nonhuman Primate Colony Behavioral Health Program of behavioral training to condition a MS to cooperate rather than experience anxiety and fear over a procedure. Here we seek to inform J Santiago*, MD Simkins you about the common threats that elicit resistance-to-handling. Also, we propose positive reinforcement training (+R) to improve welfare Comparative Medicine, Pfizer, Perth Amboy, NJ and handling-ease. Avoid any punishments, reprimands, or corrections; and perform a routine in a highly enthusiastic and Nonhuman primate (NHP) colony maintenance requires ongoing playful manner so that, when repeated multiple times, it becomes consideration of a number of factors, including the macro and micro highly predictable, safe, and pleasurable for the MS. By repetition, environments, enrichment, diet, social housing, behavior, and the MS will learn the formula: your request plus its response equals individualized medical needs of the animals. Decisions and changes its reward. If not or if the MS becomes fearful or resists to handling, are being made on a constant basis in the best interest of both the then ignore the bad behavior and go back to basics. Ignoring means animal and the care staff working closely with the animals. Often no looking (including direct, prolonged, or challenging eyes contact); times when people are present in an NHP room, the animals are no touching (including hitting and body pushing or blocking); no focused only on that person. This makes it challenging to accurately talking (including yelling, saying no or what not to do); and no observe important interactions related to social housing, partners or moving (simply stand still and with your hands on your side). Once pairs, or normal behavioral habits specific to each NHP. Assessing an calm and looking for your attention, redirect the MS by requesting a animals’ interest in enrichment devices, hierarchical interactions previously learned basic behavior such as a target touch. Then, start within a room, and many other important behaviors that play a the training process from a previous step when he was calm so that major role in the wellbeing of an NHP can also be obscured by the MS remains calm. If not, give the MS a time-out period. Before presence of staff. The key is to be able to observe the NHP with the introducing a new behavior, make sure to review and reinforce all the least amount of distractions possible to encourage whatever normal basics and the prior lessons before starting anew. It will refresh the or abnormal behaviors you are interested in observing. Cameras and MS memory and give you both confidence. Teach new rewarding a remote monitoring system have allowed us to keep an eye on our behaviors incrementally. All along, be careful as mixed signals, lack NHPs without added distractions and/or variables in the room. Over of reinforcement, and going too fast are the culprits of good training. the past 3 years, our remote monitoring system has been used mostly for observing suspected or reported abnormal behaviors in order to P13 Cesarean Rederivation Performed in house by Animal Care gain a better understanding of how often the abnormal behavior is Technicians to Eradicate Opportunistic Pathogens displayed, and whether or not the frequency is dependent on the condition of the room. Recently, we started using the remote monitor- S Graffam, J Lorabello, JR Stathopoulos* ing system more often to observe animals with alopecia, animals displaying poor social and interaction skills, the progression of Comparative Medicine Facility, Broad Institute of MIT and Harvard, animals in our habituation training program, and to determine Cambridge, MA whether or not an animal is benefitting from a particular enrichment device. It has even helped us to determine the best time to success- Upon opening, a new comparative medicine facility experienced a fully pair 2 NHPs s based on their cageside interactions with one high volume of mouse import requests from affiliated institutions. A another and how these interactions evolved over time. The availabil- number of these institutions had less restrictive bioexclusion criteria ity of remote monitoring provides us the opportunity to observe our than the standards chosen by our investigators. We sought to NHPs closely and accommodate their needs without disrupting their maintain a strict biohealth status while being able to import valuable environment. lines of mice from other institutions. Some opportunistic pathogens that are excluded (Staph. aureus, Klebsiella spp., Helicobacter) were P15 Lymph Node Fine Needle Aspiration as a Refinement for diagnosed positive on incoming health reports. Cesarean rederiva- Longitudinal Assessment of Lymphoid Tissue in Rhesus Macaques tion was chosen as the method to solve the problem. Animal care (Macaca mulatta) technicians were trained to perform the procedures in house. Three didactic training sessions were held with the team to develop an JS Wood*1, D Carnathan2,3, A Gsrcia4, S Gumber4, C Havenar-Daugh- understanding of the technical procedures, estrous cycles, and ton5,3, S Crotty5,3, G Silvestri2,3 embryo development timelines, as well as materials required. The team researched current literature and methods to develop an 1Animal Resources, Yerkes National Primate Research Center, inhouse standard operating procedure. An experienced technician Atlanta, GA; 2Emory Vaccine Center, Yerkes National Primate performed initial procedures. With continuing guidance, technicians Research Center, Atlanta, GA; 3Center for HIV/AIDS Vaccine quickly demonstrated proficiency in the techniques. To perform the Immunology & Immunogen Discovery, The Scripps Research rederivations, a colony of CD-1 outbred stocks, free of select Institute, La Jolla, CA; 4Department of Pathology, Yerkes National infectious agents and parasites, were bred to use as recipient foster Primate Research Center, Atlanta, GA;5Division of Vaccine Discovery, dams. Sequentially, donor dams were bred to give birth at e19 La Jolla Institute for Allergy and Immunology, La Jolla, CA (CRISPR lines) and e20 (traditional genetically modified lines). Donor dams were euthanized and pups were extracted under sterile Nonhuman primates are commonly used in immunology studies. conditions. Extracted pups were warmed and then passed through One aspect of these studies includes evaluating changes within an airlock to the technician to perform the fostering procedure. At lymphoid tissue over the course of an immunization series. There are wean age p21 health status is confirmed via a modified PCR rodent challenges to longitudinal assessment of the immune response in infectious agent panel. Upon receipt of negative results, genotyping rhesus macaques, including, but not limited to practical and ethical

609 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 considerations restricting the number of excisional lymph node stress and good behavior learned by positive reinforcement can biopsies that can be collected from an individual, the impact on the provide better overall animal welfare and subsequently lead to high immune response when draining lymph nodes are removed, and the quality CV data. difficulty in evaluating specific lymphoid cell subsets, particularly + germinal center (GC) B cells and CD4 T follicular helper cells (TFH). P17 Donating Rodents in a CRO Environment Fine needle aspiration (FNA) is a commonly used diagnostic tool in clinical practice. This technique allows sampling of the peripheral KM Fiala*, D Kenjebaev, CA Plews, S Snyder lymph nodes in situ using a needle and syringe. A pilot study evaluating the utility of FNA in studying GC activity in lymph nodes MPI Research, Mattawan, MI was performed. Interpretable data was obtained in 76% of the FNA samples. There was close correlation of GC-B cells and Donating rodent carcasses to a local nature center or bird sanctuary

TFH frequencies observed between FNA samples and lymph node was initiated as a means to give added purpose to animals not biopsies. Based on these results, lymph node FNA has been selected for study. Most nature centers are not-for-profit organiza- incorporated into multiple HIV vaccine studies with much success tions that are dependent upon donations of money and goods to stay and thusly provides an alternative means of evaluating specific in operation. One item that nature centers are in need of, and CROs lymphoid cell subsets over time. FNAs were performed under are uniquely qualified to donate, is rodent carcasses to be given as injectable anesthesia and all animals received pain medication to food to their birds of prey. Once approval was given for the dona- prevent any discomfort. Since September 2014, 393 FNAs have been tions by upper management and the attending veterinarian, a performed on 62 rhesus macaques. Over 97% of the samples committee was convened to create and implement a program that contained usable GC-B cell and TFH populations for flow cytometry would appease all affected departments, including security, IACUC, and gene expression. The average number of cells collected was 2.5 x finance, purchasing, safety, and in-life groups. The committee started 106 cells per FNA sample, with a range of 2x104 – 4.9x106. The length by creating a title and mission statement for the program. The team of time between FNA collections has varied, with a minimum of 6 then worked together to ensure all affected departments were days between collections. The only complication from this procedure consulted and prepared a streamlined process that would be easy to has been minor bleeding and bruising at the collection site. FNA follow while allowing the maximum number of qualified animals to presents a novel way of evaluating GC changes in lymph nodes. This be donated. To qualify for donation animals must be naïve to test method allows longitudinal study of the peripheral lymph node article administration (certain vehicles permissible as defined by the response in a single animal, which can decrease the need for, and attending veterinarian) and euthanized via CO2. Within 1 month the frequency of, excisional lymph node biopsies and lead to a reduction Raptor Food Donation Program was implemented and within 3 in the total number of animals used on certain immunology studies. months the first donations were submitted to the nature center. Tax-related donation forms are collected at each transaction to P16 Evaluation of the Effect on Heart Rate when Providing maintain accurate records for any deduction or other legal consider- Supplemental Treats after Oral Dose Administration in the ations. The program has proved beneficial in not only feeding local Minipig raptors, but also in engendering goodwill between the CRO and local communities, and helping the technical staff promote animal welfare JE Sentz* by bringing added purpose to these lives.

Surgical Services / Infusion Toxicology, Envigo, East Millstone, NJ P18 Refining Endpoints for a Rhesus Macaque Macaca( mulatta) Model of Tuberculosis A critical aspect of cardiovascular (CV) safety pharmacology studies is the collection of high-quality CV data prior to dose, during dose, KE Russell-Lodrigue*1, LA Doyle-Meyers1, R Bickham1, D Kaushal2, and post-dose administration. The minipig is extremely intelligent JL Blanchard1 and every interaction is a learning process, including stressful experiences that the animal will remember and avoid in the future. 1Veterinary Medicine, Tulane National Primate Research Center, Through positive reinforcement, a procedure can become less Covington, LA; 2Bacteriology and Parasitology, Tulane National stressful for the animal. By providing positive reinforcement, such as Primate Research Center, Covington, LA juice, after a procedure, the animal will remember the procedure as a positive experience, thereby promoting good behavior. Minipigs are Rhesus macaques (Macaca mulatta) inoculated with Mycobacterium generally motivated by food and treats (e.g. apples or pears) which tuberculosis via small particle aerosol are used as models of active and are routinely presented to provide positive reinforcement. However latent tuberculosis. In the final stages of disease, the miliary it has been observed that heart rate (HR) in minipigs increases after tuberculosis produced in this model can cause severe respiratory feeding and can remain elevated for several hours. This limits our distress and death. Veterinarians, animal care technicians, and ability to use positive reinforcement in the form of treats to promote investigators can work together to evaluate disease-related changes good behavior. Apple juice has also been used for positive reinforce- such as fever, decreased appetite, respiratory compromise, ment; however no CV data has been assessed for the effects of apple radiographically evident pneumonia, and increased C-reactive juice on HR. The objective of this study was to assess the effect on HR protein levels and use a combination of objective measurements and when apple juice was provided immediately post dose administra- subjective scoring systems to determine the most appropriate study tion. The study consisted of 2 tests. In both tests CV data was endpoint for each animal to negate the need for death-as-an-endpoint collected for 2 hours after which apple juice was presented for ~15 studies. This multimodal approach allows for consistency in secs. CV data was then collected continuously for 24 hours. In test 1 endpoints as required for scientific vigor while maintaining the no dose was administered, only apple juice was provided. In test 2 clinician’s ability to improve animal welfare by making dose (tap water) was administered via oral gavage and then apple individualized patient care decisions. juice was provided immediately post dose. This data was then compared to previously collected data (data obtained from the same P19 Identifying Correlates Between Behavioral and Physiologic animals after restraint in a dosing chair, using a bite bar, with/ Endpoints of Mice in Infectious Disease Studies without administration of Vehicle A, and did not receive apple juice post dose). The data indicated minimal impact on HR when only KJ Knapek*, K Posey-Bruner, LV Kendall apple juice was provided. When apple juice was provided immediately postdose administration, the initial change in HR was similar to Laboratory Animal Resources, Colorado State, Fort Collins, CO 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 Research with select agents in mice often result in death as an presentation of apple juice . We have demonstrated that reduced endpoint, intentional or not, as identifying appropriate endpoint

610 Abstracts of scientific papers 2016 AALAS National Meeting

criteria can be difficult to establish for the various agents and mouse Historically, our institution has supported various veterinary technol- strains. This can have a negative impact on the animal’s welfare. ogy training programs in the region by providing animal facility Establishing a means to identify early endpoint using indicators of tours to students as a supplement to their laboratory animal morbidity that can be applied to multiple agent would be invaluable medicine coursework. Over the years, the numbers of requests grew for those working with infectious disease models. A behavioral as word spread about the tours to other programs in the area. In ethogram was used to assess the effects of infection with Burkholderia order to keep up with demand, while minimizing disruption of daily pseudomallei (Bpd) and Bacillus anthracis (Banx). The ethogram activities, we elected to formalize these tours into an annual program included general activity, grooming, rearing, and orbital tightening. targeted towards veterinary technology students at all 4 of the major In addition to the ethogram, body temperatures were measured with veterinary technology programs in the region. The first program a thermal imaging camera, and the time-to-integrate to nest (TINT) occurred in May of 2015. We had a total of 22 students and staff from test was performed. The mice used in this study were on an existing all 4 of the programs in Minnesota. Due to only having 5 hours with protocol to evaluate the vaccine efficacy against Bpd and Banx. the students, the tours had to be limited to the vivarium, with only 15 Following infection, mice were observed for 5 minutes every 6-12 minutes dedicated to the workings of the research laboratory. We also hours. Mice infected with Bpd and Banx had reduced general activity, hosted lunch with a panel of Certified Veterinary Technicians rearing and grooming, (p < 0.05), and increased orbital tightening. working in various areas of our research program. The technicians These behavioral observations correlated with a reduced body spoke about their positions and the students were given an opportu- temperature. Bpd infected mice failed to integrate any nesting nity to ask questions. Feedback from the first program indicated that material, whereas Banx infected mice delayed integration of new students wanted to see more of the facilities and projects. The 2016 nesting material to their existing nest, which in some instances took event was expanded to 8 hours, and included both the Rochester, more than 24 h. The use of these observation techniques with MN and Scottsdale, AZ campuses. The extra time allowed for an multiple agents can assist investigators in assessing early time points hour dedicated to just the research laboratories besides the time spent for infectious disease studies. Further, it provides a means of more on the vivarium tour. We had 11 students and staff who attended in objective measurements, such as body temperature and integration of Minnesota, and 13 students and staff attended in Arizona. Research nesting material, that correlate to behavioral signs of disease. This Day provides ample opportunities to the students to see the roles of provides a means to identify moribund animals which can easily be veterinary technicians in research and is rewarding for our employ- overlooked in prey species. ees and staff to talk about their work. It is exciting to educate new technicians on the ever growing possibilities available to them as P20 Effects of Caretaker Behavior on Nonhuman Primate Perfor- they go through their program, graduate, and enter the world as a mance Certified Veterinary Technician.

K McGrew* P22 Dollars and Sense: When Planning a Study It Makes Sense to Know the Economic Impact of the Model You Choose Behavior, Charles River, Houston, TX LA Knarr*1, GJ Ochs2, MJ Horn1 Human interaction is an important step in gaining the trust necessary to train NHPs to cooperate. However, establishing trust can be 1Veterinary Sciences, Research Support, Envigo, Indianapolis, difficult. This study examined how technician heart rate range, IN; 2Commercial, Envigo, Indianapolis, IN measured by a wrist-wearable activity tracker, correlated to how many primates would accept a hand treat (peanuts or crackers). This Preclinical toxicology studies involving animal models require was then compared to the average results over several sessions. All several factors allowing for study completion. Researchers rely on sessions were conducted between 9 and 11 am after cleaning and historical control databases for baseline information, and may be feeding by husbandry. Results contradicted a hypothesis that animals hesitant to consider novel models; however, other factors should be responded better to calmer technicians. A slightly higher percentage considered in model selection, such as survivability, body weight, (2.5%) accepted hand treats when the technician was excited (40% and lesion incidence, which have animal welfare and financial above resting heart rate) and a decreased percentage (-2%) accepted implications. When planning a study, one must consider several when the technician was calm (within 10% of resting heart rate). factors when choosing the appropriate model to ensure a robust Normal activity range (heart rate 20% above resting) led to a -1% study and the ability to complete the study within budget. Addition- change, and an active state (30% above resting HR) showed no al considerations are cost of compound, vivarium space, and change. The test was repeated using calming exercises to lower heart technician time. An analysis of 2 rat models widely used in toxicol- rate. Lower heart rates and calm, slow movements reduced the ogy studies was performed. The Wistar Han® rat is widely used in number of animals coming up front to accept treats (-8% with female Europe as the toxicology model of choice while the CD® rat is animals and -2% with males). In an effort to determine a more commonly used in the United States. To allow readers to gauge effective means of establishing trust, the technician repeated testing economic impact of model selection between these 2 rat models, a with the following conditions: spent extra time with animals before tool has been developed to allow researchers to calculate per study human interaction (-6% change in males, +3% change in females), cost by using various internal costs associated with these studies, “baby talk” towards animals (+2% with both sexes), and nonverbal including technician time, consumables, diet, bedding, and overhead. body language (lip smacking and peering), which was the most This tool will allow researchers to compare economic impact between successful strategy with females (+3%) but not with males (-2%). This the 2 models at various compound costs and study types by allowing data suggests NHPs are most likely not demonstrably affected in a the researcher to account for individual internal costs at a per study statistically significant metric by human caretaker emotional states level. Briefly, with regards to body weight, at 104-wk of age, the when heart rate is the surrogate marker for defining the emotional CD®male rat weighs 35% more than the Wistar Han® male rat (800 state. Further work is needed in this area to determine if there are compared to 594 g), and the CD® female rat weighs 30% more than other behavioral indices affected by human caretaker emotional the Wistar Han® female rat (500 compared to 385 g). Additionally, states. body weight begins to diverge at approximately 3 months of age in the males. Further, survivability is 32 percentage points lower (35% P21 Promoting Transparency: Hosting Veterinary Technicians for compared to 67%) in the CD® compared to the Wistar Han® male Research Day and 33 percentage points lower (40% compared to 73%) in the CD® compared to the Wistar Han® female. Neo and nonneoplastic KL Thompson*, NM Gades, TR Meier 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 Comparative Medicine, Mayo Clinic, Rochester, MN lower body weight, robust nature, and improved survival rate of the Wistar Han® rat may translate into improved animal welfare and

611 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 overall cost savings for the toxicology program. The use of this tool care and use (ACU) programs and the offices charged with oversee- will aid investigators in model selection and could be considered for ing institutional animal care and use committees (IACUCs). This has future studies. made it difficult for administrators to benchmark their research oversight programs in terms of workload, scope of responsibilities, P23 Goals, Achievements, and Challenges of the Institute for and salary. In response to requests from the oversight community for Laboratory Animal Research Roundtable on Science and Welfare of this data, Public Responsibility in Medicine and Research (PRIM&R) Laboratory Animal Use gathered information from 3 sources. PRIM&R’s annual workload and salary survey (WLSS) asks animal care and use professionals for L Anestidou* crucial data on workloads, salaries, and staffing patterns. On their examination applications, Certified Professional IACUC Administra- Institute for Laboratory Animal Research (ILAR), Washington, D.C. tor (CPIA) candidates have the option to answer a series of demographic questions, which provides critical insight into those Since 2014 the National Academies’ Institute for Laboratory Animal seeking professional development opportunities. In 2016, PRIM&R Research (ILAR) has established the Roundtable on Science and conducted a job analysis survey of animal care and use professionals, Welfare in Laboratory Animal Use to foster communication among which asked a series of questions about respondents’ professional the many constituencies with strong interests in the use of laboratory roles, job functions, duties, and tasks. The information generated by animals in research. By convening representatives from government these 3 resources describes the scope of the administrative structures agencies, pharmaceutical and consumer product corporations, of programs charged with overseeing the use of animals in research, professional societies, academic institutions, scientists, and veterinar- as well as provides detailed insight into the responsibilities and ians, the roundtable’s aims have been to stimulate dialogue and knowledge base required of staff members. For example, nearly half collaboration, help build trust and transparency, and provide a new of the respondents work in IACUC offices that support fewer than 3 pathway to promote the responsible use of animals in science. We fulltime equivalent staff members; over half of these individuals have discuss the goals of the roundtable and summarize the achievements additional responsibilities with institutional biosafety committees over the past 2.5 years. Importantly, it will also analyze the chal- and institutional review boards. The majority of respondents are lenges the roundtable has overcome and the ones still ahead. highly experienced, having been involved in this field for at least 5 years. These data describing overall program structure as well as P24 The Harm in Harm-Benefit Terminology profiling ACU staff will be invaluable for benchmarking analysis, identification of gaps and needs in training and staffing, and L Kinter3, D Johnson1, LJ DeTolla*2 development of best practices in administering animal care and use oversight programs. 1Comparative Medicine, Cascades Biosciences, Sisters, OR; 2Univer- sity of Maryland, Baltimore, MD; 3Green Lawn Scientific Consulting, P26 Toward the 3Rs in Fish Toxicity Testing: Development of a Unionville, PA Fathead Minnow (Pimephales promelas) Fish Embryo Toxicity

Risk-benefit analysis is current established terminology in animal research programs to determine and appropriately mitigate potential MK Jeffries*, K Roush, JC Krzykwa, K Phillips, J Malmquist pain and distress. Harm-benefit analysis terminology was recently introduced in animal research accreditation for the same purpose. Biology, Texas Christian University, Fort Worth, TX Pain, distress, risk, and benefit are objective concepts quantifiable through clinical observation, experimental measurement, statistics, A simultaneous increase in the number of fish used for toxicity and/or probability analysis, and long used in the ILAR Guide for testing and calls to move toxicity testing towards the 3Rs has created Care and Use of Laboratory Animals, a foundation document for a need to reevaluate current test methods and identify viable design, assessment, and evaluation of animal research programs. alternatives. This goal of this project was to test the utility of the Harm/benefit is not included in the Guide, USDA-AWA, or USPHS fathead minnow (Pimephales promelas) fish embryo toxicity test (FET) Policy. Harm was introduced internationally in Directive 2010/63/ as a replacement for the currently used larval growth and survival EU as a surrogate for “suffering, pain, and distress.” Harm is a test (LGS). The objectives of this study were to compare the sensitiv- subjective, ambiguous, and emotionally loaded term about which ity of the FET and LGS and to determine whether the inclusion of well-meaning individuals may reasonably disagree. When used in sublethal endpoints (e.g., growth-related gene expression, the animal research context “harm” is jargon for pain and distress. Harm incidence of developmental abnormalities, etc.) increases the conveys inaccurate and misleading impressions that biomedical predictive power of the FET. To compare FET and LGS sensitivities, research necessarily causes substantial injury or damage to animals. estimates of acute toxicity were generated for a suite of model Further, alternative definitions of harm include implications of moral toxicants via the FET and LGS test. A comparison of the acute toxicity evil and wrong-doing that should not be associated with animal estimates generated via the FET and LGS revealed that, in most cases, research. The authors posit that harm- benefit terminology (the use of the sensitivity of the FET is similar to that of the LGS. Upon FET test the word “harm” rather than the word “risk”) (1) contributes no conclusion, several sublethal metrics were evaluated and of these advantage or improvement over risk-benefit, 2) lends support to growth, growth-related gene expression and the incidence of misinformation and misdirected beliefs of nefarious conduct, 3) pericardial edema were found to be the most commonly altered in damages the reputation of the scientific community and recruiting of response to contaminants. Overall, the results of this study show that young scientists, 4) delays critical research, and 5) should not be used the fathead minnow FET may be a viable replacement for the LGS as alternative language for or in replacement of risk-benefit terminol- and that the inclusion of sublethal metrics may improve the ogy in ethical considerations of animal research activities, including predictive power of the FET. protocol review procedures and program accreditations and evaluations. P27 Creation of Epoxy Mouse Skulls to Reduce Animal Numbers for Stereotactic Surgery Practice P25 Staffing, Salary, and Scope: Survey of Animal Care and Use Personnel MM Haney*1, C Hinkel1, A Thiessen1, J Allen1, I Deninger1, D Ohlhausen1, T Lever2 ME Luthin*, S Shriver 1Comparative Medicine, University of Missouri, Columbia, Public Responsibility in Medicine and Research, Boston, MA MO; 2Otolaryngology-Head and Neck Surgery, University of Missouri, Columbia, MO There are vast differences in how organizations structure their animal

612 Abstracts of scientific papers 2016 AALAS National Meeting

Stereotactic brain surgery is an essential tool to study neurologic complications associated with typical RFID transponders, we function in many healthy and disease states of animal models. This requested a smaller transponder and delivery trocar. This next technique requires immobilization of the animal in ear bars and generation of micro-transponder was developed by our collaborator precise coordinates to be used to allow insertion of electrodes or who specializes in laboratory animal research technology. Our cannulas into specific regions of the brain. Furthermore, screws and laboratory evaluated the animal welfare, quality of product, tissue dental cement are often used to secure protective head stages to the reactivity, and longevity of the transponder in 4 of our standard skull. For novice users, this procedure can be technically challenging mouse models. The novel transponder was implanted using a smaller and requires a considerable amount of practice before reliable results 15 gauge trocar needle (previously 12 gauge). The overall transpon- are achieved, leading to the euthanasia of additional animals from der dimension was decreased by 60% (1.4 mm in diameter and 8 mm which no data will be acquired. Our lab sought to reduce the number long). Forty mice (NOD, C57BL/6, nude, and SCID mice) were of practice animals needed to become proficient at stereotactic implanted and monitored for 30-90 days. During the evaluation, mice surgery. By using a resin mold kit, casting epoxy, and 1 representative were manipulated daily to mimic study conditions, weighed, and the mouse, we created multiple (~20), reusable practice skulls within an transponder site was frequently palpated. Upon euthanasia, the skin, hour. The process involves sacrificing a single mouse of the strain subcutaneous tissues and the transponders were excised and and approximate weight of future experimental mice. The head is evaluated for histopathology. Compatibility of the transponders with separated from the body, the skull is cleaned thoroughly, and any high-field (7 Tesla) magnetic resonance imaging and optical imaging excess tissue is removed. The mold is placed around the skull and systems was also evaluated. Less scratching at the transponder site removed, leaving an imprint in which casting epoxy is poured. After and less struggling during restraint were observed with the smaller proper drying time, the cast is taken out of the mold and can be used delivery trocar and transponder. We conclude that animals tolerated for training. These practice skulls allowed lab members to practice the transponders well and with increased ease of placement, there ear bar manipulation, burr hole drilling, anchor screw placement, was less apparent pain on implantation. We also concluded that the and dental cement application. Additionally, the practice skulls can smaller delivery trocar is less invasive as the reduction in delivery be reused after placement in acetone to remove dental cement which trocar gauge size creates a significantly smaller hole in the skin. There does not damage the integrity of the epoxy skull. Through the use of was also a decrease in transponder migration resulting in 50% fewer a resin mold kit and epoxy casting material, skull replicates can be losses of the transponder, compatibility with imaging systems, and easily made for stereotactic surgery practice. These skull models minimal tissue reactivity. The novel smaller RFID transponders drastically reduce the number of animals euthanized for practice or represent a significant refinement in methodology, resulting in a less due to procedural error. The same materials can also be used for invasive and apparently more humane method of permanent larger rodent species used in research, such as rats. electronic identification of animals. Additional studies to evaluate lifetime use of these novel transponders in day 0 and weanling mice P28 Creating a Third-Party Risk Assessment Process are underway.

MM Perez*, TL Condet, S Vaughn, TL Vandegiessen P30 Urinary Catheterization of the Female Murine Model

Animal Welfare Compliance, Zoetis, Kalamazoo, MI MM Williams*, H Ding, M Tweedle, MV Knopp

Our institution has a corporate policy that defines the approach to Radiology, The Ohio State University, Columbus, OH humane animal care and use within the facilities. This policy states we accept responsibility for conducting animal research in a humane Contrast agents can be excellent diagnostic tools. However, there are and ethical manner and expect all colleagues to treat animals with instances where the excretion method can become hazardous or respect. The policy also requires all third party research organizations impair the quality of a diagnostic scan. This is most commonly seen we engage that conduct animal-based research on our behalf, in nuclear imaging of the bladder where the genito-urethral and including animal vendors and animal transportation companies, to lower gastrointestinal visualization becomes impaired and presents a work at the same standards. To ensure third-party organizations are spill hazard. The option to express or use a needle to drain the in compliance with the policy, a risk-based process was devised and a bladder can lead to contamination of the fur/skin, surgical theater, global team formed to support the commitments described in the and an extra exposure to the handler and unfortunately does not policy. Compliance with humane animal care and use at external guarantee complete removal. The procedure presented here requires facilities is necessary for animal welfare, data quality, study integrity, a length of small tubing cut to the length appropriate for access to the and regulatory compliance. We will outline the risk assessment and imager. It is best to mark the tubing to indicate 1 centimeter of audit process used to ensure compliance with the policy. We will also penetration. The tubing should be sterilized with ethylene oxide and provide a list of materials and assessment standards used, results, lubricated prior to use. This procedure requires no metal and thus is and a summary of the improvements in the process that came from safe for use around and MRI. The procedures used can be performed our analysis of the results. with plastics that do not fluoresce and thus are safe for optical imaging. The tubes can also be shielded with radiopaque materials to P29 Evaluation of Refinement Method for Mouse Identification 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 MJ Weinstein*1, C Norton2, D Bangari3, K Singh3, X Ying4, G Hunt5, N bladder does not completely drain of its own accord. Gentle pressure Campbell5, F Sun1, ST Savage2 to the bladder should be applied just prior to scanning to assure complete evacuation. Allowing the tubing to remain during an in 1Pharmacology, Sanofi Genzyme, Cambridge, MA; 2IVRC, Sanofi vivo scan would allow for much more detailed imaging of orthotopic Genzyme, Framingham, MA; 3Pathology, Sanofi Genzyme, Framing- bladder models as well as GI evaluations. The removal or reduction ham, MA; 4Preclinical Safety, Sanofi Genzyme, Framingham, of bladder artifact allows for better dynamic contrast across the rest MA; 5Bio Medic Data Systems, Inc., Seaford, DE of the body. This, in turn, would improve the signal to noise ratio for faintly localized tracers. There are many advantages to adopting this Use of radio-frequency identification (RFID) transponder in mice is a as common practice prior to or during various imaging procedures. standard practice in our animal facility. Due to concerns with RFID The placement of the tube is simple and the materials are inexpen- transponder migration or loss from improper injection techniques sive. In conclusion, the application of a urinary catheter can and also a perceived pain and distress upon injection, we proposed drastically improve the quality of data in the preclinical community to refine the use of this technology in collaboration with our without extreme expense. Further efforts will be applied to making transponder supplier. The standard transponder provides researchers this applicable to both sexes of mice in hopes of increasing its utility with a reliable, nonreactive, and permanent method of identification in biomedical research. that facilitates rapid electronic data capture. To address the perceived

613 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P31 Improving Sevoflurane Anesthesia with Premedication of had 3 interns complete a 10-week internship in our department. Each Fentanyl-Midazolam in Laboratory Mice student brought a unique skill set, positive attitude, and willingness to learn, which contributed to their personal accomplishment and M Lipiski*1, M Arras1,2, P Jirkof1,2, N Cesarovic1 departmental productivity. Our staff welcomed the opportunity to help the students throughout their internship. The interns provided 1Division of Surgical Research, University Hospital Zurich, Zurich, an avenue towards employee engagement. Several staff members Switzerland; 2Neuroscience Center Zurich, University of Zurich, worked side by side with the students each day, mentoring and Zurich, Switzerland guiding them. The benefits were mutual, however, as our staff members were able to approach their jobs from a different point of Laboratory mice typically are anesthetized by inhalation of volatile view, that of a teacher. The staff also built trust amongst each other to anesthetics or injection of drugs. Using synergistic and additive collaborate and construct a platform for our interns to learn. Project effects of injectable drugs and volatile anesthetics, known as balanced SEARCH was an overwhelming success in our department, and is an anesthesia, allow for the dosages of each component to be decreased excellent example of how organizations can participate in outreach while inducing general anesthesia of sufficient depth with fewer side programs as a way to support their community and enhance the local effects. Here we evaluate 2 balanced anesthetic protocols, a combina- workforce. tion of Ketamine-Sevoflurane (KS) or Fentanyl-Midazolam-Sevoflurane (FMS) on their suitability for P33 Tools of the Training Trade: Mechanics of Improved Animal moderate surgical intervention and their impact on post-surgical Health recovery compared to a Sevoflurane mono-anesthesia (S). Twenty- four female C57BL/6 mice were assigned randomly to 1 of 3 CR Lockworth1, NA Monts de Oca*2 protocols: S, KS, or FMS. Injectables were administered 8-10 minutes prior to induction with Sevoflurane. A sham ovariectomy was 1MD Anderson Cancer Center, Houston, TX; 2Animal Care Opera- performed. A single dose of Carprofen was administered to all tion, University of Houston, Houston, TX animals at the end of the procedure. Heart rate and core body temperature were recorded by telemetric transmitters intra-opera- A technician training program must be developed that effectively tively and for 3 days prior and 3 days post experimental procedure. equips the technician with all of the tools necessary to provide the Intraoperative respiratory rate was determined by counting thorax highest-quality of care to research animals. To prepare technicians, movements. Body weight, food, and water intake were measured for we have used common educational methods including didactic 3 days prior and 3 days post experimental procedure. Measurements training and mentoring. But, we have also found the incorporation of revealed a decrease in heart rate and core body temperature during training aids to be particularly useful implements to further augment anesthesia in both premedicated groups. Respiratory depression was learning during routine instruction. Moreover, aids can later function evident in all groups. Painful stimuli induced respiratory alterations as references materials following training when needed most, while in the KS group but not in the FMS group. Heart rate remained stable working with animals cageside. Training tools that we have in all groups. Time to reposture and motion was 1-2 minutes after developed include algorithms for rodent case management and for Sevoflurane withdrawal in all groups. Heart rate was elevated in all large animal anesthetic emergencies, which support logic and groups for the first 12 hours following experimental procedure; reasoning in decision-making. Additionally, we have produced a changes were least pronounced in the FMS group. No changes in pocket reference card of all common health conditions that provide food and water intake were seen in any of the groups. In conclusion, technicians with a quick and handy reference while examining single low-dose injection of Ketamine did not provide sufficient animals. Finally, we developed a rodent health conditions and intraoperative analgesia and was not superior to Sevoflurane alone. management poster. Posters are placed at suite level or within Animals premedicated with Fentanyl-Midazolam showed a more biosafety cabinets for continuous reference. Consistency in training reliable anesthesia based on responses of heart and respiratory rate to with such aids, as well as the ready availability of these tools painful stimuli. A shorter and quieter induction phase and decreased cageside, has resulted in earlier health issue identification, rapid negative postanesthetic effects on the heart rate were further animal treatment and disposition, and overall consistency across advantages seen with FMS. health activities of both husbandry and health technicians. Using these tools at 1 facility, we demonstrated a 2.5 fold increase in the P32 Comparative Medicine Teams up with Project SEARCH identification of health issues by husbandry staff and a 14% decrease in the number of animals found dead. As a result of this training NJ Rindels*1, N Joyce2, DM Kirchner2, MM Watson1 program and its implements, all members of the animal care team are increasingly empowered and the quality of animal health care has 1Comparative Medicine, Mayo Clinic, Rochester, MN; 2Human been advanced, producing better research outcomes. Resources, Mayo Clinic, Rochester, MN P34 Disease Activity Index Scoring in Rodent Models of Inflamma- Project SEARCH is an internationally recognized program that was tory Bowel Disease developed in 1996. The Project SEARCH High School Transition Program is a unique, business-led, 1-year, school-to-work program NP Allotta*, A Miller, M Zhang for students with developmental cognitive disabilities in their last year of high school eligibility. The outcome is competitive, integrated Amgen, Thousand Oaks, CA employment for each student intern. The Mayo Clinic Project SEARCH site was developed as a partnership between the Rochester Inflammatory bowel disease (IBD) represents a group of intestinal Public Schools, Mayo Clinic, Vocational Rehabilitation Services, and disorders causing prolonged inflammation of the digestive tract Olmsted County Developmental Disabilities Services with support affecting 5 million people worldwide. The 2 most common intestinal from the Project SEARCH State Leadership Team. In 2015, the first disorders are ulcerative colitis (UC) and Crohn’s disease (CD). UC students were selected for this program, and the Mayo Clinic involves inflammation of the large intestine and rectum, while CD Department of Comparative Medicine became an enthusiastic partici- causes inflammation in any part of the digestive tract mostly pant. Our goal was to develop a program that would provide affecting the tail end of the small intestine (ileum). Both UC and CD students a chance to develop hands-on, job-related skills and polish generate weight loss, diarrhea, rectal bleeding, and abdominal pain. soft skills such as communication, problem solving, teamwork, and IBD pathogenesis remains unknown; however, genetics, the immune adaptability that are important for achieving gainful employment. system, and environmental factors have been linked to this disease. We also anticipated that this program would enable our employees to Animal models of colonic inflammation are a critical tool in both work with and mentor older youth with disabilities and increase research and drug development. There is a large range of animal camaraderie in the workplace. During the 2015-2016 school year, we colitis models such as spontaneous colitis in susceptible mice strains

614 Abstracts of scientific papers 2016 AALAS National Meeting

or administration of dextran sulphate sodium (DSS), a colitis-induc- only interfered with the bags even more and the large bandages ing chemical. During the administration of DSS, a disease activity prevented the skin from healing. In an attempt to find an alternative index (DAI) score is assessed to evaluate the clinical progression of to the stoma bags, ultra-absorbent maxi pads replaced the stoma colitis. To monitor the animals in the study and determine the bags. The distal end of the foley catheter was positioned dorsal to the appropriate endpoints, we established a DAI score that combined stoma. Gauze was placed between the catheter and the body for stool consistency, bleeding in stool or around anus, and health comfort. The absorbent side of the maxi pad was placed over the appearance of animal. The DAI scores are defined as follows: stool stoma and catheter. Vet wrap and elastic cloth tape was wrapped consistency 0 (normal), 1 (moist/sticky), 2 (soft), and 3 (diarrhea); around the body covering the pad. Within 24 hours, the scalded skin bleeding 0 (no blood), 1 (mild), 2 (moderate), 3 (severe); appearance 0 had healed significantly and any leakage was quickly absorbed. The (normal), 1 (ruffled fur or altered walking gait), 2 (moribund or use of pads allowed ointment application to the scalded skin and lethargic). In addition to the DAI score, body weights were collected reduced the width of the total bandage, allowing the skin to heal. The to compare weight loss from the animal’s initial weight. A DAI score pigs appeared more comfortable and bandage changes were of 6 and greater or 20% body weight loss resulted in euthanasia. The accomplished in a shorter period. DAI scoring yielded strong correlation to disease progression and did not require any invasive procedures. This process provides a P37 Gene Expression Changes and Signal Transduction Pathway method to monitor animals daily and determine timely euthanasia Alterations in Primary Human Oral Epithelial Cells Exposed to when animals reach en points. With well trained staff and a validated Smokeless Tobacco Extracts model, alignment on the DAI scoring can effectively minimize pain and distress of the study animals. R Rajapakse*

P35 Ensuring That New Laboratory Personnel Stay Compliant: An University of Virginia, Charlottesville, VA Introduction to the Rules and Regulations of Animal Research Smokeless tobacco (ST), an alternative to smoking, has gained wide PI Mireles* popularity among tobacco users. This study is conducted to determine the time course of gene expression associated with specific IACUC, Northwestern University, Chicago, IL signaling pathways in human oral epithelial cells after exposure to smokeless tobacco extract (STE). As an alternative to use laboratory With over 300 principle investigator laboratories the number of new animals as well an in an attempt to reduce use of animals for research laboratory personnel added to an animal study protocol (ASP) (3R), here we conducted this study with human oral epithelial cells. ranged from 10-30 new members a month. Some arrive with previous A differentiated layer of epithelial cell is created as a way to mimic experience from other institutions within the US and other from reasonably similar physiologic atmosphere. A dose and time-depen- institutions from other countries; however, the majority of the new dent response is observed for cell viability and cell proliferation laboratory members are new to working with laboratory animals. assays indicating that this model system is responsive to the Even though they are required to take certain online courses, and a treatment. Expressions of 84 genes representing 18 different signal hands-on session, there still seemed to be a gap in understanding the transduction pathways are quantitated. This is accomplished by requirements and techniques. So to fill that gap, the IACUC started using real-time polymerase chain reaction arrays at 1-hour, 3-hour, hosting a monthly brown bag session during the lunch hour, inviting 6-hour, and 24-hour time points following exposure to STE. Changes all the new members added to a protocol from the previous month. in gene expression are observed on many cellular processes including This ensured that they have already completed the all required cell cycle regulation, cell adhesion, inflammation, apoptosis, and training, did the hands-on requirements, and were already added to DNA breaksdown including Akt pathway activation. Short time an ASP. The session is a combination a brief lecture and an interactive exposure (1 hour) leads more genes to down regulate whereas longer Q&A session that helps engage the audience throughout the session. incubation time results in more genes up regulation. Most notable The questions are based on the topics discussed, along with any differences in the expression of genes during the course of treatment items noticed during the semiannual inspections, PAM visits, and are BCL 2A 1, BIRC 3, CCL 20, CDK 2, EGR 1, FOXA 2, HOXA 1, minor deficiencies. This program was implemented 5 months ago IGFBP 3, IL 1A, IL- 8, MMP 10, NOS 2, NRIP 1, PTGS 2, SELPLG, and and so far has been a success, with a decline in minor noted TNF-a. This study provides an insight on gene expression on oral deficiencies, understanding of the requirements, and has even epithelial cells as a result of STE exposure. encouraged long-time members (laboratory managers) to request an invite as a refresher for themselves. P38 An Analysis of Inspection Reports in Animal Facility Based on GLP, IACUC, and AAALAC Site Visits P36 Padded Piggies to the Max S Park*1, E Auh1, H Han1, J Che3, J Yun1, B Kang1,2 RK Hlavka*, A Gallardo, G Moreno, C Torres 1Department of experimental animal research, Biomedical Research University of California—Los Angeles, Los Angeles, CA Institute, Seoul National University Hospital, Korea, Seoul, Korea (the Republic of); 2Graduate School of Translational Medicine, Seoul An ongoing project required creating a stoma on Yucatan piglets, an National University College of Medicine, Seoul, Korea (the Republic externalized segment of the small intestine. A stoma bag for of); 3Biomedical Center for Animal Resource and Development, Seoul collecting intestinal fluid was secured around the stoma and National University College of Medicine, Seoul, Korea (the Republic bandaged with vet wrap and elastic cloth tape. Piglets were trained of) using positive reinforcement to walk on leashes from their cage to the clinic and stand still on the exam table for treatment. Bags were Our department obtained Good Laboratory Practice (GLP) certifica- drained at least every other day and new bandages applied. Initially, tion from the Ministry of Food And Drug Safety (MFDS) in Korea in no issues were anticipated, however the lab discovered stomas were 2003, and was fully accredited by AAALAC in 2007. Since then, we healing and the opening was constricting, preventing them from have performed several inspections in accordance with GLP, IACUC, moving on to the next phase of the project. In an attempt to maintain and AAALAC. According to the results of 10 GLP quality assess- and increase the diameter of the stoma, the veterinarian recommend- ments, 9 IACUC visits, and 2 AAALAC inspections over the last 5 ed placing foley catheters. While this worked for the diameter of the years, the total number of items noted as needing immediate stoma, it posed a problem for the stoma bags and bandaging. Regular remedial action was 732, 226, and 25, respectively. We classified these leakage of intestinal fluid occurred due to the weight of the catheter 983 items according to 6 categories, which were animal care and use, in the stoma bags, soaking through the entire bandage. Because of the equipment, environment, personnel safety, facility, and drug. acidity of the fluid, piglets’ skin became scalded. Applying ointment Indicated items about animal care and use were the highest frequen-

615 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 cy (277 of 983, 28.2%) among the categories, followed by drug (161, reduces maintenance costs of live animals and it safeguards valuable 16.4%), facility (157, 16.0%), equipment (156, 15.9%), environment lines against infectious outbreaks or natural disasters. We provide (152, 15.5%), and personnel safety (80, 8.1%). A comparison between investigators the cryopreservation service of mouse 2-cell embryos 2011 and 2015 reports indicated that environmental and equipment mostly by in vitro fertilization (IVF). As a member of the European aspects decreased from 33.3% to 9.0% and 27.8% to 7.8%, respectively. Consortium Infrafrontier/EMMA, we follow protocols distributed by But items about facility, animal care and use, drug increased from the Consortium, including the TYH + MBCD IVF protocol developed 6.3% to 27.1%, 19.2% to 28.9%, and 9.4% to 18.7%. Also, personnel at Kumamoto University. We started this work following this safety slightly increased from 3.9% to 8.4%. Environmental and protocol, which is optimized in C57BL/6 background and imple- equipment aspects were found to be improved by regular inspection ments the use of TYH + methyl-β cyclodextrin as sperm and postevaluation whereas items about facilities increased possibly preincubation medium in addition of reduced glutathione (GSH) due to a relatively decrepit facility. Also, recent social trends and supplemented fertilization medium. With this method, similar guidelines associated with animal welfare seem to affect the fertilization efficiency rates were registered in different genetic increased animal care and drug items. In conclusion, we suggest that backgrounds (24% in C57BL/6, 28% in FVB, and 22% in Balb/c). In regular inspections and postevaluation programs are necessary not 2014 we started to use the FERTIUP & CARD Medium, also only management and improvement of animal facility but also optimized in C57BL/6, and observed a relevant increase in the continuous update of standard operating procedures. Therefore, fertilization efficiency rates. Analyzing the results per genetic animal institutes should retain the standardized staff educational and background we found that, not only C57BL/6 (47%), but also FVB assessment program. (71%) and Balb/c (36%) showed noticeable improvements. We report here the results obtained in the last 3 years, which summarize all IVF P39 Assessing the Influence of Different Caging Environments and sessions done, including sessions with 0% of efficiency. In order to Ventilation Protocols In Cynomolgus Macaques (Macaca fascicu- assess the quality and viability of the frozen material we implement- laris) Using EEG Telemetry ed 2 quality controls: (1) embryo culture into blastocyst, done for every embryo freezing session, and (2) embryo revitalization of a S Authier*2,1, MV Accardi1, M Pouliot1, E Boulay2 2-cell embryos straw followed by surgical embryo transfer to pseudo-pregnant recipients. Upon a viable progeny, with the 1CiToxLAB, Laval, Canada; 2University of Montreal, St-Hyacinthe, expected genotype, the strain is considered secure and the live Canada animal resource is ended. During the last 3 years, by constantly updating IVF protocols, we have contributed to a 2-cell embryo Environment has a major impact on animal welfare but limited tools biobank of 85 secured lines, having at the moment 50 more lines in are available to quantify animal wellbeing. Monitoring of cardiovas- the cryopreservation process, and a total of 43,237 frozen embryos. cular parameters failed to identify beneficial effects of social housing in animal species such as cynomolgus macaque (Macaca fascicularis). P41 Performance-Based Approach to Environmental Enrichment This study evaluated the effect different caging and ventilation on for Mice and Rats biomarkers of anxiety in cynomolgus macaques (n = 6). Stress levels and anxiety were monitored by means of various electroencephalo- SP Swing*2, E Bartlett2, WE Nack-Lawlor1 graphic (EEG) parameters (e.g. beta power spectral analysis and individual EEG frequencies) while each macaque was either single- 1Regulatory, Taconic Biosciences, Inc., Hudson, NY; 2Veterinary or pair-housed with a familiar or an unfamiliar cagemate in either an Sciences, Taconic Biosciences, Inc., Germantown, NY AAALAC-compliant (12 days) or ETS-123-compliant cage (12 days). On occasion, extraneous stressors were introduced (e.g. an unfamiliar An environmental enrichment policy for mice and rats should fulfill staff) to determine the response to spontaneous stressors. Ventilation the objective of promoting animal welfare by defining the objects in the room was adjusted from 12 to 3 air changes per hour with and/or structures to be included in the microenvironment which will continuous monitoring of air quality (ammonium, CO2 and total encourage species-typical behaviors and engagement with their particle) and animal welfare. Spectral analysis with quantitative surroundings. Providing the appropriate materials to enhance the electroencephalography (qEEG) demonstrated that single-housed environment, rather than to create stress within the environment, is animals displayed higher levels of anxiety biomarkers throughout key to a successful enrichment program. While bedding and nesting the experiment but with progressive adaptation to the isolated state. material, chew blocks, and similar items are typically associated with At equivalent time points, pair-housed animals showed signs of desirable enrichment for mice and rats, differences between animal decreased stress/anxiety with the presence of a familiar cagemate. models suggest that the most effective forms of enrichment will vary An unfamiliar cagemate was associated with variable results as some within a species, and not only between different species. A perfor- animals showed a significant increase in biomarkers of anxiety and mance-based, or Environmental Enrichment Efficacy Evaluation stress while others presented reduced stress with an unknown (4Es), approach offers an effective means of assessing the type of cagemate. Animals remained normal with ventilation parameters enrichment best suited to support the wellbeing of a particular adjusted to maintain air quality but to prevent a “windshield factor” model. Environmental enrichment may be characterized as ineffec- from typical high ventilation rates. Our results suggest that tive where the animals ignore it, interact with it in an unproductive cynomolgus monkeys may benefit from adjusted ventilation rates. manner, or where the object/structure generates or increases Furthermore, the data from this study suggests that qEEG can be aggressive behaviors. By designing a study wherein a different form used as a sensitive and quantitative methodology to evaluate animal of enrichment is offered to each study group of a particular model, welfare in cynomolgus monkeys. observations of the animals’ interaction with and reaction to the object/structure in the cage allows identification of the best choice of P40 Cryopreservation of Mouse Two-Cell Embryos: A 3-Year species appropriate and line-specific enrichment. As a measure of Report desired performance, or efficacy, observation data is scored against performance criteria based on the targeted undesirable behavior with AS Leocádio*, M Pereira, J Bom, A Ribeiro, M Rebelo, J Demengeot, A the most successful environmental enrichment resulting in reduced Pereira observations of the malbehavior. One example studied is reduced conspecific aggression, but other behaviors such as stereotypies and Animal House Facility, Instituto Gulbenkian de Ciência, Oeiras, , overgrooming will also be evaluated. These studies enable the most Portugal effective enrichment item to be used for the given line of mouse or rat, a more economical and appropriate enrichment approach. 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

616 Abstracts of scientific papers 2016 AALAS National Meeting

P42 Operational Efficiency Yields a Robust Postapproval Monitor- 10cm balloon dilator was passed into the LES for inflation. Post ing Program dilatation, measurements of the LES reached into the normal range at 10.9 mm. While specific esophageal and LES balloons catheters are SP Swing*2, E Bartlett2, WE Nack-Lawlor1 commercially marketed, they can be costly and, in our case, not kept in stock. By using available equipment and later a specific dilation 1Regulatory, Taconic Biosciences, Inc., Hudson, NY; 2Veterinary balloon, we were able to maintain the animal in good condition until Sciences, Taconic Biosciences, Inc., Germantown, NY the scheduled study end point. This prevented the loss of valuable data and the need to enroll additional replacement animals. Post approval or other ongoing monitoring programs are among the critical activities conducted by IACUCs to ensure animal wellbeing P44 Use of C-Arm Clamp to Achieve Hemostasis after Percutane- and program compliance. The challenge is to routinely obtain the ous Coronary Intervention in Yucatan Miniature Swine (Sus scrofa) data needed to assess, and to ensure an effective review of, the animal care and use program. This balance may be difficult to strike AM Corn*, P Corts, MP Short, K Peterson, TM McCrum, AA Carter, A for programs with limited resources to extend to an audit-based Zook, PA Mount, AD Meyers monitoring approach; observations of data and practices at a colony level can also be limiting in scope. Our program maintains several Scirball Center for Innovation, Cardiovascular Research Foundation, husbandry practices that represent departures from Guide-recom- Orangeburg, NY mended practices. Initially, large-scale studies were conducted to obtain performance-based data for each practice departing from Swine are commonly used as models for cardiovascular disease and Guide recommendations. The performance criteria was defined for preclinical device evaluation. Many of these procedures require each study using the performance measures described in the Guide, percutaneous access of the femoral artery and vein along with the as they were characterized relative to the practice in question (for need for subsequent hemostasis. While vascular closure devices are example, housing density factors assessed included health, reproduc- available, the mainstay of achieving hemostasis in swine remains tion, growth, behavior, activity). Upon completion of each study and manual compression. This can be provided by hand or by several where approval to maintain the Guide-departure practice was commercially available devices designed for human patients. We approved by the IACUC, ongoing monitoring criteria were defined evaluated a system that includes a flat metal base and a plastic, based on performance metrics equivalent to study performance adjustable arm. A translucent plastic disc is attached to the tip of the criteria and routinely captured in the husbandry operations database. arm. The disc is placed over the access site and lowered to induce The IACUC has leveraged this current performance data, already and maintain pressure as the sheaths are removed. We used this recorded in the course of daily animal activities, to compare against device to obtain hemostasis of the femoral artery in Yucatan the study data; the study data became the baseline for accepted miniature swine (20 to 70kg). These animals were on an antiplatelet welfare performance. Each month the IACUC is presented with a regimen and heparinized perioperatively to maintain an active report, including graphic depictions of current performance data clotting time, (ACT) greater than 250 seconds during the procedure. compared against the baseline. Having access to this volume of data The sheath was not removed until the ACT drops below 250 seconds. in real time allows the IACUC to monitor the performance of Time of compression was based on sheath size. We have found that multiple animal colonies across a variety of criteria and provides a this device works well on animals weighing less than 50kg. As the robust assessment tool for monitoring animal welfare performance. swine grow they develop increasingly large muscles which interfere with device placement. Advantages to using the device instead of P43 Management of Lower Esophageal Sphincter Stenosis in a manual pressure include a more controllable and constant pressure Familial Hypercholesteremic Swine (Sus scrofa) application. The device also eliminates ergonomic challenges as well as hand fatigue. While an improvement over manual hemostasis, AD Meyers*1, AA Carter1, AM Corn1, P Corts1, TM McCrum1, PA proper placement and monitoring of the access site is still required. Mount1, K Peterson1, MP Short1, A Zook1, J McGregor1, SJ Popilskis2,1 We found the device led to fewer complications when used to provide hemostasis on a previously naïve vessels. Animals undergoing serial 1Skirball Center for Innovation, Orangeburg, NY; 2New York Medical femoral vessel access tended to have an increased risk of bleeding and College, Valhalla, NY hematoma formation. This may be due to use of nonstandard access locations. A deeper access site requires a more exact pressure point, A 7-month-old female Familial Hypercholesterolemic Swine enrolled which the disc cannot provide as well as a finger can. Attempts to in a peripheral vascular study fed a high cholesterol diet was place the disc at a more proximal point usually result in bleeding due repeatedly noted to have undigested material beneath the cage. On to misalignment of the pressure point or obstructed venous return exam, the animal was bright and alert with activity level and due to the high amount of pressure required in that area. Overall it hydration status within normal limits. Initial differential diagnoses seems the C-arm clamp is a good replacement for manual compres- included causes of obstruction, bowel torsion, and dysphagia. Given sion only in particular circumstances. the lack of response to diet change and medical therapy, thorough diagnostic testing was performed. When plain thoracic and abdomi- P45 The Use of Enrichment in the Management of Endemic Tricho- nal radiographs were inconclusive, a barium study was performed phyton mentagrophytes in a Chinchilla (Chinchilla laniger) Colony revealing dilation of the lower esophagus and narrowing of the lower esophageal sphincter (LES) to 2.0mm. Average LES measurements for AE Sparks*, DM LeMoine, JM Hickman-Davis swine of this size and breed are ~10mm. Potential causes of LES stenosis include chronic irritation from gastroesophageal reflux and University Laboratory Animal Resources, The Ohio State University, achalasia, an esophageal motility disorder. Regardless of etiology, Columbus, OH symptomatic treatment was pursued. Dilatation of the LES using a percutaneous transluminal angioplasty balloon dilation catheter was Chinchillas were obtained from a local vendor for use in long-term, attempted. Post-dilation, the LES measured 5.6mm. This procedure noise-induced hearing loss studies. Chinchillas were singly housed in along with gastroprotectant medications and modified feeding solid bottom cages with aspen bedding and touch contact with neigh- practices allowed for symptomatic improvement. Four weeks later, boring animals. The colony was comprised of both genders and regurgitation increased and barium study again revealed LES ranged in size from 6 to 16 animals. Chinchillas were provided a stenosis. Due to the severity of stenosis, step-wise dilation of the LES commercially available pelleted diet formulated for chinchillas and was necessary. An 8.0x40mm balloon was first passed into the LES, water via an automatic watering system ad libitum. Enrichment for inflated, and removed. Then, a second dilation was performed using the colony consisted of a hanging toy, a push toy, and weekly dust two balloons (8.0x40mm and 7.0x40mm) in a side-by-side kissing- baths with blue cloud dust. Toys were rotated weekly and animals balloon technique. After these dilations were completed, a 30mm x were given hay cubes or fresh hay daily. Routine examination

617 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 identified 2 animals with patchy scaling skin lesions and fur loss on Exhaust Air Dust (EAD) PCR testing has been investigated on a the face. Trichophyton mentagrophytes was identified by Dermatophyte variety of Individually Ventilated Cage (IVC) rack types. Previous Test Medium (DTM) culture and PCR of fur samples from both studies suggest that cage-level filtration on some IVC racks may animals. T. mentagrophytes is the most common dermatophyte in retard or prevent agent-associated DNA from efficiently passing into chinchillas. Infected chinchillas show small, scaly patches of alopecia plenums thereby compromising detection of plenum sampling. Other on the nose, behind the ears, or on the forefeet. Wood’s lamp reports evaluating 3 different rack systems suggested that the filter in examination is rarely helpful in chinchillas as T. mentagrophytes does the sentinel cages may serve as a better sampling medium than the not fluoresce under UV light. Because endemic infection of the entire actual sentinel rodent. Our facility uses an IVC rack that provides colony was considered likely, treatment of all chinchillas was cage-level filtration; however, EAD has not previously been initiated. Systemic treatment with antifungals was contraindicated evaluated nor reported in this type of rack. This caging system for experimental reasons therefore miconazole nitrate antifungal employs the use of triangular-shaped cages and is exhausted directly powder was administered in the dust bath. Medicated dust baths into the building exhaust with an air exchange rate of 15-25 ACH at were initially offered for 1 hour, 3 times per week, for 12 weeks with the cage level. At the beginning of a 3 month screening period, two additional application of antifungal powder directly to any skin 4-6 week old CD-1 female sentinels were placed in one cage on each lesion. DTM culture was negative at 6 weeks and PCR for of four racks. 10 cm3 of dirty bedding were transferred weekly to the dermatophytes was negative at 8 weeks. Antifungal powder was sentinel cage and to another cage on each rack containing no mice. A incorporated into the weekly dust bath enrichment to prevent future new filter was placed in both cages on each rack and transferred to outbreaks. Due to the zoonotic nature of dermatophytosis, personnel new cages as cage cleaning and bedding transferred occurred. At the were educated on best practices and handling techniques to reduce end of the 3 month period, sentinel mice were evaluated by risk of infection and spread to other animals. traditional screening methods and sentinel cage filters were evaluated by PCR to detect rodent pathogens. MNV and Entamoeba P46 Oral Analgesic Delivery Method Refinement to Improve were both equivalently detected by both sentinel cage filters and in Animal Welfare in DBA/1J Mouse Model the sentinel mouse. Helicobacter, P. pneumotropica, P. mirabilis, Klebsiella, Spironucleus m., and Tritrichomonas were best detected AC Allaire* using EAD PCR testing of the sentinel cage. Furthermore, the filter taken from the sentinel cage detected a substantially higher number Disposition, Safety and Animal Research, Sanofi Genzyme, Framing- of agents when compared to the filter from the cage that did not ham, MA house sentinel mice. This finding suggests that the presence of the mice may be important to agitate and transfer material from dirty An effort to refine delivery methods of orally administered butorpha- bedding onto the filters. nol in the collagen-induced arthritis (CIA) DBA/1J mouse model examined an alternative approach to improve consistency in P48 Diagnosis and Treatment of the Mite Archemyobia (Nearche- analgesic coverage. CIA triggers an autoimmune reaction causing the myobia) latipilis Found in the Brazilian opossum (Monodelphis degradation of cartilage, hyperplasia of joint space, and turgescence domestica) of limbs varying from mild to severe. Due to disease symptoms, animals require continuous, minimally invasive pain management BD Smith* during a study’s in-life phase. A pharmacokinetic study was designed to evaluate the uniformity of butorphanol delivery in a Department of Veterinary Pathobiology, Comparative Medicine, liquid gel water replacement in comparison to inhouse-made Texas A&M University, College Station, TX butorphanol “shake” at a concentration of 1 mg/ml. Butorphanol delivered in shake must be replenished daily and delivered via Routine testing for parasites, such as for mites and pinworms in an petri-dish, while liquid gel water replacement can be delivered into a animal facility, is a typical part of a quality assurance program. cage via water bottle with a volume sufficient for 4 days of animal The Guide for the Care and Use of Laboratory Animals provides for dosing. Naïve DBA/1J mice were used to mimic the CIA model. surveillance, diagnosis, treatment, and control of diseases within a Animals were allocated into 3 groups of 10: a non-dosed control, vivarium, but is widely left to the discretion of the veterinarian. butorphanol delivered via shake, and butorphanol delivered via Species which are not widely used in the research setting also present a liquid gel water replacement. Animals were acclimated to a group- complicating factor as often there is a deficiency in normal values or specific delivery method for 24 hours. Following acclimation, diagnostic methods. Such was the case encountered with Monodelphis micro-sampling of venous blood was performed at 1-hour, 3-hour, domestica, also known as the Brazilian or gray short-tailed opossum. and 6-hour post butorphanol introduction daily, for 4 days to Recently during necropsy, mites of unknown origin were recovered on measure long-term blood levels. Following shake administration, histopathology of the dermis. After failure to obtain mites via tape blood concentrations of butorphanol were variable, ranging from testing, skin scraping under inhalant anesthesia was determined to be 4.1ng/mL (+/-6.7ng/mL) to 3210.0ng/mL (+/-3920.0ng/mL) over the most successful method to isolate parasites for further classification the 4 days studied. In comparison, the fluctuation in blood concentra- and to determine colony prevalence. These mites were found tions of butorphanol after gel administration was less, with a range throughout the entire colony and identified as Archemyobia (Nearche- of 4.1ng/mL (+/-8.52ng/mL) to 199.0ng/mL (+/-111.0ng/mL). The myobia) latipilis, which were recognized in 1979 when the original data collected demonstrates that higher butorphanol concentrations opossums were imported into the United States. While mites are were achieved in the shake group, but the liquid gel delivery method generally host specific, there was concern for spread to other species of consistently maintained a uniform dosage of butorphanol throughout animals housed in the facility, including mice, rats, rabbits, and guinea the cage population. Additionally, liquid gel water replacement can pigs, however routine sentinel testing has been negative for any type be delivered up to 4 days without causing disruption to the cage. of mites. Ivermectin was initially chosen for treatment to eradicate the Further study in CIA studies is planned to determine efficacy of infestation as compared to using selemectin or moxidectin, which both analgesic administration and to determine serum levels correspon- had variable success for mite outbreaks in mice and rats. Initial low dent to clinical analgesia. dose oral treatment at 0.2 mg/kg in propylene glycol was not suitable to eliminate the mites, therefore additional routes of administration P47 Evaluation of Exhaust Air Dust PCR Testing of the Bedding and dosages were employed. The route of administration did not Sentinel Cage Filter 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 NA Monts de Oca1, L Gray2, AM Williams*2, DW Brammer2 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 1Research Models—Houston, Charles River, Houston, TX; 2Animal individuals remained negative for the mites, and treatment to the Care Operations, University of Houston, Houston, TX entire colony other than pregnant or lactating females was performed.

618 Abstracts of scientific papers 2016 AALAS National Meeting

P49 Red-Tinged Urine in Arctic Ground Squirrels (Spermophilus leukemia. A side effect of the agent is low blood cell counts and poor parryii) appetite. Mice receiving this treatment appear to experience the same side effects, leading to discontinuation of the study or premature CP Jones*2, DM LeMay1, ME Maddox1, P Austin1, EN Yu2 euthanasia. Even with additional supportive care, the health of the mice continued to decline. To continue on the study, they needed to 1Animal Care and Use Program, Division of Animal Care, Vanderbilt increase their body condition, weight, and their blood cell count. It University Medical Center, Nashville, TN; 2Pathology, Microbiology, was hypothesized that a transfusion of whole blood would increase and Immunology-Division of Comparative Medicine, Vanderbilt Uni- the blood count and aid the mice with increased health and ability to versity Medical Center, Nashville, TN thrive. We used retired breeding NSG mice as the nonsurvival donors. Blood was collected under anesthesia via cardiac puncture An adult, male, wild-caught Arctic ground squirrel (Spermophilus using a heparinized syringe. If no clots were seen, the blood was parryii) presented within days of arrival with red-tinged urine in the transfused into the recipient mouse via the tail vein. A total of 0.3ml litter pan. Urinalysis showed no red bloods cells, no hemoglobin, a blood was transfused. Immediately following the transfusion, the high pH, and struvite crystals. The animal was bright, alert, mice were released from restraint and monitored. Weights were taken responsive, and hydrated, with no other abnormal clinical findings. post transfusion as a reference to their improved health. The Normal ranges are unestablished for Arctic ground squirrel urinaly- transfused mice continued on study as their weights increased and sis, so we performed urinalyses for the entire colony to establish the anemia subsided. Increased body condition and activity was seen baseline parameters. Samples were collected from clean, unlined in all recipients. Secondary transfusions have been performed with pans of 5 male and 6 female individually housed Arctic ground similar favorable outcomes. Whole blood transfusions in mice were squirrels. Individual ages were not available, but ranged from performed solely as a treatment for animals receiving 6-thioguanine. juvenile to adult. All samples were evaluated visually, by urine All mice receiving transfusions have been able to remain on study dipstick and by sediment microscopy. Urine color varied from pale rather than halting the treatments or having to be prematurely yellow to dark orange (red-tinged). Stored samples became more euthanized. red-tinged over time. All samples were negative for red blood cells and hemoglobin. Average urine specific gravity was low (1.014) and P52 Keratoacanthoma in an Adult Zebra Finch (Taeniopygia average urine pH was high (8.5 or higher). Protein was present in the guttata) urine of all animals. Struvite crystals were common, as were amorphous crystals with brown, globular material. Red-tinged urine CM Bodi*, V Bakthavatchalu, M Siddalls, MM Patterson may be a normal finding in Arctic ground squirrels kept in the laboratory, as well as high urine pH and struvite crystals. The Division of Comparative Medicine, Massachusetts Institute of amorphous crystals with brown globular material could account for Technology, Cambridge, MA the proteinuria and red-tinged urine color in this species. An adult male zebra finch (Taeniopygia guttata) presented with an P50 Enhancing Gene Delivery in Guinea Pigs Using an Electro- ulcerated cervical mass and poor body condition. Because of its poration Device declining health, the bird was euthanized and a diagnostic necropsy was performed. A 3.0 x 4.0 mm, nodular, dark brown dermal mass C Chiedi*1,2, M Dillon1,2, DG Scorpio1 was attached to the ventral aspect of the neck. Otherwise there were no unusual necropsy findings. Histologic evaluation of the abnormal 1Vaccine Research Center, Bethesda , MD; 2SoBran, Inc, Bethesda, MD tissue revealed an unencapsulated, well-differentiated neoplasm disrupting the dermal architecture and elevating the overlying Electroporation (EP) is an innovative biologic technique in which an epidermis. The neoplasm was composed of epithelial cells arranged electrical field is applied to cells in order to temporarily disturb the in cords that were separated by fibrovascular connective tissue. phospholipid bilayer. Increasing cell membrane permeability allows Immunohistochemically, the neoplastic cells stained positive for molecules like DNA or protein to be introduced into the cell more pan-cytokeratin. Based on the histologic and immunohistochemical effectively. The ease of use, reproducibility, high efficiency, and low findings, the mass was diagnosed as an avian keratoacanthoma, also toxicity has made electroporation the method of choice for introduc- known as squamous cell carcinoma of the skin or squamous ing many types of molecules into cells including mammalian, epithelioma. This is the first reported case of a keratoacanthoma in a bacterial, yeast plant, and insect. Increasing cellular uptake is zebra finch; such tumors are rare in passerines overall. It has been particularly important to enhance the potency of test articles such as speculated that various factors (e.g., skin irritation, overgrooming, DNA vaccines. For our institute, the standard method of delivering low humidity) can exacerbate early lesions leading to keratoacan- these test articles to guinea pigs is via a simple intramuscular thoma development. Though benign, these tumors have the potential injection with needle and syringe. Since investigators achieved to become invasive. They should be carefully monitored and suboptimal test results using the standard delivery method alone, surgically removed if normal function is compromised. Keratoacan- electroporation may enhance this method by increasing cellular thomas should be added to the list of possible dermal lesions in uptake, optimizing long-term gene expression. Here, we present and laboratory-reared songbirds. illustrate the steps used to prepare the animal and perform an electroporation procedure on an anesthetized guinea pig using a P53 Clinical Management of Immunosuppressed Swine in a facility approved electroporation device. The electroporation process Transplantation Model involves a few variables, including field strength, pulse length, and pulse shape which is species and device specific. Field strength and AD Werts*1, K Kolegraff2, CR Moats1, AE Goodroe1, G Furtmuller2, G pulse length can be manipulated with in approved parameters in Brandacher2, CM Garrett1 order to maximize the efficiency of gene transfer, while pulse shape is dependent on the type of pulse generator used. 1Molecular and Comparative Pathobiology, Johns Hopkins Univer- isty, Baltimore, MD; 2Department of Plastic and Reconstructive P51 Whole Blood Transfusion in Mice Surgery, Johns Hopkins Univeristy, Baltimore, MD

CB Volpe* Reconstructive transplantation using vascularized composite allografts (VCAs) has become a promising option for patients with Division of Laboratory Animal Medicine, University of Los Ange- amputation or other devastating injuries affecting the face, trunk, or les—California, Los Angeles, CA even genitalia. Current protocols, however, require life-long immunosuppression to prevent allograft rejection, and these 6-thioguanine (6TG) is a chemotherapeutic drug used to treat regimens can lead to serious complications such as infection and

619 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 malignancy. To investigate novel strategies of immune modulation would interfere with the procedure and location for dose application. and the induction of tolerance, a VCA model in miniature swine has The most common dose site used for dermal application on the been developed which combines nonmyeloablative total body and minipig is across the dorsal area and onto the flanks of the animal, thymic irradiation with short-term tacrolimus-based immunosup- totaling approximately 10% of the total body surface area based on pression. Similar to severely immunosuppressed human patients, a bodyweight. This dose site does not interfere with the placement of major complication of this model has been life-threatening infections. the electrodes for electrocardiogram collections, however the jacket Clinical manifestations ranging from pneumonia (Streptococcus commonly used to secure the electrodes and house the transmitter suis, Klebsiella oxytoca, Pneumocystis carinii), sepsis (Enterococcus spp.), covers the entire trunk of the animal, thus creating the challenge of septic arthritis (Klebsiella oxytoca, Pseudomonas spp., MRSA) and performing both dermal dosing and electrocardiogram tracings endocarditis (Escherichia coli), to posttransplant lymphoproliferative simultaneously. By modifying the jacket design to have an opening disease (PTLD) and a wasting syndrome have all been documented large enough to allow for the entire dermal dose site to be unaffected within our model. Animals with PTLD and signs of wasting were during the collection process, but secure enough that the electrodes confirmed to have one or more viral infections (e.g., cytomegalovirus, stay in place, we are able to successfully add electrocardiogram porcine circovirus, porcine lymphotropic herpesvirus). Infection has collections on any minipig dermal toxicity study. appeared independent of the specific immunosuppression regime used, but has been traced to a specific nidus (e.g., central venous P56 Troubleshooting an Acute Complication of Cell Transfer in catheters) in several instances. Pathogen screening and exclusion has C57BL/6J Mice proven to be challenging; many of these agents are environmental contaminants or endogenous to this particular line of miniature DE Collins*, C Fry, I Bergin, JA Nemzek swine. In order to reduce the overall incidence of clinical disease, a customized preventative health and diagnostics program was Unit for Laboratory Animal Medicine, University of Michigan, Ann established. This program includes antibiotic and antiparasitic Arbor, MI prophylaxis, vaccination, and postoperative diagnostics aimed at rapidly linking complex clinical presentations to underlying disease Adoptive transfer of autologous cells offers a unique approach to etiologies. As immunosuppressed miniature swine models continue immunotherapy and is easily modeled by intravenous (IV) injection to evolve, we anticipate the clinical data established at our institution of purified cells. In IACUC-approved immunotherapy studies with over the past half decade will serve as a unique knowledge base for fibrocytes, there were no notable complications in ICR mice after tail medical doctors, scientists and veterinarians across the sectors of vein injection (1x106 cells/100ul saline). However, severe respiratory translational medicine. complications developed after autologous cell transfer in C57BL/6J mice. In total, 6 out of 21 (28% loss) mice decompensated within 2-3 P54 Sudden Death in a Black-Tailed Prairie Dog (Cynomys minutes of injection, displaying dyspnea and recumbency that ludovicianus) required euthanasia. A comprehensive literature review revealed reports of dose-dependent pulmonary thromboembolism in the CM Tansey*, JM Gary, N Gallardo-Romero, C Hutson pulmonary capillaries and arterioles of mice after IV administration of mesenchymal stem cells and collagen respectively. Fibrocytes are Center for Disease Control, Atlanta, GA 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 = An adult, 644-gram, intact, colony housed, wild-caught male 3) were submitted for histopathology and special staining. Thrombo- black-tailed prairie dog (Cynomys ludovicianus) was found dead emboli were observed affecting at least 30% of pulmonary arterioles. during morning rounds. No previous clinical signs were reported. The thromboemboli contained fibrin, evidenced by blue staining with The animal was submitted for necropsy. Upon gross examination, the PTAH. Pink granular material suggestive of platelet, cellular, or great vessels were firm and white. The stomach was adhered to the granular collagen debris was also present, but organized collagen liver by fibrosis, and was thickened and firm with multifocal mucosal was absent, as indicated by a lack of blue staining with Masson’s and serosal hemorrhages. The kidneys were shrunken with a rough trichrome. The histopathology suggests that degenerate cells or extra- subcapsular surface. Histopathology showed multifocal metastatic cellular matrix debris may lodge in pulmonary arterioles, triggering mineralization in multiple organs, including the great vessels and acute thrombosis. The reason for strain differences in the occurrence gastric submucosa. The suspected cause of the histopathologic of this complication is unknown. Several measures were put in place findings in this animal was severe renal disease, which included to ameliorate these adverse effects, including improved positive tubular degeneration and regeneration, glomerulonephropathy, and isolation of fibrocytes from primary cultures, extra filtration steps, interstitial nephritis. Differentials for these renal lesions include a and doubling of the injection volume. These combined changes chronic progressive nephropathy-like condition, bacterial infections reduced mortality to 2 out of 26 animals (7.6% loss) in subsequent such as Leptospirosis, and age-related degenerative changes. Meta- experiments. The distribution of this information may help other labs static mineralization is not commonly reported in the literature experiencing similar problems, as adoptive transfer injection on Cynomys species, but should be suspected as a sequela to renal associated mortality is not widely covered in the veterinary or disease in animals presenting with poor hair growth, hair loss, laboratory animal literature. muscle stiffness, bone deformities, nephrosis, and death. P57 Facial Abscesses in NOD-SCID Mice Weaned from a Breeding P55 Minipig Jacket Design-Enabling Simultaneous Electrocardio- Female with Abscessing Pyelonephritis gram Collections and Dermal Dose Administration DM LeMoine*, C Keller, S Lewis CL Savidge*, C Artis, A Camacho, JE Sentz, M Miyamoto The Ohio State University, Columbus, OH Envigo, Princeton, NJ Eleven NOD-SCID mice in a breeding colony managed by a single The minipig is quickly becoming a widely accepted scientific model principal investigator presented with subcutaneous nodules affecting in toxicology studies with a cardiac component, due to the similarity the muzzle, mandible, and submandibular regions. The colony was between the pig and the human heart. Because of this similarity, the housed in 9 sterile individually ventilated cages, provided irradiated ability to collect continuous electrocardiogram parameters on a rodent chow and reverse osmosis water ad libitum, and all husband- variety of minipig study designs is critical to the scientific commu- ry activities were conducted within a disinfected biosafety cabinet. nity. In the past, these critical data parameters have been avoided on Cytology, culture, and sensitivity from 1 mouse led to a diagnosis of minipig dermal toxicity studies because the design of the jacket used abscesses containing enrofloxacin-sensitive Staphylococcus sciuri. to secure the electrodes in place for electrocardiogram collections Evaluation of the breeding records revealed that all affected mice had

620 Abstracts of scientific papers 2016 AALAS National Meeting

been weaned from a single founder cage. A second founder trio and 1Biologic Resources Laboratory, University of Illinois at Chicago, its offspring comprised the unaffected half of the colony. Enrofloxacin Chicago, IL; 2Animal Care Services, University of Florida, Gaines- treatment of all affected cages was initiated, but all treated animals (n ville, FL = 13) were subsequently euthanized due to lack of response to therapy. S. sciuri was cultured from lesions in all affected mice. All Adequate monitoring during anesthesia in Old World primates is tissues from the affected founder female and representative tissues critical for adjusting and maintaining proper anesthetic depth. It is from one of the affected offspring were submitted for histopathology. essential to have an understanding of both the plane of anesthesia, as Findings in the founder female included maxillary tooth root abscess, well as the maintenance of adequate tissue perfusion during focal abscessing myositis of the epaxial muscle, unilateral abscessing anesthesia. Furthermore, anticipating the deleterious effects of an pyelonephritis, and bilateral otitis media. Lesions in the offspring individual agent in a given species helps minimize complications mouse were limited to multifocal subcuticular abscesses on the head. associated with anesthesia. Opting for combinations of inhaled and/ The simultaneous presentation of several mice with facial abscesses or injectable anesthetic agents provides flexibility to tailor anesthetic has never been seen at our institution. Staphylococcosis is considered protocols to specific animal health and experimental needs. Thirty- an opportunistic infection predisposed to by compromised immune four macaques (10.4 ± 4.6 y), male and female, Macaca status, skin wounds, and trichotillomania. At presentation, none of fascicularis, Macaca mulatta, and Macaca radiata undergoing routine these mice had clinical signs suggesting a route of exposure. S. dental prophylactic cleanings were enrolled in the study to compare sciuri is a ubiquitous organism that has been isolated from healthy continuous infusions of propofol or ketamine and midazolam (KM) laboratory mice, but has also been implicated in human and swine to inhaled isoflurane. Animals were randomly assigned to treatment diseases. We suspect that a contamination event caused disease and groups and the average anesthetic duration was 53.1 ± 17.5 m. The bacterial shedding in the founder female, leading to colonization and effects of species, sex, body weight, body condition score, age, and disease in most of the cohoused animals. Our standard husbandry anesthetic agent on extubation time, recovery time, heart rate, practices limited the infection to animals originating from that single temperature, and blood pressure were evaluated using a repeated cage, but the source of exposure remains unclear. Constant measures mixed model. There were no significant differences adherence to aseptic technique by all personnel is critical for between the anesthetic groups on either extubation (14.0 ± 11.9 m) or maintaining the health of immunocompromised mouse strains. recovery times (37.2 ± 17.3 m). Heart rate was significantly affected by the interactions between time and anesthetic agent, sex, age, and P58 Endometriosis Associated with a Malignant Tumor in a Rhesus body condition score. Heart rate was highest over the anesthetic Monkey (Macaca mulatta) period in animals receiving KM. Body temperature was significantly affected by time and the interactions between time and anesthetic EH Curran*, F Connor-Stroud, S Wilson-Cox, A Gsrcia agent and time and species, trending to be highest after 15 minutes of anesthesia in animals receiving KM. Mean arterial pressure was Yerkes National Primate Research Center, Atlanta, GA significantly affected by time and the interactions between time and anesthetic agent and time and body condition score. Mean arterial A 15-year old female rhesus macaque (Macaca mulatta) was presented blood pressure was highest over the anesthetic period in animals for physical examination in March 2014 due to abdominal bruising receiving KM (107.8 ± 25.5 mmHg), compared to isoflurane (66.8 ± and petechiation on her forearms and legs and an enlarged firm 21.5 mmHg) and propofol (49.5 ± 20.6 mmHg). The results highlight uterus. Based on clinical findings endometriosis was diagnosed. the benefit of tailoring an anesthetic regimen to the specific animal Complete blood count and chemistry were within normal limits; and experimental needs to minimize the negative physiologic effects however, the coagulation profile showed an increased partial of anesthetic agents. thromboplastin time. A few months later, the animal was reevaluated due to dysmenorrhea and recurrent abdominal bruising. She was P60 A Prospective Analysis of Causes of Hind Limb Paralysis in started on meloxicam for potential discomfort and medroxyproges- Research Mice (Mus musculus) terone to regulate her menstrual cycles. The animal was euthanized and submitted for postmortem examination in August 2015 at the E Bryant*, C Autieri, JG Fox, S Muthupalani completion of study. Postmortem examination revealed extensive adhesions of the abdominal viscera. A “chocolate cyst” was noted in Division of Comparative Medicine, Massachusetts Institute of the omentum and several dark to light red masses were present on Technology, Cambridge, MA the uterine serosa. On cut section, the uterus had multiple small white nodules. Sections of the uterus and associated tissues were Hind limb paralysis is an infrequent cause of euthanasia in research submitted for histologic evaluation. Histologically, the myometrium mice and there is a paucity of studies in the literature describing the had multifocal cystic areas and severe proliferation of round to oval pathologic basis of this clinical condition. In mice, acute hind limb cells intermixed with bizarre multinucleated giant cells, degenerative paralysis can occur from various causes, including spontaneous or neutrophils, and necrotic debris. The neoplastic cells were predomi- experimental tumors (metastatic or primary) involving the spinal nantly round and moderately pleomorphic with vesicular round cord or the brain, infectious processes, traumatic, degenerative or nuclei and eosinophilic cytoplasm with distinct borders. Similar toxic neuropathies, or anomalous causes. To better elucidate the neoplastic cell aggregates were observed within the endometrium etiopathogenesis of sporadic hind limb paralysis in mice, we and adjacent tissues. Many glands were severely dilated and undertook a multiyear intrainstitutional study to clinically identify contained an amphophilic secretory material. Immunohistochemical and perform a detailed pathologic evaluation of all mice exhibiting analysis of tumor cells was positive for vimentin and cytokeratin. signs of hind limb paresis and/or paralysis. During this ongoing Malignant transformation of endometriosis in humans has been study of nearly 30 months, we included all mice with a clinical described in numerous case reports and literature reviews. Approxi- presentation of hind limb paralysis, regardless of sex, strain, age, and mately 1% of women with endometriosis have lesions that undergo manipulation status. We have evaluated 20 mice with clinical signs of malignant transformation. In nonhuman primates, endometriosis is hind limb paralysis and 5 mice with no signs of hind limb paralysis. relatively common but malignant transformation is rare. To the The various causes of hind limb paralysis in these 20 mice were the authors’ knowledge this is the first report of malignant neoplasia following: metastatic lymphoma involving the thoracolumbar spinal associated with endometriosis in a rhesus monkey. cord (n = 8), spinal cord sarcoma (n = 2), known history of injection of orthotopic tumors (n = 3, two of which had adenocarcinoma P59 Comparison of Intravenous Anesthetic Protocols for Chemical involving the spinal cord, and the third had a poorly differentiated Restraint in Multiple Macaque Species sarcoma), vertebral body and spinal cord abscesses (n = 2, presumed bacterial), multifocal necrohemorrhagic and degenerative myelopa- EA Nunamaker*2, LC Halliday1 thy of undetermined cause (n = 2), acute neutrophilic myelitis (n = 1, etiology unknown), polyarteritis involving multiple visceral

621 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 vasculature as well as the intercostal vessels supplying the spinal logical changes were distinct from each other, clinical presentation cord and nerve roots (n = 1), and a focal epidermoid cyst causing was identical between all affected animals. Ovarian acid-fast rods spinal cord compression (n = 1) . Additionally, immunohistochemical were found in 4 out of 6 individuals that were PCR-positive analyses are being performed to further classify the neoplasms for Mycobacterium chelonae/abscessus in ovarian tissue (the two species involving the spinal cord. The data from this diagnostic study of Mycobacterium were indistinguishable by PCR). Liver PCR increased our understanding of spinal paralysis in various strains of identified M. chelonae/abscessus in 2 animals despite absence of mice and its underlying etiopathogenesis. pathology in extraovarian tissues. Neither acid fast rods nor inflammation was noted in the liver or other tissues. To investigate P61 An Approach to Spontaneous Cardiopulmonary Arrest in the source of Mycobacterium, multiple components of the housing sys- Nonhuman Primates tem were tested by PCR, and a hose containing recirculated, filtered water prior to its introduction into the system was found to be E Bryant*, JG Fox, RP Marini positive for M. chelonae/abscessus. Failure of routine decontamination of embryos prior to introduction into the colony was ruled out as a Division of Comparative Medicine, Massachusetts Institute of source of bacterial seeding into the colony. To our knowledge, this is Technology, Cambridge, MA the first time that the combination of egg-associated inflammation and fibroplasia and Mycobacteriosis has been observed to have Spontaneous cardiopulmonary arrest is a rare but serious potential contributed to low reproductive performance discovered in a problem in nonhuman primate research colonies. Although primates zebrafish colony. These data indicate that 2 distinct syndromes, have been used (albeit infrequently) as models of resuscitative egg-associated inflammation and fibroplasia and ovarian Mycobacte- therapy following induced arrest, occasionally veterinary personnel riosis may contribute to a single, clinically indistinguishable are required to respond to an unexpected spontaneous cardiopulmo- syndrome of decreased reproductive performance. nary arrest. Animals undergoing sedation or anesthesia are at higher risk for adverse events, given that the agents used to achieve P63 Cranial Vascular Hamartoma in a BALB/c Mouse sedation and anesthesia have the ability to depress pulmonary and/ or cardiac functions. Nonhuman primates are often sedated for HR Holcombe*, V Lau, P Chamberlain, JG Fox, V Bakthavatchalu routine procedures, such as physical examinations, and therefore have a higher risk for adverse events than other species that do not Massachusetts Institute of Technology, Cambridge, MA have to be sedated for handling. At our institution, we have devised a coherent, straightforward flow chart detailing the sequence of Thirty 7-week-old female BALB/c mice were obtained from a events that should take place following an arrest, based on recom- commercial vendor on March 8, 2016. A mass was noted on the top of mendations from the Veterinary Emergency and Critical Care Society the head of 1 of the mice 6 days later. The mouse was unmanipulated, RECOVER documents and human pediatric guidelines. The flow and the investigator elected to euthanize the animal without chart is included in all rooms that have the greatest potential for a additional diagnostic testing other than necropsy. Grossly, the cranial spontaneous cardiopulmonary arrest, such as areas in which mass was observed to be firm, red, roughly spherical in shape, and primates are sedated for physical exams, surgical preparation rooms, measuring approximately 9 mm in diameter. Histologic examination and operating rooms. We have also developed a standard operating demonstrated that the mass originated between the meninges and procedure for training research and veterinary staff to respond the skull. The connective tissue stroma overlying the cranium was immediately and efficiently following an arrest. In addition, we have disrupted and expanded by encapsulated densely packed bundles of developed a protocol for stocking and maintaining our emergency spindle cells separating blood-filled vascular channels. The vascular supply cart with relevant pharmaceuticals and supplies, such as channels were lined by endothelium and surrounded by smooth endotracheal tubes and catheters of various sizes, manual resuscita- muscle and collagen bundles. Based on these findings, the mass was tion self-inflating bags, and other relevant emergency supplies. As determined to be a vascular hamartoma. The primary differential part of this emergency supply cart, a short, coherent pictorial guide would be hemangioma; however, hemangiomas lack all of the depicting the locations of various items contained in the cart is supporting structural elements of a normal blood vessel. Vascular included. The goal of this management initiative is to decrease hamartomas are developmental malformations that result in morbidity and mortality of primates undergoing sedation or overgrowth of disorganized mature vascular tissue. Spontaneous anesthesia for routine procedures by implementing protocols to hamartomas are rarely reported in rodents, with only a single report achieve a fast and efficient response in the event of a cardiopulmo- of a vascular hamartoma in the uterus of a Sprague–Dawley rat. nary arrest. Although hamartomas tend to be benign and are typically considered incidental findings, the size and location of the mass in this mouse P62 Low Reproductive Performance Associated with Mycobacte- made the animal unsuitable for research purposes. rium Infection and Egg-Associated Inflammation and Fibroplasia in a Zebrafish Danio( rerio) Colony P64 Developmental Abnormalities in Zebrafish Dana( rerio)

GJ Barnett*, J Rodenfels, SR Compton, CJ Zeiss JR Owiny*

Comparative Medicine, Yale University, New Haven, CT Colorado State University, Fort Collins, CO

A colony of zebrafish (Danio rerio) experienced decreased reproduc- Zebrafish (Danio rerio) are an increasingly important model organism tive performance and increased incidence of coelomic distension in for a wide range of basic science and disease studies. One investiga- females. Two adult females were initially submitted for necropsy, tor has a small zebrafish facility containing 8 racks housing about 250 which revealed egg-associated inflammation and fibroplasia, with tanks primarily to generate embryos for developmental studies. The acid-fast rods in retrocoelomic tissue and ovaries. Forty-seven adult fish are on a recirculating system with deionized, charcoal-filtered female zebrafish from the colony were subsequently submitted for and ultraviolet-sterilized water. The system adds bicarbonate and a necropsy and PCR diagnosis of Mycobacterium species. Histopathol- sea salt mixture to maintain a pH of 7.5-8 and conductivity of ogy revealed two distinct patterns of intracoelomic disease: 1) 300-1200 µS. Water temperature is held at 23-28°C. A staff member extensive fibro-granulomatous sheets scattered throughout the setting up fish for spawning reported to the veterinary staff that coelom with multiple, discrete granulomas containing acid-fast rods several fish appeared to have reddening of their branchial region. An and 2) oocyte death within the posterior coelom, accompanied by examination by a veterinarian revealed that the fish were behaving variable oviduct obstruction. Granulomatous inflammation at the normally and water quality was within normal limits. Affected fish interface between live and degenerate oocytes was noted; however, were distributed over 5 tanks, and were transgenic on a WIK these regions did not contain acid-fast rods. While these histopatho- background. The majority of affected fish had lesions on the left

622 Abstracts of scientific papers 2016 AALAS National Meeting

opercular region, although a couple had bilateral lesions. Three fish P67 Observation and Intervention of Stereotypic Behaviors in were submitted for necropsy, and the remaining affected tanks were Raccoons (Procyon lotor) in a Laboratory Animal Setting taken offline. Differential diagnoses included trauma, possible developmental abnormalities, neoplasia, poor nutrition, branchiatis, JM Jarrell*, AE Sparks, S Lewis or an infectious case. A preliminary pathologic diagnosis included unilateral chondro-osseous malformation of the operculum in 2 fish. University Laboratory Animal Resources, The Ohio State University, The third fish had mild opercular lymphocytic dermatitis. The lesions Grove City, OH were suspected to be congenital and may represent a phenotype associated with the background or transgenic manipulation of the Standard housing systems, environmental enrichment, and species- fish. specific natural behavior is well established in common laboratory animal species. When animals that are not routinely used in a P65 Constrictive Tail Lesions in Multiple Litters of Gray Short- research setting are introduced, these principles can become Tailed Opossum (Monodelphis domestica) challenging. We received 10 purpose-bred raccoons (Procyon lotor) from a commercial furrier. Upon arrival, one raccoon was observed JM Criley*, J Ludwig, LJ Goodly engaging in a repetitive locomotion stereotypic behavior. Briefly, the raccoon would stand on its hind limbs and “weave” in a vertical University of Illinois, Urbana, IL pattern. This behavior persisted despite the provision of physical, food, and human-interaction environmental enrichment. Within a A breeding research colony of approximately 100 adult gray, few weeks, the raccoon in the adjacent cage appropriated the short-tailed opossums (Monodelphis domestica) is maintained for behavior. In an attempt to ameliorate the behavior, an ethogram- biomedical research projects focused on developmental biology. The based direct observation scoring system was developed to assess colony is maintained in individually ventilated cages designed for which raccoons were thriving in the environment and which were rats in an AAALAC-accredited animal facility. Temperature and not adapting as readily and/or displaying stereotypic behaviors. This humidity ranges are recorded daily. Animals are housed on recycled scoring system provided objective information on which animals to paper bedding with nesting jars and shredded paper towel nesting house adjacent to one another: high (positive adaptation) and low material and fed a commercial diet supplemented with treats. Over scoring (negative adaptation) animals. After 1 week, the positive an 8-year time span, 8 litters of Monodelphis presented with annular animal’s score decreased slightly as it appropriated the stereotypic tail constrictions resulting in inflammation and necrosis distal to the behavior, but the low scoring animal’s number increased, indicating lesions. Animals presented with lesions between 1 and 2 months of less frequent stereotypic behaviors. This trend continued through age and typically not all individuals in a litter were affected. week 2 of the study, but by week 3 a significant decrease to complete Response to topical lanolin therapy varied. Lesions resolved with absence of stereotypic behaviors observed in both animals which has mild scarring or with sloughing of the distal tail. The histologic been maintained. To the author’s knowledge, this is the first report of changes included acanthosis and hyperkeratosis with extensive stereotypic behaviors of Procyon lotor in a laboratory animal setting. edema similar those described for ringtail in rats. The etiology of the condition is unknown but may have environmental, genetic, and/or P68 T-cell Lymphoma in a Gilt (Sus scrofa) nutritional factors as suggested in rodent ringtail. JA Scholz*1, TR Meier1, C Frisk1, J Brazzell2 P66 Routine Cage Wash Cycle Does Not Clear DNA From Cage Lid Filters When Tested By Polymerase Chain Reaction 1Department of Comparative Medicine, Mayo Clinic, Rochester, MN; 2Marshfield Labs, Marshfield, WI JS Hubbard*, P Chen, K Boyd A 1.5-year-old transgenic gilt with a targeted disruption of the alpha Vanderbilt University Medical Center, Nashville, TN 1, 3 galactosyltransferase (GGTA-1) gene presented for thin body condition, lethargy, and decreased appetite. Prior to presentation, the Common mouse parasites in modern laboratory settings in- gilt had no major health issues and was experimentally naïve; a clude Myobia musculi, Rhadfordia affinis, and Myocoptes recent change in her environment occurred a week earlier when the musculinus (mouse fur mites) and Syphacia obvelata and Aspicularis animal’s pen mate was moved to a different room to farrow. On tetraptera (mouse pinworms). A recent dual infestation of mouse fur physical examination, the gilt was noted to have increased respira- mites and pinworms led to an evaluation of current practices tory effort with normal heart rate and body temperature. Primary regarding PCR testing for these parasites. These organisms are rule-outs included pneumonia and gastric ulceration, and the animal routinely detected by microscopic examination or by PCR analysis on was started on a proton pump inhibitor and given a long-acting samples collected directly from the mouse. However, recent studies cephalosporin. Food supplements and electrolyte water were offered advocate PCR testing of environmental samples such as plenum for supportive care. While the gilt initially showed signs of improve- swabs and cage lid filters in place of animals. Currently there is ment, condition declined over the next 4 days. The respiratory rate limited information surrounding the effect that standard cage wash persistently increased and muffled heart and lung sounds were noted has on DNA degradation or clearance from housing material and on the ventral left side. Due to poor health and lack of response to how this affects PCR results. To test the efficacy of a routine cage treatment, euthanasia was elected. Significant gross findings included wash cycle on DNA clearance from cage lid filters, we collected filter bicavitary effusion with fibrinous serositis; collapsed lungs; samples from fur mite and/or pinworm positive cages (n = 5) pre- ulceration of the esophagus and pars esophagea; multifocal firm and post-cage wash. Standard cage wash at our institution is defined white to light yellow hepatic and splenic nodules; markedly enlarged as an 8-minute cycle which reaches a minimum temperature of 180°F cervical and thoracic lymph nodes; and a pericardial abscess. and includes an alkaline wash/rinse cycle followed by an acid wash/ Histology of the lymph node masses was consistent with high-grade rinse cycle. All samples were submitted to a commercial diagnostic lymphoma. Immunohistochemistry of the neoplastic cells displayed laboratory for PCR fur mite and pinworm analysis and pre- and strong positivity for CD3 while neoplastic cells were diffusely post-cage wash results were compared. Post-cage wash results varied negative for CD20. In addition, septic peritonitis, epicarditis, and from positive (n = 2), negative (n = 2), to inconclusive (n = 1). There thoracic abscessation was observed and were suspected to originate were also 2 cases in which different agents were identified on the from bacterial translocation from the esophageal/gastric ulceration. post-wash filter when compared to the same filter pre-cage wash. Although lymphoma is considered to be a common neoplasm of Our results show that a routine cage wash cycle does not degrade or domestic pigs, it is very infrequently reported in the laboratory clear DNA from the cage lid filter. This information must be taken animal literature. Lymphoma should be considered as a rule-out for into consideration when designing surveillance testing schemes and respiratory and nonspecific clinical signs in research pigs. interpreting PCR results using cage lid filters.

623 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P69 Spontaneous Urinary Bladder Leiomyoma in a Rhesus to a nesting box would increase the temperature of the nest and Macaque (Macaca mulatta) increase the kit survival rate. Six nesting groups were evaluated over a 6-month period, which included double caging, cage board, ad lib KE Scott*, G Frydman, JG Fox, V Bakthavatchalu 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 Division of Comparative Medicine, Massachusetts Institute of nesting box (NB). The internal temperature of the nest and cage was Technology, Cambridge, MA recorded 3 days post parturition. The woodchip with no nesting box group was discontinued due to poor nest quality. Data analysis from A 19-year-old female Rhesus macaque (Macaca mulatta) involved in the remaining nesting groups, spread evenly over 35 kindlings, neuro-cognitive research for 7 years underwent a routine necropsy demonstrated higher survival rates at 2 weeks of age than historic after a terminal intracranial viral injection procedure. She was controls: crinkle paper + NB (92%), crinkle paper only (87%), crinkle socially housed and fed a commercially available standard monkey paper + woodchips + NB (83%), woodchips + NB (82%), crinkle diet supplemented with fresh fruit and vegetables. Gross examina- paper + woodchips (74%). All nesting groups had significantly higher tion revealed a diffusely thickened bladder wall and a soft internal nest temperature than cage temperature demonstrating circumscribed dermal mass proximal to the left stifle joint. Histo- thermogenic properties of all nesting materials evaluated. However, pathological examination of the urinary bladder revealed leiomyoma, crinkle paper, both with or without a nesting box, provided the characterized by the adventitia expanded by an encapsulated benign highest internal nest temperatures amongst the groups, 81°F and neoplasm. The neoplasm was composed of well-differentiated 83°F respectively. Interestingly, presence of a nest box did not make a neoplastic spindle cells arranged in fascicles. The neoplastic cells significant difference in internal nest temperatures. This study stained positive for vimentin, smooth muscle actin, and desmin and demonstrates using ~18c of nesting material with or without a negative for S-100 and pan-cytokeratin. The macaque lacked clinical nesting box will increase survival rate compared to providing ~10c signs of dysuria, pollakiuria, macroscopic hematuria, discomfort, or crinkle paper and no nesting box. Crinkle paper (18c) in particular, pain. The soft tissue mass proximal to the stifle was diagnosed as a with or without a nesting box, produced a significant increase in both lipoma. In humans, urinary bladder leiomyoma is 3 times more likely survival rate and internal nest temperature. 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 P72 Development of a Vascular Access Port Model in Woodchucks leimyoma in a rhesus macaque. (Marmota monax)

P70 A Novel Approach to Intravenous Access for Sampling and MA Batchelder*1, HA Dulac1, S Aborn2, G Marks1, J Brennan1, KD Administration in the Axolotl (Ambystoma mexicanum) Walton1

LD Cacioppo*, K Novak-Janus 1Veterinary Sciences, Bristol-Myers Squibb, Wallingford, CT; 2Univer- sity of Connecticut, Storrs, CT Research Animal Resources Center/SMPH, University of Wisconsin, Madison, WI A colony of woodchucks was scheduled to go onto a study which involved repeated IV dosing and multiple blood collection time Axolotls (Ambystoma mexicanum) have long been known as a model points. Some of the animals were wild-caught, most were aggressive, organism for regeneration due to their ability to replace and repair and all were challenging to handle or restrain for venipuncture. lost limbs and damaged organ systems. In order to elucidate the Therefore, all vascular access procedures were performed under mechanism of this ability and aim toward translational therapies, anesthesia. Because of the frequency of access required for the study, scientists have incorporated axolotls into studies using stem cells, we decided to place vascular access ports to allow dosing and epigenetics, and endocrinology, among others. Although axolotls are collecting blood without anesthesia. We incorporated knowledge and unique models for many biomedical research studies, their useful- experience obtained inhouse from implanting ports in other species ness can be stifled by difficulties with in vivo blood sampling and and information from collaborators who worked with ported drug and experimental compound administration. Here we describe woodchucks to develop a successful Woodchuck Vascular Access a novel approach to intravenous access in the anesthetized axolotl Port model. We placed arterial and venous ports in 70 woodchucks using the external jugular vein. Our technique demonstrated with patency extending up to 23 months. We will provide an consistent and repeatable collection of blood samples of scientifically overview of the surgical procedure and some unique aspects of this useful quantities. Methylene blue injection using the technique procedure in woodchucks. verified extensive perfusion of the dye throughout organ systems and peripheral tissues, indicating the technique can also be used for P73 Apoptotic Enterocytes Misidentified as Entamoeba in intravenous injection. Use of this technique results in reliable Gnotobiotic Mice with Clostridium Toxin-Mediated Dysbiosis collection of undiluted blood samples and may be useful for administration of medical treatments, experimental compounds, and contrast ME Keith*1, C Besch-Williford2, I Redelsperger3, H White1, C Cumo1 agents. 1Molecular and Diagnostic Analysis, Taconic Biosciences, Rensselaer, P71 Which Nest Is Best? Nesting Material Improves NZW NY; 2Pathology, IDEXX BioResearch, Columbia, MO; 3Veterinary Transgenic Rabbit Breeding Colony Kit Survival Rates Sciences, Taconic Biosciences, Rensselaer, NY

LA Burlingame*1, S Thurston1, J Song2, JL Lofgren1, P Lester1 Unassociated, gnotobiotic Swiss Webster females presented for routine necropsy examination with mild rectal prolapse and irritation 1Unit for Laboratory Animal Medicine, University of Michigan, Ann of the perianal area. Upon dissection, gas-distended ceca were noted. Arbor, MI; 2Int Med-Cardiovascular Medicine, University of Cecal contents had an abnormally thin consistency, with sparse, Michigan, Ann Arbor, MI granular material. Wet mount examination showed round organisms, approximately 10 microns in diameter, with a small indistinct Providing adequate nesting material is essential for rabbit kit nucleus. PCR testing of feces was negative for Entamoeba spp. Four survival. In our New Zealand White, transgenic, breeding colony, additional mice from the same isolator were submitted for histologic does were previously provided a double cage and nesting material 1 analysis of the digestive tract, wet mount analysis, fecal float week prior to parturition, including cage board, ad lib hay, approxi- analysis, and to confirm negative PCR results for Entamoeba spp. Wet mately (~) 10 cups(c) crinkled paper, and a partial enclosure under a mounts of cecal and colonic contents were positive for the same shelf (hutch). The kit survival rate at 2 weeks of age was 48%. We round organisms 10-12 microns in diameter, which contained 1 or hypothesized increasing the amount of nesting material in addition more condensed nuclei and did not display pseudopodia formation

624 Abstracts of scientific papers 2016 AALAS National Meeting

or evidence of motility. Fecal flotations were negative for protozoal The overall health and wellness of animals is of the upmost impor- cysts, and fecal PCR results were once again negative. The GI tracts tance in a research environment. Unfortunately, an internal ailment is had a normal appearance at time of necropsy. Histopathologic more difficult to recognize as opposed to an external injury. This is examination revealed mild to moderate typhlocolonic mucosal especially true of gastrointestinal ulceration in swine where sudden hyperplasia with mucosal edema and neutrophilic infiltrates. Mucus, death is a common presentation. As a cardiovascular model, pigs are bacteria, and numerous clusters of sloughed, degenerate enterocytes often placed on antiplatelet and anticoagulation therapies to prevent were present in the lumen of affected gut segments. Subsequent device thrombosis and mimic clinical situations. These study testing identified Clostridium perfringens in fecal samples from this requirements along with perioperative stress, fasting, and the isolator. Given the small size of the observed organisms and the coadministration of nonsteroidal anti-inflammatory drugs can negative PCR results, the organisms observed were most likely exacerbate the risk of gastrointestinal hemorrhage. Clinical signs of sloughed, apoptotic enterocytes. These were likely observed due gastrointestinal hemorrhage may include lethargy, generalized pallor, to C. perfringens toxin-mediated enterocyte injury, with compensatory pale mucus membranes, and darkening of stool; however these mani- crypt epithelial hyperplasia. While luminal enterocyte apoptosis festations are only noticeable after significant blood loss has occurs as a normal part of mucosal replacement, the level is normally occurred. We aimed at using a commercially available fecal occult low enough that degenerate cells are not identified in wet mount blood test (FOBT) for earlier detection of gastrointestinal hemor- analysis. Shifts in enteric microbiota (as occurs with dysbiosis due to rhage. Within the past calendar year, we screened 21 swine using this overgrowth of certain Clostridial species) can alter enterocyte method. These FOBTs are noninvasive, easy to use, and can be run turnover and result in an increase in sloughed enterocytes found in inhouse yielding results in seconds without specialized equipment. the GI lumen. Lack of motility, indistinct nuclei, and granular As a result, the time and expense of sending fecal samples to an cytoplasmic appearance are features that may be used to distinguish external diagnostic lab is bypassed. Of the two common FOBTs, these degenerate enterocytes on wet mount exams from Entamoe- guaiac and immunochemical, the guaiac FOBT is more readily ba and prevent misidentification. 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. P74 Management of a Colony of Mice with Motor Neuron Disease The reliability and use of guaiac FOBTs has been questioned in literature with studies showing a decrease in its positive predictive MC Dixon* value and with the ever increasing availability of immunochemical FOBT. However, in our facility the benefit of low cost, rapid Neurology, Drexel University, Eagleville, PA 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 Managing a colony of transgenic mice with various motor neuron there were no false negatives. When working with animals, such as disease presents a variety of complications. The 2 mouse models swine, that have a propensity for gastrointestinal ulceration, a guaiac discussed are the SOD1 G93A model of ALS and the G59S-hDCTN1 FOBT is a simple, cost-effective first step in determining if hemor- mouse model of a mutation in the dynactin complex (commonly rhage has occurred. referred to as Dynactin mice). The backgrounds of these mice are either C57BL/6, SJL/J, or B6SJL. Due to the nature of these neurode- P76 Effect of Ketamine Sedation on Maternal and Fetal Heart Rate generative diseases, the animals face challenges such as muscle in the Common Marmoset (Callithrix jacchus) degeneration, weight loss, paralysis of either a hind or front limb, and grooming problems. To remedy challenges pertaining to muscle MA Burns*, MT Lieberman, L Wachtman 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 Division of Comparative Medicine, Massachusetts Institute of event they are too weak to posture to drink from the water valve. Technology, Somerville, MA 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 The common marmoset (Callithrix jacchus) has grown in popularity as of one hind limb. End stage for the Dynactin mice qualifies as not a research model due to the high reproductive efficiency, small size, being able to right themselves after 30 seconds when placed on their and ease of handling of the species. Our institution maintains an back. Grooming also is an issue for these mice towards their end active breeding colony of marmosets with approximately 40 infants stage. They have more discharge from their eyes and trouble cleaning born per year. Estimation of parturition date is important for their eyes due to muscle loss. For this problem, the animals’ eyes and management of marmoset breeding colonies. While correlations faces are cleaned as needed using an artificial tears solution. Another between uterine size and fetal biparietal diameter (BPD) with eye problem, pertaining mostly to the Dynactin mice, are corneal gestational age have previously been described, current advances in ulcers. These ulcers tend to show up later in life for these mice (>1 ultrasound and transducer technology offer the opportunity to refine year of age). For treatment, triple antibiotic ointment is applied to the existing data. Normal fetal heart rates (FHR) in marmosets have not eye daily. Most of the time the ulcer will not completely dissipate, been established and offer a rapid, noninvasive method for evaluat- however it will stop worsening. Most of these animals with corneal ing fetal health or potential distress. Additionally, the effect of the ulcers remain on triple antibiotic ointment treatment for the rest of commonly used sedative ketamine hydrochloride on maternal and their lives. If the ulcer continues to worsen (reddening, swelling) fetal remains undefined in marmosets. We hypothesized that because euthanasia is recommended by the veterinary staff. Another ketamine is able to cross the placenta, FHR would be affected during symptom typical for the Dynactin mice is hepatomegaly and/or ultrasound exams performed under ketamine sedation. We per- splenomegaly. These issues usually affect mice over 1 year old. formed weekly abdominal ultrasounds using 22-30 MHz linear Frequently, the liver and/or spleen will have multiple tumors upon probes on 5 pregnant marmosets at various stages of gestation until necropsy. Due to the constant care these animal models require, delivery. Dams were manually restrained and given positive future researchers should be mindful of these points should they reinforcement training during ultrasounds, excluding scheduled choose to work with these animals. semiannual examinations performed under ketamine sedation (25-30mg/kg via intramuscular injection). Measurements acquired P75 Early Detection of Gastrointestinal Hemorrhage in Swine (Sus included uterine diameter, uterine lumen diameter, fetal BPD, FHR, scofa) and maternal femoral pulse rate (MPR). The date of parturition was set at 145 days for purposes of matching dams for gestational stage. MP Short*, AA Carter, AM Corn, P Corts, TM McCrum, K Peterson, A Mean and standard deviations of FHR acquired from conscious and Zook, PA Mount, AD Meyers ketamine-sedated dams were 217 + 43 beats per minute (bpm) and 172 + 27 bpm respectively. Mean and standard deviations of MPR Cardiovascular Research Foundation, Orangeburg, NY were 334 + 40 bpm and 425 + 26 bpm respectively. Both FHR and MPR were significantly lower under ketamine sedation compared to

625 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 values obtained from awake ultrasound examinations (Mann- Euthanasia was elected due to rapid decline and poor prognosis. Whitney; P < 0.01). In conclusion, ketamine sedation can decrease Necropsy findings included abundant adipose stores, segmental FHR, which may obscure signs of fetal distress. Ultrasonagraphy of mineralization of the aorta, renal cortical cysts, and a nodular mass pregnant dams habituated to manual restraint is recommended for attached to the cecal serosa. Histopathology revealed extensive accurate assessment of FHR. neutrophilic and mixed inflammation with microabscesses in the brainstem, midbrain, cerebellar white matter, and associated P77 Sustained-Release Buprenorphine Improves Postsurgical leptomeninges. There was also mixed perivascular inflammation, Clinical Condition and Does Not Affect Survival and Cytokine neutrophilic vasculitis, and fibrinoid vascular necrosis in affected Levels in a Murine Model of Polymicrobial Sepsis regions. Scattered gram-positive short bacterial rods were present amidst neutrophilic infiltrates, and were highlighted by immunohis- NL Herndon*1, S Bandyopadhyay2, E Hod2, K Prestia3 tochemical assay for Listeria monocytogenes. Molecular testing of DNA extracts from the brain tissue was also positive for L. monocytogenes. 1Institute of Comparative Medicine, Columbia University, NY, At this time, the source of infection is unknown, and all other NY; 2Department of Pathology and Cell Biology, College of Physi- animals in the colony appear clinically normal to date. Testing of the cians and Surgeons, Laboratory of Transfusion Biology, New York, animals’ commercial and supplemental enrichment feed for Liste- NY; 3Division of Comparative Medicine, New York University ria was performed by a government laboratory with negative results. Langone Medical Center, New York, NY Raw food sanitization practices have been enhanced to include washing fresh fruits and vegetables in a 200ppm chlorine solution Cecal ligation and puncture (CLP) is a commonly used technique to before rinsing with potable water. It is possible that infection may study sepsis in the murine model. Due to the invasiveness of the have been latent, with reactivation at time of clinical presentation. procedure and the impact on animal clinical condition, many Our facility sources wild-caught prairie dogs from the Southwestern IACUCs require analgesics. However, some analgesics have immuno- United States. This animal had been housed in our facility for 7 years. modulatory effects and can hinder research outcomes. We sought to Animals are evaluated twice daily by trained animal care staff and determine the effect of buprenorphine hydrochloride (Bup HCL) receive a physical examination annually. L. monocytogenes is a compared to sustained-release buprenorphine (Bup SR) on the gram-positive bacterial rod of public health significance, as it is a inflammatory cytokines, monocyte chemoattractant protein (MCP)-1 zoonotic agent capable of causing severe clinical illness and death in and interleukin (IL)-6, animal clinical condition, and overall mortality humans. More commonly isolated from ruminant species and in a murine CLP model of sepsis. We hypothesized that there would occasionally birds, this is the first case report in this species. Public be no difference in the tested cytokine levels, mortality, or animal health awareness of the potential for listeriosis in prairie dogs is clinical condition in mice dosed with either Bup HCL or Bup SR important, as these animals may be kept as household pets. analgesics following the CLP procedure. Male C57/BL6 mice were separated into Bup HCL or Bup SR treatment groups and each mouse P79 Clostridium difficile-Positive Mice: Two Barriers and Two had the identical CLP procedure performed. Mice assigned to the Outcomes 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 PL Roesch*1, SP Swing3, A Ericsson4, C Besch-Williford2, RS Livingston2 48 hours. The Bup SR group was dosed with 1 mg/kg Bup SR intraoperatively, followed by equal volumes of saline at the same 1Molecular and Diagnostic Analysis, Taconic Biosciences, Rensselaer, time points as the Bup HCL group. MCP-1 and IL-6 levels were NY; 2IDEXX BioResearch, Columbia, MO; 3Veterinary Sciences, measured at 24 hours post procedure and were not significantly Taconic Biosciences, Germantown, NY; 4Mutant Mouse Resource and different between the 2 groups. Mortality over the duration of the Research Center, Metagenomics Center, University of Missouri, study was not significantly different between the 2 treatment groups. Columbia, MO Mice were observed twice daily for clinical condition scoring by the same blinded investigator for the duration of the study. An abbrevi- The first clinical case was reported as a female NOG mouse with ated version of a validated behavioral scoring system was used to dried perianal feces, hunched posture, and a rough hair coat. At create a clinical condition scoring system, integrating physiologic and necropsy, yellow viscous fecal matter was noted in the cage and at behavioral parameters. Mice were first observed from a distance in the anus. Grossly, the colon and cecum were enlarged and edematous their home cage and assessed for movement, interaction with and contained gas and semi-formed mucoid feces. Over the next conspecifics, posture, and amount of time spent focused at the several days, many more cages in this location were identified with surgical site. Following the initial observation, mice were observed sick and dying animals exhibiting loose stool. Note that most animals immediately following the opening of the home cage lid to evaluate housed in this location were severely immunodeficient lines lacking responsiveness to cage opening, grooming, and respiratory effort. B, T, and NK cell production. Feces and blood were collected for Finally, mice were evaluated for reactiveness and exploration while culture and PCR. In addition, tissues were collected for histopathol- being handled and a clinical exam was performed. The results ogy. Blood cultures were positive for Lactobacillus murinus, a member showed a significant decrease in behaviors of perceived pain in mice of the Altered Schaedler Flora, and fecal cultures were positive in the Bup SR group at 12 and 24 hours postoperatively compared to for Clostridium difficile. Histopathologic exam of the cecum and colon the Bup HCL group. Because of the lack of significant effect on revealed mucosal changes consistent with the presence of a toxin- MCP-1 and IL-6 cytokine levels and mortality, and the superior producing bacteria. As a proactive measure, all other locations analgesic effects of Bup SR, we recommend the use of Bup SR in the housing NOG mice were tested for the presence of C. difficile by both murine CLP model of sepsis. culture and PCR. Surprisingly, another location was positive for the organism, but no clinical signs were evident. This led to the P78 Listeria monocytogenes in a Wild-Caught Indoor-Housed hypothesis that differences in the gut microbiome may be pivotal to Black-Tailed Prairie Dog (Cynomys ludovicianus) the clinical outcome. To test this hypothesis, fresh fecal samples were collected from both sick and healthy animals housed in multiple NL Lukovsky-Akhsanov*, BL Skinner, G Lathrop, JR, j hayes, J Ritter, locations and sent for microbiome analysis. Results demonstrated M DeLeon-Carnes that the animals from the location exhibiting severe clinical signs had a relatively simplified microbiome while the location where animals Center for Disease Control, Atlanta, GA remained healthy had a more diverse microbiome that included the presence of Lachnospiraceae, a family of bacteria often associated with An indoor and group-housed adult, prairie dog (Cynomys ludovicia- colonization resistance. These results suggest that the composition nus) was found acutely obtunded, exhibiting a Schiff-Sherrington and diversity of the intestinal microbiome of immunodeficient mice posture. During an examination, body condition score was 4.5 out of play a significant role in protection from dysbiosis due to C. 5, rectal temperature 86.4°F, and paddling in all limbs began. difficile and the development of subsequent clinical signs.

626 Abstracts of scientific papers 2016 AALAS National Meeting

P80 Enrofloxacin Site Reaction after Subcutaneous Injection in P82 Blood Volume Measurement in Rhesus Macaques by 18F-Albu- Chinchillas (Chinchilla chinchilla) min and PET Imaging

RM Burns*1, EL Sheldon1, EL Horn-Ranney2, MC Rodriguez1 RA Byrum*1, S Chefer1, J Solomon2, D Thomasson1, P Sayre1, J Seidel1,5, R Reeder1, K Hagen1, F Basuli3, R Swenson3, X Zhang3, PB 1Eastern Virginia Medical School, SoBran Bioscience, Norfolk, Jahrling1,4, RF Johnson4, M St Claire1 VA; 2Tympanogen, Inc., Norfolk, VA 1Integrated Research Facility at Fort Detrick, Division of Clinical Enrofloxacin (ENR) is a common antimicrobial used in the laboratory Research, National Institute of Allergy and Infectious Diseases, animal science field. ENR is a fluoroquinolone antibiotic with broad National Institutes of Health, Frederick, MD; 2Clinical Research spectrum for both gram-negative and -positive bacteria. It is available Directorate/Clinical Monitoring Research Program, Leidos Biomedi- for oral administration and for intravenous, intramuscular (IM), and cal Research, Inc., NCI Campus at Frederick, Frederick, subcutaneous (SQ) injections. ENR is a favorable off-label choice for MD; 3Imaging Probe Development Center, National Heart, Lung and exotic and laboratory animal species. A Chinchilla (Chinchilla Blood Institute, National Institutes of Health, Rockville, MD; 4Emerg- chinchilla) project was approved for the use of ENR SQ with a dosage ing Viral Pathogens Section, National Institute of Allergy and of 10mg/kg and a concentration of 2.27%. Injections were given SID Infectious Diseases, National Institutes of Health, Frederick, for 5 to 7 days, alternating injection sites on each side of the back. MD; 5Molecular Imaging Program, National Cancer Institute, There were no signs that the ENR could have been tainted and no National Institutes of Health, Bethesda, MD 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 Currently, multiple methods are available to measure blood volume injection site. The lesions started as round and red in color with (BV) using dyes or serum albumin labeled radioisotopes. These inflammation and subsequent hair loss at the injection site. The methods require repeated blood sampling over an extended period of lesions resembled a hematoma and in some cases ulcers. Lesions time. Use of 18F labeled serum albumin (18F-ALB), a novel blood pool ranged from a few millimeters to about 1 centimeter in diameter. imaging probe, measures BV without repeated sampling. Since the Immediate treatment was provided using topical antimicrobial probe is retained stably (1 hour post-injection) in the circulatory ointment (triple antibiotic) and wound dressing powder. Complete system after intravenous injection, use of this tracer as a positron resolution took an average of 21.5 days (6-45 days). Biopsies and emission tomography (PET) blood-pool imaging agent is feasible. In histopatholoy of injection sites were ordered after the project was this pilot study, we evaluated 18F-ALB application for BV measure- completed. The pathology report indicated the presence of healed ment in nonhuman primates by PET. Three male Rhesus monkeys skin lesions with decreased hair follicles supporting a clinical (Macaca mulatta) were injected intravenously with 18F-ALB (0.37–0.48 diagnosis of a local tissue reaction of the injected solution. Similar mCi/kg) and underwent up to three whole body 18F-ALB-PET and injection site reactions have been described as transient, localized CT scans using Gemini PET/CT scanner (Philips Healthcare). The tissue reactions resulting in necrosis, for both SQ and IM injections in whole body volume of distribution (VT) was calculated using PET horses, cattle, pigs, rodents, and rabbits. To our knowledge this is the imaging as the ratio of total 18F-ALB radioactivity in the body to 18 18 first time it is reported as described in chinchillas. the F-ALB concentration in the left ventricle (LV), VT=Total F-ALB / LV 18F-ALB, with the concentration in the left ventricle representing

P81 Development of a Pain Scoring System for Ruminants in arterial blood concentration. The VT was also evaluated by assessing Biomedical Research the concentration of 18F-ALB in venous blood samples collected at the end of PET scans by scintigraphy. 18F-ALB concentration in the LV RA LaFleur*, J Izer, R Wilson determined by PET was in agreement with that in venous blood samples assessed by scintillation counter (r2=0.87, P = 0.0003). The

Pennsylvania State University, Hershey, PA VTs 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 Effective pain management is integral to the use of animals in ml/kg when using scintigraphy of venous blood samples. These data biomedical research and laboratory animal medicine. A common are in agreement with previously published BV estimates in rhesus assumption is procedures which are painful to humans will also be macaques. In this study, the total BV in NHP was determined for the painful to animals. Although this principle is helpful in determining first time by PET imaging. The data suggest that detection of changes which procedures will require analgesics, additional challenges lie in in BV using 18F-ALB PET could be useful for assessing animal health determining if the analgesic regimen is effective and when pain and disease progression in animal models of disease. medications can be discontinued. In order to overcome these challenges, it is important to consider species-specific indicators of P83 Managing Abnormal Behavior in a Rhesus Macaque (Macaca pain and distress. Ruminants manifest pain in subtle ways and some mulatta) through an Individualized- and Multifaceted-Approach biomedical protocols may require ruminants to be housed in a Focusing on Preventative Treatment nontraditional environment which makes typical measures of pain in these species such as lameness and flock behavior difficult to RJ Mistretta*, M Carey, B Sullivan evaluate. Several publications exist on pain evaluation for ruminants but these focus on lameness or pain following castration or tail Animal Behavior and Enrichment, BIOQUAL, Inc., Rockville, MD docking in a farm environment. Therefore, we have developed a pain scoring system specifically designed for ruminants in biomedical Management of abnormal behavior through an individualized and research. The pain scoring system uses behaviors that can indicate multifaceted approach focusing on preventative treatments can be pain in ruminants such as bruxism, restlessness, excessive vocaliza- used in an attempt to reduce or eliminate instances of severe tion, kicking at the abdomen, and abnormal posture combined with self-injurious behavior (SIB) in nonhuman primates. A 15-year-old objective measurements such as heart and respiratory rate. After the rhesus macaque (Macaca mulatta) designated L862 with a history of score is generated, a decision tree is used to help guide further action. performing severe SIBs required additional behavioral intervention Sheep in our facility were scored at least twice daily in the immediate to increase the animal’s wellbeing and study availability. L862 had a postoperative period and the system was effective in identifying medical history including 11 instances of SIB requiring veterinary several animals with increased pain scores that benefited from attention from 3/12/2008 to 12/10/2012. Through detailed observa- administration of additional analgesics. We believe this pain scoring tions of possible triggers for abnormal behaviors an individualized system is an easy to use tool which can guide veterinary and animal Abnormal Behavior Management Plan (ABMP) was created. The care staff in the assessment and alleviation of pain in ruminants used management plan focused on transitioning the animal’s treatment in biomedical research. methods from reactive (responding to SIB events such as medications, e-collar, etc.) to preventative (avoiding SIB events such as

627 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 positive human interaction, positive social group construction, etc.). imaging) in the presence of experimentally controlled coronary artery Solutions were designed to alleviate each hypothesized trigger for stenosis and different severities of FFR, a system was needed that these behaviors and continuous evaluations were performed in an would respond to the changing aortic pressure during the stenosis to attempt to track progress and continuously improve the ABMP. It maintain the required distal coronary pressure in Yorkshire pigs (Sus was hypothesized that through detailed individualized behavioral scrofa domesticus). The experimental coronary artery stenosis was management, focusing on preventative treatment, the triggers for induced during pharmacological vasodilation with adenosine. A abnormal behavior could be dealt with and the instances of self- programmable pump was paired with a data acquisition system to injurious behavior would be reduced. The statistical data obtained create a real-time feedback system to inflate or deflate a balloon from comparing the instances of SIB prior to the implementation of occluder around the left anterior descending artery (LAD). The data the ABMP and after the implementation of the ABMP displayed acquisition system was used to simultaneously and continuously significantly less SIB (P = 0.01, α=0.05). The p value correlates with measure the aortic pressure and the distal coronary artery pressure both the observed decrease in percent chance of SIB post plan (24G catheter). The desired FFR was programmed as a separate implementation from 14% to 5% and the observed reduction of channel and calculated as a percentage decrease of the aortic abnormal behavior during human interaction sessions from 11% to pressure. A series of event triggers and digital outputs from the data 0%. The average months between major SIB events was increased acquisition system combined with a custom program regulated the from 5.0 months to 13.3 months with the current count at 31 months rate of infusion or withdraw of the syringe pump to achieve the and counting since the last event. Through an individualized and desired reduction in FFR as part of a continuous feedback loop multifaceted approach to behavioral management it has been system. The syringe pump is connected to the balloon occluder possible to eliminate the occurrence of abnormal behaviors such as which is around the LAD to maintain the desired stenosis that will self-injurious and self-directed behaviors in the case of L862. provide the desired FFR during the acquisition of the perfusion scan. The end result is a precise stable method to maintain a partial P84 Evaluation of Trimethoprim-Sulfamethoxazole in Medicated occlusion of the LAD during the acquisition of cardiac perfusion with Gel Formulation in Mice contrast enhancement. The system has successful used in over twenty simultaneous MRI and FFR experiments. S Satheesan*, TW Adamson, R Ermel P86 Combined Inapparent Zoonotic Diseases in an Awassi Ewe City of Hope, Duarte, CA (Ovis aries): Implications for Health and Biosecurity Management

The use of immunocompromised and transgenic mice has increased SM Prattis* the risk of bacterial infections; however individual dosing of Agriculture, American University of Beirut, Beirut, Lebanon medications for prolonged duration is not always a feasible option in laboratory rodents, particularly when a large number of animals Sheep (Ovis aries) have historically served as experimental subjects. need treatment. The administration of antibiotics, and in general As a result of their physiologic parameters, ovine models have been medications, as oral formulations is desirable as it results in developed to study orthopedic disease models, fetal physiology and decreased handling and less stress to the animal while also saving surgical intervention, lipid metabolism, feeding behavior, germplasm time for research and animal care staff. Recently, there have been cryopreservation, drought and temperature adaptation, and to serve many gel products that are available to facilitate the delivery of oral as sentinels of environmental chemical disease onset. It can be medication and provide supplemental support to laboratory animals. difficult in locations outside of the United States to obtain animals In our study, we assessed the efficacy of a commonly used broad- from flocks that have been raised using standard laboratory spectrum antibiotic combination trimethoprim–sulfamethoxazole husbandry and management principles, and that are specific-patho- (TMP-SMX) when administered in a medicated gel formulation. gen free in health status. The most common local sheep breed in C57BL/6 mice received medicated gel for up to 7 days, and daily Lebanon is the Awassi sheep, which are drought-resistant, nomadic, medicated gel consumption was measured by weighing the gel cup fat-tailed sheep that originated in the Syro-Arabian desert of for each cage of mice for the course of the study. There were 4 southwest Asia. In this study a thin Awassi ewe was identified with a experimental groups (4 animals each), each receiving the medicated body condition score of 2 that was detected at shearing. The ewe was gel for a different length of time: Group 1–24 hours, Group 2–48 then sacrificed and necropsied. This ewe was born and reared onsite hours, Group 3–72 hours, and Group 4–96 hours. At the end of the as a part of a 75-head Awassi sheep and Baladi goat mixed herd in predetermined administration period, blood was collected (AM) to residence on a farm that also had previously housed dairy cattle and measure the antibiotic plasma concentrations. These levels were then poultry. Mycobacteria avium subspecies paratuberculosis was compared to control groups that received a fresh cup of medicated identified, and acid fast (+) organisms were present in intestinal gel daily. Plasma concentrations of TMP-SMX were well above the lesions. The Rose Bengal assay detected a Brucella-positive reaction in

MIC90 values for common pathogenic bacteria in all groups, with the the ewe only, which was accompanied by suspect liver lesions. We levels varying from 4.41 ± 4.0 µg/mL to 23.58 ± 6.4 µg/mL. Our also identified Echinoccocus granulosa, Clostridium perfringens, Staphy- results indicate that the consumption of TMP-SMX medicated gel by lococcus, and Streptococcus species, and parasitic skeletal muscle cysts C57BL/6 results in therapeutic plasma concentrations of the most consistent in appearance with either Toxoplasma gondii or Neos- antibiotic with a recommended changing frequency of every 3-4 porum caninuminfection. Molecular PCR amplification for Torque Ten days. Virus, Blue Tongue Virus, Epizootic Hemorrhagic Viral Disease, and Foot and Mouth Disease was performed on the ewe and herds and P85 Real-Time Induction of Specific Fractional Flow Reserve was negative. We review the disease implications for public health During MRI Perfusion Studies Using a Programmable Infusion and biosecurity management on an experimental farm. To date Pump and Data Acquisition System implementation of routine governmental test and eradication programs and consistent border control is limited in developing SM Kozlov*1, GW Daniel2, JL Taylor1, A Arai2 countries and those with contiguous ongoing conflict zones.

1NHLBI/ASR, National Institutes of Health, Bethesda, MD; 2Ad- P87 Echocardiography Screening of Nonhuman Primates with vanced Cardiovascular Imaging, NHLBI, Bethesda, MD Asculted Heart Murmurs

Fractional flow reserve (FFR) is defined as the ratio of blood pressure TA Swanson*1, H Pisharanth2, B Morenko Campbell1 distal to a coronary artery stenosis divided by the pressure proximal to the stenosis during maximum coronary blood flow as typically 1Comparative Medicine, Pfizer Worldwide Research, Groton, induced by adenosine. In order to validate quantitative cardiac CT; 2Animal Resources Core, Nationwide Children’s Hospital, perfusion measurements using cardiac MRI (magnetic resonance Columbus, OH

628 Abstracts of scientific papers 2016 AALAS National Meeting

Cardiac auscultation of nonhuman primates (NHP) during a routine must be considered. Because it is a common occurrence for research- physical exam may sometimes reveal a heart murmur. The presence ers to request additional tail tissue to perform the genetic analysis, of a murmur must be taken into consideration when assessing the we wanted to determine if it is beneficial to the animal to have 1 large animal’s suitability for toxicology and safety pharmacology studies. biopsy or a small biopsy with a second biopsy later, if required. We Echocardiography of these animals often does not reveal any also wanted to ascertain at which age the procedure causes the least valvulopathy or other cause of the murmur. We theorized that this detriment to the growth of the animal. For this study 2 inbred mouse finding, which can be transient and/or positional, is physiologic in strains, FVB and C57Bl/6, 8 mice per gender per strain for a total of origin in our NHP population. Our goal was to perform a thorough 48 mice, including the control group, were used. Mice were subjected auscultation and echocardiography exam, including B-mode images to either a single biopsy of 5 mm or 2 biopsies, both 2 mm in tail tip of heart, pulse wave, and color Doppler analysis, on animals with length, that were taken several days apart. Single biopsies were taken audible murmurs to generate a knowledge base on the incidence, from mice at either days 5, 10, or 21. For mice that underwent dual relevance, and possible causes of the murmurs. In our study to date; sampling, biopsies were performed on days 5 and 21, days 10 and 21, NHPs with a grade 3 or higher murmur had echocardiographic days 10 and 28, and on days 21 and 28. Animals over the age of 14 evidence of a valve insufficiency either in the mitral or tricuspid days were anesthetized with Isoflurane for the procedure. Mice were valves. Animals with a grade 2 or below often have transient weighed at 1 week of age through 16 weeks of age. We chose to use murmurs that may be positional or due to other factors (hemocon- the weight gain comparison to ascertain the wellbeing of the animals centration or cardiac implant), but no evidence of insufficiency was because it is a noninvasive measurement that does not interfere with observed in this group. Murmurs with grade 2 or higher were also physiologic parameters in the neonatal mice. The comparison of the noted to have statically significant greater left ventricular diameter. weekly weight gained over the 16-week period revealed no signifi- As a result of our findings, we propose that any animal with cant variation of weight gain for all treatment groups, suggesting that suspected murmur be screened by echocardiography, and those dual small tail-tissue sampling through at least 4 weeks of age is not found to have documented valvular insufficiency not be used in detrimental to the growth of the animal when compared to mice that studies with cardiac endpoints. In addition, normal nonhuman underwent a single, larger sampling of tissue. primates have a wide range of functional cardiac parameters according to previously published information. We found that our P90 The Use of an Antibacterial Cleanse and an Antifungal animals diagnosed with murmurs were well within those normal Corticosteroid in Resolving Ulcerative Dermatitis Cases ranges, specifically ejection fraction and fractional shortening. VN Frazier*, J DeGraff P88 Ultrasound Monitoring of Fetal Guinea Pig (Cavia porcellus) Development for Maternal-Fetal Vaccine Programs Department of Laboratory Animal Research, Duke University, Raleigh, NC TA Swanson*1, D Church1, P Lewis1, HR Devantier1, D Yates1, I Scully2 Antibacterial cleansing treatment chlorhexidine followed by an antifungal corticosteroid ointment has been a common treatment for 1Comparative Medicine, Pfizer Worldwide Research, Groton, many years to manage ulcerative dermatitis (UD) on research mice. CT; 2Vaccines Research, Pfizer, Pearl River, NY Recently clinical veterinarians suggested terminating use of chlorhexidine and replacing with a cleansing treatment better suited Guinea pigs (Cavia porcellus) continue to evolve as a vital species for the facility’s needs. A new therapy of an antibacterial cleanse supporting research involving maternal-fetal disease transmission. containing active ingredient hypochlorous acid, followed by Maternal immunization to infer passive protection to the fetus or standard application of antifungal corticosteroid ointment containing neonate is gaining momentum in the field of vaccines research and active ingredients nystatin, neomycin sulfate, thiostrpton, and development. The ability to serially monitor fetal development in a triamcinolone acetonide was suggested for treatment of UD. To noninvasive, nonstressful manner could serve to provide data determine effectiveness of the new treatment plan husbandry staff supporting a correlate of protection in maternal vaccine efficacy scored all UD cases found in C57BL/6 research mice, or mice studies. The guinea pig was specifically chosen as the most appropri- with C57BL/6 background, immediately upon discovery. UD scoring ate small animal species to support this type of model development was based on a standard scoring system creating a calculated severity due to the length of gestation (68 days), as well as being closely score percentage derived from scoring the scratching number, translational in implantation process (interstitial) and placentation character of lesion, length of lesion, and regions affected. Higher type to human which has a significant impact on maternal/fetal scores reflect more severe cases. The effected mice were then treated antibody transfer. Guinea pigs were mated and shipped on embry- by gently cleansing the ulcerated area with antibacterial-soaked onic day 1 (e1). Ultrasound imaging was performed 2-3 times each gauze followed by a thin layer of antifungal corticosteroid ointment. week using light manual restraint. We found no ultrasound evidence This treatment was performed daily and cases were scored weekly or of pregnancy prior to day e17. On or after e17 we were able to track when considerable improvements were seen. During the trial period development of individual fetuses over the length of the develop- UD cases were diagnosed and treated. Of these cases, cases originally ment cycle. Heartbeats were visible and measureable after day e19. scored at above 40% were closed and considered completely healed Specific morphologic parameters including heart rate, crown-rump by a clinical veterinarian within a 2-week time. Some cases originally length and brain/skull size were serially monitored, as well as scored above 60% healed in a longer time period (4 weeks). Based on resorbing embryos and still birth rate, to establish baseline data on this information, we can confidently conclude that early diagnosis of normal fetal development and pregnancy outcomes in healthy, UD, treating with an antibacterial cleanse followed by antifungal unchallenged animals. corticosteroid ointment does effectively resolve clinical cases of ulcerative dermatitis within a reasonable time period. P89 Repeated Tail-Tissue Sampling Does Not Affect the Growth of Mice versus Single Sampling P91 1H NMR-Based Metabolomics Profiling to Investigation of Metabolism Changes in Urine and Serum of Cynomolgus Ma- TS Highbaugh*1, KL Stewart1, A Dawson1, AL Fletcher1, M Suckow2 caques (Macaca fascicularis) Subjected to Air and Road Transportation 1Freimann Life Science Center, University of Notre Dame, Notre Dame, IN; 2University of Minnesota, Minneapolis, MN X Pan*, Y Chang, X Zeng

The use of tail biopsy samples for the genotyping of genetically Shanghai InnoStar Bio-Tech Co., Ltd., Shanghai, China engineered mice (GEM) is common among research laboratories. For the wellbeing of the animal, the age of the animal and the sample size Stress is increasing markedly in modern society. Many types of physi-

629 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 ologic and socioeconomic stresses have adverse effects on human considered a best practice and is widely accepted as a means to better health. We aimed to study the effects of transport stress on cynomol- animal welfare and psychologic wellbeing. However, few investiga- gus monkeys and molecular changes after transport. This study tions on methods and approaches to overcome the specific challenges included 6 cynomolgus monkeys that were subjected to road and air of introducing adult male Macaca mulatta have been accomplished. transport for 8 hours from Hainan to Shanghai. To assess alterations An online survey targeting individuals directly involved in in metabolite levels in the serum and urine due to combined pairing Macaca mulatta was distributed to determine which factors transport, we applied a proton nuclear magnetic resonance spectros- are perceived to be the most critical when selecting adult male rhesus copy (1H-NMR)-based metabolomics strategy in conjugation with the to introduce, and which methods are associated with a greater rate of orthogonal projection to latent structure with discriminant analysis successful long-term pairs. The survey was sent to approximately 187 (OPLS-DA) method. Serum and urine samples were collected during individuals at 93 different institutions in 5 different countries (USA, pretransport, arrival, and recovery. NMR-based metabonomics Canada, UK, Germany, and China) with a survey response rate of revealed that serum levels of lactate, alanine, glutamate, glutamine, 15% completed and 37% partially completed. Survey users, 17 out of pyruvate, myo-inositol, creatinine, and urea were higher posttrans- 24, reported that identifying primates having different ranks was an port, while serum levels of isoleucine and proline (P < 0.05) were important selection criteria and that dynamic, larger cages increased lower after transport. Additionally, marked changes in urine their perceived rate of successful pairings. No significant correlation metabolites, including increased levels of lactate, glycoproteins, was evident between the cage sizes used for introductions to the rate pyruvate, citrate, choline, creatinine, α-ketoglutarate, taurine, and of successful pairs formed. Methodical stepwise introductions were allantoin, and decreased levels of isoleucine, leucine, creatine, the most commonly implemented practices (78%), using quad cages myo-inositol, 3-hydroxybutyrate, and hippurate were observed after (25%) giving new pairs horizontal and vertical space. Users perceived 24 hours recovery compared with 1-week recovery. These novel lip smacking and mutual grooming as successful prognostic findings indicate that cynomolgus monkeys subjected to transport indicators, but lunging and the presence of food aggression were stress have a distinctive metabolic status, including altered energy reported as negative prognostic indicators. The results from this metabolism, amino acid metabolism, lipid metabolism, and perturba- survey are intended to aid institutions in achieving pair-housing tions in gut microbiota. The information obtained is important for success of adult male-male rhesus macaques by consolidating best prevention or intervention of transport stress to improve health and practices from laboratories in multiple countries. welfare. P94 Bridging Nonhuman Primate Husbandry and Research P92 A Simple and Reliable Protocol for Induction of Superovula- Workflows with Task-Specific Information Tools tion in New Zealand White Rabbits AW Legasse*2, GD Lawrence1 X Peng* 1Director’s Office, Oregon National Primate Research Center, Comparative Medicine, Penn State University, Hershey, PA Portland, OR; 2Oregon National Primate Research Center, Portland, OR Genetically modified rabbits, especially transgenic rabbits, have been increasingly used as animal models for a variety of human diseases Hardware and software technologies are developing at a ferocious and as bioreactors for production of pharmaceutical proteins. To pace. Servers procured today are twice as fast as those deployed in date, the majority of transgenic rabbits have been produced by micro- 2010, while the latest software technology allows some applications injection of a DNA construct into the pronucleus of fertilized eggs at to be developed, deployed, and in production in as little as two single cell stage. Therefore, the first step for production of transgenic weeks. While these changes are tremendously promising, dividends rabbits is the capability to obtain enough fertilized eggs via hormone for the quality and efficiency of work remain stubbornly unremark- induced superovulation. Numerous protocols have been reported to able in many biomedical research institutions, where monolithic induce superovulation by using pregnant mare serum gonadotropin record systems constrain access to, interaction with, and benefits (PMSG) or follicular stimulating hormone (FSH) with various doses, from, institutional data resources. Putting a new face on these injection route and time points. However, these protocols have systems, one more tailored to user needs versus developer expedi- shown some disadvantages, such as significant number of non- ence, promises to unlock substantial gains in productivity and job responders, wide variation in number of oocytes produced by satisfaction. We set out to test this by developing a set of task-specific individual rabbits, large number of abnormal eggs unsuitable for computational tools according to what was required by users injection, and requirement of multiple injections (FSH, twice a day performing those tasks. We targeted the intervention at an especially for 3 days). Here we report a simple and reliable protocol for complex workflow spanning laboratory, clinic and husbandry inducing superovulation in New Zealand White (NZW) rabbits. The domains in nonhuman primate (NHP) pathobiology involving 700 rabbits were given a single intramuscular injection of PMSG (120 IU) animals, 40 IACUC protocols, 60 experimental schedules, with more at 1:00PM on day 1, followed by mating and a single intravenous than 100 individuals involved at some point in mission-critical work. injection of human chorionic gonadotropin (hCG, 150 IU) at 1:00PM We asked workers to describe their needs at the individual and task on day 4. Fertilized eggs at single cell stage were harvested at levels, and then used state-of-the-art programming technologies to 9:00AM on day 5 for microinjection. In this study, a total of 358 rapidly build computational tools that did precisely what they had fertilized eggs were collected from the PMSG and hCG injected described. This is in contrast to conventional approaches that train rabbits (n = 11) with 21 to 54 eggs from different rabbits (32.5 + 3.0). the user to apply an institutional system in very particular and Results of this study indicate that a stable induction of superovula- inflexible ways of working, where there is a cost of training, and a tion with a reasonable number of eggs can be achieved by this simple relatively larger software-development expense that comes with protocol. maintenance and upgrade cycles in complex institutional systems. We expected that tools developed in this way, embedding the user’s P93 Factors Contributing to Successfully Pair-Housing Adult Male insight and understanding of their work, would also dramatically Rhesus Macaques (Macaca mulatta) enhance efficiencies, work products and job satisfaction. Over the course of a few weeks we deployed a collection of tools to “scaffold” AA Smith*1,2 the pathobiology workflow, refining them in an evolutionary way based on experience in use. Initial results indicate a savings of almost 1Production and Population Health, Royal Veterinary College, Potters 30% in the cost operations (3.2 FTE), along with reduction in the Bar, United Kingdom; 2Center for Comparative Medicine, Massachu- frequency of irregularities, error and “stress events”. setts General Hospital, Boston, MA

Pair housing nonhuman primates in the laboratory setting is

630 Abstracts of scientific papers 2016 AALAS National Meeting

P95 Naked Mole Rats (Heterocephalus glaber): Short-Term Housing conduct a similar study examining current environment conditions and Husbandry of a Nontraditional Animal Model within rat IVCs.

A Gallagher*, C Sims, DL Coble P97 Novel Wild Squirrel Tree Hideaway to Aid in a Research Setting Unit for Laboratory Animal Resources, The Ohio State University, Columbus, OH AS Glenn*

Naked mole rats (Heterocephalus glaber) present unique housing and Comparative Medicine, University of Maryland, Baltimore, MD husbandry challenges when maintained in the biomedical research setting. Naked mole rats belong to the order Rodentia and family We have designed a novel hideaway for wild-caught Gray Squirrels Bathyergidae which is characterized by small infraorbital foramina (Sciurus carolinensis) to try and replicate a more natural setting while into which the masseter muscle does not enter. Naked mole rats are using them for research. We sought to design a structure that would particularly resistant to neoplasia which makes them useful for replicate a natural tree with a nesting hole. What we designed was a research on aging, metabolic regulation, development, analgesia, and user-friendly structure that could go through the cage washer after behavior. A novel caging system was created using repurposed each use and with slight modification could be used as a cat perch plastic feed bins (24” x 16” x 15”) and a filter paper lid. Each system once the squirrel study had concluded. We designed our structure contained 3 socially compatible male naked mole rats for an using PVC material that was easy to cut and simple to put together. IACUC-approved study. Enrichment toys were provided to replicate The trunk of the tree was made with 4” PVC that was connected to the tunnel systems, toilet, and sleeping areas used by these animals an “H” base also using PVC material. The H base was also construct- in the wild and to provide opportunities for chewing. Initially, aspen ed using 4” PVC with flat end 4” caps for stability. We overlaid the wood shavings were used for bedding, but this was replaced with a trunk of the tree using corrugated black drainage pipe to allow the paper bedding following clinical observations of dermatologic squirrel to climb up the smooth PVC surface and added ¾” pipes to changes on the limbs and tails. Cages were changed every 2 weeks the trunk as branches. This allowed the squirrel to climb up to the top with spot changes occurring twice weekly. Development of per diem of the tree with ease. At the top of the tree we attached a nesting box costs, maintenance of temperature and humidity, determination of to give the squirrel a hide-a-way as was needed for our study. Once floor space allotment, and provision of an optimal diet were released into the secure room the squirrels immediately climbed the addressed for these naked mole rats. Our facility charged a per diem tree and went into the nesting box. The squirrels were allowed to similar to conventionally housed rats to maintain the naked mole come and go into the nesting box as they willed and were seen rats. The temperature of the animal room was maintained at 85 ± 4° F climbing down the tree to roam around the secure testing room. We and the animals’ primary enclosures were misted with reverse suggest that this design is successful as a new tool in keeping osmosis (RO) water twice daily to maintain appropriate microenvi- squirrels stimulated in a more nature environment during testing in a ronment humidity. Sweet potatoes soaked in water were provided as research facility. the primary diet and water source, but animals were also supplemented with additional low calcium fruits and vegetables. In P98 Study Considerations for Vibration-Induced Behavior Changes summary, we describe techniques used to overcome the unique in Mice husbandry and housing challenges when maintaining naked mole rats in a biomedical research facility. A Garner*, RP Reynolds, JN Norton

P96 Environmental Monitoring to Support Extended Cage-Change Division of Laboratory Animal Resources, Duke University Medical Frequency in Individually Ventilated Mouse Cages Center, Durham, NC

AM Swanson*, JW Grimm Minimizing noise and vibration in animal facilities is a concern of critical importance. Previous studies have shown that vibration in Psychology, Western Washington University, Bellingham, WA particular can have a wide range of adverse effects in rodents including impaired immunity, altered neuroendocrine function, We measured temperature, humidity, and ammonia levels inside changes to cardiovascular parameters, and decreased reproductive individually ventilated mouse cages (IVCs) to support extending efficiency. Decreases in reproductive efficiency can heavily impact the cage cleaning to every 14 days using compacted cellulose 1/8” viability of breeding colonies and research results. Despite docu- bedding. Different social housing densities (1, 3, 6, and dam with mented adverse effects, there are no established vibration exposure litter) were studied with C57/B6 mice of both sexes. Routine cage limits for rodents. Therefore our goal has been to determine vibration changes are required in order to limit ammonia levels within the thresholds at which female C57Bl/6 mice show alterations in normal mouse microenvironment, but the frequency of which can negatively behavior. Prior to being exposed to vibration, mice were first affect both animal and human welfare. Cleaning less frequently can acclimated to the testing environment to ensure they were reacting to help improve negative stereotypic behaviors in rodents and limit the vibration and not the novel environment in which they were staff exposure to allergens, ergonomic injury risk, and zoonotic tested. Mice were video recorded immediately after being placed in a diseases. Most cage change intervals have been set based on visual cage on the shaker and at various time points up to 24 hours. cues indicating a dirty cage and the odor of ammonia, which humans Behaviors demonstrated in video recordings from each time point can detect at 0.4 ppm, far below the suggested maximum within the were reviewed, scored, and categorized as active, inactive, or mouse microenvironment. This suggests that it may be possible to maintenance every 10 seconds during a 10-minute period. Within the extend the time between cage changes without exposing animals to first 3 hours mice exhibited significantly more active behaviors when high levels of ammonia. Previous literature supports the ability to compared to both inactive/maintenance behaviors. By 6 and 24 extend mouse cage changes up to and past 14 days while housed in hours inactive/maintenance behaviors were more frequent but the IVCs, dependent on the type of bedding substrate used, and amount difference was not statistically significant. Once an adequate of air changes per hour. For this study, 50 ppm was the maximum acclimation period was established mice were then exposed to level of ammonia allowed. The highest average ammonia level vibration at frequencies between 30-100 Hz and accelerations observed during the study was 5 ppm in the cage housing a dam and between 0.1-2.5m/s2. Mice did not demonstrate a behavioral change litter. It was determined the cage change frequency for all mouse until the acceleration of vibration was 2m/s2 or above. These results cages within the facility could be extended to every 14 days and suggest that a longer acclimation period may be needed for behav- possibly even longer. There was a significant (P < 0.5) effect of ioral testing relative to acclimation times noted in the literature and ammonia level variation by condition and all results were found to that relatively high acceleration of vibration may be necessary to be well under the allowed maximum of 50ppm. The next step is to cause behavior changes in mice.

631 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P99 Let’s Get Certified! Keeping Employees Motivated and Driven P101 Comparison of Individual Ventilated Cage Rack Cleaning Methods: Tools for Exhaust Air Dust Testing AA Gyles*, M Anderson, K Lucas, P Trice, L Keener A Dodelet-Devillers*1,2, J Canale2, CL Perkins3, A Jimenez2, J SoBran Inc, Bethesda, MD Gourdon2, KS Henderson3

The laboratory animal science industry offers a variety of avenues for 1Medecine Veterinaire, Universite de Montreal, Montreal , Cana- career growth and employee development. Management faces the da; 2Comparative Medicine and Animal Resource Centre, McGill, challenge of continuously motivating their employees to achieve Montreal, Canada; 3Charles River Laboratories, Wilmington, MA AALAS certification, which will assist the employee to be equipped with the appropriate qualifications for advancement within the field. Rodents housed in individually ventilated cages have been routinely Many institutions promote and, in many cases, require certification monitored for pathogenic agents by the use of soiled bedding from AALAS to qualify for promotional opportunities. Based on our sentinels. Wanting to take advantage of the recent advances in pass/fail stats we felt the need to assist employees by providing an exhaust air dust testing as an alternative to sentinels unfortunately inhouse AALAS certification training program. Developing an option left us with historical fur mite results that do not reflect the current for making AALAS certification achievable for each employee is a state of our animal facility. In this study we tested 4 different cleaning useful strategy in promoting career growth and advancement. The methods (rack wash only, mechanical wash only, mechanical and first step in creating a program is developing a syllabus that will rack wash, and mechanical with a bleach solution and rack wash) to maximize each student’s capabilities, identify subject experts to best determine the most effective way to remove residue fur mite provide the training, identify any potential adult learning challenges, DNA, bacterial DNA (Pasteurella pneumotropica) and rodent nonspe- and develop a schedule that is feasible with a convenient location. cific DNA. We tested by PCR assay before cleaning and after We’ve found that smaller inhouse AALAS certification preparation cleaning, and determined that all 4 ways were effective in removing courses are more desirable as they create more employee engagement fur mite DNA. A mechanical wash with a bleach solution alongside and interaction. Incorporating various tools such as chapter quizzes, rack wash was most effective to eliminate the dust containing Pasteu- crossword puzzles, and games into the training syllabus greatly rella pneumotropica and rodent DNA. With this knowledge, we are assist with the connection of a variety of learning styles. These tools now able to fully implement an environmental health monitoring maintain employee interest and reiterate information learned that program. may have otherwise been forgotten. One-on-one review/training should be offered to individuals that are perhaps struggling with P102 Comparative Analysis of 2 Health Monitoring Programs in an reading and technical comprehension. Providing this attention IVC Mouse Facility: PCR versus Conventional Screening provides the employees with a sense of comfort, thus creating self-confidence and a drive to succeed. As a result, employees are K Schlarmann2, MC Pils1, H Riedesel3, B Pasche*2 inspired and empowered. They are gaining knowledge, enjoying the training, and achieving AALAS certification. It’s paramount in 1Mouse-Pathology, Helmholtz Centre for Infection Research, understanding that each student is different and training options Braunschweig, Germany; 2Central Animal Facility, Helmholtz Centre should be flexible to achieve maximum success. The inhouse AALAS for Infection Research, Braunschweig, Germany; 3Central Animal training program has increased our staff pass/fail rates by 20.2%. Facility, University Medical Center Goettingen, Göttingen, Germany

P100 Electronic Rodent Colony Management Made Simple Health monitoring programs using sentinels are being criticized for their inability to detect pathogens with low submission rate. Here we A Dollar*, M Rammling report our practical experience with a PCR-based health monitoring in 2 separate barrier facilities, 1 of which is operated as a specified Insourcing Solutions, Charles River Laboratories, Orlando, FL and opportunistic free (SOPF)-breeding unit with about 5,000 cages. The other one with about 1,000 experimental cages is run under In many laboratory animal facilities the task of rodent colony incomplete SPF conditions with Helicobacter, Norovirus, and Pasteu- management is the responsibility of the animal care staff (ACS). rella pneumotropica being tolerated. Both facilities are fully equipped When commercially available electronic systems are not in place, with IVCs. During this study the animals’ health status was manual means to create and exchange colony management informa- monitored quarterly by PCR of pooled environmental swabs taken tion, such as new litter logs, wean reports, and service requests can from exhaust prefilters of all IVC blowers and fecal samples taken cause excessive paper trails and delays. Having an efficient docu- from 26 dirty bedding sentinels, which are kept in pairs of 1 mentation process in place is crucial to ensure successful colony immunodeficient (B6.129S7-Rag1tm1Mom/JHzif) and 1 immunocompe- management. Our program has streamlined the process by creating tent CD1 (Crl:CD1(ICR)) female used for 6 months. These results an electronic New Litter Log form (eNLL) which can easily be were compared to those of the conventional health monitoring using implemented in other animal facilities to enhance the service serology, bacteriology, parasitology, and pathology of 160 CD-1 dirty provided to the principal investigator (PI), research staff, and bedding sentinels which were shipped quarterly to an external veterinarian. The processes of handwritten documentation and diagnostic lab. Whereas in the SOPF breeding unit no mouse manual distribution via mailboxes for new litter logs, wean reports, pathogens could be detected, all the above mentioned tolerated and service requests, was eliminated. Instead, an eNLL form in PDF pathogens were found in the experimental unit with both methods. format was created to facilitate these processes electronically and The presence of ß-hemolytic Streptoccoci, Staphylococcus aureus, directly from the animal holding room via laptops (wi-fi or ethernet). Klebsiella oxytoca, Proteus mirabilis, Helicobacter spp., and Pasteurella This simple PDF form can easily be customized to any site-specific pneumotropica could be identified with environmental swabs but not needs and processed via any email provider or even integrated into a in fecal samples. The PCR-based health monitoring significantly web application such as a content mangement system, which enables reduces the number of sentinels and cuts the overall costs by half. It additional features on workflow management. In order to further is an excellent method to monitor the health status of barrier expedite the distribution of colony management information via the maintained animals and shows an increased sensitivity compared to email application, our program has developed standardized eNLL conventional methods. The reduction of the number of live animals email formats, along with task specific email distribution lists. By necessary for examination is an active contribution to animal welfare developing this system and creating the eNLL form, the rodent and the principle of the 3Rs. 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.

632 Abstracts of scientific papers 2016 AALAS National Meeting

P103 Assessment of Drinking Water Quality in Storage Water Incyte, Wilmington, DE Bottles and Carboys in the Vivarium The industry standard to provide adequate, sterile drinking water to W Gately* severely immune-compromised mice requires multiple steps of water and equipment preparation. This includes ultra-filtering water Comparative Medicine, Pfizer, Andover, MA supplied to the animal facility, water acidification (pH ≤ 3), filling the water bottles, followed by autoclaving the batch to be supplied to the Providing high-quality drinking water is essential to both the welfare animals. This is the current process used for NSG mice housed in our of laboratory animals and the scientific integrity of studies. Provision animal facility. The NSG mice are our most sensitive strain suscep- of water in bottles continues to be the most common method of water tible to disease caused by organisms considered to be commensal in delivery in rodent colonies. We provide high-quality, filtered reverse immune-competent mice, and we wanted to explore a more osmosis (RO) water to our animals in glass water bottles. RO employs simplified and refined process for supplying water to our NSG mice. an ultrafiltration membrane through which water flows under This would be a 1-step process of chlorinating water (5 to 6ppm) in a pressure. Up to 99% of inorganic compounds including toxins, sealed drinking pouch thus replacing the acidification process. We pyrogens, heavy metals, and microorganisms are removed from the first tested the sterility of the unused acidified/autoclaved water and filtered water. The filtered water is collected into bottles, some of unused chlorinated water pouches. Test results proved both waters to which are used same day. The remaining bottles and carboys filled be sterile and appropriate for NSG mice. We next tested used with water are stored for use over the next 7 days. The Guide requires acidified/autoclaved water and used chlorinated pouches to that periodic monitoring of water be done for pH, hardness, and determine if sterility was maintained with normal use Sterility was microbial or chemical contamination, but does not state how long maintained in both samples. Our test results indicate that the refined different types of clean water (RO, tap, etc.) can be stored safely. We process of chlorinated water pouches could provide an appropriate routinely store RO water in glass bottles for up to 7 days, though the water source for NSG mice. 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 P106 Big Efficiency Gains in Census Management with Radio for RO water stored in glass bottles and carboys, we measured pH, Frequency Identification Technology 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 CD Ranns*1, PA Sinnett-Smith3, RL Menner2, BM Stewart2 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 1Comparative Medicine, Pfizer, Inc, Cambridge, MA; 2Business to our water bottle storage practices.The data that was collected is on Technology, Pfizer, Groton, CT; 3Comparative Medicine, Pfizer, par with what we do currently on site now. We are currently Sandwich, United Kingdom revaluating the data and study to determine if it can be duplicated. Census management can be a time-consuming process for operations P104 Reducing Aggression in Mice with Addition of Cage Dividers personnel in large vivarium facilities. We partnered with colleagues in the Business Technology department to find a more efficient BR Tallent*1,2, J Lifshitz1,2, T Parekh1 solution for census management to improve accuracy and compliance, decrease the resources required to manage the census, and fully 1Child Health, University of Arizona College of Medicine, Phoenix, integrate with the existing animal ordering and stock management Phoenix, AZ; 2Translational Neurotrauma Research Program, Barrow system already in place. The first step was to reevaluate and Neurologic Institute at Phoenix Children’s Hospital, Phoenix, AZ reengineer the census business process establishing a standardized process across all sites. Radio Frequency Identification (RFI) Mice fight for hierarchical dominance, resources, territory, breeding, technology was adopted as the primary tool of the process. All cage or aberrant behavior. Reducing aggression has long been the goal of cards now include an embedded chip and antennae which contain many institutions and investigators. The advent of environmental animal and cage identification data. Partnering with a third party enrichment has helped, but is often limited due to cost, housing, or vendor, disposition boxes were created with radio frequency research parameters. The end result of fighting is injury, which identification scanners which read the chip on cage cards deposited requires isolation of animals, thereby increasing costs, with an impact in them after the animals are euthanized at the end of a study. The on research studies. Further, animal models of traumatic brain injury dataset created from the scanner is sent to the animal ordering and (TBI) have led to increase in aggressive tendencies in mice, particu- stock management system where the euthanized animal cages are larly following the stresses associated with cognitive and motor removed from the census hourly. Using the disposition boxes reduces function testing. Wherein behavioral assessment and stress are both recording a disposition from 84 seconds per cage to 1 second per key components of our paradigm, our goal is to develop a method of cage, a significant time savings for operations personnel. Cage counts reducing aggression that does not involve isolation of animals, drugs, in the vivarium are captured using hand-held radio frequency or other behavior modifying techniques that push responses away identification readers which substantially reduce the time spent from “normal.” Additionally, the solution cannot incur undue costs collecting this data compared to hand count or optical bar code or work for laboratory animal staff. Therefore, we conducted scanners previously used by Comparative Medicine. An automated preliminary observational studies of group-housed mice within report then compares the cages scanned with the active cages in the standard disposable mouse cages with the addition of custom built animal ordering and stock management system, highlighting any dividers. Mice cohorts were videotaped and reviewed for quantity variance. Comparative Medicine personnel can review the report, and severity of aggressive behavior, in comparison to cages without assess reasons for the variance, and take direct action to remediate dividers. Marked reductions in aggressive behavior were observed in variances in the report by clicking on the appropriate button. The cages containing dividers. The cage dividers, which provide burrows report automation was able to reduce reconciliation time by 54% and and common access to food/water for mice, are a simple, cost-effec- continues to improve as experience with the tools is gained. tive means to reduce aggression in group-housed mice. Overall, animal welfare is predicted to benefit from compartmentalization P107 Yellow to Green: Exploring the Possibility of Composting within a single cage, without obscuring observation or restricting Soiled Animal Bedding access to food/water. M Stoltz1, C Paulson*1, JD Reuter1, E von Bleichert3, K Ramirez P105 Evaluating the Quality of Chlorinated Water for Use with Aguilar2, M Lapham3 Severely Immune-Compromised Mice 1Office of Animal Resources, University of Colorado—Boulder, BP Long*, R Diamond, KA Adams, D Shuey Boulder, CO; 2CU Green Labs, University of Colorado—Boulder,

633 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Boulder, CO; 3Environmental Health and Safety, University of detecting Mycobacterium, especially M. abscessus, which was only Colorado—Boulder, Boulder, CO detected in the detritus samples. M. fortuitum was detected in all sample types, but M. chelonae was detected only in the sentinel fish, An academic institution has a campus-wide initiative to promote detritus, and biofilm from the sentinel tank. The DNA copy number sustainability through CU Green Labs (CUGL). CUGL uses a team for the Mycobacterium spp. was consistently the highest in the detritus approach to minimize the use of energy, water, and material goods sample from the system tank. This study data supports that a health without compromising research integrity or safety. In January 2015, monitoring program for zebrafish via PCR should include sentinel CUGL, collaborating with Environmental Health & Safety (EH&S) fish, moribund fish, and detritus samples for the most comprehensive and the Office of Animal Resources (OAR) initiated a feasibility study analysis of a given RAS. to assess the practicality of composting soiled animal bedding. It was estimated that vivaria, supporting a modest cage population of 4,000 P109 Transcending Challenges during a Departmental Restructure: rodent cages, generated around 52 tons of soiled animal bedding Fostering Autonomy and Communication through Technician- annually (mainly hard wood shavings). Propitiously, the institution Lead, Cross-Functional Teams already had an arrangement with a composting facility to receive compostables generated on campus. Samples of soiled bedding were CM Curci*, KM McDonald submitted for review to ensure compliance with local, state, and federal regulations. Biologic, chemical, and other hazards were Division of Laboratory Animal Resources, University of Pittsburgh, assessed for compost compatibility by the IACUC and EH&S during Pittsburgh, PA protocol review as these items must be segregated into different waste streams. Next, we established performance metrics to identify Our organization recently restructured, resulting in partially biocompatible collections bags, determine how many bags of soiled hybridized cagewash, animal care, and veterinary technical positions. bedding were generated daily and traced OAR workflow routes, Potential benefits included enhanced integration between units, including compost holding areas. These factors were not found to be increased efficiency, and development opportunities for staff through obstructive of our goals. The animal care staff were trained on diversification of skill sets. However, employees vocalized fear of segregating compostables as it was crucial that bio/chemical hazards changing performance expectations and reporting lines. Six months were sent out via independent waste streams. Plans are underway for post restructure, in our largest site (N = 40), we observed decreased modification of loading dock areas to expand waste holding morale; 21% of staff actively engaged in extra-departmental job capabilities, which was the greatest challenge because of the cost, searches. We also observed a decline in efficiency. In response, we restricted space, and impact to various groups. Bedding ultimately surveyed staff. Positively, 79% reported they intended to stay with will be sent to an anaerobic digester, which will convert the waste the department for greater than 2 years, and 87% felt there was good into useable products such as renewable natural gas, high value team environment. However, staff reported dissatisfaction with time organic amendments, and fertilizer grade products. Establishing a management between units, 51% felt overworked, and 48% felt a program to recycle material animal waste requires a team effort from need for improved communication from management and general- multiple departments, vivarium staff, and institutiuonal support. It is ized ambiguity. To address staff concerns and improve the goal of this program to reduce the environmental impact of understanding of restructure goals, we implemented a 6-week vivaria waste and minimize use of municipal landfills by incorporat- project, using 5 cross-functional teams, comprised of cagewash, ing animal bedding into the current campus compost waste stream. animal care, and veterinary technical staff. Technicians recently delegated leadership positions in the restructure were assigned as P108 Health Monitoring Programs for Zebrafish Danio( rerio) by team captains. Captains lead the groups and facilitated charter goals PCR Testing including attendance, efficient scheduling, volunteering, providing positive feedback to others, and generating ideas for process CL Perkins*1, C Lawrence2, KS Henderson1 improvement. Incentive for participation was based on a point system, and teams were provided weekly standing updates. The 1Charles River, Wilmington, MA; 2Boston’s Children’s Hospital, project improved assignment volunteering from 59% before Boston, MA implementation to 95% post implementation. Staff was more efficient despite increased volunteering. There was 1 resignation during the Zebrafish (Danio rerio) are used as models for research in genetics, project, but otherwise we observed no increase in overtime. Staff was disease, toxicology, and behavior studies. To maintain research more integrated, as 60% independently reported an appreciation for integrity and reproducibility, health monitoring programs should be another unit’s responsibilities. Lastly, staff was more autonomous implemented for zebrafish colonies so that they are consistent with and communicative, leading to improvements such as sanitization standard practices for other laboratory animal models. In our study, a scheduling between cagewash and animal care. The project success- simulated health monitoring program using environmental samples fully facilitated job comprehension and autonomy, but final surveys and fish from a tank rack at an academic institution was evaluated revealed a need for additional efforts to improve bilateral communi- using qPCR assays for the detection of 17 pathogenic bacteria, cation between staff and management. parasites, and viruses. Each fluorogenic real-time PCR assay was designed to be specific for the target agent based on sequences P110 Comparison of Vaporized Hydrogen Peroxide and Parafor- available in GenBank or sequencing performed in our laboratory. maldehyde Gas Decontamination on High-Efficiency Particulate Internal controls for nucleic acid isolation efficiency and PCR Air Filter Banks inhibition were included in the protocol. Baseline PCR testing was performed on a subset of the sentinel fish population, prior to CM Brown*1, J Gibbs Erwin1, S Crossfield2 introduction into the system, and all results were negative. The remaining fish were added to standard sentinel tanks. After 6 months 1Animal Resources, University of Alabama at Birmingham, Birming- exposure to the effluent water in a recirculating aquaculture system ham, AL; 2Occupational Health and Safety, University of Alabama at (RAS), qPCR testing was performed on pooled sentinel fish, as well Birmingham, Birmingham, AL as pooled moribund fish, detritus, and biofilm sample types from the same RAS. Detritus and biofilm samples were submitted from both Decontamination of high-efficiency particulate air (HEPA) filter the sentinel tank and the pooled system tanks for separate evalua- banks is required for high-containment facilities. Filter bank tion. The parasite P. neurophilia was detected in the moribund and decontamination may be performed prior to HEPA filter change in sentinel fish only, and was not detected in any environmental response to a spill of infectious agent outside primary containment or sample. The DNA copy number detected for P. neurophilia was other reasons based upon a risk assessment. When decontaminating approximately one log10 higher in the moribund fish than the with paraformaldehyde gas or vaporized hydrogen peroxide (VHP), sentinel fish. The environmental samples were preferential for a complete kill of the organisms on the biologic indicators must be

634 Abstracts of scientific papers 2016 AALAS National Meeting

documented 3 consecutive times to be accepted as the approved compared to other domestic pigs. This gene enables them to survive method of decontamination. Biologic indicators were placed in 3 when food is scarce, but also predisposes them to developing locations inside the filter bank: the supply port, downstream side of metabolic syndrome. This adaptation makes them a useful animal HEPA filters, and downstream side of HEPA filter at the exhaust port. model for human metabolic syndrome and its associated disease Fourteen decontamination cycles were run; 7 using vaporized processes. In a research project with Ossabaw swine, pigs were fed a hydrogen peroxide and 7 using paraformaldehyde gas. Six of the 7 high-fat diet to induce metabolic syndrome over an extended VHP runs were successful as evidenced by the biologic indicators, duration (~17 months). Multiple husbandry and veterinary chal- which showed no growth for 7 days. The failure was attributed to a lenges were encountered during the study period. The clinical faulty biologic indicator, where the positive control did not grow. All problems associated with the study-induced obesity which required 7 of the paraformaldehyde runs failed. After the initial failure the additional management included: skin fold dermatitis, loose stool, concentration of paraformaldehyde was increased and a blower was laminitis, and possible neurologic effects. Maintenance of the pigs for introduced to the system to circulate the gas through the filter bank an extended duration was also problematic as the growth of their for different periods of time. All of the biologic indicators grew canine teeth or “tusks” mimic that of their feral cousins. Both sexes within 48 hours. Consistently demonstrating 100% kill of the biologic are born with deciduous canine teeth and permanent tusks erupt at indicators is required for HEPA filter change. Of the 2 methods about 7 to 13 months of age. To prevent injury to both humans and evaluated, vaporized hydrogen peroxide was shown to be the most animals alike, a routine teeth-trimming program was implemented to consistent and efficient method of decontamination. Paraformalde- manage the length of the tusks. In summary, long term maintenance hyde gas failed to achieve decontamination of the HEPA filters, thus of Ossabaw swine on a high fat diet results in multiple husbandry making it unacceptable for use in our facility. related and clinical challenges; however, appropriate management of this valuable animal model can be achieved with diligent care. P111 Cage Card Wars P113 Successful Housing of Pigmy Rattlesnakes (Sistrurus CL Yates*1, S Lunt2,1, E Jackson1 miliarius) in a Laboratory Setting

1Laboratory Animal Resource Center, Southern Methodist University, DM LeMoine*, CL Freed, ML Holding Dallas, TX; 2Office of Research and Graduate Studies, Southern Methodist University, Dallas, TX The Ohio State University, Columbus, OH

On a daily basis, animal care technicians and other vivarium users Laboratory housing of any species must take into account the navigate a multitude of animal holding room and cage-specific physiologic and behavioral requirements of that species, personnel requirements. These requirements can range from biosafety-level safety, and research needs. Housing of venomous snakes in particular designation, nonstandard diets, and specialized husbandry to requires consideration of specific environmental requirements, environmental enrichment for study-specific cages. Additionally, proper handling, safety precautions, and emergency plans. Our breeding projects can require supplemental cage-level tracking and institution has maintained a group of 13 pigmy rattlesnakes documentation. It is critical that visual identification and labeling (Sistrurus miliarius) for venom composition research, requiring the systems are developed to effectively communicate these require- establishment of an IACUC-approved satellite housing space. Prior ments. Through the implementation of these systems inconsistencies to housing venomous snakes, drains and heating units were covered are reduced, communication is improved, and efficiencies in animal with screens and a floor sweep was attached to the bottom of the care and disease model management in any animal care and research door to prevent snakes’ egress from the laboratory should they program can be gained. Our facility has developed a systematic cage escape their primary enclosures. A relationship with the local card program to eliminate overuse of cage cards and provide emergency room was established, including the development of an organization and conformity. This system consists of a series of 1’’ x emergency SOP. Following these preparations, wild-caught gravid 1’’ adhesive, colored labels, various colored 4’’ x 5’’ and 3’’x 5’’ females were obtained and adults and their offspring have been transparent cards, and card stock of 2.5’’ x 5’’ standard facility card, successfully maintained for 8 years. Snakes are individually housed 3’’ x 5’’ breeder cage card, and 3’’ x 5’’ sick and dead animal notices. on newspaper with pine bark bedding which was spot changed as Each rodent housing cage is furnished with either a standard facility needed. Standard plastic reptile cages with opaque walls and a card or a breeder cage card. Additionally, every rodent housing cage padlocked sliding transparent front were sanitized monthly. The is supplied with a 4’’ x 5’’ clear transparent overlay. The transparent room temperature is maintained at 70-80°F year-round with a 12:12h overlay serves a place holder to affix the labels. The facility issued light cycle and full-spectrum lights. Bedding is misted as needed to cage cards serve as a helpful reference source for obtaining informa- maintain optimum humidity at the cage level. Fresh water is tion about an individual cage. The colored overlays assist with provided ad libitum and, as part of the study design, snakes are fed providing more of a comprehensive understanding of what should frozen-thawed mice, lizards, or frogs sourced from a collaborator be focused on during the daily health monitoring process. Lastly, the once weekly. Hides or exam glove boxes are provided for additional labels affix to the transparent cards to provide direction for specified shelter. For personnel safety, snakes are transferred into a secure instructions. All components of the cage card notification system that plastic holding container during routine husbandry. Snake hooks, is in place at our institution work in a collaborative effort to aid in tongs, and transparent restraint tubes are used for restraint and seamless communication of special instructions, treatment regimens, handling is restricted to trained individuals with a minimum of 2 and post surgical and breeder cage requirements at the cage level. people present during any procedure. Only 1 enclosure unlocked at a time. Multiple levels of security restrict access to the housing space to P112 Husbandry and Clinical Management of a Unique Species: approved personnel only. There are specific risks associated with The Ossabaw Swine housing venomous species, however this pigmy rattlesnake colony has been maintained with minimal clinical concerns, no observable C Sims* behavioral abnormalities, and no injuries to personnel.

The Ohio State University, Columbus, OH P114 Disposable Water Delivery Systems Role in Contingency Planning The domestic pig is one of the most valuable animal models used in biomedical research today because of its biologic, physiologic, and DK Joseph*, J McNair, JM O’Donnoghue, ED Allen, LJ DeTolla anatomic similarities to human beings. The Ossabaw pig breed is biologically unique having been shaped by natural selection in an Comparative Medicine Program, Department of Pathology, Univer- isolated island environment. They possess a “thrifty” gene which sity of Maryland School of Medicine, Baltimore, MD enables individuals to store significantly higher amounts of body fat

635 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Contingency and disaster planning in accord with the USDA APHIS in static cages. Although only a few differences in blood chemistries directive and Office of Laboratory Animal Welfare is an essential and counts were observed, these differences may be significant component of an animal care and use program that should account depending on the research goals and should be considered before for all aspects of animal health and wellbeing. In the face of any moving animals into a new caging type. These findings further disaster, 1 of many contingencies is provisions for uncompromised suggest that the fecal microbiome and breeding performance are and uncontaminated water. Water is delivered to research facilities minimally impacted by caging type or whether mice are moved into through a municipal supply or well reliant on utilities like power to a different type of cage. render water potable and to convey water to the site where it is consumed. Furthermore, research facilities equipped with water P116 Single Technician Setup and Husbandry Of Germ-Free Mice treatment and purification systems also rely on site utilities to further Housed in a Positive-Pressure Caging System refine and treat water and to bring water to the point of use. Laboratory animals maintained on automated watering systems DM Urbonas*, J Alderman and/or water bottles and the associated equipment used to process water delivery systems are affected by utilities outages. Despite Immunobiology, Yale University School of Medicine, West Haven, CT possible provisions for emergency power to water delivery and processing systems, there are many times limitations on the duration Conventional flexible film and semi-rigid isolators require a great emergency power may be sustained. For example, fuel-driven deal research space. Labor costs and lead time to ready an isolator for emergency generators may be operational for a limited duration in occupancy are significant. Positive-pressure caging offers a revolu- the absence of fuel or unless the fuel supply is re-established or tionary alternative, as the set up and husbandry of the cages can be replenished. Our institution’s contingency plan provides for the use can be performed by a single technician. Reprocessing of the cages of disposable water delivery systems that provide a viable solution can be done in a single day, significantly reducing the lead time for water delivery under these conditions. Automated watering necessary for study setup. Cage components are washed, double- systems and bottle filling equipment can be easily adapted to feed wrapped in a surgical pack style, autoclaved using the facilities water from an elevated container relying simply on the transfer of established gnotobiotic cycle, and assembled under a biosafety water from the disposable water delivery system and gravity. cabinet that has been pretreated with a concentrated solution of Producing and maintaining a sizable inventory of water is a practical chlorine dioxide. Rodent chow is processed in the wire bar assembly contingency approach and one that can be used to deliver water to all of the cage bottom. Packs containing empty water bottles and sipper species regardless of the native water delivery system used. tubes are also delivered into the BSC. Flasks of water and bagged HEPA filters are submerged in the sterilant before entering the BSC. P115 Moving Mice from Static Caging to IVC: Effects on Microbi- One sampling cage, including forceps and sample collection tubes, is ome, Breeding Performance, and Blood Chemistry included with every cage change event to allow for cage sampling. With all cage components under the BSC, a sealed container of DR Goulding*1, PH Myers1, CA McGee1, S Hackney1, J Locklear2, SD germ-free mice is submerged in sterilant and placed into a running Peddada3, DM Kurtz2, TL Blankenship1 biosafety cabinet. The technician then dons sterile surgical attire and begins to assemble the cages including installation of a HEPA filter in 1Veterinary Medicine Section, Comparative Medicine Branch, the cage lid. Prior to securing the lid assembly, mice are transferred National Institute of Environmental Health Sciences, NIH, DHHS, into the cage bottoms. The cages are visually inspected for proper Research Triangle Park, NC; 2Quality Assurance Laboratory, closure, removed from the BSC, and docked on the rack. For cage Comparative Medicine Branch, National Institute of Environmental changing, soiled cages are removed from the rack, submerged in Health Sciences, NIH, DHHS, Research Triangle Park, NC; 3Biostatis- sterilant, and entered into the BSC where clean equipment had tics and Computational Biology Branch, National Institute of already been introduced. The technician dons sterile surgical attire Environmental Health Sciences, NIH, DHHS, Research Triangle Park, and uses the packs to perform the cage change and sampling. New NC cage bottoms containing bedding, food, and enrichment are changed out weekly. Water bottles are changed every 2 weeks. Cage lids with Our facility has migrated from static microisolation cages to a new HEPA filter are changed every 4 weeks. Soiled cages are individually ventilated cages (IVC) and it was of interest to deter- broken down and processed through a tunnel washer. During this mine if changes in physiologic parameters might occur. We evaluated evaluation, cages were sampled and changed weekly. PCR analysis of differences in mice housed in the two types of caging as well as the fecal pellets indicated our ability to maintain the germ free differences in mice born in one type of caging and moved to the environment after 7 cage changes. An alternative to conventional other. Fecal microbiome, breeding performance, blood chemistries, flexible film and semi-rigid isolators this system offers a time saving, and complete blood counts (CBCs) were the parameters selected for staff conserving, cost effective alternative for conducting both short evaluation. Twelve breeding pairs of 8-week old C57BL/6 mice each and long term gnotobiotic studies. were set up in static cages and in IVC. Offspring were weaned into their respective caging types. Two weeks following weaning half of P117 Collaboration and Innovation: Developing a Device to Help the litters born into static were moved into IVC and half born into Track Mice after Blood Sampling/Dosing IVC were moved into static. Breeding pairs remained together until 3 litters were produced. Litter to litter interval and litter size at MF Lazar, D Forteza* weaning were documented. At 6-weeks of age 8 males and 8 females from each group were selected from the first litters and euthanized LAR, Merck, Kenilworth, NJ and blood collected for serum chemistries and CBCs. Feces were collected from all cages for microbiome composition 3 weeks after Dosing and/or bleeding multiple rodents occupying a single cage migration from one cage type to the other. No significant differences can prove challenging, especially if the animals are not individually in litter size at weaning or interval were observed between or among identified. Investigative staff may resort to shifting mice to an empty the groups. A significant increase in Lactobacillus spp. in feces of mice cage. If it is a clean cage this could result in extra labor and materials housed in the IVCs compared to the static cages was observed; and be impractical if the animals are dosed daily. If transferred to a however, the relative abundance in both groups was considered too cage or container previously occupied by other mice on study, this low to be biologically relevant. Male mice housed in ventilated could increase the risk of potential cross-contamination, may be caging had slightly elevated alkaline phosphatase. Hematocrits were stressful to the animals, and could therefore negatively impact study significantly higher in both sexes that had their caging type switched results. It is for these reasons that a multifunctional team consisting from static to IVC compared to mice that remained in static cages. of veterinarians, animal care management, investigative staff, and Significantly fewer monocytes in both sexes were observed in those bioelectronics engineers was put together to develop a solution to that were moved from static to IVC compared to mice that remained this problem. Based on group discussion and feedback from

636 Abstracts of scientific papers 2016 AALAS National Meeting

investigator needs, the team designed a device that would be used to needleless catheter was evaluated by several staff where it was divide the home cage into 2 separate spaces. Investigators could start appreciated that the catheter did not need to be disassembled prior to their dosing/bleeding procedure with all mice on one side of the use and performed in the same manner as the traditional catheter. No divider and then move them to the other side after the procedure, all negative impact was seen in the nasal wash return volumes. The within the home cage. Design parameters included lightweight and needleless catheter is a custom order item so special attention must sanitizable material, as well as a way to keep it in place on the cage. be paid when preparing for a study which uses nasal washing. While Bioelectronics engineers recommended building usable prototypes there is a minimal cost increase for this special unit, the added via a 3D printer to be evaluated by the team and tested by a select benefits to using a needleless catheter over the traditional catheter group of investigators. After a series of design modifications and cancel out the added expenditure. The needleless catheter stays in the retesting by investigators, the final design consisted of a divider sterile package for an increased length of time leading up to the made of stainless steel that would hang from the cage top via rails procedure. Staff labor is more efficient by negating the need for that fit on the cage lip. The collaborative team effort resulted in a disassembly and preparation of the catheter for the procedure. Less novel device that met the objectives and was readily accepted by the waste is deposited into a sharps container. And most importantly, an investigative staff. unnecessary health hazard to staff has been eliminated since no sharps are used in the procedure. P118 Pinworm Syphacia sp. Decontamination through Efficient Team-Based Action Plan P120 Husbandry of Wild-Caught Arctic Ground Squirrels (Sper- mophilus parryii) P Kathy, MF Lazar, D Forteza* DM LeMay*1, J Farley1, BS McGinnis1, CP Jones1, EN Yu1, ML Riess2 Laboratory Animal Resources, Merck, Kenilworth, NJ 1Division of Animal Care, Vanderbilt University, Nashville, TN; 2An- Historically our facility has been rodent pinworm Syphacia sp. free. esthesiology and Pharmacology, Vanderbilt University Medical All incoming rodent vendor health reports are reviewed prior to Center, Nashville, TN receiving animals into the facility. Unfortunately, due to a break at a vendor facility, we received 21 suspect positive rodent shipments Arctic ground squirrels (Spermophilus parryii) possess unique over a 3-month period. A task force was immediately assigned for hibernation physiology that lends itself to biomedical research, but the development of an action plan to address the potential impact of are not available from a commercial vendor for study in the pinworm eradication from our facility. The action plan was laboratory setting. Our institution was presented with the challenge developed by representatives from the affected departmental areas of developing a husbandry plan for this unique wild-caught species coming together to assess the problem, develop solutions, and in our research facility. We based our husbandry plan on contribu- communicate to all key stakeholders the steps to be taken on tions from the researchers, animal care staff, leadership, and executing the plan. The action plan allowed us to identify the affected collaborative information from other institutions that have housed areas by assessing initial housing of suspect positive animal orders, these animals in the past. A modified rabbit rack with water bottles animal transfers, and analysis of staff traffic flow patterns. At least 6 and automatic watering were utilized to individually house the of the orders were found to be positive, impacting 7 rooms over 2 animals. A high-fat rodent diet was chosen as the main nutrition floors. Rodent tape test, fecal samples, and environmental swabs source. Enrichment provided included crinkle paper bedding, a hide were taken to confirm the presence of pinworms. Decontamination tube, toys, and fresh produce and treats on a daily basis. Pan liners procedures were established for caging, scientific equipment, and the were changed twice per week, and racks were sanitized every 2 overall facility. The action plan was presented to the research weeks. To simulate the natural decline in daylight hours wild Arctic community to demonstrate an approach that would have minimal ground squirrels experience as summer ends, the light:dark cycle was impact on their research. The implementation of the action plan was initially 12:12 hours, and then each day the lights were turned off 8 successful in eliminating pinworms form the facility. Since pinworm minutes earlier than the day before. While at our research facility, the eggs can remain viable for long periods in the environment, the Arctic ground squirrels gained weight, acclimated to housing and rodent colony continues to be monitored through tape and fecal test husbandry procedures, and utilized environmental enrichment to ensure eradication. At the 20-month time-point the facility remains devices provided. We were able to successfully house this wild- pinworm free. caught species by modifying our current equipment and husbandry processes. P119 A Custom-Devised Catheter System and Platform Collecting Apparatus Refines Nasal Wash Procedures in Ferrets P121 Managing and Monitoring the IVC Microenvironment: Sensor Placement Differences and Suggested Best Practices DA Pfefferle*, J Hess, J Lucas J McNair*, DK Joseph, JM O’Donnoghue, ED Allen, LJ DeTolla Vivo, Battelle Memorial Institute, Columbus, OH Comparative Medicine Program, Department of Pathology, Univer- Nasal washes are routinely conducted to detect viral load shedding, sity of Maryland School of Medicine, Baltimore, MD antibody titers, or other analysis. Historically, a standard intravenous catheter is used to conduct the nasal washing, requiring that the The use of building automation systems (BAS) to manage heating, sharp stylet be removed by hand prior to conducting the procedure. ventilation, air conditioning, electrical, lighting, safety, and other This posed an unneeded sharps hazard, created excess sharps waste, systems affords institutions the ability to effectively and centrally and required increased labor time in prepping the catheter. Increased monitor and manage while also affording control of energy costs handling of the catheter also creates a greater potential for decreased without sacrifice to human comfort and to control the laboratory sterility of the catheter prior to the procedure. A needleless catheter animal macroenvironment. BAS monitoring and managing labora- was sought to increase safety and sterility and to decrease waste and tory animal room systems are usually based on strategic and labor. A commercially available option was not found so facility staff engineered placement of individual sensors as a single point of worked with an outside vendor to develop a needleless catheter. reference. However, managing these systems at this level does not Additional refinements to the nasal wash procedure included necessarily reflect the microenvironmental conditions within the designing a custom elevated and easily sanitizable platform and animals’ primary enclosures, particularly temperature and humidity. sample cup holder to be used during the nasal wash procedure to aid In 2014, Veterinary Resources rolled out a standalone wireless with sample collection. The ergonomic design of this set up helps the environmental monitoring solution to monitor, report, and alert technician position the ferret so sample collection can be obtained husbandry management personnel of real-time temperature, comfortably and naturally for both handler and ferret. The use of the humidity, and lighting values and critical deviations. Placement of

637 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 temperature sensors at strategic locations within the secondary (70-74 °F). The need of replenishing feed and water between cage enclosure intended to be representative of intracage temperatures changes may cause interference of intracage microenvironment and resulted in disparate results compared to BAS values. Further introduce unnecessary stress to animals. Husbandry management evaluation of temperature sensor placement within occupied and hypothesized that housing NU(NCr)-Foxn1nu mice at their unoccupied intraventilated rodent housing systems (IVC) as well as thermoneutral zone of 84-86°F will not only decrease their feed and IVC exhaust plenums revealed temperatures higher than reported at water intake, but also reduce the frequency of replenishing feed and the room level and different than those values reported by the BAS. water between cage change. NU(NCr)- Foxn1nu mice were acquired Studies were also carried out to assess the comparative temperature from an inhouse breeding colony. Using sterile caging on an IVC measurements between occupied IVC cages and their associated system, the mice were housed in autoclaved cages, complete with exhaust plenums. Results revealed temperature values were highly bedding, diet, and nesting material and provided with correlated and indicates sole reliance on BAS and secondary hyperchlorinated water pouches. The study was carried out over the enclosure values may not represent the best solution for monitoring course of a 4-week period with cages changed every 14 days. One set and managing microenvironmental temperature and humidity. The of mice were housed at ambient room temperatures (72 ±2°F) and study also indicates placement of sensors in IVC exhaust plenums is another set were housed at the lower threshold of their equally representative of occupied intracage microenvironmental thermoneutral zone (82 ±2°F). Food and water consumption was values and may be the appropriate basis for managing and monitor- measured every other day. Athymic mice housed under ambient ing these conditions. 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 P122 Labor-Cost Analysis associated with Disposable Water thermoneutral conditions consumed an average of 10 gr./mouse/ Delivery Systems: A Matured Assessment day and 8 ml. water/mouse/day, comparatively reducing both food and water consumption. NU(NCr)-Foxn1nu mice housed at ED Allen*, DK Joseph, J McNair, JM O’Donnoghue, LJ DeTolla thermoneutral temperatures eliminated the need to replenish food and water and eliminated interference of intracage Comparative Medicine Program, Department of Pathology, Univer- microenvironment by normalizing their feed and water intake. A sity of Maryland School of Medicine, Baltimore, MD little warmth can go a long way.

A medical school introduced a disposable water delivery system for P124 Suitable Identification Method for Transgenic Hemophilia laboratory mice in 2004. Extensive time and motion studies were Type A Mice carried out to define labor costs specifically associated with the existing water delivery technologies at the time (both automated J Forget1, L Bernier1, EE Arlund*2 watering systems and water bottles) and to establish a comparative cost basis. The same principles of labor-cost analysis were applied to 1Charles River, Senneville, Canada; 2Somark Innovations, German- establish true labor costs associated with the new delivery system. town, NY This analysis revealed the fully loaded labor cost, inclusive of inhouse production, transportation to the use locations, and waste Reliable identification of laboratory animals is critical to safety disposable yielded a labor cost of $0.005/cage/day. Since 2004, there assessment study conduct to generate quality data. Various identifi- have been substantive process changes and program enhancements cation methods are available, including ear tags, manual tattoos, and that warranted revisiting a labor-cost analysis. Local and remote radio frequency identification devices (RFID); however, most of these water storage strategies were changed to address the assurance of a are not suitable for transgenic Hemophilia Type A (HA) mice as this continuous and available water supply. The dynamics associated model has less than 1% of normal factor VIII activity and exhibits with early cage retirement prior to the IACUC-approved 14 day cage prolonged clotting times. Minor lesions induced by ear tags, RFIDs, change interval, changes in housing densities, and expansion of or manual tattoos have resulted in excessive blood loss leading to genetically altered mouse lines were reviewed. An increase in the death within few hours. An automated tail tattoo system was number of breeding cages generated a need for additional cages. evaluated for its suitability. The system provides a controlled depth Husbandry staff cage intervention to mitigate unsatisfactory of needle penetration and delivery of the ink to the mid dermal layer micro-environmental deviations and a host of other unaccounted for of the skin. Injury to blood vessels is avoided thereby alleviating risk factors were identified. The results of this cost analysis revealed of hemorrhage in these hemophilic mice. This method was evaluated inconsequential increased labor costs translating to a revised true on 210 transgenic HA mice that were 6 to 7 weeks of age at time of cost/cage/day. The focus of this publication is to show all labor costs identification. Animals were observed for up to 7 weeks during associated with a mature program using a disposable water delivery which time there were no clinical observations associated with the technology, how those costs were captured, and inclusion of all labor identification procedure, confirming absence of substantial damage costs associated with the use of this system. The potential for other to blood vessels at the set depth of penetration. Legibility of the peer institutions to adopt this solution is evident in the queries we tattoo was maintained throughout this 7-week period. The automat- have received. This analysis and use metrics ultimately may benefit ed characters of the LabStamp system also eliminated risks of those institutions, gaining insight into true labor costs of a relatively ambiguity in characters which may exist with manual tattoo young disposable water delivery solution. inscriptions. This method was therefore considered to provide an appropriate identification method for use with transgenic HA mice. P123 A Little Warmth is Good for Nude Mice P125 Microbial Contamination of Purified Rodent Diets SC Nance*, JM O’Donnoghue, ED Allen, T Coksaygan, LJ DeTolla F Adsit*, J Locklear, TE Whiteside, T Ward, DM Kurtz Comparative Medicine Program, Department of Pathology, Univer- sity of Maryland School of Medicine, Baltimore, MD Quality Assurance Laboratory, NIEHS, Research Triangle Park, NC

In an ever evolving industry of managing animal research facilities The term “purified diet” can be misleading to the consumer, in one of the greatest challenges faced is minimizing disruption of mice particular, to laboratory investigators lacking a working knowledge between cage-changing cycles to prevent the introduction of of the laboratory animal food industry. Some may assume by the variables on research outcomes. Currently, most intraventilated term purified that these diets are free of extraneous chemical and caging (IVC) systems allow for a 14-day cage changing cycle at which microbial contamination. However, a purified diet is defined by the feed and water last for 14 days in a cage of 5 adult mice. However, American Institute of Nutrition as diets that are formulated with a athymic nude mice (NU(NCr)-Foxn1nu) consume more food and more refined and restricted set of ingredients. Such ingredients water due to higher metabolic demand under ambient temperature frequently include isolated proteins (casein), refined carbohydrates

638 Abstracts of scientific papers 2016 AALAS National Meeting

(sucrose and corn starch), and oils (soybean oil). While this definition m2 (740 mm by 790 mm) that provided the expansion of floor area. assures chemical purity, it does not address microbial purity. These Furthermore, we additionally installed new functions for user diets are often not amenable to being autoclaved due to the labile convenience and animal enrichment as follows. The height of perches nature of their ingredients, and some investigators avoid the use of was lowered 100 mm from the floor considering the species-typical ionizing radiation out of concern for chemical changes that may behavior and sitting height. The sitting height is approximately 50 cm result from that process (e.g., lipid peroxidation). Thus, it is incum- in 8kg NHPs. Also, width and number of perches were modified bent upon the investigator to understand what microbial agents may from 130 mm (2 bars) to 56 mm (3 bars) for preventing from tangling be present in unsterilized purified diets that may be undesirable for tether lines. A newly small door in the front side facilitated conve- consumption by their specific animal model. As an example, nient approaching and the blood collection for researchers. investigators respiratory biology may wish to avoid the introduction Removable holder for heat lamp or fluid in the side frame increased of species such as Klebsiella pneumoniae to their animal colonies. We efficiency at the space arrangement additionally. Trimmed edges in examined a number of commonly used purified diets by enrichment the floor panel facilitated water cleaning and sanitation for eliminat- in Rappaport-Vassiliadis broth to obtain microbial isolates after ing excrement. Visual barriers which were printed with trees and streaking for isolation on Brilliant Green agar. The type of microbial forest for optical stimulation affected social housing and naturalistic agents most often detected were then identified via sequencing and environmental enrichments. Above changes were based on the represented at least 5 different families with Enterobacteriaceae being compatibility between old and new design frame because compatibil- most commonly encountered, including documented opportunistic ity of components are convenient in inventory management and pathogens. minimized additional cost in cage maintenance aspects. Although improvement in NHP cages was primarily due to revised guideline P126 Factors Affecting the Vocational Calling of Laboratory Animal associated with floor area, it also enhances animal wellbeing, Care and Research Employees research convenience, and cost-efficient management.

GP Boivin*1, RJ Markert2 P128 Practical Experiences with PCR-Based Health Monitoring Compared to Classic Methods 1Laboratory Animal Resources, Wright State University, Dayton, OH; 2Internal Medicine, Wright State University, Dayton, OH H Riedesel*, A Brockmann, V Reupke, J Sichelstiel, G Hindermann

A survey was conducted to determine the factors affecting the Central Animal Facility, University Medical Center Goettingen, vocational calling of laboratory animal care and research workers. Goettingen, Germany The survey was comprised of 56 questions in 4 groups: passion, job stability/happiness, work volition, and demographics. We hypoth- A reliable health monitoring program (HMP) is essential for the esized that individuals who worked in the field a longer time, were health maintenance of rodent colonies in lab animal facilities. In the older, had higher education levels, were involved with AALAS, and past 2 decades this has mainly been achieved by screening dirty in higher positions in their organization were more likely to indicate bedding sentinels for rodent pathogens by classic serological, a calling to the laboratory animal care field. We also hypothesized microbiological, and parasitological methods. The validity of dirty that job satisfaction and classifying one’s job as a calling were bedding sentinels, especially in rodents housed in individually positively related to organizational support and work volition. ventilated caging (IVC), is being critically questioned in some recent Forty-four percent of respondents categorized their work as at least studies, which clearly show that the transmission rate for some partially a calling. Those working at a higher level in the position of important pathogens is poor. Furthermore, such a sentinel program laboratory animal technician and in the organization were more requires a large number of animals and is therefore space consuming, likely to view their work as a calling. Increasing education level was costly, and a potential animal welfare issue. During the past few related to work being a calling. Overall, vocational calling was associ- years PCR-based health monitoring methods (PCRHM) have been ated with higher pay, but technicians were the only subgroup where developed which open up new possibilities. Last year it was decided calling and higher pay were significantly related. Vocational calling to change our dirty bedding sentinel HMP for a PCR-based, and job satisfaction were significantly associated with organizational sentinel-free program in our rodent facility consisting of SPF-barriers support. For our sample of workers in the animal care field other and several conventional units with an average daily census of about factors analyzed were not related to work being considered a calling. 30.000 rodents. Mice were mainly kept in IVC and rats in open Leaders in the field of animal care may find our survey results caging. The housing units differ regarding the health status: valuable as they strive to adapt their organization’s structure to the conventional units are positive for Norovirus, Helicobacter spp., Pas- perceptions of their workforce with regard to their sense of calling. teurella pneumotropica and gut protozoa, while another one unit harbors MHV. All these pathogens which formerly have been P127 The Improvement, Consideration, and Suitability of Nonhu- detected by classic methods, are now easily and regularly identified man Primates Caging by PCRHM in pooled environmental swabs (dust from the exhaust ventilation pipes or exhaust prefilters of the IVC blowers) and in H Jung*1, K Roh1, S Park1, E Auh1, J Yun1, H Yu2, B Kang1,3 pooled animal swabs (fecal pellet, oral, anal, and fur swabs). This new HMP results in a reduced work load, significantly lower costs, 1Department of Experimental Animal Research, Biomedical Research and saving of more than 600 sentinel animals per year which allow Institute, Seoul National University Hospital, Seoul, Korea (the more than 150 free cages to be made available for scientific use. In Republic of); 2Department of Ophthalmology, Seoul National conclusion, PCRHM presents an efficient, fast, and reliable means for University Hospital, Seoul, Korea (the Republic of); 3Graduate School routine health monitoring in rodent facilities. It is a contribution to of Translational Medicine, Seoul National University College of the 3Rs in terms of replacement of all sentinel mice, a methodical Medicine, Seoul, Korea (the Republic of) refinement, and significantly reduces the costs of a HMP. Further- more, a kind of standardization of health monitoring can be achieved Our institution provides infrastructure and support for a large if a specific profile is being used which will facilitate animal exchange number of biomedical research projects using nonhuman primates between different institutions. (NHP) as models for human disease. We introduced NHP cages according to the Guide in 2009. In 2010, an updated edition was P129 Technical Support for the Maintenance of a Guinea Pig (Cavia published and standard of floor area was changed from 0.54 m2 to porcellus) Myopia Model Colony 0.56 m2. In addition, some problems and necessity of improvement were found in old cages in convenient aspect. At first, the new cages JA Stewart*, AD Pucker, JM Hickman-Davis 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 The Ohio State University, Columbus, OH

639 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Proper management of a guinea pig (Cavia porcellus) colony to P131 Decontamintion Validation of Duct Work Associate with produce and sustain pups for myopia induction experiments is vital Biosafety Level 3 Labs for success. Some concerns with producing the model included the need for pigmented eyes, source of breeders, number of breeder JW Sweeley* pairs, average litter size, level of noise exposure, design and maintenance of myopia induction hoods, additional technician University of Georgia, Athens, GA training, and type of bedding used. Pigmented eyes and a susceptibility to myopia are essential for the model. Guinea pigs were Decontamination is vital to the operation of Biosafety Level 3 (BSL-3) obtained from 3 separate sources (government, private institution, facilities. Multiple proven decontamination methods are available, and commercial breeding facility) however only 2 of the sources each having their own pros and cons. Validating that these decon- showed a susceptibility to myopia. Animals were housed in taminations are successful is easy with the use of biologic indicators commercially available caging with aspen bedding and automatic (BIs) placed throughout the decontamination space. However, some water systems. Enrichment consisted of music, a hut, and a toy with spaces such as the duct work from the room to the associated high the addition of a novel item every week. Nutritional enrichment was efficiency particulate air (HEPA) filters can pose a problem when provided daily in the form of fruits or vegetables according to a attempting validation. Validation for this space is needed if any work varied schedule. Approximately 3 females per source were set up for or repairs are need for the duct work. Our instiution has developed a breeding with a consistent average litter size of 3.6 ± 1 for all strains. novel way to achieve this validation. During the preparation of the Pup mortality was < 1% for the private institution and commercial space for decontamination BIs are placed within the duct work using source animals; however, mortality for the government strain was a flat drain auger. First, the length of the duct work from the room to 38%. The high mortality rate was attributed to music played in the the HEPA bank is determined. Second, BIs are placed at a distance of room, as once it was discontinued, the mortality rate decreased every 10 feet on the flat drain auger. The flat drain auger is then dramatically. To generate myopia, a soft, cloth hood was designed to placed into the duct work until it reaches the HEPA bank. The room hold a lens over one eye. Hoods were applied to pups only at 24-72 and duct work are then decontaminated using vaporized hydrogen hours after birth. Pups were acclimated to hoods for 1 day prior to peroxide (VHP). This is accomplished by inputting the VHP directly lens placement. Hoods did not interfere with normal movements and into the room through ports in the mechanical area and exhausted did not appear to disturb pups or sows. Low dust crinkle paper was through a decontamination port at the terminal end of the duct work, used for bedding following hood application to prevent foreign right before the HEPA bank. After the decontamination is complete particles from being trapped under the lens. Lenses were cleaned the BIs are retrieved and incubated to determine the successfulness of once every 24 hours for the 8-day study timeline. In addition to basic the decontamination. This method has been used several times with husbandry, technicians were given special training to give technical great success. Having validation that the duct work has been support for hood management. Technical support above husbandry successfully decontaminated allows work and repairs to be done in needs for this colony amounted to 2-3 hours per week including between studies. weekends and holidays. The successful identification of susceptibility to myopia in two of the guinea pig strains is the direct result of P132 Single Chickens Need Enrichment careful collaboration between the researchers and animal care team. JA Blair* P130 A Novel Design for Minipig Protective Jackets Office of Animal Resources, University of Missouri—Columbia, J Lovaglio*1, K Thrall2, K Tyrrell3 Fulton, MO

1Rocky Mountain Laboratories, Hamilton, MT; 2SNBL USA, Everett, Maintaining the physiologic needs of our chickens, even though they WA; 3Pacific Northwest National Laboratory, Richland, WA 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 Miniature pigs are a common large animal model used for a wide and enrichment is vital to maintaining the health of these birds. variety of research projects ranging from toxicology to surgical Identifying the materials to be used was determined by choosing models to wound healing. Many of these projects require the use of sanitizable and safe items, which could easily be purchased at a local garments or other means to protect critical study components, such store, and food items chosen were already approved for chicken as drug application sites, surgical sites, and/or bandages from consumption in our facility. The goal was to encourage more foraging animals that are known to rub on cage bars, cagemates, and any behavior with positive interaction, which were criteria considered a other items in their environment. A number of techniques have been success. Three variations were made with the materials chosen and used over the years for such studies, and many with great success. purchased. Two were used with food, while the third was visual. The When presented with a project that necessitated the bandaging of first 2 were plastic bottles with holes cut in it, which food material minipigs and protection of those bandages for several months, we could be pecked out of the inside. The third had glass marbles in a embarked on developing a method that would allow us to complete plastic bottle of water. During observations, the food had the most the project with the least impact on the minipigs. A multitude of interaction and had the most success. The chickens seemed to look bandage fixation and protection methods were tried, however, all forward to the treats after a few times of being offered. The nonfood failed for 1 reason or another. Thus, we set out to design a new set-up was a nice change to a hanging mirror and will continue to be protective jacket. After careful review of the positive and negative used within the enrichment rotation. The chickens were observed attributes of available techniques, several prototypes were developed interacting with it, but did not have the level of anticipation as the and tested until a satisfactory design was discovered. The new jacket food/forage enrichment did. Overall, the added enrichment for our is simple in design and construction, is machine washable, accommo- chickens was a success based on the interaction by the chickens and dates some variation in body conformation, incorporates regular and materials were easy to aquire. stretch fabric and Fabric hook-and-loop fasteners closures, and is worn with a collar. The novel design allowed us to exquisitely protect P133 The “Baa”sics of Sheep Flocking the bandages of the animals on study (up to 75 days) with very little impact on the everyday activities of the minipigs. Additionally, JL Volkmann*, C Evans, CA Buckmaster animals quickly acclimated to wearing the collars and jackets. We believe this novel design may be useful for other research projects Center for Comparative Medicine, Baylor College of Medicine, faced with similar challenges. Houston, TX

When sheep are pair-housed in standard stalls grazing and ruminating behaviors are limited, relative to what is seen typically in natural

640 Abstracts of scientific papers 2016 AALAS National Meeting

settings when sheep are in groups of 5 or more. We converted a large c-section rederivation and adoption by GF foster mothers, with a rodent room into a flock room used to socially house all of our sheep high rate of success. All isolators follow a health monitoring program in an effort to stimulate more normal flock behaviors in our animals. that consists of microbiology testing in house once a month and a Constructions costs for this project were minimal and included FELASA annual panel every 6 months. Major concerns are to avoid mounting hay bale bags, swine scratchers, and feed and water contaminations and technical problems, such as isolator breakdown. containers on the wall to maximize square footage for roaming and To prepare for such situations, strains are always housed in 2 grazing. A livestock gate was installed a few feet from the entrance to isolators. In addition, 1 isolator is always kept as a backup for speed provide a small ante space for caregivers to observe animals before expansion of the affected colonies. The gnoto/axenic facility has entering their living space. Over the past 13 months, we have highly specialized and dedicated personnel. It is integrated in the observed an increase in walking while grazing and ruminating animal house facility, which allows resource optimization such as behavior for our six sheep that are currently housed in the room. equipment (sterilizers, washing machines), basic supplies (diet, Sheep usually ruminate while resting on their sternum with one or bedding, and beverage), human resources, and general facility both front feet tucked under their bodies. When pair-housed our workflow (staff and animals circulation). 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 P136 Using a Novel Disposition Box to Improve Daily Accuracy of and housing 6 sheep together we are able to improve their wellbeing A Vivarium Census Database and make them better research models. JA Maher*1, JP Rodriguez2, SA Mischler1 P134 Got Milk? A Guide to Effective Milk Extractions in Mice 1Comparative Medicine, Pfizer, Inc, Pearl River, NY; 2Charles River JL LeGrand*1,2, S Himmelfarb1, T Pilant2,1, Y Belkaid1 Laboratories, Wilmington, MA

1National Institutes of Health—LPD , Bethesda, MD; 2SoBran Inc., Animal research facilities need accurate and timely information about Bethesda, MD their animal population. This information is obtained via censuses, where a physical count is made first of the cages/animals in the Passive immunity is the short-term immunity that results from the facility, and then the results of the count are used to update a introduction of antibodies and/or immune cells from one person or previously existing census database. The database is accurate on the animal to another. In nature, this occurs from mother to neonate day of the census, but in subsequent days it may differ from the real during breastfeeding and across the placenta during fetal develop- population by as much as 10 to 15%. As cages/animals are eutha- ment. However, it is not well understood what types of cells are nized, these changes are not updated in the database due mainly to involved in this process. In order to analyze the cellular contents of constrains on the researchers’ time. We sought to solve this problem breastmilk and its role in passive immunity, we have developed a by automating the database update process at euthanasia time by technique that allows us to obtain a high yield of breastmilk from a using a “disposition box,” which is a plastic box with an RFID scanner mouse with minimal discomfort for the animal. Here, we show that at the top and a slot through which the cage cards were fed. As the using oxytocin and by taking advantage of the natural progressive animals were euthanized, the cage card was fed through the box slot, milk development cycle we can optimize milk output collection. the cage/animal identification was read by the RFID scanner, and the Furthermore, we also found that using a 20uL pipette with tip gave information was passed to a communications circuit at the bottom of us the perfect amount of suction with minimal cell disruption during the box. A program then passed the animal/cage card information to this collection method. Now that we have developed this technique, the database, which marked that animal/cage as euthanized. It was we can collect measurable volumes of milk to be studied and gain theorized that these real-time updates would reduce the differentials further insight into how passive immunity is passed in the breastmilk between the census database and the real vivarium population, to offspring and the physiologic consequence of this transfer to short improving the accuracy of the database. We now have data for 9 term and long-term immunity. 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, P135 Implementation of a Gnoto/Axenic Facility: A Case Report when no disposition box was in use. Median PD values (number of cages/animals dispositioned at census time as a percent of the total J Bom*1, K Berman2, A Ribeiro1, M Pereira1, AS Leocádio1, M Rebelo1, number of cages in the Vivarium) were 9.1% for 2014, 7.7% for 2015, J Demengeot1 and 2.7% for 2016. Data to date indicates that using disposition boxes significantly improved the accuracy of the census database. 1Animal House Facility, Instituto Gulbenkian de Ciência, Lisboa, Portugal; 2Baylor College of Medicine, Houston, TX P137 Intracage Ammonia Levels in Cotton Rats Cages: Effects of Cage Density, Age, and Bedding Sterilization Our germ-free (GF)/axenic facility started in 2005 and features rigid walls isolators for axenization and maintenance of mutant and JA Maher*1, JP Rodriguez2, J Phillips1, SA Mischler1 wild-type mouse strains of different genetic backgrounds. The facility is open to the international community through the EMMA/ 1Comparative Medicine, Pfizer, Inc, Pearl River, NY; 2Charles River Infrafrontier consortium. In 2013, a gnotobiology facility was Laboratories, Wilmington, MA implemented for experiments with GF animals. Each cage is a microisolation, allowing multiple studies on the same rack, providing Cotton rats are useful models for the study of respiratory diseases, full bioexclusion for maximum animal protection, excluding but they are high strung and difficult to handle during routine cage-to-cage contamination. SOPs and GF sentinels were implement- procedures such as cage changes. Reducing the frequency of cage ed to validate our working system. These animals are manipulated change (initially 2 times per week) could reduce the stress on the exactly the same way as experimental animals, and their health status animals and the handlers. No intracage environmental quality is checked monthly for quality control. Experimental animals are information, however, was readily available for cotton rats to help microbiologically tested in the beginning of the experiments and the make this decision. We conducted a series of studies to obtain such respective control animals at the end. The use of this system for information using intra cage NH3 concentration as the criteria of gnotobiology experiments allowed free space in the isolators and environmental quality. We studied the effects of the number of cotton expanded the number of GF animals produced. Currently, the gnoto/ rats per cage (2 or 3) and/or their age (7, 14, 18, or 22 weeks at the axenic facility is running with 7 isolators, 2 systems, and is breeding start of the test). Ammonia levels were measured daily for 7 days 4 different axenic strains. The facility has a capacity of 400 deliverable with a Drager x-am 7000 meter equipped with an NH3 sensor animals and at least 2 EMMA/Infrafrontier research projects calibrated to detect levels between 0 and 200 ppm. The measure- concluded per year. Axenization of new strains is performed by ments were done by introducing the sampling probe through the

641 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 automatic watering system port of the cage in test 1, and through the noted. Results showed more pronounced pinna vessels after heat or cage’s lid water port in the other tests. NH3 production rate was ace versus no treatment, more rapid 2mL arterial blood draw after lower (P < 0.001) in cages with 2 animals than in those with 3 rats heat versus ace or no heat, and much more rapid arterial versus ((<20 ppm versus ~60 ppm at test’s end). Older animals had higher venous blood draw. Catheter persistence varied with dressing used, initial ammonia levels (P < 0.01) but the rate of NH3 production with with dressing 1 > dressing 2 > dressing 3 at 6 hours and dressing 2 > time was similar for all ages. No adverse histopathological findings dressing 1 > dressing 3 at 24 hours. Most catheters drew blood at 6 were observed in the nasal tissues of the rats. Study 3 sought to hours, but few at 24 hours due to clots in catheter stem or hub. determine if using irradiated bedding would slow down the Dressing characteristics varied. Dressing 1 was transparent, flexible, production of ammonia in the cage but no differences were found difficult to affix, and edges rolled. Dressing 2 was translucent, due to the use of sterile bedding. The results of our studies indicated flexible, easy to affix, and remained adhered. Dressing 3 was opaque, that if cotton rats were housed 2 per cage, the cages could be changed less flexible, easy to affix, but edges detached. In summary, heat once each week without adversely affecting the animals. produced the optimal combination of visible pinna vessels and rapid arterial blood draw. Dressing 1 and dressing 2 were optimal for P138 Slip Lead Program for Positive Reinforcement Training visibility and adherence, respectively. Our comparative data on treatments, vessel types, and dressings provide information intended J McMahon*, SS Rapa, S Rubino, B Deschenes, R Flores, K Noorbehesht to help optimize blood sampling in research rabbits.

Pfizer, San Diego, CA P140 Reducing Social Housing Complications in Adult Female New Zealand White Rabbits (Oryctolagus cuniculus) through The benefits of physical activity are evident in most pet dogs and Application of Male Rabbit Urine many enjoy going for a walk. Walking offers the dog an opportunity to exercise, reduce stress, explore, gain mental stimulation, and it KM McDonald*, K Hoffmann promotes a sense of bonding with the handler. With this in mind, we proposed to broaden our canine environmental enrichment program Division of Laboratory Animal Resources, University of Pittsburgh, and mimic the benefits of dog walking by introducing slip lead Pittsburgh, PA training within the boundaries of our research environment. A slip lead was selected over a leash and collar to eliminate the need for In accordance with the Guide, social animals should be housed with collars on dogs. A shaping plan was developed, with the final compatible conspecifics. However, species, sex, and age greatly behavior requiring the dogs to accept lead placement, walk with influence socialization success and injury risk. Illustrating these minimal tension to anteroom, get on a scale and wait, then get off the challenges, when socially housing adult female rabbits at our scale when released and walk back into kennel with minimal tension. institution, we observed a discouraging 12% separation rate, due to The project was piloted with 1 group of 6 dogs and 1 group of 10 fight wounds, over a 6-month period. Subjectively, we noticed greater dogs. One-hour sessions were scheduled over 2 weeks so each dog success with female groups when they were housed proximal to a received approximately 6 to 10 minutes of training daily. Of the 2 male. We hypothesized that like some rodent species, male urine and scenarios, we found it most beneficial to work with the dogs in its associated sex hormones, may affect female rabbit behavior. To groups of 6 providing them with 10, 10-minute sessions. The cage evaluate the effects of male urine, we assessed research naive, 2-year cards of dogs that completed training were marked with orange dots old, female, New Zealand White rabbits (N = 36). The animals were to differentiate trained and untrained animals. The shaping plan singly housed at the vendor, and upon arrival at our facility, were implemented was effective for training most of the dogs in the pilot. socially housed in pens (25 sq. ft.), in groups of 4. No intervention Slip lead training has improved ergonomics for our canine techni- was given to the control room (n = 16), and in the treatment room, cians by decreasing the need for lifting and carrying dogs. It has rabbits (n = 20) were marked with 0.5 ml of male rabbit urine, provided a stimulating environment for the canines and increased 1:1 immediately before socialization. To evaluate the frequency of time with the handler. Several apprehensive dogs were also noted to dominant and affiliative behaviors, we video recorded the control be more confident following training. The results of this pilot indicate and treatment rooms for one hour during initial housing, and at one that slip lead training is a valuable addition to an environmental 1 post arrival. We also measured fecal cortisol levels that correspond- enrichment program for canines. An added benefit is improved ed to stress before shipment, and 2, 7, and 10 days post arrival. Our ergonomics for the comparative medicine staff and the animal users. results indicate no significant difference in cortisol levels between the groups over time. However, the urine marked group engaged in P139 Optimization of Blood Sampling from the Rabbit Pinna 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 KA Hough*1, IM Washington1,2 (F(1, 19) = 14.420, P = .001). Contrarily, the control group exhibited significantly more dominant behaviors at intake, such as mounting 1Magee-Womens Research Institute, Pittsburgh, PA; 2SoBran (F(1, 35) = 5.577, P = 0.027), and incurred significantly more injuries

BioScience, Pittsburgh, PA 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 Rabbits are used for blood sampling in toxicology, pharmacokinetic, animals, 37.5% of the control were removed from groups due to and antibody production studies due to ease of handling, ample fighting, suggesting that male rabbit urine application may improve blood volume, and accessible pinna blood vessels. This IACUC- social housing success with sexually mature females. approved work provides a comparative study of methods used for blood sampling from pinna vessels to help researchers optimize P141 The Effects of Socialization and Playtime on Antibody blood sampling in rabbits. Adult (~3kg; N = 24) female New Zealand Production in Long-Term, Single-Housed New Zealand White White rabbits (Oryctolagus cuniculus) were treated subcutaneously Rabbits (SC) with ketamine, xylazine, and glycopyrrolate for a surgical implant study. The right pinna was treated topically with lidocaine/ K Wearsch*, P Lincoln, J Villano prilocaine and photographed. Rabbits were treated for 10m with 0.5mg/kg acepromazine (ace) SC, heat (~90°F), or no treatment, and Unit for Laboratory Animal Medicine, University of Michigan, the pinna rephotographed. After topical alcohol and povidone- Westland, MI iodine, a 24G catheter was placed in central auricular artery or lateral marginal vein. Dressing (Tegaderm®, Hypafix®, or Moleskin®) was Rabbits are used in biomedical research as an animal model for affixed over catheter. Time to withdraw 2mL blood was recorded and various physiologic and immunologic processes. Until its 2011 catheter flushed with 1mL heparinized saline. At 6 hours and 24 edition, the Guide did not address the behavioral and psychologic hours, catheter persistence, blood draw, and dressing adherence were implications of single housing in rabbits. The most recent Guide indi-

642 Abstracts of scientific papers 2016 AALAS National Meeting

cates that single housing of social species may only be justified based BSC filters can provide a steady breeding ground for these pests. The on experimental requirements or veterinary related concerns. Thus, ATS is where our psocid infestation occurred. To help with the psocid animal care and use programs are faced with the challenge of remediation, we lowered our humidity set point from 50% to 40% meeting this standard vis-à-vis minimizing unwanted effects on within the room. To resolve our psocid infestation, we implemented a research studies. Many investigators have had singly housed rabbits weekly disinfection process of the internal pieces of the ATS or BSC prior to the establishment of the new guideline, and older rabbits that which includes a thorough vacuum detail and sanitizing with Accel have not been exposed to socialization may not interact well with TB. The psocid lifecycle is usually completed within 1 month and other rabbits thus posing a dilemma. In this retrospective study, 5 adults can survive up to 6 months. Once the eggs are laid they will adult female New Zealand White rabbits used for antibody produc- begin to hatch in 1-2 weeks and development from egg to adult takes tion that had been housed alone for 4 years since young were approximately two weeks. The weekly sanitization breaks the psocid introduced to one another for pairing attempts and supervised lifecycle by exterminating the female psocids before they can lay playtimes. The process rendered no successful pairs for long-term eggs, which are resistant to traditional means of sanitization. By housing but allowed weekly group playtimes in a large open pen maintaining this process, we have not had further infestations. with dividers. Supervised socialization attempts and playtimes that Psocoptera are manageable with proper sanitization practices. presumably caused stress in these rabbits did not adversely alter antibody production, with titer values consistently staying at or P144 Assessment and Modification of a Mechanical Restraint above 105. To conclude, if rabbits cannot be socially housed, then Device for the Prevention of Genital Manipulation in Singly open pen playtimes can serve as an alternative source of enrichment Housed Male Cynomolgus Macaques (Macaca fascicularis) without affecting antibody production. Socialization and playtimes can then be adopted into the animal care program for single-housed KR Lambert*1, A Dean2, SS Lankford2, JK Williams2, E Mitchell1 rabbits. 1Animal Resources Program, Wake Forest School of Medicine, P142 Validation and Refinement of Sanitization Practices in a Winston-Salem, NC; 2Wake Forest Institute for Regenerative Facility Housing Ferrets Medicine, Winston-Salem, NC

K Noorbehesht*, JM David In the laboratory environment, jackets have been utilized for approximately 4 decades for the protection of surgical sites, Comparative Medicine, Pfizer Inc., San Diego, CA 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 Sanitizing animal housing and equipment is essential to securing the amount of available information on its use, function, design, health and wellbeing of laboratory animals. The sanitization process assessment, and modification is lacking when compared to the use of for ferret racks at our institution includes an initial 10-minute soak a jacket alone. In order to successfully create a primate animal model with hot water, followed by 5 minutes of scrubbing, and finally, a for the regeneration of the lower urinary tract, it was necessary that 45-minute rack washer cycle using 180F water, an acid descaler, and male cynomolgus macaques (Macaca fascicularis) wear a jacket with an alkaline detergent. We evaluated the procedure to determine if skirt postoperatively. The use of the jacket with a skirt was to prevent any steps in the process could be modified to improve efficiency, or access of the surgical site and inadvertent damage or early removal conversely, if steps needed to be added to achieve satisfactory levels of the urinary catheter. Each animal was fitted with a commercially of cleanliness. The sanitization process was assessed using an ATP available nylon mesh jacket. A plastic skirt was attached to the luminometer, a device that has been successfully used at other outside of the jacket to prevent self trauma or manipulation of the facilities, and Replicate Organism Detection And Counting (CON- genital area, urinary catheter, and/or surgical sites. Each animal was TACT PLATES) plates, the industry standard for sanitization allowed a period of at least 5 days acclimation with the jacket/skirt monitoring. We were able to significantly reduce our rack washer combo prior to surgery. During this acclimation period, each animal cycle from 45 to 35 minutes and eliminate usage of the acid descaler was closely monitored to ensure the device posed minimal stress and while still demonstrating Colony Forming Unit (CFU) and Relative impact on daily behaviors. The acclimation period allowed us to Light Unit (RLU) values consistent with levels considered sanitary by identify and address challenges the animals may face postsurgically. industry standards. Additionally, by cross validating RLU readouts Some of those challenges include the prevention of dermatitis or with CFU counts from the CONTACT PLATES plates, we are now excoriations, varying sizes of individuals requiring jacket/skirt able to evaluate a variety of our sanitization processes quickly and application, the ability to assess surgical sites, and administering objectively using the luminometer. Our improved rack sanitization injectable medications once in the animal was placed in the jacket process may provide a useful baseline process for facilities currently and skirt. With slight modifications based on individual challenges, housing, or planning on housing, ferrets in the future. we believe the jacket and skirt provides an effective means of mechanical restraint postsurgically that still allows the animal to P143 Routine Sanitization Scheduling Inhibits Psocid Population have adequate use of his environment and the ability to engage in within Biologic Safety Cabinets and Animal Transfer Stations most species-typical behaviors.

KA Bozek*1, CC Sem1, MC Debrue2 P145 Husbandry of Laboratory-Housed Egyptian Fruit Bats (Rousettus aegypticus) 1Comparative Medicine, Pfizer, San Diego, CA; 2Comparative Medicine, Pfizer, Cambridge, MA KM Pincolini*1, CE Ferrecchia1, H Chum1, C Roberts1,2

Biologic safety cabinets (BSCs) and animal transfer stations (ATSs) 1Office of Laboratory Animal Care, University of California, Berkeley, are an essential part of research facilities striving to maintain a Berkeley, CA; 2University of California, San Francisco, San Francisco, Biosafety Level 2 (BSL-2) environment. With the recurring receipt of CA animals and other miscellaneous products packaged in paper-based containment, the opportunity for nuisance such as Psocoptera to We house a variety of unique species, each requiring specific infiltrate is frequent. Psocoptera is an order of insects commonly husbandry, enrichment, and safety protocols. While housing of bats known as booklice that primarily feed on starch based household in zoological parks is quite common, but new to us, we developed items like grains, wallpaper glue and book bindings. Psocoptera standards specifically for housing a wild-caught breeding colony of thrive in conditions higher than 50% humidity, and desiccate in bats in an indoor laboratory facility. The Egyptian fruit bats (Rouset- warm and dry conditions. As per the Guide for the Care and Use of tus aegypticus) that instigated these protocols are used in research Laboratory Animals, animal facilities should maintain a humidity level studies investigating the neural basis of complex spatial behavior, the between 30-70%. When room humidity is higher than 50%, ATS or neural basis of social behavior, and vocal communication and

643 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 learning. Developing daily husbandry practices that allowed for observed to have buphthalmia, an enlargement of the eyeball, but effective sanitation but minimized the potential impact on breeding had no record of this being reported despite daily health observa- and research were essential. During the initial quarantine period, the tions. This resulted in a recommendation to better improve our unknown health status of the colony necessitated the implementation program for daily animal observations. In response, HERSLAW was of added personal protective equipment as well as rabies vaccination created. HERSLAW (head, eyes, respiration, skin, limbs, appetite, and safety training for all personnel. Furthermore, transitioning a waste) is an acronym used to describe an animal health assessment group of bats from the wild to a laboratory setting involved method for technicians to use while making daily animal observa- providing environmental enrichment that promoted species-typical tions. This acronym guides the animal care technician to ask certain behaviors, such as foraging, in order to maintain a lean, freely flying, questions when looking at particular areas of the body. Is the head breeding colony. Examples of current enrichment devices include tilted? Are the eyes fully open and clear? Is the breathing normal? metal parrot skewers for fruit, nonhuman primate Prima-Hedrons, What is the appearance of the animal output and is it a normal and faux leaf fencing. Thus far, the fruit bat colony has thrived under amount? In recent years more focus to capture behavior as a these conditions, with a very promising breeding season in progress. significant indicator of health has prompted modification of As the field of bat research continues to grow, the lessons learned HERSLAW to HERBS LAW (head, ears/eyes, respiration, behavior, through development of husbandry, enrichment, and safety protocols skin, limbs, appetite, waste). In addition to the incorporation of ears, presented here will prove valuable for other institutions housing bats we also added behavior. Behavior can be a significant indicator of in a laboratory setting. potential abnormal health issue that is not visually apparent. For example, repetitive rubbing of the same spot on the arm could be P146 Evolution of a Nonhuman Primate Enrichment Program indicative of a laceration they are trying to rub away. Some things to ask are: Are they spinning or eating feces? Are they biting them- KI Graika*1, D Toomey1, T Rodriguez1, NA Monts de Oca2, M selves? These are examples of abnormal behaviors that should not be Cabrera1, CR Lockworth1 accepted as personality but reported for further assessment. Since the implementation of the HERSLAW method, we have not received any 1Department of Veterinary Medicine and Surgery, MD Anderson further recommendations for improvement regarding our daily Cancer Center, Houston, TX; 2Charles River Laboratory, Houston, TX animal health observations. In addition, this method has helped to refine our process and improve animal welfare by ensuring that all We continuously strive to improve animal welfare and wellbeing and animals across the vivarium are being examined in a uniform are committed to producing high-quality research, which can only be consistent manner. achieved through physiologically and psychologically sound research subjects. For many years, we maintained a nonhuman P148 Down and Dirty with Rotifers: Daily Care to Support a primate (NHP) enrichment program that met minimum standards. Zebrafish Colony Common provisions for occupational enrichment, produce, and documentation were followed. Nevertheless, after examining the KM Burritt*, S Lowry, CA Johnson program, it became clear that although we were meeting basic requirements, there were numerous opportunities for development University Animal Care, University of Arizona, Tucson, AZ and improvement within the program. With effort and innovative ideas gathered from within our team and from colleagues at nearby One of the main concerns when it comes to caring for any animal institutions, we were able to dramatically transform our program colony is providing healthy and nutritious food. The majority of into one that more closely meets the behavioral needs of the NHP feeds are standardized diets that are manufactured in a manner that colony under our care. The support of our innovative program was ensures the highest quality available. These diets are an efficient and only possible through the addition of infrastructure necessary to cost-effective way to feed most colonies. This is not the case when sustain its additional requirements. To ensure success of this new and beginning a larval zebrafish colony. It has been shown that providing complex primate enrichment program, we implemented new live feed rotifers between the ages of day 5 to 30 results in bigger, processes and resources in order to ensure its functioning without healthier zebrafish, and more fish survive to adulthood. While increasing personnel. We were able to do this by investing in providing rotifer diets has shown these positive outcomes, it also significant upgrades and additions to our enrichment preparation comes with more time spent caring for the live feed. We have facility to meet our requirements of space, ergonomics, and versatil- developed a streamlined process to ensure a healthy, disease-free ity. Additionally, we developed an efficient logging system, coded source of rotifers are always available. Here we show the innovative calendars to quickly and efficiently manage tasks, and visual husbandry care for the rotifer colony. Daily husbandry care activities standard operating procedures for enrichment preparations and include a 30% water exchange in each tank and 2 daily feedings, as personnel training support. This foundation permitted the augmen- well as maintaining a marine mix water supply at an appropriate tation and enhancement of the entire behavioral management salinity and pH. A constant supply of 15 ppt marine mix water is program at our facility. We were able to increase options and provide maintained by testing the pH with a test strip and using a refractom- novelty, standardization, and structure by implementing a rotating eter each time a new batch of water is mixed up to ensure the correct system for the management of the occupational, nutritional, and salinity. Weekly husbandry care includes samples sent to pathology, sensory enrichment devices. Moreover, we expanded our social where the total number of rotifers are counted, as well as the number enrichment, positive reinforcement training, and behavioral of active rotifers and the number of rotifers bearing eggs. Other tasks monitoring programs. Together, all of these changes resulted in a such as changing rotifer floss and watching for low-performing air more complex, yet efficient program, and most importantly, stones are also crucial to keeping a rotifer colony excelling. The floss improved animal wellbeing for our NHPs. placed in the tanks catches unwanted contaminants and extra, uneaten food. Once an air stone becomes clogged and begins to P147 HERBS LAW: An Animal Health Assessment Method 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 KM Vera* 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 Alcon, Fort Worth, TX weeks, a new rotifer colony is started, ensuring there is always a healthy growth in our rotifer culture. While performing daily animal observations, animal care technicians look for any abnormality to assess animal health and wellbeing. P149 Extended Use of Nylon Rodent Chews Resulted in Cost Assessing the same animal every day can make the abnormal seem Savings with No Detrimental Effects in Sprague–Dawley Rats normal, and undesired behaviors may be accepted as personality traits. Some years ago during a facility inspection, an animal was L Danner*, R Varada

644 Abstracts of scientific papers 2016 AALAS National Meeting

Comparative Medicine, Pfizer Inc, Andover, MA peroxide (VHP) and chlorine dioxide were compared for use as cold sterilants. While both VHP and chlorine dioxide effectively sterilized Environmental enrichment for laboratory animals has beneficial the isolators, we determined that, due to isolator material incompat- effects on their welfare and health. The data generated from ibility with VHP, chlorine dioxide was the more appropriate sterilant environmentally enriched animals has been shown to be less variable for flexible film isolators. While carrying out sterilization protocols, and of higher quality. Rodents rely in large part on their olfactory occupational exposure levels of VHP and aerosolized chlorine cues to navigate in their natural environment. Enrichment devices dioxide were found to be well below permissible exposure limits for such as nylon chews and wood blocks have been shown to reduce both chemicals. Twenty-four hours following chlorine dioxide anxiety and stress in rodents. In this study we evaluated whether the fogging of the isolators, a mold trap consisting of autoclaved food, use of a commercially available nylon rodent chew can be prolonged caging material, and sterile water was placed in the isolators for without affecting the health of the rats. Sprague–Dawley Rats (5-6 sterility testing. Sterility of the isolators was verified by culturing week old) were pair-housed in cages with a loose animal bedding swabs of the isolators to confirm that sterility had been achieved. made of alpha cellulose and ad libitum diet with portion-controlled Flexible film isolators present operational challenges that semi-rigid nesting material. We provided the chew (one/cage) to 3 groups each and rigid isolators do not, but do offer advantages in terms of cost containing 8 cages (n = 16 rats) and assessed the chews at 4-, 8- and and ease of adaptability. Generating reliable, quality data from 12-week intervals for their bacterial load, debris accumulation, and gnotobiotic mouse colonies is only possible when sterility is properly percent usage. We also examined rats for their CBC/chemistry at the maintained. Matching the sterilization technique to the isolator and above intervals and measured their body weights weekly. The material type is crucial to success, and methods presented here help bacterial load as well as debris accumulation on the chews peaked at to standardize and optimize workflows in these isolators. Research 4 weeks and dropped to lowest levels by 8 weeks. The percentage use was conducted in compliance with all Federal requirements. The of the chews as measured by their weight peaked at 12 weeks. In views expressed are those of the authors and do not constitute addition, we saw no significant changes in RBC, WBC, liver enzymes, endorsement by the U.S. Army. and body weights. Therefore, we suggest that chews can be safely used for extended periods up to 8 weeks as enrichment devices for P152 The Establishment of Specific Pathogen-Free Ducks in China rats. L Han*1, J Han2, L Qu1, H Chen1 P150 Did Your Brooder Fail You? Get the Pail and Have Happy Quail! 1Harbin Veterinary Research Institute, Harbin, China; 2CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources, LN Ward*, T Hanna, M Quinn, A Jackovitz Institute of Animal Science, CAAS, Beijing, China

Army Public Health Center, Bel Air, MD Ducks are one of major domesticated poultry species for meat, egg, and feather production. Thus duck diseases are of great concern to Complications from housing Japanese quail (Coturnix japonica) in duck production industry, especially after the birds have been galvanized steel brooders led study staff to brainstorm and develop confirmed to be a combine host for highly pathogenic avian influenza an alternative housing option. Disadvantages to the galvanized steel virus which can spread from nonpathogenic ducks to humans. brooders included poor visibility of the chicks, and since the flooring Furthermore, several duck and human vaccines are produced and was constructed of a wire mesh material, there was potential for qualified by using the regular duck eggs which may carry pathogens. chick/personnel injury. “Quail pails” were constructed from Therefore, specific pathogen-free (SPF) ducks are urgently required 54-gallon tote boxes, transforming the plastic storage solution into a both for avian disease research and biologic products. In 2005, free-range chick set-up. Modifications included drilling holes in the hatching eggs of great parent generation of Shaoxing White Shell I totes to accommodate a carboy for delivery of drinking water and ducks were introduced for developing SPF ducks in an isolated replacing the solid plastic lid with mesh for increased air circulation. environment. The ducklings and adult ducks were kept in duck Advantages to these pails included easy observation of the chicks special isolators with HEPA all through their lives and fed with and soft bedding was used as alternative to wire mesh to prevent radio-sterilized feeds and acidified drinking water. Sera were injury to chicks/personnel. Since their development, quail pails have regularly detected at a 3-month interval and serum antibody-positive housed nearly 500 chicks. No animal welfare or husbandry issues individuals were weeded out. After have being bred and purified for occurred during their use. successive 8 generations, the duck population is proved to be free from 10 major pathogens including duck plaque virus, duck hepatitis P151 Sterilization Methods for Flexible Film Isolators Used for virus (DHV), avian influenza (AIV), Salmonella pullorum, Pasteurella Housing Gnotobiotic Mice multocida, Newcastle disease virus, reticuloendotheliosis virus, avian reovirus, mycoplasma, and avian adenovirus group III. Meantime, 2 Y Gonzalez2, JM Koontz1, CL Horton3, BC Dancy1, JA Lewis1, LM genetic lines of B and Q were selectively bred according to the Caffo*3 phenotypes of egg shell colors (white or cyan) as well as genotypes at 18 genomic microsatellite markers. Four families, named as B1 to B4, 1U.S. Army Center for Environmental Health Research, Fort Detrick, were specifically selected following selective mating among MD; 2Excet, Inc., Fort Detrick, MD; 3Oak Ridge Institute for Science homozygote birds screened from genomic sequences of a transporter and Education, Fort Detrick, MD associated with antigen processing gene located within major histocompatibility complex region. Animal experiments showed that Gnotobiotic animals have become an increasingly popular research B2 family was more susceptible to DHV and B3 induced higher AIV model, yet standardized methods and procedures for animal HI antibody titers than the other 3 families. At present the scale of husbandry have not been established. We have assessed and breeder duck stocks are about 500. We believe that these unique SPF developed methods to sterilize and maintain gnotobiotic conditions duck genetic materials enrich laboratory animal resources and within flexible film isolators. A combination of sterilization methods provide valuable standard animals for avian science research. was required. Caging materials, rodent food, water, and all necessary experimental tools and reagents were autoclaved or chemically P153 Enhancing Husbandry and Laboratory Personnel Safety by sterilized to ensure elimination of exogenous microbes prior to the Creation of a Dedicated Animal Safety Coordinator transferring materials into the isolators. Gamma irradiation and electron beam irradiation processes were employed for items that L Steiner*, VA Hill, RC Dysko were not compatible with liquid or vapor contact. Modifications to gaskets, tubing, transfer cylinders, and sleeves were made to reduce Unit for Laboratory Animal Medicine, University of Michigan, Ann chances of breaches within the isolators. Vaporized hydrogen Arbor, MI

645 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Health and safety issues are often identified during facility inspec- chewing enrichment as represented by increased individual body tions performed by the IACUC and AAALAC. The Occupational weights, but has mixed results on reducing food grinding behavior. Health Department (OHD) at a large, decentralized institution is Overall, this study demonstrates environmental enrichment that often unable to provide routine visits to all locations to ensure a promotes nesting behavior in mice can be used to help decrease food robust health and safety program for all individuals who work in the grinding behavior leading to improved animal welfare and wellbeing animal research facilities across campus. In an effort to enhance the while decreasing management costs. occupational health and safety component of the animal care program (ACP) and to more directly address and prevent health and P155 A Novel Mouse Individually Ventilated Cage Design safety concerns, the centralized husbandry unit created the position Demonstrating Extended Cage Change Interval, Reduced Airflow, of Animal Safety Coordinator (ASC). The ASC serves as a liaison and Compensation of Modeled Water System Leaks between the ACP and the institution’s OHD. This person advises facility supervisors/managers on various health and safety issues but L Kramer*, M Bailey, M Semenuk is not an official inspector. As such, interactions are primarily collegial and collaborative, and not punitive. Duties include (but are Lenderking Caging Products, Millersville, MD not limited to) assessment of appropriate use of biologic indicators, ensuring appropriate placement and flushing of eyewash stations, A novel individually ventilated cage (IVC) design, where air flows sanitation validation of both mechanical cage washing and hand through the bedding, was used in a study of female C57BL/6J mice. washing of supplies, annual verification of animal transfer stations to The study used 27 cages containing 5 mice per cage, and, another 9 ensure correct operation for personnel protection, and monitoring cages containing 2-timed pregnant mothers and their litters. Cage Personal Protective Equipment (PPE) for both effectiveness and change intervals were extended past 21 days using 30 air changes per application. Previously, these tasks had been occurring as separate hour (ACH). Slow and fast water system leaks were simulated by functions over several different job classifications. By assigning these spraying 5 mL and 30 mL of water (respectively) on the bedding responsibilities to a single individual, we have centralized these roles through a sampling port. The serology panel was completely and have a dedicated staff member to prioritize the occupational negative. Blinded histopathology samples of the lungs and turbinates health and safety component within the ACP. The most important in the nasal passages were all within normal limits with 1 exception aspect of the ASC is that the ACP now has a person who goes into the in the control group which showed slight inflammation in the lungs, facilities on a regular basis and interacts with the staff to support but no necrosis. This included pups reared their entire lives under safety and look for areas of risk. The OHD creates the policies and this novel airflow schema. Ammonia concentration data was enforces them in a formal capacity. The ASC is their partner in the gathered using a Matheson Kitagawa gas sampling system through field to understand how the policies are being applied and where the the sampling port located 1 inch above the bedding in the cage front. institution has inconsistencies or liability. After 6 months of full-time Histology and serology data demonstrate that the novel airflow activity, our ASC has contributed to long overdue improvements to design did not compromise mouse health under an extended cage the program along with a much more efficient and effective means of change interval. Water bottle leaks garnered surprising results of addressing health and safety concerns. lower ammonia concentration as compared to positive controls.

P154 Can Type of Environmental Enrichment Help Reduce Food P156 Effects of Partial Bedding Changes on Inter-Male Aggression Grinding in Mice? in Mice

LH Webb*, A Pacio G Bhade, LV Kendall*

Research Triangle Park, Mispro Biotech Services Corporation, Laboratory Animal Resources, Colorado State University, Fort Durham, NC Collins, CO

We questioned if different types of environmental enrichment can Group housing of male mice can lead to increases in aggressive affect or reduce food grinding in research mice. Food grinding is a behavior, and cage cleaning is a source of disturbance that may common problem observed among CD-1 mice in our animal exasperate this problem. This study tested if differences in bedding program. It occurs when rodents chew food pellets, but do not composition affected stress levels of mice. This could potentially consume them, resulting in excessive food particles (orts) in the cage develop new cage-cleaning methods that lower stress levels of bedding. Food grinding leads to more frequent cage changes laboratory mice which can aid other research projects in which stress is attributing to increased animal stress, as well as higher food and a variable that needs to be controlled. Sixteen cages in 4 groups were management costs. The hypothesis is that food grinding occurs due made with fresh bedding, food, and water. Five male mice were placed to boredom, composition of diet, or lack of natural behavior into each cage. Behavior was observed daily for the next 2 weeks. opportunities. According to previous published studies conducted to Aspen chip bedding was changed at the beginning of the third week evaluate if the causes of food grinding were behavioral or nutritional using the same cage. The first group received 100% fresh bedding; the deficits, it was concluded that change in diet or additional nutritional second received 25% old bedding and 75% new bedding; the third enrichment (i.e. sunflower seeds) was most effective at reducing group received 50% old bedding and 50% new bedding; and the fourth overall food grinding behavior. However, as is the case in most received 75% old bedding and 25% new bedding. Behavior was animal studies, change in diet or added edible enrichment is observed daily for the following week until the next bedding change. considered an unacceptable study variable. In our animal program, a Mice then received new cages with fresh bedding for 1 week then the modification to environmental enrichment is the initial approach to study repeated in a cross over design so each group received each address stereotypical behaviors. This study compared mice known bedding treatment. Individual instances of sexual aggression (SA), for grinding (CD-1 male and females) with mice not known for nonsexual aggression (NSA), vocalization (V), bar gnawing (BG), and grinding (C57BL/6 males). Mice were given a different enrichment bare spots in bedding (BS) were recorded over a 30-minute period. type each week, starting with a single product followed by combina- There was no difference in the SA observed the day of the change; tions of products for 1 week each. Food consumption, individual however, during the week, the SA was significantly greater in the mice body weight, and visual ort rating scale were measured over the that received 100% fresh bedding in a used cage (p < 0.01). There was 13-week study. Preliminary data results suggest that a single no significant difference in the other behaviors observed. It was noted enrichment product offered has little to no impact on food grinding as the male mice aged there was a significant increase in SA and NSA behavior. Mice had increased weight gain when offered nesting (p < 0.01) regardless of the amount of partial or fresh bedding used. enrichment as compared to enrichment that promoted chewing These results demonstrate the current cage cleaning method (new behaviors, which resulted in weight loss. This suggests that, when cages and fresh bedding) leads to fewer instances of SA, NSA, and BG given a single product, mice prefer nesting enrichment versus than reusing the cage with complete or partial bedding changes.

646 Abstracts of scientific papers 2016 AALAS National Meeting

P157 The Effects of Lower Protein Diet on Growth, Survivability, P159 Implementing a Sanitization Verification Program for PI and Lesion Incidence in Aged Male Sprague–Dawley Rats Testing Chambers and Surgical Equipment

MJ Horn*1, BD Mickelson2 M Nigro*, LS Bird

1Veterinary Sciences, Research and Support, Envigo, Indianapolis, Rutgers University, Piscataway, NJ IN; 2Teklad, Envigo, Madison, WI ATP (adenosine triphosphate) testing is being used on an increas- Two-year toxicology studies and some types of age-related research ingly routine basis for testing the effectiveness of cleaning in animal involve maintenance of cohorts of animals for extended periods of rooms and cage washer sanitized items in our facilities. Our time. Survival above a specific threshold at study completion is department wanted to expand this practice to other areas of live desirable and may be required for regulatory reasons. Diet restriction animal use, as well. A large portion of the research conducted within is one method used to increase survival; however, this is resource our university is behavior based, which requires the use of different intensive and raises animal welfare considerations. Another behavior chambers, devices, and equipment. Many scientific lab staff approach to consider is the use of a lower-protein, lower-energy also perform surgical procedures with mice and rats within their density diet. This study used a cohort of 200 male Sprague–Dawley lab’s benchtop space, as opposed to inside the animal facility. In rats maintained on a 14% protein diet from 8 to 104 weeks of age. compliance with SOPs, each lab is required to maintain the cleanli- Body weight was monitored monthly while survival was reported ness of their equipment both in between each animal and at the weekly. At the culmination of the 2-year study, animals were completion of their work. In order to verify that their equipment submitted for necropsy including histopathology of select organs. used for live animals is being cleaned and sanitized thoroughly, our Neoplastic and nonneoplastic lesions were characterized, and blood department developed a sanitization verification program. The and urine were analyzed for complete blood count/serum chemistry program needed to be accurate and reliable but also timely for both and urinalysis, respectively. Data from the study were compared to the veterinary technicians performing the verification and the data collected in a previous study where male Sprague–Dawley rats scientific staff. Our veterinary technician team began the process by were maintained on an 18% protein diet under similar conditions. taking an inventory of the scientific staff’s behavior and surgical One hundred and four-week survivability was numerically but not equipment. Based on this information and further discussion with statistically improved in animals maintained on 14% (68.5%) our veterinary staff, we decided to biannually sample 10-25% of each compared to 18% (63.3%) protein diet. Additionally, body weight was type of equipment depending on the total amount. We use the values decreased by nearly 120 g (20% body weight reduction) at 104-wk of preprogrammed into the ATP device for our numerical result and age (P < 0.05). Thymus, spleen, liver, heart, testes, epididymis, and any result higher than 150 (“good”) is an automatic fail. Our brain tissue weight (normalized to brain tissue weight) were veterinary technicians communicate with the PIs and scientific staff significantly different due to diet (P < 0.05) at 104 weeks. Neoplastic to schedule the initial testing date, provide sanitation policy lesion incidence was also reduced with the 14% protein diet in both information, and to answer any questions. If results are unsatisfac- pituitary gland and pancreas. In conclusion, the use of a lower tory, a training session is organized for the PI staff, which includes protein/lower energy density diet decreases body weight, may education on acceptable solutions, contact time, and cleaning enhance survivability, and reduces neoplastic lesion incidence technique. Once a satisfactory result is achieved, the items tested are without the use of diet restriction in long-term studies. This is a added into our regular biannual testing schedule. viable and simple option for long-term diet maintenance of outbred male Sprague–Dawley rats. P160 Constant Antibiotic Treatment Reduces Breeding Prosperity of MyD88/TRIF Double Knockout Mice in a Specific Opportunist P158 Cross-Fostering Mice to Produce “Clean” Colony for Research Pathogen-Free Mouse Facility

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

Office of Animal Resources, University of Missouri, Columbia, MO 1Mouse-Pathology, Helmholtz Centre for Infection Research, Braunschweig, Germany; 2Central Animal Facility, Helmholtz Centre Cross-fostering is a technique that may be used to eliminate certain for Infection Research, Braunschweig, Germany; 3Central Animal diseases in research rodents that may be difficult to eradicate by Facility, Universitätsmedizin Göttingen, Göttingen, Germany medication alone. When a principal investigator has a need to establish a new colony in a higher health status (cleaner) vivarium, MyD88 knockout mice are deficient in the recognition of bacterial cross-fostering is an accepted process to eradicate certain diseases, and viral infectious agents due to the lack of toll-like receptor (TLR) including Helicobacter sp. In simple terms, cross-fostering is the signaling. It is widely assumed that these mice cannot be bred and removal of pups from the biologic dam shortly after birth and maintained without constant antibiotic treatment because of their placement of the pups with a surrogate dam, another nursing dam susceptibility to infections. However, the need of this chronic that is free of the disease requiring elimination. The process requires antibiotic treatment has not been demonstrated for Specific Oppor- careful timing to ensure the foster attempt is successful. Close tunist Pathogen-Free (SOPF) housing conditions. We have transferred observation of the breeding female for a vaginal plug is first needed several MyD88 knockout mouse strains to our SOPF mouse facility. to determine the exact due date of the pups. Subsequently, a Breeding pairs received constant antibiotic treatment (trimethoprim- surrogate dam that will deliver pups a day before the infected dam sulfonamide). Twenty-five to 50% of the breeding females died due to must be available or purchased. Using a surrogate that is a different endometric/urogenital infections with Enterococcus faecalis. The color than the original mouse is helpful in being able to differentiate isolated Enterococcus faecalis strain was highly resistant against pups. Following deliberate protocols during transfer of pups from various antibiotics. In order to investigate the possible protective their dam to the surrogate is necessary to ensure disease control and effect of the antibiotic treatment, 3 groups of breeding pairs (n = 6 acceptance of the foreign pups. For example, making sure there is no females) of the MyD88/TRIF double knockout strain (B6.129P2- bedding or nesting material on the pups before moving them to the Myd88tm1Aki-Ticam1tm1Aki) were constantly treated via the drinking surrogate’s nest and having the surrogate urinate on the new pups water with placebo, trimethoprim-sulfonamide, or enrofloxacin. The helps the acceptance process. Following a successful transfer of pups, study was setup double-blinded: neither animal care takers nor weaned animals should be tested to determine that the cross- veterinarians were aware of the group-assignment. Surprisingly, fostering process eliminated the agent of interest. We have used blinded clinical and pathohistological assessment revealed an cross-fostering successfully in starting a “clean” colony free increase of endometritis in trimethoprim-sulfonamide treated of Helicobacter sp. numerous times. animals. The difference in enrofloxacin and placebo treated groups was not significant. These data suggest that the antibiotic treatment

647 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 with trimethoprim-sulfonamide is deteriorating the health status of cages can represent over 50 percent of the total rack weight when the animals and promoting endometritis in breeding MyD88/TRIF fully occupied. Push forces were significantly lower than pull forces ko females. Enrofloxacin treatment did not display any advantage except for Hard 70 wheels. The hardness of the wheels and bearing compared to placebo. In order to decrease the risk of further types significantly influenced both initial forces. Pull/push forces resistance development, the application of antibiotics needs to be with Soft 95 were significantly higher than with Hard 70 and Hard 75 reduced. Moreover, antibiotic treatment effects the microflora and wheels. We also observed that any damaged wheels, floor imperfec- might thereby influence experimental results. Thus, we conclude that tions or objects impeding rack movement could greatly influence the constant antibiotic treatment of MyD88 mouse strains should be initial pull/push forces. Using the same standard conditions on racks omitted in SOPF facilities. equipped with Hard 75 wheels, we evaluated the effect of 2 floor types, namely smooth epoxy and rough methyl methacrylate (MMA) P161 Temperature/Humidity Data Logger during Animal Exporta- coated floors on both initial forces. Pull forces were similar but there tion/Transportation was a tendency for greater push forces on the MMA floor (NS). Overall, these results stress the importance of carefully choosing MC Rodriguez*, R Cook wheels to minimize initial pushing and pulling forces and prevent work related injuries. Eastern Virginia Medical School, SoBran Bioscience, Norfolk, VA P163 A Case Study on Implementing an Animal Management Transportation within or between institutions is a common procedure Software where laboratory animals are routinely removed from their highly controlled macro and microenvironments. Controlled environment, D Latterich*, E Schouten, L Belen, JD Reuter, M Leblanc including stable within acceptable ranges for temperature and humidity (T&H), are important and regulated conditions. However, ARD, Salk Institute, La Jolla, CA T&H are rarely monitored closely by shipping couriers despite the possible adverse physiologic effects when basic parameters are lost. Electronic systems are commonly used to streamline animal Our institutional exportation SOP now requires a T&H data logger management. Some institutions choose in house developed software (DL) to monitor environmental parameters during any exportation. while others purchase off-the-shelf solutions, but all face similar We use a data logger that looks like an ordinary USB memory stick. implementation challenges that require careful planning. Our With the help of a customized casing we are able to place the device department operates over 45,000 ft2 of vivarium including 15,000 inside the primary transport enclosure. The DL provides continuous mice IVC and supports 40 labs and over 700 animal users. In 2015, monitoring and recording of T&H during the transit of our sensitive we purchased and implemented an electronic system to manage packages and can be set to users preference on how often data is animal ordering, bar code census, and recharges. We identified 3 obtained, acceptable ranges for parameters, and even an audible critical areas that would impact the success of implementation: user alarm for when ranges are breached. Once a shipping container adoption and training, system performance, and internal support arrives to its destination, the logger is shipped back to us in a resources. Because users are diverse with different levels of computer self-addressed envelope. The DL allows us to verify the T&H inside skills, we estimated a prolonged implementation timeline lasting at the cage during transport and confirmed that it was maintained least 1 year. We adopted a phased approach, rolling out the within acceptable levels. This helps us asses the quality of the courier animal-ordering module first to accustom users to the system and service and inform the receiving institution of any situation. In gradually added the remaining functions with extensive training at addition, the audible alarm could alert the driver when deviations each phase. Second, we strove to use as closely as possible out-of-the occurs. The only drawback to this DL is the lack of real-time box functions and minimize custom changes. It lessened the burden monitoring or distance alarm. Technology offering text or email alerts of database maintenance and occurrence of glitches as well as exist, as well as GPS and video. Institutions have to study the streamlined software upgrades. We also built interfaces with the feasibility of such considerably more expensive equipment. human resources and finance department databases to automate data flow. Furthermore, we conducted exhaustive testing on each function P162 Effect of Wheel and Bearing Types on Initial Pulling and to minimize system kinks and met individually with lab managers Pushing Forces on Ventilated Racks and end users to identify process conflicts and make changes early. Third, an effective technical support team including members from GK Palma*1, B Becker1, SJ Graciano1, SC Adams1, JD Reuter2,1, M the vendor and various company departments was assembled to Leblanc1 coordinate efforts throughout the process. In retrospect, the phased approach was strategic to secure user confidence in the new 1ARD, Salk Institute, La Jolla, CA; 2Office of the Vice Chancellor for software. Significant time spent on system testing paid dividends Research, University of Colorado—Boulder, Boulder, CO and minimized hiccups. While the project team could not anticipate all facets of system or user issues, the company and vendor allocated Mouse colonies in barrier facilities are often housed on ventilated the necessary resources to resolve ad hoc problems and support racks that can weigh over 1.000 lbs and must be pushed and pulled users throughout the process. In conclusion, having a collaborative repeatedly to accomplish normal tasks. Selecting the right rack team of key stakeholders, proper training resources, and a wheels is often overlooked and may result in work-related injuries. customized plan were essential to the successful implementation of Using a digital force gauge, we evaluated the effect of different the software. wheels and bearings on the initial push and pull forces to displace ventilated racks. Three wheels of increasing hardness were evalu- P164 Reduction of Animal and Technician Stress via an Efficient, ated: 1) 95 durometer Shore A with roller bearings (soft 95); 2) 70 Simple Method of CO 2 Euthanasia for Mice durometer Shore D with ball bearings (Hard 70) and, 3) 75 durometer shore D with ball bearings (Hard75). Casters were identical on all MM Trogdon*1, JW Hensley1, GE Kissling2, K Laber3 racks and wheels had the same diameter and thickness. Experimental conditions were strictly standardized. Forces were applied 3 times at 1Animal Resources Section, Comparative Medicine Branch, National waist height in a horizontal plane with a constant floor slope Institute of Environmental Health Sciences, NIH, Research Triangle (<1° variation) on an epoxy-coated floor and with an initial wheel Park, NC; 2Biostatistics and Computational Biology Branch, National position aligned to the applied force. Overall, none of the racks Institute of Environmental Health Sciences, NIH, Research Triangle required an initial force exceeding the Liberty Mutual Research Park, NC; 3Office of the Chief, Comparative Medicine Branch, Center standards for workers of both sex. Except for pushing with National Institute of Environmental Health Sciences, NIH, Research Hard 75 wheels (R2=0.61, p<0.0001), the initial push/pull forces were Triangle Park, NC not correlated with the number of cages on the rack even though

648 Abstracts of scientific papers 2016 AALAS National Meeting

With the continued high use of genetically altered mice, the need for Laboratory animal facilities cannot function without significant euthanasia of nondesirable genotypes persists. To accommodate the support from personnel that do not directly interact with the animals. large numbers of rodents used and to maximize available space, Any disruption to this requisite support has the potential to institutes are using individually ventilated caging (IVC) systems. profoundly impact operations in a manner that is both expected and Following recommendations that rodents should be euthanized in unexpected. Indoor air quality is a prominent concern in laboratory their home cage whenever possible, we have come up with a simple, animal science with research animals and workers in animal facilities efficient method of delivering CO2 that reduces not only the both participating as stakeholders of this ongoing discussion. Our procedure time, but the stress experienced by both the mice and the administrative support area had experienced ongoing air quality technicians. Using a manufacturer’s IVC transport rack, and building concerns since 1974. In 2012, in an effort to address the accumulating on the use of our multiport Plexiglas manifolds, we used a horizontal and significant health and safety concerns, personnel with offices in multiport manifold obtained from a home brewing supplier, some the administrative area were provided with alternative space in flexible polymer tubing, and stainless steel sipper tubes cut from another building on campus. The administrative area was the main bottle caps. Removing only the water bottle from cages on the entry point and communication hub for the animal facility. Its transport rack, we were able to simultaneously euthanize 5 cages of relocation had the potential to cause logistical complications rodents by delivering a concentrated distribution of CO2 in surrounding facility access, security, communication, and the compliance with AVMA guidelines, and caused no visible change in ordering of products and animals. Eight weeks before the planned animal behavior or animal activity. In addition, we reduced the relocation, biweekly meetings were scheduled and included overall time of the euthanasia process by at least 2 minutes per cage. administrative, technical, veterinary, and facility management staff to For an 80-cage transport rack, this equates to a reduction of develop action plans to mitigate any disruptions that could be approximately 30 minutes. foreseen. Once the relocation occurred the importance of continued biweekly meetings became apparent to ensure emerging issues did P165 Preclinical Immuno-Oncology Models—Husbandry and not progress into functional obstacles. Two months after the move the Housing Considerations biweekly meetings reduced to monthly meetings. Due to the proactivity and innovation of the Campus Animal Facilities and MM MacBride* Animal Facility Support team, meetings are currently held on an ad hoc basis. The initiation of a “grand rounds” that includes adminis- Taconic Biosciences, Hudson, NY trative, technical, veterinary, and facility management staff has allowed for ongoing face-to-face dialogue between colleagues The growth in immunooncology research, in which the body’s own working in different buildings and provides an effective forum for immune system is recruited to fight a tumor, has prompted a shift in resolving logistical issues on an ongoing basis. preclinical animal models used for oncology research. While traditional cell line xenografts in immunodeficient models are still P167 Subfloor Radiant Heat: A Novel Approach to Animal widely used, they are generally not appropriate for immunooncology Warming studies as they lack critical immune cell types required for immunotherapy efficacy. Immunooncology researchers have turned to 2 MA Gregory*, B Gien model systems: syngeneic tumor models in immunocompetent inbred strains and mice engrafted both with a humanized immune Surgery, Envigo, Indianapolis, IN system and a tumor. Facility managers and veterinarians involved in these types of studies must consider various factors. For syngeneic Maintenance of normal body temperature minimizes cardiovascular models, what health standard is acceptable or preferable? Under- and respiratory disturbances caused by anesthetic agents and is of standing the microbial status of inbred mice used and being particular importance in small animals where the high ratio of consistent is critical. Inbred mice with higher health statuses are now surface area to body weight may lead to hypothermia. Therefore, to readily available, and these should be housed in immunodeficient or improve surgery outcomes, supplementary heat is provided to high-barrier rooms rather than conventional rooms in order to animals during and after surgery. We recently began searching for an maintain that health status. Humanized models are severely alternative to our current system. There are many different methods immunocompromised mice reconstituted with a human immune to provide supplemental heat, but most are not designed to provide system that has functional limitations. As the base model is so heat support for prolonged surgeries and recoveries. We were also immunocompromised, they are exquisitely sensitive to opportunistic looking to provide a heat gradient during recovery so the animals agents and require special housing and husbandry, including very can escape the heat if they wanted to, but this is difficult to do with strict bioexclusion processes. All materials must be sterile and aseptic most of the available products. Noisy motors, leaking pads, and technique must be used. Robust health monitoring is critical to connections have been a constant issue when using our current distinguish between infectious processes and graft-related issues. The heating source. We decided to approach the problem from a new severely immunocompromised base models used for humanization direction using a radiant heat product designed for under-floor may be susceptible to health problems caused by agents not on heating sandwiched between 2 sheets of clear acrylic. The acrylic typical exclusion lists, so veterinarians must keep an open mind provided support and protected the heating element, as well as during diagnostics. Pooled fecal samples are tested monthly for making it sanitizable. We connected the heating element to a bacterial agents, and sentinels which have been exposed to dirty programmable thermostat which allowed us to set a schedule and bedding from humanized mice are used for health testing. We use monitor the temperature. The width of the heating element, both immunocompetent C3H, as well as humanized mice which have combined with the overall width of the acrylic, provided a wide not engrafted sufficiently as sentinels. While humanized mice are not thermal gradient, allowing the animals to find a place to feel suitable for serology, using them as sentinels provides a 3Rs benefit comfortable. When compared to our current heating source the initial as mice that would otherwise be discarded have a further value, and cost savings are not substantial, but the lower maintenance cost and they may be more sensitive for parasitology and microbiology. longer life of this new heating source allows savings to continue to grow year after year. The enhanced monitoring and scheduling P166 Framework to Mitigate Complications Resulting from the capabilities, worry-free operation, and better in-cage environment Relocation of the Administrative Support Area Out of a Vivarium make this an excellent solution for peri and postsurgical animal warming. MW Brunt*1, J Ferguson2, M Zoethout2, R MacQueen2, ME Fowler1 P168 The Use of Braided Cloth as a Pig Enrichment Device 1Campus Animal Facilities, University of Guelph, Guelph, Cana- da; 2Animal Facility Support, University of Guelph, Guelph, Canada KR Lambert, MG Kimmer*, E Mitchell

649 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Animal Resources Program, Wake Forest School of Medicine, P170 Scrub Access and Availability Project Winston-Salem, NC D McNeill1, L Schliep1, NM Gades*1, J Benedetto2, C Ehrler1, S Moe1, J Pigs are highly intelligent animals that need lots of stimulation in Posvenski1, R Waldron1 their environment to meet the species behavioral needs. Despite stereotypes, pigs prefer interacting with devices that are relatively 1Comparative Medicine, Mayo Clinic Arizona, Scottsdale, AZ; 2Uni- clean and free from fecal material. Hanging enrichment devices is a versity of North Carolina—Chapel Hill, Chapel Hill, NC good strategy to provide pigs with the opportunity to interact with clean devices in a laboratory environment. In developing a toy Our department was not meeting its goal of a complete set of scrubs rotation for pigs housed at our facility, we studied the use of a for every DCM employee every day of the week. The Guide states braided cloth hanging on the inside of the pig enclosure as a potential that soiled attire should be disposed of, laundered, or enrichment device for laboratory pigs. Each singly housed pig was decontaminated by the institution. We reorganized scrubs by size, provided with a braided cloth hung on their enclosure every creating a color-coded reference chart. Scrub disposal bins were morning and the cloth was removed in the afternoon. Observations numbered, and soiled scrubs counted at the end of each day. Clean occurred twice per day: once in the morning after device presentation scrubs were counted upon receipt, and compared with the vendor and once in the afternoon prior to device removal. We found that count sheet. Scrub-need worksheets were placed in the locker rooms most of the pigs were interested in the devices when first placed on and were completed by research staff and DCM employees. Data the enclosure, but interactions typically decreased by the afternoon. were collected from May 2 through August 1, 2014. An online survey On average, the pigs spent approximately 37% of their time of scrub users was completed. Results showed a significant difference interacting with the devices in the morning, and only 9% of their time in the number of clean scrubs received compared with what was in the afternoons. We also found that the interactions with the listed on the vendor count sheet, with the difference ranging from -5 braided cloth device appeared to decrease over the course of the to 30; tops and bottoms were counted separately and categorized by week with repeated exposure. On average, the animals spent 78% of size. The scrub need worksheet identified a lack of 2X large scrub their time interacting with the devices on day 1, but only 18% of their tops (n =3), 2X large bottoms (n =1), and X-small bottoms (n = 1). The time interacting with the devices by day 5 in the observations directly scrub survey identified that scrubs tops were worn more often than after provision. Even though the device did not maintain novelty for bottoms. We identified 2 critical areas for improvement in our long, it still received more interactions from the animals than the system. First, the need to reconcile the vendor count sheet with the other cage enrichment we regularly provide. A challenge we faced actual number of scrubs received. Secondly, the need to order more included finding the right thickness for the braid to prevent easy tops than bottoms, based on usage. We continue to do periodic destruction and encourage use, and this is a focus for ongoing monitoring of the scrub inventory using the tools developed above. studies. P171 Making an Animal Facility Emergency Response Plan P169 Novel Enrichment for Sheep (Ovis aries) Used in Biomedical Integrated, Dynamic, and Accessible Research NR Young*1, P DeLuca3, GP Langan1,2 MR Lowery*, P Vertz 1Animal Resources Center, The University of Chicago, Chicago, Teaching and Research Animal Care Services, University of Califor- IL; 2Surgery, The University of Chicago, Chicago, IL; 3Emergency nia, Davis, Davis, CA Management, The University of Chicago, Chicago, IL

Sheep (Ovis aries) are an excellent research model, as the anatomy The Guide requires animal facilities to have an emergency response and physiology have been well defined. Sheep have been shown to plan (ERP) and for the plan to be integrated into the overall recover quickly from general anesthesia and invasive surgery. We institutional emergency management program. The importance and regularly house sheep that are used in biomedical research. The functionality of these plans becomes evident during the response to animal care program’s mission is to ensure humane care and use of an emergency. There are several key components to a successful animals in teaching and research and to assist with the advancement emergency response plan. The ERP has to be able to easily evolve of scientific knowledge and the future of research goals set forth. with changes in the animal program and it needs to be frequently Environmental enrichment enhances animal wellbeing by providing tested and modified. In addition, the plan needs to be readily animals with sensory and motor stimulation. Traditional enrichment accessible during an emergency. To address these important factors, techniques for sheep include providing a “buddy” sheep so their we developed a new ERP program to improve the functionality of social needs are met. While group-housing sheep and providing our plan. The improvements included the use of even minor events forage material meets most of their needs, not all sensory and motor as drills of the ERP with subsequent dissemination of lessons learned stimulation needs are being met by just these items. Normal to all members of the program. Quick action guides were developed grooming behavior in sheep includes rubbing or scratching them- and posted in several locations which highlight the most important selves on areas such as trees, fences, or walls. This behavior is information to know during an emergency. In addition to the hard especially popular with sheep that shed their wool each year, which copy ERP, an ERP dashboard was created on commercially available is the typical breed of sheep that we house and maintain. With this in project management software to allow more accessibility and ease in mind, we designed a novel enrichment device that we call a “sheep updating. This new system has improved the efficiency and scratcher.” We used ordinary material such as angled broom heads effectiveness, as well as increased the number of people who are able that were installed in metal frames that mounted to the walls of to respond to emergency situations. sheep pens. There was an initial adjustment period for some sheep to become comfortable with the new item in their pen, but after this P172 A Novel Pin Hole Sipper Tube for Mouse Water Bottles initial adjustment period these scratchers became heavily used by all of the sheep in our facility. These scratching units give the sheep a DW Mallon1, NS Linde*2, JD Chinchilla2, TJ Plemons1, J Raber1 chance to engage in species-specific behavior in addition to their interactions with other sheep. This low-cost and easy-to-manage 1VRRS, National Eye Institute, Montgomery Village, MD; 2Priority enrichment method has exceeded our expectations and given our One Services, Alexandria, VA sheep a better environment to thrive in. This enrichment device would also work for other livestock species that have the same With the goal of transitioning to a 14-day rodent cage change behavior of rubbing and scratching on trees or fences. 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

650 Abstracts of scientific papers 2016 AALAS National Meeting

used daily with our current water bottle and sipper tube system in large variation in their performance. Enlarged seminal vesicles in multiple cages with 5 adult mice over the course of 14 days. We male mice or splenic tumors were found in approximately half of found that we needed to reduce the amount of water wasted when dead animals at autopsy. Mice with abnormalities in various organs the cage was moved or animals played with the sipper tube. In are mostly over 20 months old. Additionally, our results show that addition, we needed to increase the capacity of the bottle from 16 oz some alteration of the intestinal flora may occurs at around 12 to 18 oz to ensure enough water was always available. Working with months old. Various age-related changes (such as the motor some of our vendors, one vendor created a 3 inch long sipper tube performance, the intestinal flora, or pathologies) are found in 12-20 with a 1/32nd inch diameter pinhole and while another increased the months old B6N mice, therefore a possible turning point of senes- water bottle capacity by 2 oz while maintaining a proper fit in the cence in this strain could occur during these ages. We will focus on cage. Upon arrival, we tested each sipper tube pinhole for uniformity these specific life phases and continue to analyze various parameters by using a NIST traceable pin gage. After we verified the desired in detail. tube specifications, we slowly implemented the new system; rack by rack and room by room over the course of 3 months all the while P176 Mobile Data Collection Workstation: Design and Implemen- monitoring results during a 14-day cage change. In conclusion, we tation are able go 14 days between cage cages, reducing labor costs and improving animal welfare with a 90% reduction of wet or saturated P Sherfinski*1, M Peterson2, R Smith3, J Johnston4 cages due to water loss or rodent manipulation of the sipper tube. 1Small Animal Toxicology, MPI Research, Mattawan, MI; 2Purchas- P173 How Routine Husbandry Can Provide Opportunities for ing, MPI Research, Mattawan, MI; 3Information Technology, MPI Socialization and Bonding in a Minipig Facility Research, Mattawan, MI; 4Equipment Compliance, MPI Research, Mattawan, MI ML Salerno, B Grambo, BH Jasmin, N Navratil* Data collection is the core of any nonclinical study work. Providing Marshall BioResources, North Rose, NY researchers an efficient workstation for electronic data collection is a must. Often, the workstation is adapted from a cart with an entirely Socialization enhances animal welfare, strengthens the human-ani- different purpose. Our facility has used a media cart designed for a mal bond, and reduces stress experienced by both animals and television and retrofit to contain our required components. We caretakers. Socialization at our facility begins at birth, and minipigs determined this construction left many characteristics desired for our receive positive human interaction throughout their entire time in end users, maintenance personnel, and for our management. The cost our facility. As a breeding facility with thousands of animals to care of maintenance of the workstations was too costly in both labor and for, we also need to balance the need for socialization with the other components. The existing design was difficult to clean as the husbandry and related tasks we must perform each day. Therefore, materials and configuration often prevented more industrial means we have implemented ways to incorporate positive human interac- of cleaning. The layout was inefficient, and there was not enough tion into our routine husbandry procedures. This includes physically workspace where appropriate and excess space outside of the reach entering the animals’ space and interacting with the minipigs when of the end user. Instruments used on the workstation were poorly we perform simple daily tasks such as feeding, washing, and secured, leading to damage. Several common repairs to the computer weighing of the animals, as well as when we apply topical treat- or cart components were time consuming. In addition, the configura- ments. We use clickers at feeding time, and we also provide positive tion of the workstation required the repair technician to either bend and reinforcing touches to our sows before and during parturition, down or crouch while providing service. After researching appropri- and to the litters soon after birth. Combining socialization with ate materials stakeholders discussed the issues with the current routine husbandry tasks creates positive benefits for both the animals design. The positive features of the previous design were discussed and the staff without requiring many extra resources. and a new design was drafted. A modular configuration was adopted to address several of the issues of the previous design. After having P174 Breeding Mice and their Various Changes with Aging: Toward the design fabricated, the workstation was sent out for pilot work Clarifying the Mechanisms of Senescence with the end users. They provided feedback to improve the scale platform, an accessory work surface, and the placement of the N Ogiso* transponder reading system. We made changes and the workstation was sent out for more testing. After months of operation the Laboratory of Research Animal, National Center for Geriatrics and redesigned workstation was determined to have the features and Gerontology, Obu-city, Aichi, Japan functions necessary for additional deployment. The following improvements were accomplished by the design. The workstation Our facility has kept many naturally aged animals (mice and rats) for can now be quickly separated from the electronic components and gerontology and geriatric studies. If a scientist conducts a study with the remainder processed through cage wash equipment. Several com- these aged animals, it is important to know what changes will occur ponents of the workstation were replaced to keep maintenance costs in them with aging. However, a clear criterion for defining of aged down. The retention of the instruments was reengineered to limit risk mice has not been established. In this study, we evaluate various to those devices. The size of the workstation was reduced to make characteristics as senescence indicators in naturally aged mice kept in transportation and use in tight quarters more efficient. The configura- our facility. Four-week-old male and female mice (C57BL/6N) were tion in conjunction with components reduces time during obtained from Japan SLC and were kept over their lifetime. Physi- maintenance and reduces the need for maintenance staff to bend ologic (measurement of body weight, food consumption, and down or crouch while performing repairs. survival rates), behavioral (the rotarod tests were conducted every 3 months), and morphologic analyses (autopsy and histologic examina- P177 Efficacy of Supplemental Heat Options for Supportive Care in tion) were performed. Additionally, the intestinal flora of mice was Flooded Mouse Cages also examined. Body weight peaks at 18-19 months old in male (approximately 46.0g) and at 14-16 months old in female (approxi- P Chamberlain*, JS Kilpatrick, KE Scott, JG Fox mately 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 Division of Comparative Medicine, Massachusetts Institute of old and show a similar decrease to that in the other research Technology, Cambridge, MA 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 Flooding is a common problem when housing rodents and can occur and then continues to decline. Male mice are more strongly affected in caging using either water bottles or automatic watering systems. by body weight in aging (R = -0.76, p < 0.01), while female mice have Depending on the severity of the flooding and duration of time

651 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 before the mice are removed from the flooded cages, supportive care discuss best procedures for change management processes in may be required for full recovery. One of the most common methods different institutions. of supportive care is to provide supplemental heat to the mice once they are placed in a dry cage; however, dedicated cage heating P179 Retirement for Research Nonhuman Primates devices are cumbersome and inconvenient to use in a large vivarium setting. Our facility commonly uses 2 methods to provide supple- R McAndrew*1, J Lingo VanGilder2, K Carter3 mental heat: placing an air-activated chemical hand warmer under the cage or placing nitrile gloves filled with hot-water under the cage. 1Bioengineering, Arizona State University, Gilbert, AZ; 2Phoenix Our rational for taking these procedures were that hot-water filled Children’s Hospital, Phoenix, AZ; 3Mount Sinai Health System, New gloves increase the ambient temperature in the cage more quickly, York, NY while hand warmers provide a more sustained heat source, and that a combination of the two will provide an immediate and sustained Though retiring lab animals to a sanctuary is a great way to heat source for the rodents. In this study, 6 full cage setups with acknowledge their service and express gratitude for their contribu- hygrometers placed inside were placed in a change station in an tions to the scientific community, this can often be difficult empty animal room for 18 hours. Temperature and humidity within undertaking. Aside from the considerable costs associated with the cage setups were recorded every 30 minutes for 2 hours before residential animal care, researchers are faced with selecting a suitable the heat sources were added. Three treatments of 2 ventilated cages facility for providing for the wellbeing of the animal for the remain- (n = 6) each were tested. The treatments included 2 hand warmers der of its life, often under the potential threat of backlash from placed underneath the cage on one end (n = 2), 2 hot-water filled animal rights groups. For some species, such as mice, rabbits, dogs, gloves placed in an empty cage bottom with the home cage placed on or cats these challenges are allayed by their status as companion top of the gloves (n = 2), and a combination using both hand-warm- animals and a more widespread integration throughout society. ers and hot-water gloves (n = 2). Temperature and humidity within Sanctuaries exist for these animals outside the realm of research. In the cage setups were recorded every 30 minutes for 4.5 hours. Initial contrast, nonhuman primate (NHP) adoption must contend with data (n = 6) revealed that all treatments provide at least a 10 degree higher costs for care and limited range of choices to provide it. The Fahrenheit increase in temperature for up to 4.5 hours. Treatments Research Animal Retirement Foundation was created to provide the with hot-water gloves had the highest temperature at the 30-minute necessary financial resources for NHP retirement initiatives, as well time point while hand-warmers alone maintained a higher tempera- as advocating for a paradigm shift in perspectives on end-of-career ture at 4.5 hours. The results indicate that hand-warmers are the best options for these animals among the research community at large. We option for providing consistent supplemental heat as supportive care have applied for nonprofit status in Arizona, and will begin over a 4.5 hour time period. In future testing, the type of bedding will fundraising efforts once our application is accepted. Once estab- be examined for effect on ambient temperature. lished, investigators from around the country will be able to apply for funding to be used for the retirement of their research animals. P178 The Process of Animal Diet Harmonization: Big Trouble Selection criteria will be based on the health and behavior of the following Good Intentions animal and potential for positive outcomes. In addition to providing direct financial support, our efforts will also be focused on outreach PF Pohlig*, B Zevnik to the science community as well as the general public, promoting positive attitudes toward research that upholds the ethical responsi- In Vivo Research Facility, CECAD, University of Cologne, Cologne, bility to provide animals with a life outside the lab. Germany P180 An Assessment of Nesting Material and Ocular Health in An animal facility network consists of several animal housing units Nude Mice managed by 3 site managers. The network has heavily intensified its efforts to identify synergies. In the past we developed structural RL Kitsberg*, C Hedrick, S Lewis concepts for centralization and training, mutual exchange of SOPs, implementation of database exchange, and identifying common University Laboratory Animal Resources, The Ohio State University, vendors for technical equipment maintenance and diet. Before this, Columbus, OH every institution has negotiated their own conditions leading, for example, to the use of 3 different mouse diets in the various facilities. Nesting material is a basic environmental enrichment requirement Unfortunately, this is accompanied not only with financial but also for mice at our large academic institution. Providing the appropriate scientific discrepancies. Mice exchanges are performed regularly type of nesting material for various strains of mice can prove between the different institutions; however problems can arise challenging when attempting to maintain a level of consistency without a standardized mouse feeding. We therefore decided to start within multiple large facilities. Previously, commercially available a process of harmonization to find an appropriate standard diet compressed cotton squares have been used as the nesting material of suitable for use in every animal facility on the campus. During an choice and used with ¼” corncob bedding in individually ventilated extensive assessment process, different diet distributors were invited caging systems. With this standard cage setup, a higher incident rate to discuss diet compositions, logistical issues, and financial aspects. of nude mice were reported with varying degrees of conjunctivitis, Furthermore, food acceptance tests were performed with the mice, blepharitis, and/or debris in either 1 or both eyes. It was hypoth- followed by handling tests. Other considerations included figuring esized that this higher incident rate was due to small dust particles out the practicability during daily use such as autoclaving and from the nesting material and that nude mice lack of eyelashes to development of dust and handling inside the barriers. Finally, a joint assist as a barrier against it. To test this theory, 162 cages of nude decision was made for one company for the production and delivery mice were provided either a single compressed cotton square or 8 of the animal diet. Of course, the diet change needed to be performed grams of brown crinkled paper, which is reported by the manufac- in close cooperation with the scientists. Additionally, in parallel to the turer to have little to no dust. Each cage of nude mice were change, our facility started a study to prove how the change of diet individually evaluated every 2 weeks for 12 weeks and given a score would affect mouse holding and breeding. However, after the first of 0 to 3 with 0 having no issue and 3 presenting swelling, redness, trial period of about 3 months, information was derived from the and ocular debris. Data shows that there is a fewer number of ocular study and from investigators feedback about negative effects on health concerns (70.98%) for the nude mice that received the crinkled breeding performance and offspring survival in wildtype and paper. Following further investigation regarding nesting material transgenic mouse strains, all on C57/Bl6 background. This resulted and nude mice an institutional change may be needed to prevent and in an, at least temporary, a switch back to the former diet and a reduce ocular health concerns in nude mice. second refined study, which is currently ongoing. We would like to present this extensive evaluation process, highlight difficulties, and

652 Abstracts of scientific papers 2016 AALAS National Meeting

P181 A Novel Method to Provide Thermal Heat Support to P183 Do Your Employees Feel Stressed and Underappreciated? Primates RA See*1,2, C Mathieu1, S Paynter1,2 CA Carrier, R Samaniego* 1The University of Chicago, Chicago, IL; 2Charles River, Chicago, IL Covance Research Products, Alice, TX Our animal resources center (ARC) consists of several types of Thermal heat support is needed for many reasons when providing employees: cage wash technicians, facility attendants, animal care care for debilitated animals. Some examples are thin or dehydrated technicians, special services technicians, supervisory staff, adminis- animals, newborn animals, or animals recovering from anesthesia or trative staff, certified veterinary technicians, and veterinarians. All surgery. We provide hospital care for nonhuman primates, many of work and interact with each other and with investigators on a daily which need thermal support at some point during their treatment. basis. All are faced with a wide variety of issues every day. Whether Many different types of heating methods have been used in the past customer service, husbandry, or special services, they must deal with such as electric blankets, warm-water recirculating blankets, stress on a regular basis. The mission of the ARC is to take care of individual incubators, and forced-air warmers. However, each type customers and animals. Who is taking care of employees? One way has particular pros and cons when working with primates. Manufac- to address the issue was to develop an employee program that builds tured incubators are ideal because they are generally made with a culture of recognition and that yields higher levels of employee smooth nonporous materials and offer temperature, humidity, and engagement, retention, and satisfaction. It is easy to lose sight of the oxygen control. These incubators also provide secure containment for workforce and expect employees to just perform without question. nonhuman primates. Space and cost was a factor when we consid- But in reality, in order for employees to be successful and feel happy ered the number and type of incubators needed in the hospital. To performing their duties, they must be recognized for the work that take maximum advantage of our forced-air warmers, an inhouse they do. We believe that happy or satisfied employees are more likely incubator was made out of recycled 30-gallon drum barrels in a PVC to perform at a high level. An effective recognition strategy helps frame. A forced-air warmer is attached to the unit and simultane- create employees who are more productive, more creative, and more ously warms the four animal enclosures. Another inhouse unit was customer service oriented. Small tokens of appreciation and kind made out of 55-gallon drum barrels for larger animals. The units are words go a long way in thanking employees for their dedication and small enough to move, easy to clean, and 2-4 animals can be housed hard work. It is also a reflection of the unit’s overall culture. If a unit in them and receive thermal support. It has been a cost-effective professes that people are its greatest asset, it should also be ready to efficient means to provide thermal support to our primates in rooms invest, recognize, and reward people in visible ways. Based on this not designed for banks of recovery cages. information, we came up with the RAT Pack (Recognize Acknowl- edge Treat). Once implemented, we were able to receive feedback P182 The Feasibility of Introducing High-Quality Nesting Material showing a significant increase in employees’ attitudes and morale. In to Mouse Colonies addition, the RAT Pack is particularly appreciated because it is composed of employees who have input in figuring out ways to R Gainer*, JM Cadillac, E Dohm, SC Cartner recognize their peers.

Animal Resources Program, University of Alabama at Birmingham, P184 Swine Swaddlers Birmingham, AL RD Moore*, L Roberson This study explored the feasibility of implementing easy-to-use, preportioned shredded paper nesting products that provided 2 Office of Comparative Medicine, University Of Utah, Salt Lake City, different substrates, and could readily be integrated into husbandry UT practices. It was thought that cost would be prohibitive, since cost is typically a stumbling block when considering changes to husbandry The swine model is frequently used in research for different studies practices. Unfortunately, the cost of a preportioned shredded paper such as new instrumentation, implants, and organ functions. The pig nesting pack (preportioned pack) was nearly twice the amount of the is an intelligent animal, as well as strong and stubborn. Protecting a compressed cotton squares currently being used, suggesting the only surgery or implant site can be challenging. Originally using way a switch could be made would be to raise per diems, which was commercially available stretch covers for this purpose, we found that not an option. The compressed cotton squares were added to every trying to put the animals head and limbs into premade outlets were clean cage prior to autoclaving and distribution to the animal difficult, especially with a 60kg+ swine. We needed to find a similar, housing room. The provision of autoclaved pre-portioned packs was but easier method of clothing these animals. We created what we call instead moved to the animal housing room, so that only cages “swine swaddlers.” We omitted the outlets for the legs and head, and lacking enrichment were provided with new packs. Cages that sewed on straps to either buckle or Fabric hook-and-loop fasteners already had a shredded paper nest at cage change simply had the the clothing in place. One fastening point is around the neck to keep nest transferred to the new cage, thus avoiding the expense of the clothing secure, and one around the abdomen to keep it across providing new enrichment every cage change and reducing the cost the back of the swine. The swine swaddler kept a postoperative/ to equal or less than the compressed cotton squares. Introduction of bandage site clean and dry on the swine model, while eliminating the preportioned packs began in one animal room, and then extra stress and restraint, providing comfortable clothing for the expanded to 1 room per building. This provided time for adequate animal and less time and staff for placement. staff and investigator education. Campus wide implementation is now underway. It was determined that the preportioned packs do P185 Transmission of Pseudoloma neurophilia in Zebrafish (Danio not need to be replaced more frequently than once every other cage rerio) When Using a Mass Spawning Chamber 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 SM Peneyra*4,5, J Cardona-Costa4,5, J White4,1, C Whipps2, ER significantly decreased for both overall case reports and specifically Riedele3, NS Lipman4,5, C Lieggi4,5 fighting cases by as much as 50% or more in the 9 months since implementation. Investigators have also anecdotally reported 1Laboratory of Comparative Pathology, The Rockefeller University, improvement in reproduction. We determined it is possible to switch Memorial Sloan-Kettering Cancer Center, and Weill Cornell Medical to a high-quality nesting material with an equal if not lower cost than College, New York, NY; 2Center for Applied Microbiology, SUNY- the original enrichment. Moreover, dramatic decreases in the overall ESF, Syracuse, NY; 3Department of Epidemiology and Biostatistics, clinical cases and specifically fighting cases were seen in the colonies Memorial Sloan-Kettering Cancer Center, New York, NY; 4Tri-Institu- observed. tional Training Program in Laboratory Animal Medicine and Science,

653 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

The Rockefeller University, Memorial Sloan-Kettering Cancer Center, cubicle room; however, there was temperature variability between and Weill Cornell Medical College, New York, NY; 5Center of Com- the top and bottom rows. The ventilated cages can go static for up to parative Medicine & Pathology, Memorial Sloan-Kettering Cancer 3 days without adverse effects on the mice. We determined that when Center, and Weill Cornell Medical College, New York, NY needing to provide supplemental heat to a few cages, the microwaveable heat pad would be the preferred choice over the hand warmers. Pseudoloma neurophilia, a microsporidium that primarily infects When a large number of cages require supplemental heat, a portable neural tissues, is one of the most common pathogens in laboratory heater placed inside a cubicle appears to be a viable option although zebrafish. Infected fish can transmit P. neurophilia vertically; however, cages would be limited to the top 7 rows of the rack to minimize the horizontal transmission during spawning is more common. It is impact of temperature variability. Temperature evaluations would unclear if risk of parasite transmission is different when spawning also be necessary at different facilities to account for unique HVAC zebrafish in individual spawning tanks as compared to mass spawn- operating parameters. ing chambers, and if this risk is correlated to the number of spawning events. In addition, disinfection protocols have not been evaluated to P187 A Unique Barricade Solution to Stop Flooding Conditions determine if any could decrease or eliminate the risk of transmission following spawning. The present study was conducted to evaluate P. SM Saverino*, K Silva, M Murchison, BR Therault neurophilia transmission from a known infected colony (prevalence = 52%) to uninfected fish and their offspring when using 2 different Animal Resource Center, University of Chicago, Villa Park, IL spawning apparatuses (n = 30 per group). Infected and uninfected fish were allowed to spawn in either a mass spawning chamber Flowing water in an animal housing facility may occur due to faulty or breeding tanks for 1 or 3 spawning events. Uninfected fish and equipment, animal mischief, human error, or environmental factors. offspring were evaluated for infection 15-weeks later by histopathol- While some potential overflow water situations may be identified ogy and Luna stain. In addition, we sought to develop an effective when only small puddles are on the ground, others can consist of protocol for disinfecting the mass spawning chamber between uses unrestricted flowing water creating flood situations. Regardless of to eliminate P. neurophilia spores. Devices were either allowed to dry the size, all facility water situations need to be handled in a prompt, for 1 hour or were disinfected with one of the following protocols timely fashion. Often unique circumstances resulting in free water for 10 minutes after infected zebrafish spawned for 1 hour (n=5 per require a bit of creativity in finding prompt, innovative solutions. method): reverse-osmosis water, 90-110 ppm bleach (pH = 6.8 - 7.5), Our campus has encountered animal facility errant water on 1.0% Virkon Aquatics, 75 ppm Wescodyne, or 175 ppm Wescodyne numerous occasions arising from a variety of causes. Typically, the Plus spray. Results demonstrated increased risk for transmission to influx of water stops after only a few minutes and facility staff are uninfected fish when spawning occurs in a mass spawning chamber quickly able to clean up the water and associated soiling. On one (100% infected after 3 spawning events), as compared to the other recent occasion, however, one of the pipes in the maintenance space groups (0-7% infected). The parasite was not detected in offspring cracked and water was slowly flowing into the hallways of the from these spawning events regardless of spawning device or vivarium. Once the water reached the hallway, it flowed directly into number of spawning events. The Wescodyne Plus spray, bleach soak, laboratory spaces storing delicate electronic equipment. With no and Wescodyne soak were 100% effective at eliminating P. neurophilia sandbags on hand, a swift solution was needed to control the spores as determined by PCR following spawning. It is important to flooding while campus maintenance crews worked to find a prevent or minimize spread of this pathogen as there is no treatment resolution short of widespread water shut-off. Technicians quickly and infections can cause morbidity and mortality with the potential gathered available supplies: a pair of used, discarded scrub pants, zip to confound research results. 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 P186 Use of Hand Warmers and a Portable Heater to Provide with the absorbent bedding material. Once they were filled, the waste Supplemental Heat in an IVC System was similarly secured. The flood barrier pants were placed in front of the maintenance spaces out of which the water was flowing and S Hackney*1, A Dickerson1, DR Goulding1, PH Myers1, CA McGee1, J continued to absorb the water for several hours while the cause of the Locklear2, TL Blankenship1 flood was being resolved. Having the flood barrier pants in place allowed our technicians to continue with their work throughout the 1Veterinary Medicine Section, Comparative Medicine Branch, day, requiring only periodic checks and twice-daily swap-out to National Institute of Environmental Health Sciences, NIH, DHHS, ensure the flood pants were continuing to control the errant waters. Research Triangle Park, NC; 2Quality Assurance Laboratory, This technique has subsequently been used on numerous occasions Comparative Medicine Branch, National Institute of Environmental throughout all of the campus animal holding facilities during times Health Sciences, NIH, DHHS, Research Triangle Park, NC of flood situations and has helped to reduce costs in labor and equipment damage. Supplemental support is an important aspect of laboratory animal medicine. Our facility has recently transitioned from a rodent static P188 Occupational Health and Animal-Pathogen Status Are caging system to a ventilated caging system which has required us to Maintained with Decreased Use of Personal Protective Equipment evaluate how supplemental heat was provided to rodents. The in 2 Rodent Facilities objective of this study was to evaluate more efficient ways to provide supplemental heat to cages in a ventilated caging system. Air-activat- SJ Danon*1, D Coomber2, M Lavin3, M France4 ed 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 1BRC, Faculty of Medicine, UNSW, Sydney, Australia; 2Faculty of temperature levels were evaluated. We compared this to static Medicine, UNSW, Sydney, Australia; 3UNSW, Sydney, Austra- microisolation cages on a cart which had a 24-hour hand warmer or a lia; 4France Consulting, Haberfield, Australia microwaveable heat pad placed underneath. Additionally, we evaluated the efficacy of a portable heater placed inside a small A center operates 2 specific pathogen-free (SPF) rodent facilities that animal room (cubicle room) to warm the ambient and intra cage followed the same husbandry procedures but had operated with temperatures. Heat from both types of hand warmers did not different procedures for use of personal protective equipment (PPE). significantly raise intra cage temperatures when applied to the PPE procedures in Facility 1 were based on historical convention and bottom of the cages or when placed in a static environment. The heat included mandatory use of hair nets, surgical masks, shoe covers, from the hand warmers seemed to dissipate too quickly to be of any gowns, and vinyl gloves. In Facility 2, choice of PPE were based on benefit. The microwaveable heat pads did significantly raise tempera- evidence-based risk to staff and animals and involved only gowns, tures in a static environment. The portable heater raised the intra nitrile gloves, enclosed shoes, and optional P2 face masks. Both cage temperatures significantly on a single- sided rack inside a facilities complied with Australian animal research regulations, and

654 Abstracts of scientific papers 2016 AALAS National Meeting

while both met the required levels of pathogen exclusion and chemicals and biologic agents. Initial user feedback revealed user containment, there was a need for operational uniformity. we sought fatigue and overheating with our initial PPE choices (one yellow to implement the same PPE requirements across both facilities. Risk spunbonded polypropylene gown worn under one blue polyethyl- analyses were conducted in relevant aspects of facility operation and ene-coated polypropylene spunbonded gown), resulting in reduced included consideration of biologic hazards, allergens, cytotoxic user compliance. We set out to establish comfortable alternatives that hazards, noise, and radiation exposure. The most appropriate PPE minimize the risk of exposure to different types of liquid hazards by was matched to each risk based on Standards Australia criteria, physically testing the permeability of various protective gown/ animal research legislation, and scientific literature. Results of the sleeve combinations using a commercially available UV-fluorescent analysis were communicated to all stakeholders and changes in PPE liquid. One standard yellow spunbonded polypropylene gown with implemented on an agreed date. Live sentinel screening programs, white impermeable nonwoven spunbonded olefin sleeves provided conducted every 3 months, monitored animal health status 1 year maximal user comfort and appropriate liquid barrier protection in 2 prior to, during the PPE change, and for a further 18 months after the events determined to carry the highest risk of liquid exposure when change in PPE. The risk analysis indicated that the PPE regime in handling animals (mouse urination and liquid smear). Engineering Facility 1 was unlikely to make an effective contribution to the standards currently in place in our animal rooms (handling animals protection of users or animals and may even increase the risk of exposed to biohazardous and chemical agents in biosafety cabinets, pathogen spread. For general operations, the only PPE considered housing animals in individually ventilated cages) should absorb essential were those used in Facility 2. Other PPE were required on a remaining risks not addressed with PPE. Overall, functional case-by-case basis. Live sentinel testing conducted prior to, during, performance of specific PPE used in hazardous agent studies should and post PPE change showed no change in disease status in either be evaluated prior to use. Additionally, PPE standard operating facility. The benefits of removing unnecessary PPE included less procedures should take into account ease of use for the animal confusion among users who worked in both facilities, significant cost handler to ensure proper compliance. saving, reduced risk of pathogen spread, and better protection of facility users while still maintaining SPF status. P191 The Effects of Mainstream Media on Public Perception of a Baboon Research Program P189 Using Positive Reinforcement Training with Dogs Makes the Job Easier TJ Holbrook*

SA Iliff*, J Amador, L Zacniewski, GM Savastano Comparative Medicine, The University of Oklahoma Health Science Center, Newalla, OK Merck & Co., Inc., Kenilworth, NJ This study investigates the effects of mainstream media on public Implementation of positive reinforcement training (PRT) programs perception regarding a university’s baboon research program. A can be beneficial in research settings, reducing the need for manual survey was administered with participants recruited using the restraint or anesthesia and enhancing personnel and animal safety, researcher’s network of contacts. The survey contained 2 actual which can decrease stress for animals and staff. PRT was employed mainstream media clips covering the baboon program. One clip with a colony of beagles to gain their voluntary cooperation on portrayed the program positively and the other negatively. Partici- husbandry procedures. An animal behaviorist provided an overview pants viewed each clip then answered questions immediately after and demonstrated paws-on training. One technician was selected to related to the baboon program. The survey was counterbalanced. train existing pairs of run-housed beagles. Three desired behaviors Results of a mixed model ANOVA revealed a main effect for both were selected: animal comes to trainer, sits, and returns to kennel. media type and a main effect for media presentation order on public Verbal commands and criteria were established and animals were perception of the program. There was not a significant interaction trained 5 days per week for 5 minutes per animal per session. between media type and media presentation order. Based on the Implementation of PRT led to all beagles enrolled in the program results from this research, I suggest the university employ crisis successfully exhibiting behaviors on command. The dogs’ coopera- management strategies and techniques in order to mitigate further tion has enhanced staff satisfaction and made the job of cleaning damage caused by the negative exposure regarding the baboon easier and more efficient. Additional benefits include exercise for the program. Although the decision to phase out the baboon program dogs and more social interactions with other dogs and staff. The dogs has been made, the process could take several years to accomplish. are currently being trained on behaviors that will assist in obtaining University officials must recognize that the media and animal rights weights and transferring to kennels for room sanitation. This groups are likely to continue to monitor the baboon program, and expansion of the dogs’ repertoire is based on their recognition and there is continued risk for media exposure concerning the baboon response to the foundational behaviors. program and the university’s other lab animal research programs. It is important to keep in mind that a snowball sample was used. P190 Development of Personal Protective Equipment Require- Participants were not randomly chosen so the results may not be ments for Handling Animals Exposed to Hazardous Chemicals and representative or generalizable of the whole population. The Biologic Agents participant sample was limited. Only the researcher’s contacts were used so it is possible that some of the participants may have had TE Noe*, BS Blank, A Brubaker, K Yager, B Reddyjarugu, B Collins, previous knowledge of the crisis situation prior to taking the survey EK Daugherity which could have influenced their perception on the situation prior to taking the survey. There was only 1 media clip used for each slant Cornell University, Ithaca, NY of media coverage. The positive media clip only used some of the image restoration strategies. It is unclear if results would replicate for There is no specific regulatory guidance to govern institutional other image repair strategies. requirements for personal protective equipment (PPE) choice when handling animals exposed to hazardous chemicals and biohazards. P192 Programmatic Design of a Safety Program at a Primate Center Researchers are usually well-acquainted with the chemicals and biologic agents they are using, but animal husbandry staff and T Fountain* weekend veterinary care staff are less likely to be aware of the specific risks associated with individual agents. Little is known about Yerkes National Primate Research Center, Atlanta, GA the risks associated with animals excreting chemical compounds after exposure. We are standardizing hazardous agent signage at the There are numerous challenges to address in an occupational health animal housing room level and developing requirements for and safety program when working in a primate research facility. Haz- appropriate PPE when handling mice exposed to hazardous ards are present in every area ranging from animal care, facilities

655 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 management, as well as research labs. These hazards need to be winters. Macroenvironmental humidity within animal facilities identified both from the research aspect as well as the daily opera- exhibited seasonal variability and values recorded from in-room tions of managing an animal facility. In order to achieve this, it is digital hi-lo hygrometers were frequently below the range recom- necessary to have an effective safety program. We have developed a mended by the Guide as noted on accreditation and IACUC comprehensive safety program managed within the Safety Office semiannual evaluation reports. We sought to determine for our which is located onsite at the facility. The office is comprised of a IACUC the accuracy of digital hi-lo hygrometers compared to registered nurse who serves as the Safety Officer; the occupational centralized environmental monitoring systems (CEMS) and the health manager; the training and compliance coordinator; the actual microenvironmental humidity experienced by pair-housed containment manager; and the biohazard waste technician. The CD-1 mice static and individually ventilated cages (IVC). Humidity ability to have the occupational health, training, compliance, and data was collected using 3 separate methods, an in-room digital hi-lo other aspects of a safety program located within the facility serves to hygrometers, intracage humidity data loggers, and CEMS, from remove barriers to safety and moves the center towards an effective February through April 2016, in 3 rooms in 2 separate buildings. safety program to meet the unique challenges of working in research, Humidity was recorded once daily for digital hygrometers and every particularly with nonhuman primates. Other methods in support of 6 hours for other methods. Data was analyzed using a 1-way an effective safety program at the center include the development ANOVA. Data showed a significant difference between digital and maintenance of standard operating procedures, a safety hygrometers (M= 30.1%, SD=2.2) and CEMS (M= 42.7%; SD=0.8; P = committee with representatives serving from various departments .0001). In addition, the microenvironmental humidity measured in and a quality assurance program. In addition, the Safety Officer static cages was significantly higher (M= 69.9%; SD=1.7) than that serves on several University committees, such as IBC and IACUC. measured by the CEMS (M= 43.2%; SD=0.28; P = .0001). Intracage The structure of this safety program facilitates interdisciplinary IVC humidity (M=45.8%) was significantly lower than that of static collaboration with personnel who become invested in this program, cages (M=69.95%; SD=1.7; P = .0001). Microenvironmental humidity which ultimately promotes the incorporation of safety in all aspects experienced by mice in static caging was higher than relative of the center. humidity of the secondary enclosure and micoenvironmental humidity experienced by mice in individually ventilated caging more P193 A Novel Cage Retrofit Design to Allow Social Housing of closely approximated the relative humidity of the Rabbits macroenvironment. Based on these findings we recommended that use of in-room digital hi-lo thermometers/hygrometers be S McBride*, M Whalen, WR Kingsbury, JA Scholz, C Frisk, TR Meier discontinued in areas where CEMS existed.

Comparative Medicine, Mayo Clinic, Rochester, MN P195 Genotyping of Cryopreserved Mouse Lines

According to the Guide, social animals must be socially housed unless A Navis*, E Kalkbrenner, PL Roesch, AV Perez 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 Taconic Biosciences, Rensselaer, NY allow social housing. We wished to modify our existing cages to allow social housing and avoid the cost of purchasing new cages. Minimizing animal use is an essential component of laboratory Our goal was to develop a system with a gate that could be opened animal research. For mouse lines that are not in use, freezing sperm for social housing and easily closed when needed. The gate mecha- samples of the line for later recovery is an attractive way to reduce nism needed to be independent and not directly attached to the cage animal usage. While this method is a powerful way to minimize so the cage could be disassembled for cleaning. The mechanism for animal use, it also introduces a number of challenges for later opening, closing, and securing needed to be simple for ease of studies. One primary challenge is identifying the genomic character- operation while minimizing parts needed for future maintenance or istics of these lines prior to their recovery. For live animals, tissue repairs. Finally, the type and size of the socialization opening needed samples are easy to obtain. However, for frozen lines, critical to be appropriate for a wide range of rabbit sizes. We designed a information may be out of date or incomplete, adding uncertainty to triangular (“A”) shape gate mechanism that slides between 2 a costly recovery procedure. Here we describe a method for side-by-side cages. We decided upon a 7-inch opening and worked analyzing the genotypes of sperm samples to improve quality with a local metal fabricator to design a prototype that could easily control. The method is a direct comparison of 3 different loci in the be slid into place using existing frame structure of the rabbit rack. cryopreserved sperm samples, archived tissue samples of known Two prototypes were made: one with a 7” round hole and one with a genotype, as well as samples from the background control strains (B6 7” square hole. We piloted the protypes with pairs of adult New and FVB). To validate the method, we genotyped 5 sperm samples Zealand White and Dutch Belted rabbits. Both breeds of rabbit were and 3 related tissue samples using real-time PCR. Each sample was easily able to use 7” hole with no preference for the square hole or the tested for the SNP markers CRB1 and PDE6B, and for the presence of round hole. We chose the square hole because it would be easier and a Cre-Luciferase transgene. We used this information to confirm that more cost effective to fabricate. The 1 modification that was made the sperm samples carry the desired transgene and that they after the pilot study was the addition of the 1” holes to the sliding contained markers from the proper background. Our genotyping suc- gate so that if the rabbits needed to be housed individually housed, cessfully identified the genotype of the frozen samples and live they could still have visual contact without nose contact when it was tissue. This method can provide valuable quality control information in the closed position. We are now successfully using the cages to for frozen sperm and could also be extended to analyze other aspects house social pairs and trios of rabbits. The cage modifications were a of the line including the background strain. This analysis improves key component in developing our rabbit social program while quality control procedure for existing and future cryopreserved lines providing a budget-friendly solution. to help prevent the generation of excess research animals while also avoiding the duplication of costly work. P194 Differences in Humidity Readings across 3 Common Testing Devices P196 Comparison of Animal Welfare Issues and Vaccinia Virus Expression Kinetics of NanoLuc and Firefly Luciferase Marker ZK Dietz*, K Clark, A Rodecap, N George, WA Hill Genes in TC-1 Tumor-Bearing Mice

Animal Care Unit, University of Kansas, Lawrence, KS RS MacGill, AR Alfaro*, J Lallibert, NC Peterson, S Robbins

According to the Guide, the acceptable range of relative humidity is Laboratory Animal Resources, MedImmune, Gaithersburg, MD 30% to 70% for most mammals. Situated in Lawrence, Kansas we experience a relatively dry climate with hot summers and cold Bioluminescence has become a valuable tool for measuring in vivo

656 Abstracts of scientific papers 2016 AALAS National Meeting

endpoints such as PK/PD relationships, tumor growth, and postulates that IL-6 may be involved in stress associated social metastases. One advantage of bioluminescence imaging is its impairments. application to longitudinal studies using nonlethal quantitation, thereby greatly reducing intraanimal variability and the number of P198 Turkeys as a Model for Flexor Tendon-Related Research animals to be euthanized for these types of studies. Here we report on the comparisons of vaccinia virus (VACV) expressing a new AM Vrieze*1, A Kadar2, R Reisdorf1, A Thoreson1, S Moran2, P bioluminescent protein, NanoLuc (VACV-NL), or firefly luciferase Amadio1, C Zhao1 (VACV-FL) in regards to technical feasibility and vector expression kinetics following intratumoral injection into TC-1 tumors, a murine 1Orthopedic Surgery, Mayo Clinic, Rochester, MN; 2Mayo Clinic, model of human papilloma virus-associated cancers. We measured Rochester, MN transgene retention at the site of injection and expression kinetics following intratumoral injection of VACV-NL or VACV-FL into TC-1 Canines are commonly used for flexor tendon-related research, tumors. TC-1 tumors were established by injection of 20,000 viable however there are ethical considerations associated with their use as cells into the left footpad of C57Bl/6 mice. Initial comparisons they are companion animals. The chicken is another model com- between IP and IV administration of NanoLuc’s furimazine substrate monly used, having a major disadvantage regarding size that found IP administration was lethal in a portion of the mice, and prevents performing complex surgeries on. We hypothesized that the resulted in nonspecific abdominal luminescence. IV administration of turkey will be a suitable large animal model for flexor tendon furimazine was well tolerated and the bioluminescent signal was research, and designed an in vitro comparative trial to evaluate all localized to the tumor. However, NanoLuc bioluminescent imaging models. We compared the canine, chicken, and turkey model to the was complicated due to the short transition period between IV human using the following cadaver parts: third digit from the dog administration of furimazine and the start of imaging (<1 min.), and forepaw, the third digit from human hands, and the third digit of great care had to be taken to avoid excessive heating while dilating turkey and chicken feet. Comparing all 4 species anatomy, structure, the tail veins. Repeat IV administration via the tail vein was and biomechanical properties by means of the digits range of motion, marginally well tolerated, but often resulted in scarring and work of flexion, tendon excursion, and by a simple suture pull out sometimes loss of the tail. In contrast, bioluminescent imaging of strength.The anatomy and structure comparisons identified the VACV-FL was technically uncomplicated. In direct comparisons turkey superiority to the canine and chicken in several aspects. The between VACV-NL and VACV-FL delivered at 1e7 plaque forming turkey digit was much larger than the chickens and it was not units intratumorally, similar expression kinetics were noted. webbed like the canines. Work of flexion for the turkey was similar to However, the VACV-NL had greater half-life relative to VACV-FL. human and canine. The cross-sectional area, and the gliding This is most likely due to the greater half-life of NanoLuc relative to resistance of the turkey tendon was much closer to the humans than firefly luciferase. These results indicate bioluminescence imaging is a its counterparts as well. The canine had a significantly higher simple suitable means to monitor VACV expression in oncology studies. suture pull out strength; this was explained by the fibrocartilage Firefly luciferase was technically easier to use, had fewer animal abundant in the canine tendon, different than human and turkey. The welfare concerns, and is likely the most relevant for studying vector turkey had a significantly lower range of motion than human. We expression kinetics. were able to confirm our hypothesis that the turkey flexor tendon is similar to human in tendon size, shape, and biomechanical proper- P197 Early-Life Stress Alters Correlations between IL-6 and ties; only faltering when it comes to arc of motion. In vivo studies Depression Behaviors will need to be conducted to determine if the turkey model is suitable in terms of husbandry and tendon healing. A Hicks-Nelson*, B Nephew P199 Identifying Canine Flexor Tendon Repair Failure on a Biomedical Science, Cummings School of Veterinary Medicine, North Pressure Sensing Walkway Grafton, MA AM Vrieze*1, R Reisdorf1, A Thoreson1, P Amadio1, S Moran2, C Zhao1 Postpartum depression, anxiety disorders, and disabling mood disorders are increasingly common and impacting mothers and their 1Orthopedic Surgery, Mayo Clinic, Rochester, MN; 2Mayo Clinic, children more frequently than ever. However, most depression Rochester, MN research in animals only includes males. This limitation has led to significant gaps in our knowledge of these disorders. Recent studies Using canine model for flexor tendon-related research is common as from other laboratories indicate the significant role of the immune the anatomic, structural, and biomechanical properties of the canine system in what our laboratory has shown to be transgenerational flexor tendons are comparable to humans. Developing novel behavioral effects. Naturally occurring increased levels of IL-6 in techniques to evaluate digit function following flexor tendon repair mice have been associated with stress susceptibility, leading to a or graft surgeries in live dogs versus sacrificing is crucial. It will depression-like phenotype following social stress. The current study decrease the number of animals used by longitudinally evaluating investigated the role of cytokines in the effects of chronic social stress the digit functions, and also provide accurate force distribution, time, (CSS) on rat F1 offspring behavior. The CSS model of postpartum and pressure values of individually treated digits of the paw. The aim depression and early-life stress depresses maternal care in F0 of this study was to evaluate dog digit function associated with flexor generation dams using an intruder male to antagonize the dam, tendon repair on a newly developed pressure walkway system. The simulating neglect and establishing a cycle of deficient social and second and fifth flexor digitorum profundus (FDP) tendons from 11 maternal behavior in the F1 and F2 generations. We hypothesized dogs were transected at the proximal interphalangeal joint and that impaired social behavior of F1 juveniles would be associated repaired with modified Kessler technique. The pressure sensing with increased peripheral IL-6 concentrations. There was a strong walkway was used to assess the paw pressure while walking before correlation (r2 =.86, p < 0.001) between social investigatory bouts and repair surgery and 14-21 days post repair. Dogs were pretrained to IL-6 for control juvenile F1 females that was absent in their CSS walk on the system; a camera captured their movements enabling counterparts. Similarly, there was a moderately strong correlation identification of each cycle of gait. At least 10 passes were collected between IL-6 and both self-grooming frequency (r2 =.46, P = .04) and on the walkway per visit and each pass yielded up to 2 gait cycles. duration (r2 =.49, P = .03) only in the juvenile F1 control females. In Due to active motion, the repairs ruptured, which was confirmed 42 conclusion, CSS disrupts the robust associations between social- and days postoperatively in all 11 dogs. The peak pressure, average depression-associated behaviors and basal IL-6 concentration seen in pressure, and contact area of the surgical second and fifth digits were control F1 juvenile females. This correlation, while not yet a direct decreased compared to the second and fifth digits of the nonsurgical cause-and-effect relationship, further advances the immune system paw measurements. However, the central pad pressure increased in as a reservoir of targets for future therapies for mood disorders and the surgical paw compared to the nonsurgical paw, which indicated

657 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 that dynamic pressure was altered due to FDP tendon repair failure. and suture dehiscence upon fixation, and excessive use of technician The new pressure walkway system is a valuable tool when assessing time. Stapling the skin to the underlying muscle was the most the paw and digit function during the dogs’ gait cycle, which would effective methodology in terms of time and consistency, albeit at an be beneficial outcome measurements for other flexor tendon related increased cost in materials. Studies using the stapler method in research using the canine model. conjunction with exact anatomic localization of injection sites at dosing had significantly (p < 0.05) improved recovery of injection P200 Inhibin Antiserum for Superovulation of Genetically sites upon histopathological evaluation when compared to other Engineered Mouse Lines methods. Tissue immobilization by securing skin and muscle edges with a surgical stapler at all cut margins, in conjunction with multiple A Dasari* section sampling, results in more accurate and complete sectioning for histopathology evaluation of IM dose sites, and is associated with Product and Program Management, Taconic Biosciences, Rensselaer, decreased overall use of resources including technician time. The NY instituted measures also help ensure consistent and accurate collection of IM injection sites, and support high-quality tissue Assisted reproductive technologies have become indispensable for assessment. efficient production and management of mouse and rat breeding colonies. Recent advances in superovulation technique, coadministra- P202 Assessment of Buprenorphine Sustained-Release Efficacy in a tion of Inhibin anti-serum+equine chorionic gonadotropin (IASe), Rabbit Survival Surgical Model followed by human chorionic gonadotropins (hCG) at 48-hour interval reported to have increased the oocyte yields by 3-4 times A Zanetti*, S Putta, C Franciozi, S Louie, M Humayun, D Casebolt from prepubescent wild type C57BL/6 mice. However, to date there has been very little data on using IASe for superovulation of Department of Animal Resources, University of Southern California, genetically engineered mice (GEM) and its application in a large- Los Angeles, CA 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 In rabbits, buprenorphine instant release (IR) is commonly used to hours apart per animal) or IASe (0.2 ml of Hyperova and 7.5 IU of alleviate pain, especially in surgical models. However, maintenance HCG, 48 hours apart per animal) methods were compared for oocyte of IR therapeutic levels requires redosing which significantly yields/oocyte donor, fertilization rate, and development to term. The increases unnecessary animal handling, restraint, and noncompliance oocytes generated by IASe method were fertilized in vitro following issues postoperatively. We evaluated the most effective dose of the standard IVF protocols in mouse, and the 2-cell embryos sustained-release (SR) formulation of buprenorphine in rabbits, as generated were implanted into the oviducts of 0.5dpc pesudopreg- indicated by adequate drug plasma concentration and satisfactory nant recipients for in vivo development to birth. IASe method analgesia, thus improving the standard of care. We hypothesized that produced a significantly higher number of oocytes per oocyte donor single subcutaneous administration of 1 of 3 SR concentrations than standard superovulation method. IASe method improved the (SRLow-0.06 mg/kg, SRMedium-0.15 mg/kg, SRHigh-0.3 mg/kg; n overall efficiency of rederivation by increasing the numbers of = 3 males per group) would provide up to 72 hours of pain relief and embryos and live pups generated per oocyte donor used in IVF. more predictable behavior and pain associated parameters than IR Improvement in efficiency of rederivation of mice through IVF-ET (0.05 mg/kg BID) in a rabbit knee defect model. To evaluate pain and technologies reduce the number of oocyte donors required. In associated parameters, body weight, fecal output, grimace, and summary, we believe IASe in combination with meticulous planning lameness scales were used to score pain based on daily assessment and execution of large scale derivations will help us with striking a obtained before and within 72 hours after surgery. Results showed balance between our commitment to 3R, and high-quality services to there was no skin reaction to any of the tested SR concentrations. researchers. Postoperative assessment also revealed no significant changes in body weight across groups. Daily fecal output was higher in rabbits P201 Improving the Collection Accuracy of Common Intramuscular treated with SRLow and SRMedium than IR and SRHigh. Single Injection Sites for Histopathology in Rabbits right limb lameness and signs of pain (grimace scale) were only observed in rabbits treated with IR (24 hours) and SRLOW (24-48 AL Doan*, M Ashley, B Harmsen, C Hollinger, K Nelson, DVM, PhD, hours). Consequently, only rabbits treated with SRLow received DACVP meloxicam (0.3 mg/kg PO SID) within the first 48 hours. Pain was

not observed in SRMedium and SRHigh group. As a conclusion, MPI Research, Mattawan, MI these preliminary results indicate that 0.15 mg/kg of SR buprenorphine (SRMedium) provided effective analgesia and safely manages Intramuscular (IM) dose sites are commonly used for vaccine safety pain in rabbits enrolled in the selected surgical model. studies because of rapid and uniform absorption of test compounds. Rabbits are a common model because they mount a robust immune P203 Targeted Buprenorphine Sustained-Release Analgesia for response to vaccine administration and are large enough to test full Guinea Pigs 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 A Zanetti*, S Putta, S Louie, D Casebolt plane, allowing pathologists to most reliably assess test compound effects. However, collection of consistent high quality IM injection Department of Animal Resources, University of Southern California, sites for histopathology in rabbits is impeded by the loose attachment Los Angeles, CA of rabbit skin to the underlying muscle, resulting in unacceptable margins of error for IM dose site collection with routine procedures. Sustained-release (SR) formulations of buprenorphine were recently We hypothesized that improved postmortem skin immobilization introduced into the veterinary market to minimize redosing, and specimen collection methods would improve the accurate restraint, and noncompliance issues. Therefore, studies addressing collection of rabbit IM dose sites for histopathology. Use of multiple the SR efficacy, safety, and pharmacokinetics (PK) in laboratory sections through externally marked injection sites improved species are becoming common within the laboratory animal histopathology evaluation, but was insufficient. Multiple methods of community. In guinea pigs, however, such studies remain lacking. In skin and muscle apposition were attempted, including suturing, an attempt to fulfill the gap, we assessed the PK of buprenorphine SR pinning to a cork board surface, and stapling. Pinning the excised (0.15-0.6 mg/kg SQ once) and instant release (IR-0.025-0.1 mg/kg SQ muscles and skin to a cork board prior to fixation or suturing muscle BID) in 8 (4 males and 4 females) healthy Hartley guinea pigs using a to overlying skin had suboptimal outcomes, with tissue deformation validated liquid chromatography with tandem mass spectrometry

658 Abstracts of scientific papers 2016 AALAS National Meeting

(LC-MS). At the same time, we also monitored animals for safety Aspiculuris tetraptera continues to be a problem in rodent vivariums based on physiologic parameters (i.e. weight, heart rate, respiratory partly due to difficulties in parasite detection. While PCR testing is rate, and body temperature) and the presence of potential adverse highly sensitive, it is expensive and does not always provide effects (i.e. anorexia, injection site reaction) using noninvasive immediate results, so many institutions rely on passive fecal flotation methods. According to the results, physiologic parameters were as a quick inhouse exam for diagnosing A. tetraptera infections. In within normal range with less than 10% body weight loss observed order to increase the sensitivity of this test, we examined multiple in all guinea pigs during the first 2 days post buprenorphine parameters to determine the optimal test protocol. A 30-minute administration. Pharmacokinetic analysis of IR and SR revealed the soaking period prior to a 15-minute fecal flotation time allowed fecal time to maximum concentration (Tmax) to be around 1 hour. At this pellets to soften and facilitated efficient egg isolation. We also time point, the maximum concentration of the drug in the plasma of evaluated the impact of time of day, sample size, age, sex, and social healthy guinea pigs was 2.41±0.56 ng/ml for IR and 1.7±0.13 ng/ml housing on egg isolation. No evidence of cyclical egg shedding was for SR. Twelve hours post administration, the plasma concentration found, and while larger fecal sample sizes did not result in more eggs of drug was 0.35±0.07 ng/ml (IR) and 1.06±0.57 ng/ml (SR). isolated, incidence of false negative exams was reduced. The most Buprenorphine SR remained within the detectable range for at least eggs were isolated from 8- and 12-week-old mice, and as mice aged, 72 hours. All together, SR (0.3 mg/kg SQ once) provided sustained numbers of eggs isolated declined. Overall, sex and social housing levels of the drug in the plasma of healthy guinea pigs for up to 3 did not impact the number of eggs isolated. Finally, examination of days with no major adverse effects to the animals. Further investiga- multiple diagnostic tests (fecal flotation exam, direct examination of tion is needed to determine if this SR concentration can be safely cecal and colonic contents, and fecal PCR) revealed no single test was used to alleviate pain associated with survival procedures involving definitive, indicating multiple tests may be required to successfully guinea pigs. screen mice with low pinworm burdens. These findings provide guidance in sample selection, collection, and processing to efficiently P204 Development and Validation of a Bleeding Time Assessment detect A. tetraptera. Model in the Nonhuman Primate P206 Automated Blood Collection for Arterial Blood Gas Sampling AM Lelkes*1, J Sheehan1, T Ramani1, K Conde-Knape2, T Ziegelhofer1 in Unrestrained Monkeys

1Envigo, East Millstone, NJ; 2Johnson and Johnson Innovation, D Hopper4, BM Kemmerling*1, P Kruzich4, L Kinter5, T Riggs3, K London, United Kingdom Bigge6, S Kurtz1, C Kreilein3, N Suttles2, M Shelton3

Bleeding time assessments are used in drug development to 1Veterinary, Bioanalytical Systems, Inc., Mt. Vernon, IN; 2Animal determine the efficacy, safety, and dose response of an anticoagulant. Care, Bioanalytical Systems, Inc., Mt. Vernon, IN; 3Clinical Pathology Many animal models have been described in literature; however , Bioanalytical Systems, Inc., Mt. Vernon, IN; 4Toxicology , Bioanalyti- there are surprisingly few studies on suitable methodologies in cal Systems, Inc., Mt. Vernon, IN; 5GLP Scientific Consulting, nonhuman primates. Whereas using a template bleeding method in Unionville, PA; 6Chemistry, Bioanalytical Systems, Inc., Mt. Vernon, the skin may seem straightforward, it has been shown to be IN unreliable for drugs that do not interfere with platelet function such as Factor Xa inhibitors. This project aimed to develop and validate a Measuring the impact of drug-induced changes in respiratory reliable and reproducible bleeding time assessment model in function and acid-base status requires serial monitoring of blood pH, nonhuman primates for the investigation of factor Xa inhibitors. pCO2, PO2, and HCO3.. Blood sampling during standard cardiovascu- Apixaban (0.1 and 1 mg/kg) was used as a positive control to lar radio-telemetry studies is less than optimal due to repeated investigate internal organ bleeding techniques. Organ bleeding disturbance of subjects. This study assessed the feasibility of serially requires a complete coagulation cascade response and agents that collecting respiratory blood gases, pH, and HCO3 in cynomolgus interfere with coagulation factors such as factor Xa inhibitors monkeys with automated blood collection and to detect acute (Apixaban) can be better studied using an internal organ bleeding acidosis and alkalosis in conscious monkeys under cage conditions methodology. The liver was selected due to ease of access, consis- similar to radio-telemetry studies. Four tethered cynomolgus tency of bleeding, and ability to quantify total amount of blood loss. macaques (2/sex) were administered IV infusions of saline (0.75 mL/

An incision of 2mm x 10mm was made in the right liver lobe using a kg bolus plus 0.125 mL/kg/min for 20 min), NH4CL (250 mg/kg scalpel blade. Using filter paper and gauze, timed blood collection over 20 min), HCO3 (1680 mg/kg over 20 min), and remifentanil (12 from the incision site was performed until no further bleeding was µg/kg bolus plus 2 µg/kg /min for 20 min) in a modified Latin observed. Weights of the filter paper and gauze were also taken to Square design wherein each animal received test article on different record volume. A positive control pilot study with 4 adult male days. Serial carotid arterial blood samples (400 µL) were withdrawn cynomolgus macaques showed significant differentiation of bleeding by programmed automated blood collection (Culex-L ABC) at time between saline and Apixaban. In the main study, 3/sex baseline, then from 5 minutes to 360 minutes post-infusion via a cynomolgus macaques per group (saline, 0.1 mg/kg Apixaban, 1 vascular access port. Samples were collected and analyzed for mg/kg Apixaban), were used to evaluate the feasibility of the pCO2 and pO2, pH, and HCO3 (RapidPoint 405 Analyzer).. Baselines selected liver bleeding method. The liver methodology developed values for all parameters were within normal ranges. NH4Cl resulted in fairly consistent bleeding times that allowed for discrimi- produced metabolic acidosis (pH -0.2 units, HCO3 -50% by 5 min) nation between 2 different dose levels of Apixaban, a Factor Xa followed by compensatory respiratory alkalosis. NaHCO3 produced inhibitor. It allowed for bleeding to be recorded in a quantifiable way rapid metabolic alkalosis (pH +0.25 units, HCO3 +100% by 5 min) by recording both time and volume of blood loss. This experiment followed by compensatory respiratory acidosis. Remifentanil resulted in a robust model for evaluating bleeding time at different produced rapid respiratory acidosis (pH -0.21 units, HCO3 -15% by 5 dose levels of a Factor Xa inhibitor in nonhuman primates. min) followed by recovery with dissipation of respiratory depression. This study demonstrated the feasibility to collect serial respiratory P205 Optimizing Inhouse Sample Collection and Processing for blood gas, pH and HCO3 concentrations under experimental Identification of the Mouse Pinworm Aspiculuris tetraptera conditions compatible with cardiovascular radio-telemetry, and thereby to combine ICH S7A core battery respiratory and AE Goodroe*2,1, VK Baxter1, J Watson2,1 cardiovascular function assessments within a single study while supporting the 3R’s of animal research by decreasing animal use. 1Molecular and Comparative Pathobiology, Johns Hopkins Univer- sity, Baltimore, MD; 2Research Animal Resources, Johns Hopkins School of Medicine, Baltimore, MD

659 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P207 Corynebacterium bovis Infection of Immunocompromised high infection rate of MuAstV in the laboratory mouse colonies in Mice Variably Impacts Tumor Xenografts Taiwan, and MuAstV-NTU1 is currently the only variant characterized molecularly outside the USA. CM Santos*1,2, A Chandler1,2, A Valdivia1,2, P Villanueva1,2, S Camargo1,2, T Williams1,2, C Surratt1,2, M Ross1,2, S Montgomery1,3, D P209 A Comparison of 2 Buprenorphine Quantitative Methods Darr1 CA McGee*1, F Lih2, PH Myers1, DR Goulding1, TL Blankenship1 1Lineberger Comprehensive Cancer Center, University of North Carolina—Chapel Hill, Chapel Hill, NC; 2Division of Laboratory 1Veterinary Medicine Section, Comparative Medicine Branch, Animal Medicine, Chapel Hill, Chapel Hill, NC; 3Department of National Institute of Environmental Health Sciences, NIH, DHHS, Pathology and Laboratory Medicine, Chapel Hill, Chapel Hill, Research Triangle Park, NC; 2Mass Spectrometry Research and Chapel Hill, NC Support Group, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC Corynebacterium bovis (C. bovis) is a common, gram-positive bacterium frequently encountered in research vivaria, particularly in those The analgesic buprenorphine is commonly used to alleviate pain in housing immunocompromised mice. Although nonpathogenic in laboratory rodent models undergoing surgical procedures. Many immunocompetent animals, C. bovis clinically manifests as a dosages, routes, and frequencies have been described. The therapeu- hyperkeratotic dermatitis in naïve, immunocompromised mice. tic levels of buprenorphine in mice have been reported between 1ng/ While it is often anecdotally reported that C. bovis infection decreases ml–8ng/ml depending on the level of pain associated with the xenograft tumor take rates and subsequent tumor growth profiles in procedure. Two primary methods have been identified in the nude mice, published data on this as well as in other laboratory animal science literature for measuring buprenorphine in immunocompromised strains is lacking. To determine if C. bo- serum. A commercially available forensic enzyme linked immunosor- vis infection had an effect on tumor take and growth rates, we bent assay (ELISA) has been described to quantitate buprenorphine; subcutaneously inoculated commonly used carcinoma cell lines however it is marketed as only a qualitative assay. Liquid chromatog- (MDA-MB-231 [human breast adenocarcinoma] or SKOV3 [human raphy-mass spectrometry (LC-MS) is the gold standard of ovarian clear cell adenocarcinoma]) into either nude mice buprenorphine quantification. We compared these 2 methodologies (Crl:NU(NCr)-Foxn1nu) or NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) for accuracy and precision in detecting buprenorphine concentrations mice with or without active C. bovis infection. C. bovis status was in the serum of mice receiving either standard buprenorphine HCl determined using a combined skin/buccal swab prior to cell (Bup) or a commercially available sustained release buprenorphine inoculation and at the study endpoint. C. bovis-positive and -negative (SR-Bup) product. A single dose of Bup or SR-Bup was administered colonies were housed in separate rooms and C. bovis status was by the subcutaneous route. Mice were euthanized at designated time evaluated weekly by exhaust air swabs. Tumor presence and growth points over 24 hours and blood was collected. Each sample collected was monitored twice weekly for 60 days using caliper quantitation. was used for both the ELISA and LC-MS assay. We also measured For nude mice, both tumor take and growth rates for both tumor cell buprenorphine metabolites in each sample by LC-MS. The ELISA lines were significantly reduced in C. bovis-positive mice in compari- data had greater variability than the LC-MS data. By comparing the son to C.bovis-negative mice. However, for NSG mice, which are LC-MS buprenorphine both with and without the combined more severely immunocompromised, tumor growth and take rates metabolites to the ELISA data we determined the ELISA detects the were not significantly altered by C. bovis status. Our study provides glucuronides which can falsely elevate the level of buprenorphine. evidence that C. bovis infection can negatively impact carcinoma While the glucuronides have been described as biologically active the xenograft models, but that the impact varies across strains, likely due antinociceptive effect has been reported to be significantly less to the nature of immunocompromise. Our findings have important compared to buprenorphine when evaluated by tail flick latency or implications to the impact of C. bovis in tumor xenograft studies and tail withdrawal tests. The ELISA may, therefore, give a false management. representation of analgesic level.

P208 Identification of Novel Murine Astrovirus in Taiwan P210 Challenges in Establishing Enhanced Gut Microbial Diversity in Laboratory Mice C Su*1, C Wan1,2 DR Montonye*1,2, C Smith2, A Ericsson3,4, CL Franklin3,4 1Graduate Institute of Molecular and Comparative Pathobiology, National Taiwan University, Taipei, Taiwan; 2Animal Resource 1Comparative Medicine, University of Missouri, Columbia, MO; 2Vet- Center, National Taiwan University, Taipei, Taiwan erinary Pathobiology, University of Missouri, Columbia, MO; 3Mutant Mouse Resource and Research Center, University of Mouse astrovirus was first identified in mice in 2011. Lately, the Missouri, Columbia, MO; 4MU Metagenomics Center, University of surveillance data have indicated that murine astroviruses (MuAstV) Missouri, Columbia, MO are highly prevalent in laboratory mice in U.S. and Japan. However, the existence of murine astroviruses and prevalence of infection are Mice are widely used in biomedical research, and optimizing still unclear in laboratory mouse colonies in Taiwan. In this study, translatability of mouse models to recapitulate human disease identifying the MuAstV-like virus in Taiwan and surveying the processes is highly desired. It has been speculated that the diversity infection status in laboratory mouse colonies were pursued. First, the of the gut microbiota (GM) of laboratory mice has decreased over the fecal or tissue samples from different strains of mice of various breed- past several decades through practices such as rederivation that were ers and research institutes (n = 9) in Taiwan were screened for designed to render research mice free of opportunistic and overt MuAstV by RT-PCRs. Molecular surveillance results showed that microbial pathogens. Unfortunately, this lowered GM diversity may 57.9% mice were positive for MuAstV, and 7 out of 9 facilities (77.8%) not accurately replicate the GM diversity seen in the human were contaminated. Comparison with the RdRp sequences of population. As differences in microbial diversity have been shown to MuAstVs revealed that the isolates identified in our study (MuAstV- influence disease phenotypes of rodent models, it is conceivable that NTUs) were closely related to the previously-published MuAstVs producing mice with a microbial diversity that resembles that in with 92.4%~97.4% similarity. One MuAstV-NTU isolate was humans could result in more translatable model phenotypes. We molecularly characterized and designated MuAstV-NTU1. Compari- sought to establish laboratory mice with a more diverse GM than is son of the nucleotide and protein sequence alignments revealed that currently available. To this end, 40 mice from 2 local pet stores were the MuAstV-NTU1 was different but closely related to the previously obtained and the GM characterized using next generation sequenc- characterized murine astrovirus and suggested to be a variant of the ing. This analysis showed that these mice had significantly higher same virus species. The present study has demonstrated a distinctly GM diversity than contemporary laboratory mice with an average

660 Abstracts of scientific papers 2016 AALAS National Meeting

74.8% increase in the number of operational taxonomic units (OTUs). Due to their highly sensitive sense of smell and trainability, giant As expected, these mice were also contaminated with a variety of pouched rats (genus Cricetomys) are garnering increasing interest pathogens including, but not limited to, Mycoplasma pulmonis, mouse from the scientific community for applications such as landmine and hepatitis virus, mouse parvovirus, mouse rotavirus, fur mites, tuberculosis detection. Since 1997, organizations such as APOPO in pinworms, and Cryptosporidium sp. To rid mice of these pathogens, Morogoro, Tanzania have operated training and captive breeding the following strategies were employed: treatment with fenbendazole programs to use the giant pouched rats’ talents. However, despite (in feed for 5 months), moxidectin (twice topically 8 weeks apart), their increasing use in multiple African countries and their natural azithromycin (50 µl orally once daily for 4 weeks), cessation of ubiquity in this region, there remains a paucity of publications and breeding, and test and cull. Following this regimen, 5 mice were normative data regarding these animals. With the development of rendered free of adventitious pathogens. Unfortunately, analysis research colonies likely to increase, so too will the need for baseline revealed that the number of OTUs decreased by 65.5% and the GM normal values from healthy individuals and populations against closely resembled that of contemporary laboratory animal mice. which clinical cases may be compared. The present study sought to These findings demonstrate how quickly and dramatically a diverse establish reference intervals (RI) for complete blood counts (CBC) GM can be lost using common procedures designed to eliminate and serum biochemistry and to characterize hemocytological pathogens. Moreover, these highlight challenges faced in creating a characteristics in the southern giant pouched rat (Cricetomys ansorgei). more translatable GM in laboratory mice. Seventy-three clinically healthy animals (35 male, 38 female, 3 juveniles) were wild-caught in Tanzania, and transferred to standard P211 Liquid Microsampling in Rats and Mice laboratory rodent housing conditions at our institution. General physical exams, body condition scoring, and phlebotomy via ventral DN Bilgo*1, PM Mielke1, A Ledvina2, S Ploch3, C Scheuerell4, L tail artery were performed under isoflurane anesthesia. Two Geiger5 milliliters of blood were collected via 23 g needle into microtainers containing EDTA or lithium heparin, stored on cold packs, and 1Animal Operations, Covance, Madison, WI; 2Bioanalytical, Covance, processed by the Animal Health Diagnostic Center Clinical Pathol- Madison, WI; 3Toxicology Operations, Covance, Madison, WI; 4As- ogy Laboratory within four hours of collection. Automated CBCs sociate Pharmacokineticist, Covance, Madison, WI; 5Metabolism, were compared to manual differentials (erythrocytes, leukocytes, Covance, Madison, WI platelets) performed by a DACVP-boarded pathologist, with absolute leukocyte counts calculated from the manual differentials. Addition- Popularity of the dried blood spot method has declined somewhat ally, cytochemical staining was performed to identify the specific due to various regulatory and technical challenges. Consequently, the staining characteristics of various leukocyte types. Leukocyte use of small liquid samples (microsampling) has been gaining staining characteristics were relatively uniform among male and popularity regarding serial sampling in mice and for nonclinical female individuals. The reference interval values for CBCs and serum toxicokinetic (TK) analysis. The benefits of microsample collections biochemistry in the Southern giant pouched rat will be presented. include the reduction of total number of satellite (toxicokinetic) rats and mice (including transgenic strains), ethical use regarding the 3Rs, P213 Establishment of a Home-Cage, In Vivo Method of Rabbit and other potential cost savings. Challenges and inconsistencies have Urine Collection been previously noted while processing serum and plasma samples using glass capillary-based techniques. Therefore, in this study, a DL McEntee*, K Borg, V Markiewicz novel microsample technique was used to obtain and process samples. Female rats [Crl:CD(SD)] and mice [2 strains were used, Drug Safety Research and Development, Pfizer, Inc., Groton, CT Crl:CD1(ICR) and RasH2 (Hemizygous)] were administered a single gavage dose (150 mg/kg for rats and 100 mg/kg for mice) of In preparation for a rabbit study in which stress biomarkers were to acetaminophen. Rats were bled at approximately 0.5, 2, 4, 8, and 24 be measured, a method to collect urine was developed through a hour post dose from a tail vein for the microsample (50 µL). collaborative and innovative effort to ensure an appropriate urine Additionally, for comparison, blood was also collected via jugular sample could be obtained from multiple rabbits at a time in their vein for standard sample volume collections (0.5 mL). Mice were home cages. Study design required serial urine sample collections at serially bled at approximately 0.5, 4, and 24 hours post dose from a 8-hour or 16-hour intervals over a 48-hour period. Samples were saphenous vein (50 µL). Blood microsamples (for serum and plasma) required to be maintained chilled and protected from light during the were collected from rats and mice using a 50uL plastic blood tube via collection intervals, while minimizing contamination by food, feces, capillary action. Blood was then dispensed using the internal piston and drinking water. Several variations were tested and modified, of this plastic blood tube into a trumpet shaped tube with a push cap. resulting in a reliable and repeatable method of collection. This The trumpet shaped tube was placed in a small carrier tube for ease method primarily consisted of installing a grommet in the home-cage of centrifugation. The serum and plasma (target volume of 10uL) was tray, pitching the tray towards the grommet, and running a plastic harvested using a pipette. Samples were processed and evaluated for tube from the grommet to a collection flask enclosed in a cooler. As a acetaminophen. Results indicate exposure to acetaminophen in result, samples were collected from every animal at sufficient female rats was similar between serum and plasma matrices and volume, quality, and storage conditions (chilled and protected from between jugular vein and tail vein collection methods. This indicates light). Causing minimal stress to naturally sensitive rabbits while the collection site does not impact the results. Exposure to acetamino- effectively collecting quality urine samples allows for greater phen in female mice was similar between serum and plasma matrices opportunities and for research to be conducted while at the same and between Crl:CD1(ICR) and RasH2 (Hemizygous) strains. In time ensuring animal welfare. conclusion, these results indicate this microsample blood collection method allows for sufficient volumes of serum and plasma to P214 Administration of an Angiotensin II Receptor Inhibitor, evaluate toxicokinetic exposure. Collagen Expression, and Breast Cancer Progression in an Orthotopic Murine Model P212 Establishment of Hematological Reference Intervals and Hemocytological Characteristics in a Colony of Wild-Caught D Dréau* Southern Giant Pouched Rats (Cricetomys ansorgei) Biologic Sciences, University of North Carolina, Charlotte, NC DA Jeffery*1, L Brandt2, P Baneux1, BS Blank1, E Silvela1, BS Singh1 There are nearly 140,000 cases of breast cancer diagnosed each year 1Center for Animal Resources and Education, Cornell University, with 40,000 deaths caused by the disease. Although treatment Ithaca, NY; 2College of Veterinary Medicine, Cornell University, regimens have greatly improved the patients’ outcome, no treatment Ithaca, NY is currently available for triple-negative breast cancer tumors. The

661 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 tumor microenvironment, in particular through the promotion of a Microbiology, Immunology, and Pathology, Colorado State Univer- sustained local inflammation, angiogenesis, and extracellular matrix sity, Fort Collins, CO remodeling, has been shown to play a critical role in breast tumor progression toward metastasis. Here we investigated the potential of Chronic wasting disease (CWD) is an endemic and highly transmis- the angiotensin II receptor inhibitor losartan in preventing breast sible disease of deer, moose, and elk of North America. It is caused by cancer progression in an immunocompetent orthotopic murine propagation of specific misfolded proteins called prions, which cause model of breast cancer. Following the orthotopic implantation of the fatal neurodegeneration. CWD may be transmitted in various blood triple-negative mammary tumor 4T1-RFP cells in the mammary fat cells, saliva, urine, and feces, contributing to its increasing spread pads of Balb/c female mice, the tumor growth was monitored across North America. B cells and platelets have been found to overtime using caliper and fluorescent imaging. Mice were dosed harbor prions in CWD infection; in Scrapie, a prion disease of sheep, orally with either losartan (12-15 mg/kg/day) or vehicle. After 30 monocytes and T-lymphocytes are associated with infective prions. days, primary tumors were analyzed for alterations in the matrix and Prions are typically classified as self-proteins, and are therefore not especially the presence of collagen. Differences between groups were considered to stimulate an immune response. However, data tested by repeated measure ANOVA and post-hoc tests with p < 0.05 collected by our group indicate that immediately postinfection, there a priori. Additionally, changes in extracellular matrix composition, is a sharp spike in prion concentration, followed by a slow increase especially collagen content were determined. The daily treatment throughout disease progression. Thus we hypothesize that certain with oral low-dose losartan was not associated with any loss of types of blood cells attempt to combat CWD infection, and that this weight or noticeable behaviors changes. However, primary tumor enables prion-infected cells to be partially cleared/sequestered by the mass were significantly smaller as determined by caliper measure- immune system acutely. Flow cytometry will be used to tease apart ments and by fluorescence evaluation (P < 0.05). Of note, the the role the different blood cells play in CWD infection. Flow fluorescence decreased as the tumor size increased especially in the cytometry enables the use of fluorophore-stained cell surface treated group. Furthermore, losartan was associated with a signifi- antibodies to identify blood cell types, and detect the presence of cant decrease in collagen concentration within the extracellular prion surface antigen. There is no white-tailed deer hybridoma; matrix of the primary tumor (P < 0.05). In summary, low-dose therefore, ovine or bovine antibodies that consistently cross-react losartan per oral prevented collagen deposition within 4T1 tumor with cervid cells were identified. Blood was collected from four mass and limited the growth of the triple negative 4T1 mammary infected white-tail deer (Odocoileus virginianus) in our research herd. cells in an orthotopic murine mammary tumor model. The blood was processed to isolate platelets and peripheral blood mononuclear cells. These cells were stained with various anti-ovine P215 An Analysis of Growth Factor Concentrations in Rabbit and anti-bovine antibodies (CD4, CD8, CD14, and CD41) that were Platelet Rich Plasma Intended for Accelerated Ligament Healing conjugated to a fluorophore. Cell populations were then analyzed with flow cytometry. We concluded that the selected anti-ovine E Milchling*1,2, N Phillips3, L Goodrich3 antibodies cross-reacted reliably with the cervid blood cells. These antibodies, and others yet to be identified, will be used in future flow 1Atlantic Veterinary College, Charlottetown, Canada; 2Laboratory cytometry studies to determine how various blood cells impact the Animal Resources, Colorado State University, Fort Collins, CO; 3Or- pathogenesis of CWD in white-tailed deer. thopaedic Research Center, Colorado State University, Fort Collins, CO P217 Presence of G-Protein Coupled Receptor Gprc6a in the Human, Mouse, and Sheep Placenta Platelet rich plasma (PRP) is an excellent source of growth factors that have therapeutic indications for many soft tissues, including ES McWhorter*, R West, Q Winger, G Bouma ligament healing. The concentration and type of growth factors in PRP, however, are variable between samples and species. By Biomedical Sciences/Laboratory Animal Resources, Colorado State associating the quantity of growth factors to various PRP samples, University, Fort Collins , CO we should provide key information for selecting the preferred PRP preparation to promote ligament healing. Since platelets release Impaired placental development and function can lead to intrauter- growth factors via their granules, we hypothesize that PRP samples ine growth restriction (IUGR), and negatively impact fetal containing greater concentrations of platelets will have higher development and can lead to preterm birth. The placenta is critical in concentrations of growth factors. To test our hypothesis, growth embryo and fetal development and survival and is recognized as a factors were quantified by ELISA analysis in 4 New Zealand White major endocrine organ during pregnancy. Metabolic or environmen- rabbit samples (blood, platelet poor plasma (PPP), PRP containing 2 tally induced hormone changes also affect ruminant pregnancies, and times the concentration of baseline platelets (2XPRP), and PRP can lead to pregnancy failure, placental dysfunction, and embryo containing 4 times the concentration of baseline platelets (4XPRP). mortality. A number of pregnancy disorders associated with placental This study quantified the levels of 4 prominent growth factors abnormalities and dysfunction are associated with increased including fibroblast growth factor-2 (FGF-2), vascular endothelial circulating serum androgens and increased androgen receptor (AR) growth factor (VEGF), transforming growth factor 2β (TGF-2β), and levels in affected placental tissue The classic genomic effects of platelet derived growth factor (PDGF). Growth factors FGF2, VEGF, androgens involve translocation of the androgen-AR complex to the and TGF-2β increased as the concentration of platelets increased nucleus, leading to regulation of gene transcription. However, between PPP, 2XPRP, and 4XPRP groups. There was no change in androgens also have rapid nongenomic effects. Recently, GPRC6A, a PDGF between the groups. The clinical significance of this remains G protein-coupled receptor, was found to bind testosterone and unknown. Subsequent studies will explore the possible correlation dihydrotestosterone. GPRC6A is known to bind Osteocalcin (Ocn), an between rabbit ligament healing and the growth factor quantities osteoblast-specific hormone, and plays a role in bone calcification injected into the ligament. Histology will be performed to asses the and calcium ion homeostasis. Ocn also binds GPRC6A in the degree of ligament healing. This work aims to improve the reliability pancreas, leading to beta cell proliferation and insulin release, as well of PRP preparations so more consistencies can be seen in accelerated as stimulating testosterone production by binding Leydig cells in the ligament healing across all species. testes. Furthermore, Ocn levels increase during pregnancy, crossing the placenta to prevent neuronal apoptosis in the growing fetus. We P216 Identifying Antibodies that Cross-React with White-Tailed hypothesize that GPRC6A plays a role in mediating the non-genomic Deer (Odocoileus virginianus) Blood Bells for Use In Flow effects of androgens in the placenta. To test this hypothesis, we first Cytometry describe the presence of GPRC6A as well as AR in placental cells. Studies conducted in our laboratory show the presence of both ES Lee*, C Mathiason GPRC6A and AR in the human, mouse, and sheep. Using the telomerase-immortalized (h-TERT) first trimester human trophoblast

662 Abstracts of scientific papers 2016 AALAS National Meeting

cell line Swan-71, mouse d12.5 placental tissue, as well as ovine d55 sensing telemeter (HD-XG, DSI™) placed in the carotid artery, sham placental tissue, we identified the presence of both GPRC6A and AR surgery, and nonsurgical controls. Excepting weight loss, no protein by Western blot. These findings suggest that androgen significant postoperative complications were noted in telemeterized signaling may occur through both genomic and nongenomic and sham surgery animals. Telemeterized animals lost 15.6 percent pathways in the placenta. Ongoing experiments include treatment and sham surgery animals 5.2 percent of pre-surgical body weight ovine embryos with lentivirus that will allow us to knock-out postoperatively which returned to baseline values at 42 and 18 days GPRC6A in the placenta to determine a possible phenotype. respectively. Results from OGTT tests, including the effects of a sodium-glucose co-transporter 2 (SGLT-2) inhibitor, were similar P218 Filobacterium rodentium, a New Name for CAR Bacillus between all groups and data captured by telemetry was not different Rodent Isolates from that obtained with a glucometer. Data from 11 out of 12 telemeterized animals was well correlated (R2 > 0.75), and 1 animal F Ike*1, A Kajita1, A Yoshiki1, M Sakamoto1, T Kokubo2 poorly correlated (R2= 0.34) with glucometer results. Bland-Altman plots suggested individual variation in agreement between glucom- 1RIKEN, Tsukuba, Japan; 2National Institute of Radiologic Sciences, eter and telemeter blood glucose values. Continuous blood glucose Chiba, Japan measurements from the telemeters were possible for 70 days postdevice implantation. These data suggest the HD-XG telemeter The gram-stain negative filamentous bacterium isolated from rodent can be used in mouse and that implantation of the device does not pneumonia, formerly known as the cilia-associated respiratory significantly impact the DIO phenotype. Data from the telemeter bacillus (CAR bacillus), induced chronic respiratory disease in mice correlates well with glucometer results although significant individu- and rats. CAR bacillus was initially named in 1985 according to the al variation can occur. finding of its infection sites and morphology, and recently given a scientific name, Filobacterium rodentium gen. nov., sp. nov. in Filobacte- P220 Physiologic, Behavioral, and Histologic Responses to riaceae fam. nov., on the basis of biochemical, phenotypic, and 16S Different Euthanasia Methods in C57BL/6NTac Male Mice rRNA analysis. Here we look at the fundamental nature required to give a name to CAR bacillus. Strain SMR-CT of CAR bacillus was originally isolated from the rat and cultured in Vero E6 cells culture GP Boivin*1, M Bottomley2, PA Schiml3, L Goss4, N Grobe5 supernatants. All cultures were performed at 37°C in 5% CO2 95% air humidified chamber. Morphologic changes were monitored under 1Laboratory Animal Resources, Wright State University, Dayton, phase contrast microscopy. Biochemical reactions were determined OH; 2Mathematical and Microbiological Sciences, Wright State with the Rapid ID 32A anaerobic identification kit. Fatty acid methyl University, Dayton, OH; 3Psychology, Wright State University, esters were extracted and processed to specify fatty acid profiles. Dayton, OH; 4Neuroscience, Wright State University, Dayton, Previously obtained full genome data of SMR-CT were used to OH; 5Pharmacology and Toxicology, Wright State University, Dayton, deduce 16S rRNA sequence and DNA G+C content. Phylogenetic OH analyses were carried out by using the neighbor-joining method and the maximum likelihood method with Bootstrap resampling. Rodent euthanasia using exposure to increasing concentrations of T SMR-C showed microaerobic, nonspore-forming, motile (gliding) CO2 has come under scrutiny due to concerns of potential pain without flagella, gram-stain-negative, argentophilic, filamentous during the euthanasia process. Alternatives to CO2 such as isoflurane rods. The doubling time was 20–24 hours. When cultured in and barbiturates have been proposed as more humane methods of ultralow-attachment flasks, cells were 0.8–0.9 x 8.3–10.0 µm in size euthanasia. We examined 3 commonly used euthanasia methods in and presented singly in the planktonic state. Cells in conditioned mice: a commercially available, nonsterile solution containing medium on glassware grew in sessile state and made a netlike pentobarbital sodium and phenytoin sodium administered by

structure on glass surface. The dominant cellular fatty acids were intraperitoneal injection (150mg/kg), CO2 inhalation (15%, 30%, 50%, iso-C15:0 and anteiso-C15:0. The DNA G+C content was 47.7 mol%. and 100% chamber replacement rate), and isoflurane anesthesia (5% T SMR-C and closely related strains of CAR bacillus rodent isolates with oxygen flow rate at 1L/min) followed by CO2 inhalation (50% formed a novel family-level clade in the phylum Bacteroidetes with chamber replacement rate). Mice were implanted with telemetry high bootstrap support (98–100 %). From these results, a new units to measure heart rate, blood pressure, and movement. Mice scientific name,Filobacterium rodentium, was proposed for strain were videorecorded during all euthanasia procedures. Videos were SMR-CT of CAR bacillus. The type strain is SMR-CT (=JCM 19453T, analyzed for activity (hopping, walking/running, sedentary, =DSM 100392T). standing/rearing), breathing pattern (normal, cessation of breathing), ataxia, face wiping, grooming, recumbency/cessation of walking, P219 The Effect of Blood Glucose Telemeter Implantation on Oral and loss of muscle tone/nose resting on the bedding. Plasma was Glucose Tolerance Testing and Glucose Monitoring in Diet-In- collected for ACTH analysis and lungs for histologic analysis. The duced Obese Mouse nonsterile solution containing pentobarbital sodium and phenytoin sodium led to significantly lower elevations in ACTH (P = 0.0006) *1 1 1 2 GJ DeMarco , X Chen , C Salatto , S Tiesma than isoflurane and CO2 euthanasia. There were no significant consistent differences between euthanasia techniques in the telemetry 1Pfizer Inc., Cambridge, MA; 2Data Science International, St. Paul, recorded cardiovascular or activity changes. Behavioral differences MN were limited to time until ataxia and recumbency and elevated frequency of face wipes in the mice euthanized with the solution Oral glucose tolerance testing (OGTT) and serial blood glucose (SBG) containing pentobarbital sodium and phenytoin sodium (P < 0.001). monitoring are important tools in basic diabetes research and the Euthanasia with 50% or 100% CO2 chamber replacement rates was preclinical development of new treatments for diabetes. Although the fastest method of the 3 techniques. Histologic damage mouse models are critical to diabetes research, their small blood characterized by mild to moderate perivascular and peribronchiolar volume and marked stress response to restraint presents significant edema was significantly more severe in CO2 euthanized mice. We challenges to generating reliable and consistent OGTT and SBG data. conclude that use of CO2 with or without isoflurane is an acceptable Since telemetric technology can often mitigate the challenges of euthanasia method. Isoflurane or euthanasia by the solution physiologic data collection in mouse, the purpose of this study was containing pentobarbital sodium and phenytoin sodium are better to evaluate the effects of an implantable telemetric glucose sensor suited when studies rely on analysis of lung histology. 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

663 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P221 Characterization of Transdermal Pharmacokinetics of P223 New TK-NOG Mice as an Optimized Platform for Liver Ketoprofen in Sinclair and Gottingen Minipigs Humanization

A Stricker-Krongrad, GF Bouchard*, D Brocksmith, M Zhong, J Liu H Suemizu*1, K Kawai2, Y Ando1, Y Higuchi1, H Hashimoto1, H Nabekawa3, M Nishiwaki1,4, M Okazaki1,4, M Yamamoto5, M Goto6, R Sinclair BioResources, LLC, Auxvasse, MO Takahashi6

Our objective was to characterize the transdermal pharmacokinetics 1Laboratory Animal Research Department, Central Institute for of a ketoprofen cream formulation in the Sinclair and Göttingen Experimental Animals, Kawasaki, Japan; 2Pathology Analysis Center, breed of minipigs and compared it to published human data. Central Institute for Experimental Animals, Kawasaki, Japan; 3In- Minipigs have a fixed skin tightly attached to the subcutaneous Vivo Science Inc., Kawasaki, Japan; 4CLEA Japan, Inc., Fujinomiya, tissues similar to that in humans, which make them a preferable Japan; 5ICLAS Monitoring Center, Central Institute for Experimental model for dermal studies. Three minipigs per gender per strain Animals, Kawasaki, Japan; 6Animal Resources and Technical (9.1-9.9 months old) were used in the study. Four mL of 2.5% Research Center, Central Institute for Experimental Animals, ketoprofen cream, (100 mg Ketoprofen, a human therapeutic dose), Kawasaki, Japan were applied on an area on the back of each minipig that was approximately 3% of the total body surface area. No adverse To address issues related to interspecific differences between animal reactions were observed in the animals. Blood samples were collected models, we developed an animal model with a reconstituted liver at 4, 8, 12, 24, 28, 32, and 36 hours post dose. The plasma samples containing human hepatocytes (humanized-liver TK model). were analyzed with API 4000 LC/MS/MS system with d3-labeled However, the disadvantages associated with this model were male ketoprofen as a labelled internal standard. The linear range of sterility and low transgene expression in females. To overcome these quantitation for ketoprofen was 50 to 25600 ng/mL. The PK disadvantages, we developed a new humanized-liver model based parameters were calculated with PKSolver. Individual animal data on immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice that were analyzed with the non-compartmental analysis. Transdermal specifically expresses herpes simplex virus type 1 thymidine kinase pharmacokinetic parameters of ketoprofen were (N = 6; mean gene mutant clone30 in the liver using a transthyretin gene promoter +/- SD; Sinclair versus Gottingen): t1/2 (h): 12.2 +/- 2.0 versus 11.7 (Ttr-HSVtk mutant30 transgenic NOG; NOG-TKmut30). We +/- 3.9; Tmax (h): 8.0 +/- 2.5 versus 9.3 +/- 2.1 and Cmax (ng/m): established 3 transgenic lines (#4, 5, and 10). Male NOG-TKmut30 94.3 +/- 65.6 versus 173.1 +/- 71.8. Although ketoprofen was shown transgenic mice in each line were fertile and reproduced normally, to be absorbed better in Göttingen than in Sinclair minipigs in the transmitting the transgene in expected Mendelian ratios. average plasma concentration time curves, no statistically significant Furthermore, male and female NOG-TKmut30 mice aged 5 weeks difference was observed between all PK parameters. These data showed severe liver injury by ganciclovir (GCV) injection. One suggested that ketoprofen has a similar ADME process in both million viable human hepatocytes were transplanted intrasplenically minipig lineages. into liver-injured NOG-TKmut30 mice, and serum levels of human albumin (hAlb) were monitored every 4 weeks. Engraftment of P222 Troubleshooting Cre Activity in Off-Target Tissues: Lessons human hepatocytes in all transgenic lines was confirmed by detection Learned from a Villin- cre Mouse Model of hAlb in serum. The recipients produced more than 10 mg/mL hAlb eight weeks after transplantation. In these mice, almost 95% of HR Holcombe*1, D Puglisi1, G Gong1, E Bryant1, B Horwitz2, JG Fox1 the hepatocytes were positive for anti-HLA staining, suggesting that the recipient liver was replaced by the transplanted human hepato- 1Massachusetts Institute of Technology, Cambridge, MA; 2Brigham cytes. The NOG-TKmut30 model has several advantages over the and Women’s Hospital, Boston, MA traditional TK model. Firstly, overcoming male sterility will facilitate large-scale production of recipient transgenic mice. Secondly, the The Cre-loxP recombination system is a common method for absence of differences in GCV-induced liver injury between sexes generating conditional knock out mice, with gene deletion being will enable us to produce twice as many humanized-liver mice. We dependent on the tissue specificity of the promoter driving Cre anticipate that the NOG-TKmut30 mice, in which production expression. However, many Cre driver lines show aberrant excision efficiency of humanized-liver mouse is significantly improved, will activity that leads to deletion of target genes in unexpected tissues, become an optimized platform for generating a humanized-liver resulting in mosaic and even germline deletion. Here we describe model for studying drug metabolism, toxicology, and liver regenera- inconsistent excisional activity in Villin-cre transgenic mice driving tion. 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 P224 The Plant Flavonol Rutin Suppresses Human-Amylin pair consisting of a Cre+ female and Cre- male were mated, and their Misfolding in Vitro and Increases the Lifespan of Diabetic offspring genotyped for Cre and floxed IL22R. Genotyping of the first Human-Amylin Transgenic Mice 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 J Aitken*1, K Loomes1,2, i Riba-Garcia3, R Urwin3, G Prijic1, A of 2 pups and 3 of 9 pups, respectively, that were negative for the Phillips4, A Phillips1,2, P Barran4, A Dowsey3,5, G Cooper1,3 floxed IL22R allele. Our hypotheses were that both parents harbored one il22r allele that had undergone germline recombina- 1School of Biologic Sciences, University of Auckland, Auckland, New tion (il22r fl/D) and that the pups that were negative for the floxed Zealand; 2The Maurice Wilkins Centre for Molecular Biodiscovery, IL22R had undergone germline recombination of both al- University of Auckland, Auckland, New Zealand; 3Centre for leles (il22rD/D). These hypotheses were supported by regenotyping Advanced Discovery and Experimental Therapeutics, University of the mice using primers that differentiated between floxed and Manchester, Manchester, United Kingdom; 4Michael Barber Centre recombined IL22R. Sequencing the PCR product from il22rD/D mice for Collaborative Mass Spectrometry, University of Manchester, confirmed that exon 2 of the IL22R was deleted. Subsequent breeding Manchester, United Kingdom; 5Department of Electrical Engineering pairs were genotyped and confirmed to be il22r1flox/flox. Genotyping and Electronics, University of Liverpool, Liverpool, United Kingdom litters born to the new breeders demonstrated that spontaneous generation of il22rflox/D pups occurs in some, but not all, litters, with a Diabetes exerts an enormous toll on patients, their families and frequency of 14-30% within the litter. The colony is currently being caregivers, and health-care delivery systems globally. Type 2 diabetes expanded, and additional genotyping and immunofluorescence (T2D) is the most prevalent form of the disease, accounting for 90% staining for IL22R will be performed to further characterize Cre or more of all diabetes globally. Currently, available therapeutic recombinase activity in this line. Effect of ectopic deletion on study approaches have not stemmed the alarming increase in numbers design and expected outcomes will be highlighted. affected and there is a need for more effective therapeutics that can

664 Abstracts of scientific papers 2016 AALAS National Meeting

suppress the causative disease mechanisms. Human amylin (hA) is reduced energy expenditure to that of their controls. In conclusion, the main component of islet amyloid in diabetic patients. It is a small human amylin regulates body weight in hA-transgenic mice by protein that can spontaneously misfold and aggregate to form increasing core body temperature and elevating energy expenditure. structures that cause β-cell death and onset of diabetes. Amylin- mediated cytotoxicity has been identified as a potential target for P226 Baseline Cardiac Troponin I in Gottingen Minipigs developing new anti-diabetic molecules that might act by suppress- ing β-cell death. The aim of this study was to identify natural JA Scotto*1, S Hulet2, N Vincelli2, D Miller2 compounds that suppress human amylin misfolding and to test their efficacy in vivo in a human amylin transgenic mouse line, which 1Veterinary Services, Edgewood Chemical and Biologic Center, APG, overexpresses human amylin in the pancreatic β-cells. These MD; 2Operational Toxicology, US Army Edgewood Chemical and transgenic mice spontaneously develop diabetes and mirror many Biologic Center (ECBC), APG, MD aspects of the human disease. We identified rutin, a small, orally active compound present in the human diet which is also used as a The use of cardiac troponin I (CTnI) as an emergency medicine dietary supplement, as an inhibitor of hA misfolding. In vitro, rutin biomarker indicative of acute myocardial infarction is generally bound to hA, suppressed its misfolding, and reverted misfolded hA accepted, with the threshold typically set at the 99thpercentile value back towards the physiologic form. Rutin was administered from for the population. Over several studies we were able to collect blood weaning in the drinking water to hA-transgenic mice (n = 11) versus samples from a large number of young adult male Gottingen water-treated hA-transgenic mice (n = 13). In rutin-treated hA-trans- minipigs to assess baseline CTnI levels. We noticed a difference in genic mice, measurements of glucose, fluid-intake, and body-weight results of cardiac troponin I between K2EDTA and Lithium heparin showed that oral rutin-treatment slowed diabetes progression by tubes. We collected samples in both tubes to compare. All of the lowering the rates of elevation in blood glucose (P = 0.030), slowing animals used in these studies had a cannula surgically implanted into deterioration of diabetes symptoms to death (P = 0.014) and the right external jugular vein which was used for whole blood stabilizing body-weight (P < 0.0001). Glucose tolerance, as measured collection. A subset of these animals had DSI telemetry transmitters by ipGTT at 60 days post weaning, was improved in rutin-treated surgically implanted. All baseline blood samples were collected after compared with water-treated hA-transgenic mice (P = 0.042). In the animals recovered from surgery. For the first analysis, an aliquot conclusion, the dietary flavonol rutin, which suppresses hA-aggrega- of whole blood from each pig was split equally into lithium heparin tion in vitro, doubled the lifespan of diabetic mice (P = 0.011) by a (LiHep) tubes and K2 EDTA and analyzed for CTnI using i-Stat median of 69 days compared with water-treated diabetic mice. cartridges. A significant difference (P = 0.032) was identified in the CTnI levels in samples taken from the same animals (n = 48) when P225 Overexpression of Human Amylin in Transgenic Mice Raises stored in LiHep tubes vs. K2 EDTA tubes, with the portion stored in Core Body Temperature and Increases Energy Expenditure which LiHep tube reading significantly higher than the portion stored in the Can Be Reversed by Leptin Replacement K2 EDTA tube. Blood samples from an additional 61 minipigs were collected solely into LiHep (total n = 109) tubes and blood samples J Aitken*1, C Walker1, G Prijic1, S Johnson1, G Cooper1,2 from another 20 minipigs were collected solely into K2 EDTA (total n = 68) tubes and assessed for CTnI levels in the same manner. A 1School of Biologic Sciences, University of Auckland, Auckland, New significant difference (P = 0.019) was again identified when compar- Zealand; 2The Centre for Advanced Discovery and Experimental ing the 2 larger datasets. No significant difference was identified in Therapeutics, University of Manchester, Manchester, United baseline CTnI levels (all in LiHep tubes) in animals that had Kingdom surgically implanted telemetry transmitters (n = 54) when compared to animals without transmitters (n = 55). Mean CTnI in the large Diabetes is now pandemic. Globally, more than 380 million people LiHep dataset (n = 109) was 0.0233 ng/ml ±0.00642 SEM (Range have diabetes, of whom 90% have type 2. If no action is taken to stop 0.00-0.67 ng/ml). To calculate the 99th percentile for the dataset a the ongoing increase in prevalence then, in 20 years, there will be 592 nonparametric analysis of the population was performed. In this million people living with diabetes (1 in 10 of all adults). Effective analysis the 25th (Q1) and 75th (Q3) percentiles were calculated. The interventions are required that can prevent the progression of the interquartile range (IQR) was calculated as Q3-Q1. The exclusion disease. In order to do this, it is first necessary to understand the limit was calculated as Q3 + 3(IQR) with a result of 0.12 ng/ml. This molecular basis of diabetes. Human amylin (hA) is the main analysis resulted in the exclusion of 2 data points from the data set as component of islet amyloid in diabetic patients. It is a small protein statistical outliers.There was a significant difference of the CTnl that can spontaneously misfold and aggregate to form structures that values from blood collected from K2EDTA and lithium heparin cause β-cell death and onset of diabetes. Amylin also functions as a tubes. There was no difference in the animals that had surgically satiation signal and has long-term anorectic and weight reducing implanted telemetry transmitters. The 99th percentile of the resultant actions. We have developed hA-transgenic mice which selectively dataset (n = 107) was calculated as 0.0792 ng/ml. For comparison, the express human amylin in their β-cells. These mice undergo progres- recommended 99th percentile for marker biologic significance in sive islet damage and develop spontaneous diabetes. The aim of this humans for the same i-stat cartridges is 0.08 ng/ml. study was to investigate the effect of over-expression of human amylin in hA-transgenic mice on physiologic parameters such as P227 Lactose Dehydrogenase Isozymes as Biomarkers for the weight, blood glucose, core body temperature, energy expenditure, Progression of Tuberculosis Infection in Guinea Pigs (Cavia and endocrine hormones. Blood glucose, weight, and core body porcellus) temperature were measured weekly and indirect calorimetry was performed in approxately 100-day-old hA-transgenic and control S Murray2, JH Kopanke*1, W Williams1, A Izzo1, LV Kendall1 mice. Food intake, activity, O2 consumption, CO2 production were measured and respiratory quotient and energy expenditure were 1Microbiology, Immunology, and Pathology, Colorado State Univer- calculated. hA-transgenic mice (n = 24) showed elevated blood sity, Fort Collins, CO; 2College of Veterinary Medicine, North glucose from weaning (P < 0.001) leading to diabetes at 100-150 days Carolina State University, Raleigh, NC of age. They weighed significantly less (P < 0.001), and had significantly higher core body temperatures (P < 0.001) and energy Lactate dehydrogenase (LD) is found throughout the body, with expenditure (P < 0.05) compared to their control littermates (n = 15). some tissue groups producing signature variations in LD isozyme Amylin and insulin serum levels were significantly higher (P < 0.01; (LD1-5) content. Damaged cells can release LD into the blood P < 0.05) while leptin and adiponectin levels were significantly lower resulting in altered serum isozyme values that may serve as (P < 0.05; P < 0.01) in the hA-transgenic mice compared to their diagnostic markers. Previous studies have demonstrated that serum controls. Leptin plays a central role in appetite regulation and energy LD3 levels increase in humans infected with Mycobacterium tuberculo- expenditure. Acute leptin replacement in hA-transgenic mice sis. Guinea pigs (Cavia porcellus) are a useful model for the study

665 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 of M. tuberculosis; however, there are no known biomarkers that allow Identifying biomarkers is a powerful strategy to detect and study an adequate assessment of disease progression. This study investi- disease progression in ALS. Glial fibrillary acidic protein (GFAP) is gated whether M. tuberculosis infection in guinea pigs produces a LD an intermediate filament protein that is expressed by a number of isozyme profile that may be used to track infection severity. Blood cells related to the central nervous system including glial cells and was sampled from 5 guinea pigs prior to M. tuberculosis infection and ependymal cells. We hypothesized that GFAP represents a biomarker 4 weeks postinfection. Electrophoresis was performed on serum of disease progression in ALS rats. We first determined whether using a lactate dehydrogenase specific electrophoresis kit to separate GFAP expression was increased in the skeletal muscle of symptom- and visualize the 5 LD isozymes. Densitometry data was analyzed atic and end-stage SOD1-G93A rats. Immunohistochemical staining from digital scans of the dried gels allowing comparison of isozyme and Western blotting analyses revealed a significant increase in GFAP levels. Guinea pig total serum LD averaged 185 IU/L (range: 86–378 expression throughout disease progression in the hindlimb muscle of IU/L) pre infection and 132 IU/L (range: 67 – 218 IU/L) SOD1-G93A rats. Next, we asked whether serum GFAP levels were postinfection. LD constituent isozymes (LD1:2:3:4:5) were found to be elevated in end-stage ALS rats. We measured GFAP concentrations 35%:22%:10%:3%:30% in preinfection serum samples and by ELISA and found a correlation between increased GFAP and 45%:24%:9%:2%:21% in postinfection serum samples. LD1 was disease progression in SOD1-G93A rats. Our results implicate GFAP elevated an average of 12.3 IU/L (+/-12.1 IU/L) and LD5 fell and as a potential biomarker for ALS and will allow for further investiga- average 16.1 IU/L (+/- 22.3 IU/L). The consistent increases in LD1 tion into the specific roles of GFAP during ALS disease progression. and the frequent decreases in LD5 suggest a correlation between disease advancement and serum LD isozymes. This could provide a P230 Leptospira Infection of Mice via the Conjunctiva reliable diagnostic to gauge the progression of M. tuberculosis infections, and provide the information necessary to determine endpoints JP Sullivan*1, L Richer2, H Potula2, N Nair2, M Gomes-Solecki2,3 that minimize animal pain and discomfort. 1Comparative Medicine, University Tennessee Health Science Center, P228 Scratching Responses in C57BL/6, DBA/2, BALB/c, and CD1 Memphis, TN; 2Microbiology Immunology and Biochemistry, Mice with and without Epidermal Injury University of Tennessee Health Science Center, Memphis, TN; 3Im- muno Technologies Inc., Memphis, TN JL Sargent*, CV Lohr, HE Diggs Leptospirosis is a potentially fatal zoonosis transmitted by reservoir Oregon State University, Corvallis, OR host animals, such as mice. These reservoir animals harbor pathogenic leptospires in their renal tubules and shed the bacteria in urine Whereas early investigations into ulcerative dermatitis (UD) focused continuously or periodically for a period of a few months up to on the possibility of a primary dermatopathology, several recent several years. We analyzed the outcome of infection of C3H-HeJ mice studies have advocated scratching behavior as a primary driver for with Leptospira interrogansserovar Copenhageni via the conjunctival UD. The aim of this study was to assess whether B6 mice exhibit route and tracked a number of readouts for 15 days postinfection excessive scratching under resting conditions or when provoked by (dpi). Approximately 60 ul of a 108/mL culture of Leptospira placed epidermal barrier disruption. We hypothesized that B6 mice would onto each eye of the mouse led to bloodstream dissemination of the exhibit more spontaneous scratching behavior and that B6 mice spirochete on days 5 to 11 which was followed by urinary shedding would be more pruritic after mild epidermal barrier injury compared on days 7 to 15 postinfection. In contrast to the controls, significant with the other strains and stock tested. The behavior of the retired body weight was lost in mice infected with Leptospira from days 12 to breeder female C57BL/6J, DBA/2J, BALB/cByJ, and Crl:CD1 mice 15 postinfection with an average loss of 10% (dpi0 v dpi15). The was videotaped over several 60-minute sessions. Behavior filming acquired humoral response to conjunctival infection led to the occurred at 17:15 and at 07:00 the next morning, prior to (baseline) production of Leptospira-specific IgM and IgG. Leptospi- and after tape-stripping to initiate epidermal barrier disruption. ra dissemination led to colonization of the kidney (~104 Leptospires/ Scratching duration was recorded as brief (less than 3 seconds) or mg tissue) and triggered inflammation as determined by increased prolonged (3 seconds or longer), on the basis of observations during levels of mRNA transcripts of KC, RANTES, MIP-2, TNFa IL1b, IL6, a pilot study. In contrast to the hypothesis, B6 mice did not scratch and IL13 as well as reduced levels of IL17a mRNA in kidney tissue. significantly more frequently or for longer durations than the other mRNA levels of IFNg, IL4, IL5, IL17, IL23, IL10, and iNOS were not types of mice tested under both resting conditions and when different from controls. In this study, we show that pathogenic L. challenged by tape stripping. In fact, B6 mice showed the lowest interrogans serovar Copenhageni disseminates and colonizes the average scratching frequency at baseline and following epidermal kidney of mice after inoculation via the conjunctival route. injury. B6 mice demonstrated increased scratching behavior after Replication of one of the three natural modes of transmission of epidermal barrier disruption, but the increased scratching did not human leptospirosis in mice allows this species to be used for surpass the rate or duration of scratching in the other types of mice studying all natural modes of transmission. tested. These findings do not support the idea that a strain-related tendency toward exaggerated scratching behavior under resting or P231 Effect of Housing and Colony of Origin on Immune Pheno- epidermal barrier disruption conditions predisposes B6 mice to UD. type of Rhesus Macaques

P229 Glial Fibrillary Acidic Protein: A Potential Biomarker in a Rat J Kramer*1,2, J Lane3,2, J Whitney4 Model of Amyotrophic Lateral Sclerosis 1Leidos Biomedical Research, Bethesda, MD; 2New England Primate Research Center, Southborough, MA; 3University of Washington, J Jeffrey*, I Smit-Oistad, J Van Dyke, M Suzuki Seattle, WA; 4Beth Israel Deaconess Medical Center, Boston, MA

Comparative Biosciences, University of Wisconsin—Madison, Nonhuman primates remain an important laboratory animal species Madison, WI and are commonly used in biomedical research. Despite physiologic similarities to humans that may make them favorable to other Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease animals in certain biomedical applications, there are significant characterized by specific loss of motor neurons in the spinal cord and scientific challenges to their use. Unlike many other laboratory brain stem. For the last decade, our group has used a rat model of animal species, there is considerable variation not only in their ALS, extensively, to understand the complexity of this devastating genetics, but also origin, husbandry background, clinical history, and disease. The specific rat model we used is the NTac:SD- other traits. These factors may not be considered by scientists or Tg(SOD1G93A)L26H from Taconic Biosciences. Currently, there are veterinarians when animals are assigned to research studies, which limited options for treating ALS and further investigation of the may result in unexplained results and increased animal usage. To disease etiology and ALS disease progression needs to be completed. evaluate the effect of housing and basic husbandry practices on the

666 Abstracts of scientific papers 2016 AALAS National Meeting

rhesus immune system, we obtained blood samples from group- P233 The Effects of Isoflavones on Estrogens Levles during the housed SPF rhesus macaques in 3 facilities that, among other factors, Onset of Puberty of Male Wistar Rats raised their animals in different environments. In facility 1 (n = 18), animals were free to roam a large space and had direct contact with S Caceres Ramos1, L Peña2, P Millan1, C Perez Garcia3, I Diez Prieto3, the natural environment. In facility 2 (n = 10), animals were also JC Illera*1 raised outdoors, but in a confined corral on a clean substrate. In facility 3 (n = 16), animals were raised indoors on bedding. Animals 1Fisiologia Animal, Universidad Complutense de Madrid, Madrid, were of similar age, sex, and SPF status, but we did not control for Spain; 2Medicina y Cirugia Animal, Universidad Complutense de other variables such as long-term clinical history. We performed flow Madrid, Madrid, Spain; 3Medicina, Cirugia y Anatomia Veterinaria, cytometry to evaluate inflammatory cell subsets and a serum Universidad De Leon, Leon, Spain cytokine analysis. Animals raised indoors had increased percentages of total, central memory, and effector memory CD4+ lymphocytes Isoflavones are phytoestrogens that have a similar structure to the compared to both groups of animals raised outdoors. In contrast, the estrogen 17β estradiol. Therefore, they can affect hormonal modula- frequency of activated (CD38+/DR+) CD4+ and CD8+ lymphocytes tion and some studies have described the association between were significantly increased in both groups of outdoor animals exposure to phytoestrogens and hormonal-related disorders in compared with animals raised indoors. Both groups of outdoor- children. There are commercially available isoflavone-containing housed animals also had increased inhibitory cytokines including products that have estrogen-like effects such as milk with added IL-10 and IL-1Ra compared to animals housed indoors. Together, genistein. It has been shown that a continuous administration of these findings indicate that colony of origin can significantly alter the isoflavones to prepuberal Wistar male rats can delay the onset of immune phenotype of rhesus macaques and that husbandry practices puberty. However, it is still not clear whether isoflavones have effects may contribute to this effect. Researchers should consider colony of on the reproductive and the endocrine systems under normal dietary origin when choosing animals and assigning them to study groups. intake and overdose. The aim of this study is to determine how the 2 most important isoflavones, genistein and daidzein, affect estrogen P232 In Vitro and In Vivo Flutamide Effects on Cell Proliferation levels and the onset of puberty on male prepuberal rats, by analyzing and Tumor Growth Associated with Steroid Hormone Secretion in the 17β-estradiol and estrone sulphate levels in serum with a Human and Canine Inflammatory Breast Cancer Cell Lines competitive enzyme immunoassay (EIA), previously validated for this species. One-hundred twenty five male prepuberal Wistar rats S Caceres Ramos1, L Peña1, MJ Illera1, B Monsalve1, W Woodwar2, J divided in 7 groups, 1 control group and 6 experimental groups, Reuben3, JC Illera*1 were orally daily administered a high and low dose (HD and LD respectively) of genistein, daidzein, and a mixture of both isofla- 1Fisiologia Animal, Universidad Complutense de Madrid, Madrid, vones, over 5 weeks. Higher estrogen levels were detected in all Spain; 2Radiation Oncology, MD Anderson Cancer Center, Houston, experimental groups compared with the control group from the third TX; 3Hematopathology, MD Anderson Cancer Center, Houston, TX week of experiment. In control group at the third week there was a significant decrease of estrogen levels associated to the onset of Inflammatory breast carcinoma (IBC) is a special type of breast cancer puberty. However, in LD groups, a similar decrease was found at the with a poor survival rate. The investigation on the role of androgens fourth week of the experiment indicating a delay in the onset of in breast cancer is dramatically increasing in recent years in an puberty in these groups. Moreover, HD groups did not show this attempt to find new anti-androgen therapeutic strategies. We studied decrease, making undetectable the onset of puberty in these animals. the effects of the anti-androgen drug flutamide on cell prolifera- We can conclude that oral administration of isoflavones in male rats tion, in vivo tumor growth and steroid production in canine and affect to the gonadal axis increasing estrogen levels and causing the human IBC triple negative cell lines. Canine cell line, IPC-366, was delay of the onset of puberty, and consequently, could cause cultured in free steroid DMEM/F12 media. Human cell line SUM149 physiologic and developmental problems. was maintained in free steroid Ham›s F-12 media. Cell cultures were exposed to flutamide treatment. Flutamide concentrations added to P234 Disruption of Folliculin Interacting Protein-1 Prolongs the culture media were: 5 µM, 10 µM, and 15 µM for 72 hours and Survival and Improves Response to Therapy in a Mouse B Cell proliferation rates were assayed daily. Additionally, IPC-366 and Lymphoma Model SUM149 xenotrasplanted mice were used for in vivo assays. Experimental groups of 5 female mice each were divided in a control JA Ramirez*1, M Tsang1, H Park1, D Margineantu2, D Hockenbery2, H group and 3 treatment groups (administrated orally with a solution Gu3, D Raftery3, B Iritani1 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 1Comparative Medicine, University of Washington, Seattle, determination in culture media and tumor homogenates WA; 2Clinical Division, Fred Hutchinson Cancer Research Center, (pregnenolone (P5), progesterone (P4), androstenedione (A4), Seattle, WA; 3Department of Anesthesiology and Pain Medicine, testosterone (T), dihydrotestosterone (DHT), 17β-estradiol (E2), and Mitochondrial and Metabolism Center, University of Washington, estrone sulphate (SO4E1)) were assayed by EIA previously validated. Seattle, WA In vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. In vivo flutamide Folliculin interacting protein-1 (Fnip1) is a cytoplasmic protein provoked a tumor size reduction in around 55-65% (IPC-366) and originally discovered through its interaction with Folliculin (Flcn), a 50-60% (SUM149) of mice treated. In IPC-366 xenotrasplanted mice, tumor suppressor mutated in human Birt-Hogg Dubé syndrome. metastasis rates decreased compared with control group. P5, P4, Fnip1 and Flcn interact directly with the master metabolic regulator SO4E1, A4, T, and DHT hormonal secretion to culture media of cell AMP kinase (AMPK), an enzyme that stimulates energy/nutrient lines was increased with flutamide addition in contrast to E2 levels production and inhibits energy/nutrient consumption in response to that decreased in the treated groups. In tumor homogenates of low ATP. Using Fnip1-deficient mice, we previously determined that treated mice, high levels of P5, A4, T, and SO4E1 were found. DHT, Fnip1 is required for the development of B lymphocytes, and for B P4, and E2 levels were lower compared to control group. Flutamide lymphocyte transformation induced by the Myc oncogene in a reduced cell proliferation in vitro, in vivo tumor growth and caused murine model of human Burkitt’s lymphoma. We hypothesize that significant changes in steroid hormone secretion by increasing T inducing Fnip1 deficiency in the tumor cells will prolong survival of production leading to lower E2 levels. These results support future the host and improve the response to treatment. In this study, we therapy approaches of human and canine IBC triple negative breast used the Cre-LoxP system to conditionally disrupt Fnip1 in primary cancer with anti-androgen drugs. 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

667 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 occurring B cell lymphomas fromFnip1floxedEµ-MycMx1Cre mice. The During active secretion, the gastric epithelium generates ion floxed Fnip1 gene was inducibly deleted in tumor cells from these gradients that depend on limited paracellular transport of ions across mice by intraperitoneal injection of polyinosinic-polycytidylic acid the mucosa. This paracellular “barrier” to permeability is created by (PolyIC; 5-10 mg/kg) to induce Cre expression from the Mx1-Cre tight junctions (TJs), which are regulated, in part, by the gut (interferon-inducible Cre) transgene. We found that conditional microbiota. TJs are disrupted and contribute to mucosal pathogenesis knockdown of Fnip1 significantly delayed lymphoma onset and in Helicobacter pylori infection. Infected male insulin-gastrin (INS- prolonged survival following chemotherapy (cyclophosphamide, 150 GAS) transgenic mice have greater pathology than infected female mg/kg IP). Mass spectrometric analysis revealed that inhibition of INS-GAS mice. Development of pathology is delayed in H. pylori- Fnip1 significantly altered the representation of metabolites involved monoassociated INS-GAS mice compared to H. pylori-infected in glutaminolysis, nucleic acid metabolism, antioxidant, and serine gnotobiotic INS-GAS mice co-colonized with select species of Altered biosynthesis pathways. Using flow cytometry, we found that Schaedler Flora (ASF). In ASF colonized INS-GAS mice, bacteria that disruption of Fnip1 increased apoptosis in primary Eμ- would normally colonize the large intestine also colonize the Myc lymphoma cells in vivo and also increased metabolic demand by stomach. We sought determine whether the paracellular barrier in significantly increasing both mitochondrial number and membrane male mice is more permeable than in female mice and to determine potential. These results suggest that inhibition of Fnip1 may provide the role of gut microbiota in this process. For this, transepithelial a novel strategy to improve response to chemotherapy in lymphoma resistance (TER), potential difference (PD), and the rate of dextran patients, in part by sensitizing tumor cells to cytotoxic and metabolic flux was measured in 4-month-old male and female germfree (GF) stress. 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-) P235 Joint Angles and Limb Alignment of the Humerus and Radius secretion into the stomach lumen. All studies were done with acid in Nonchondrodysplastic and Chondrodysplastic Feline Breeds blocking agents in the buffer. The results showed GF female INS-GAS mice had a significantly higher TER and negative PD compared to GF KL Chesney*1, LA Lyons2 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 1Veterinary Pathobiology, University of Missouri, Columbia, suggest that female GF INS-GAS mice maintain a tighter gastric MO; 2Veterinary Medicine and Surgery, University of Missouri, barrier and maintain more Cl- in the gastric lumen than do all other Columbia, MO 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 Orthopedic disease, once thought to be a rarity amongst felines, has both groups have a higher TER and negative PD than males of their recently been shown to affect a large portion of their population. respective groups. These results support that there is a difference in Most senior-age felines have widely variable osteoarthritis, and the the composition of TJs in male versus female mice and also support incidence of angular limb deformity and chondrodysplasia is on the that the microbiota, modeled here using ASF, make gastric epithelia rise, the latter characteristic of the Munchkin breed and its variants. less tight. Future research will evaluate specific differences and Treating these conditions in felines is difficult due to the lack of regulation of gastric TJ function in male and female mice. published normal joint angle and limb alignment values. One cadaveric study has examined normal values for the feline pelvic P237 Comparison of Pharmaceutical versus Chemical Grade limb, and no data exists for the forelimb. The goal of this study was Streptozotocin and Bolus versus Multiple Low-Dose Regimens in to develop a range of values for the normal joint angles and limb Induction of Diabetes in C57bl/6 Mice alignment of the radius and humerus in nonchondrodysplastic mixed-breed felines to characterize the abnormal limb orientation of KP Storves*1, L Lacefield2, K Buac1, J Hutcheson1, SB Harvey2, S the chondrodysplastic feline breed, the Munchkin. We hypothesized Sanchez2, U Blas-machado2, N Chronos1, S Chavan1 chondrodysplastic cats would have significantly different limb values than nonchondrodysplastic cats. The entirety of both forelimbs, 1ViCapsys, Athens, GA; 2University of Georgia, Athens, GA inclusive of the shoulder joint and carpus, were radiographed in the frontal and sagittal planes in 12 adult, mixed-breed, nonchondrodys- Streptozotocin (STZ) is an alkylating agent that destroys insulin plastic cats under sedation. Radiographs were analyzed to determine producing cells and is used in research for diabetes induction. normal ranges of frontal and sagittal plane anatomic axes and joint Animal welfare regulations stipulate that pharmaceutical grade angles using a methodology for alignment quantification and agents be used instead of chemical grade unless scientifically justified preoperative surgical planning described in human surgery, the and IACUC approved. Models using STZ often do not result in 100% Center of Rotation of Angulation (CORA). The same procedure was induction of diabetes and result in toxicity. Dosing regimens in the performed for chondrodysplasic cats. Nonchondrodysplastic cats literature range from 1 bolus dose to multiple low doses. Previously show narrow, normally distributed ranges for each limb value. our collaborators have used 200mg/kg once IP and achieve roughly Chondrodysplastic cats show significant differences (P < 0.05) for 9 of 80% induction of diabetes with varying amounts of mortality. 12 mean limb values, the most significant of which is the CORA of Chemical grade STZ requires reconstitution with sodium citrate the radius at 10.04 ± 1.30; and 29.56 ± 6.27 (P = 0.0002) for normal and tightly pH buffered to 4.5 and use within 30 minutes of reconstitu- chondrodysplastic radii, respectively. Ranges for normal feline radial tion. Pharmaceutical grade STZ has an extended shelf life (12 hours) and humeral joint angles and limb alignment can be used to and is more stable suggesting that it may be a safer product for use. characterize newly developed chondrodysplastic breeds and for We hypothesized that pharmaceutical grade STZ would provide a angular limb and traumatic injury correction when there is bilateral greater induction percentage with less adverse effects than chemical deformity. Many treatment modalities, such as dome osteotomy, are grade STZ. Our second hypothesis was that multiple low doses heavily reliant on appropriate preoperative planning, and verified would result in less toxicity than a single bolus dose. We used 50 normal values for treatment will be vital for the future of feline ortho- male C57BL/6 mice. Groups 1 and 3 received 3 doses of pharmaceu- pedic treatments. tical and chemical grade STZ at 83mg/kg intraperitoneally (IP), respectively. Groups 2 and 4 received 1 dose of 250 mg/kg IP P236 Gender and Microbiota Effects on Gastric Mucosa Tight pharmaceutical and chemical grade STZ, respectively. Groups 5 and 6 Junctions in Gnotobiotic INS-GAS Mice were controls. Animals were evaluated over 6 weeks by body weight and glucose readings. All animals in both the high-dose pharmaceuti- KE Scott*1, TJ Caron1, MT Whary1, JG Fox1, SJ Hagen2 cal and chemical-grade STZ groups required early euthanasia due to poor clinical conditions and severe weight loss. All animals in the 1Division of Comparative Medicine, Massachusetts Institute of low-dose pharmaceutical and low-dose chemical groups survived for Technology, Cambridge, MA; 2Beth Israel Deaconess Medical Center, the duration of the study without clinical signs or weight loss. All Boston, MA animals in both groups achieved and maintained blood glucose

668 Abstracts of scientific papers 2016 AALAS National Meeting

levels above 200mg/dL for the extent of the study. The animals in the Bordetella hinzii is a gram-negative rod-shaped bacterium. Consistent low-dose chemical STZ group maintained higher blood glucose with other members of the genus Bordetella, it resides in respiratory levels as a group than the pharmaceutical grade animals maintained. pathways and has been reported to contribute to respiratory Histopathology revealed hydronephrosis, hepatocellular necrosis, inflammation. Up until 2008, our laboratory occasionally isolated an and changes in the pancreas in all animals treated with STZ. Changes organism from laboratory mice which was consistently identified were similar regardless of grade of STZ or dosing regimen. These as B. avium because of the similar biochemical profile. There are results indicate that there is no significant difference in toxicities reports of isolates consistent with B. avium biochemically, but which between chemical and pharmaceutical grade STZ and administration also metabolized alkali to malonate and had marginally distinct 16s of either form at a high bolus dose results in severe clinical signs sequence, both which were consistent with B. hinzii. As a result of requiring euthanasia. Based on the consistently elevated glucose this information, our laboratory identified multiple isolates that were levels and lack of clinical signs in the multiple low dose chemical consistent with the B. avium biochemical profile, but had a 16s grade STZ group (group 3), we have adopted this regimen as our sequence similar to B. hinzii. We further sequenced a portion of the of method for diabetes induction of C57BL/6 mice for future work. bacterial RNA polymerase β subunit which contained a larger set of unique nucleotides compared to closely relatedB. spp. which P238 The Role of Platelets in the Pathogenesis of Cytomegalovirus accommodated the development of a highly specific fluorogenic Infection in Mice PCR. This assay was added to our routine PCR screening panels to determine the prevalence of B. avium. To further understand the JK Brockhurst2,1, K Najarro1, CG Cryer2,1, S Guerrero-Martin1, Y Su3, pathogenesis and detection by PCR and bacteriology, we intranasally R Boger3, KA Metcalf Pate*1 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 1Department of Molecular and Comparative Pathobiology, Johns controls). Minimum to mild bronchitis was observed in inoculated Hopkins School of Medicine, Baltimore, MD; 2School of Veterinary mice of all strains, with the interesting exception of bronchitis being Medicine, University of Pennsylvania, Philadelphia, PA; 3Division of absent in the Nu/Nu. Tracheitis and rhinitis was minimal in CD-1, Pediatric Infectious Disease, Johns Hopkins University, Baltimore, min-mild in B6 and NU/NU, and mild-moderate in SCID mice. MD Nasal aspirate, bronchial lavage fluid, and fecal pellets were positive by PCR, and oral swabs, lung tissue, external genitalia, and cecum Cytomegalovirus (CMV) a herpesvirus that is excluded from many contents were mostly negative by PCR. Approximately 2-3% of field specific pathogen-free animal facilities. It is difficult to eradicate samples received for PCR quarantine and routine screening panels without rederivation because it forms a latent state in tissue myeloid received 2013-2016 were positive for B. hinzii with the majority being cells and the mechanisms underlying the development of this latency detected in transgenic mouse lines. In conclusion, our investigation are incompletely understood. Platelet decline is characteristic of the identified B. hinzii as a prevalent primary pathogen contributing to acute stage of many viral infections, including CMV, and can be respiratory inflammation in both immunocompetent and partly driven by platelet sequestration in associations with mono- immunodeficient strains. cytes and endothelial cells. Such associations affect the characteristics of the myeloid monocyte and the endothelial-organ border, and may P240 Functional Validation of Tensin2 SH2-PTB Domain by change the ability of the monocyte to become infected and invade CRISPR/Cas9-Mediated Genome Editing into tissues by causing the monocyte to adopt an activated Ly- 6Clo phenotype. We hypothesized that platelet decline and K Marusugi*1, K Nakano1,2, R Kaneda1, H Sasaki1, J Kimura4, R platelet-monocyte aggregate (PMA) formation would occur in Yanobu2,3, T Okamura2,3, N Sasaki1 CMV-infected mice, and that platelet depletion would decrease viral load in the salivary gland and spleen. Male BALB/c mice were 1Laboratory of Laboratory Animal Science and Medicine, Faculty of infected with 3 X 106 plaque forming units of murine CMV on day 0, Veterinary Medicine, Kitasato University, Towada, Japan; 2Depart- and bled for complete blood count every other day to longitudinally ment of Laboratory Animal Medicine, Research Institute, National track platelet numbers over 21 days. The lowest platelet counts were Center for Global Health and Medicine, Tokyo, Japan; 3Department observed on day 3 of infection, and were followed by a rebound of Infectious Diseases, Section of Animal Models, Research Institute, thrombocytosis on day 8 (N = 5, P = 0.04). To determine whether National Center for Global Health and Medicine, Tokyo, Japan; 4Lab- sequestration in PMAs contributed to this decline, additional mice oratory of Anatomy, Department of Biomedical Sciences, Graduate were infected and euthanized on days 0, 3, 8, or 14 for blood and School of Veterinary Medicine, Hokkaido University, Sapporo, Japan tissue harvest to quantify PMA formation by flow cytometry (N = 5 per timepoint). PMA formation, especially Ly-6Clo PMA formation, Podocytes are terminally differentiated and highly specialized cells in decreased rather than increased at all timepoints following infection the glomerulus that form a crucial component of the glomerular compared to baseline (P = 0.05), indicating that PMA formation was filtration barrier. The ICGN mouse is a model of glomerular not contributing to platelet decline during acute infection. A subset dysfunction that shows gross morphologic changes in the podocyte of mice were treated with platelet depleting antibody at the time of foot process accompanying proteinuria. Previously, we demonstrated infection to determine the effect of platelets on viral entry into tissues that proteinuria in ICGN mice might be caused by the deletion at days 0, 3, 8, and 14 (N = 5 depleted and 5 undepleted per mutation in the tensin2 (Tns2) gene (designated Tns2nph). To test timepoint); organ viral load was quantified by plaque assay and whether this mutation might cause the mutant phenotype, we compared to undepleted controls. Three of 4 undepleted mice created knockout (KO) mice carrying Tns2 protein deleted of its the demonstrated higher levels of CMV in the spleen than the depleted C-terminal Src homology domain and phosphotyrosine binding mice. Platelets therefore may enhance the ability of CMV to enter into (SH2-PTB) domain (designated Tns2ΔC) via CRISPR/Cas9-mediated tissues such as the spleen through associations with cells other than genome editing. Tns2nph/Tns2ΔC compound heterozygotes monocytes. and Tns2ΔC/Tns2ΔC homozygous KO mice displayed the podocyte abnormality and massive proteinuria similar to those seen in ICGN P239 Detection and Pathogenesis of Bordetella Hinzii in 4 mice, indicating that these 2 mutations are allelic. Further, this result Commonly Used Mouse Strains suggests that SH2-PTB domain of Tns2 is required for podocyte integrity. Tns2 knockdown in mouse podocyte cell line significantly KS Henderson*1, KR Pritchett-Corning2, P Momtsios1, VS Dole1 enhanced actin stress fiber formation and the cell migration. Thus, this study provides evidence that alteration of actin remodeling by 1Research Animal Diagnostic Services, Charles River, Wilmington, deficiency of Tns2 causes the morphologic changes in podocytes, and MA; 2Office of Animal Resources, Faculty of Arts and Sciences, subsequent proteinuria. Harvard University, Cambridge, MA

669 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

P241 Photoreceptor Proliferation and Dysregulation of Cell Cycle Bup, 1mg/kg SQ, on day 0 and again approximately 90 hours later. Genes in Early Onset Inherited Retinal Degenerations Half of the animals (n = 3) underwent ischemia reperfusion cardiac injury via thoracotomy (ketamine/xylazine anesthesia) followed by a KL Gardiner*1,2, L Downs2, A Berta-Antalics2,3, E Santana2, G repeat thoracotomy (isoflurane anesthesia) for stem cell therapy 4 Aguirre2, S Genini2 days later. The control group (n = 3) underwent the same anesthetic protocol, but no surgery. Serum buprenorphine levels were deter- 1Pathobiology, University of Pennsylvania, Philadelphia, PA; 2Section mined at 12-14 time points (0 to 8-10 days) after the first dose. Serum of Ophthalmology, Department of Clinical Studies, School of chemistry screens, before the first procedure and preceding the Veterinary Medicine, University of Pennsylvania, University of second dose of SR Bup, indicated no underlying renal or hepatic Pennsylvania, Philadelphia, PA; 3Augenklinik Uniklinik Erlangen, impairment. Rats in the control group had serum buprenorphine Erlangen, Germany 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 Mitotic terminally differentiated photoreceptors (PRs) are observed 150 hours (>6 days) after the second dose. In rats that received in early retinal degeneration (ERD), an inherited canine retinal surgery, serum buprenorphine levels remained above 0.5 ng/ml for disease caused by mutation in the NDR kinaseSTK38L (NDR2). In 100 hours (> 4 days) following the second dose of SR Bup. Area this study, we examined whether PR proliferation may also occur in under the curve analysis revealed elevated serum buprenorphine other early onset inherited retinal diseases to determine if common levels in the three days following the second dose compared to the 3 molecular pathways were involved. Retinas and retinal pigmented days after the first dose in the control group (P = 0.001). These results epithelium (RPE)/choroid samples were obtained from dogs that suggest that protocols with multiple survival surgeries within close were either normal, xlpra2, rcd1, or ERD-affected at timepoints time proximity may need to consider altering the dose and timing of before cell death peak (induction: 3 weeks), at cell death peak repeat SR Bup administration. (execution: 5–10 weeks), and during sustained but reduced cell death rate (chronic cell death: ≥ 14 weeks). Samples were analyzed by P243 A Novel Model of Regional Hepatic Arterial Infusion in immunohistochemistry and Western blot analysis, and by real-time Hepatitis-Infected Woodchucks (Marmota monax) with Hepatocel- PCR (qRT-PCR; n = 3 per timepoint per condition) for evidence of PR lular Carcinoma proliferation, as well as differential expression of genes involved in cell cycle regulation, PR and eye development regulation, and LI Curtin*1, M Kim2, C Powers2, S Sexton1, A Gudkov3, A Purmal4, R HIPPO/LATS1 pathway. We show that a proliferative response Iyer5 similar to that observed in ERD, but lower in magnitude, occurs in 2 other early onset disease models, xlpra2 and rcd1 by immunolabeling 1Laboratory Animal Shared Resource, Roswell Park Cancer Institute, with PCNA and phospho-histone H3 antibodies. Proliferating cells Buffalo, NY; 2Department of Surgical Oncology, Roswell Park Cancer were determined to be rods. Statistical significance of differentially Institute, Buffalo, NY; 3Department of Cell Stress Biology, Roswell expressed retinal genes (P < 0.05; fold change FC>+/-2) was assessed Park Cancer Institute, Buffalo, NY; 4Incuron LLC, Buffalo, NY; 5De- with an unpaired t-test. Expression of the cell cycle-related partment of Medical Oncology, Roswell Park Cancer Institute, genes RB1 and E2F1, as well as CDK2,4,6 was up-regulated, but Buffalo, NY changes were mutation-specific. Changes in cyclin expression differed across all genes, diseases and time points analyzed, althoug The woodchuck (Marmota monax) is the only large animal model of h CCNA1 and CCNE1 expression increased with age in the 3 models, spontaneous hepatocellular carcinoma (HCC) in the context of suggesting that there is a dysregulation of cell cycle gene expression chronic hepatitis infection. This makes it ideally suited for preclinical in the 3 diseases. Unique to ERD, however, are mutation-specific studies of antineoplastic therapies. Intravenous chemotherapy for changes in the expression of NDR kinases and Hippo signaling surgically unresectable tumors that are limited to the liver is often members with increased expression of MOB1 and LATS1 in the mired by undesirable side effects and low response rates. Regional newly generated hybrid rod/S-cones using Western blot analysis. cancer therapy through a hepatic artery infusion pump (HAIP) takes Our data raise the intriguing possibility that terminally differentiated advantage of the first-pass effect owing to the liver’s unique blood normal PRs are kept from dividing by NDR2-MOB1 interaction. supply and the fact that liver tumors are fed exclusively by arterial Furthermore, they provide the framework for the selection of flow. HAI has been used extensively in primary and metastatic candidate genes for further investigation as potential targets of human liver tumors, albeit, with mixed clinical results. The purpose therapy. 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 P242 Prolonged Therapeutic Levels of Sustained Release Buprenor- tumors underwent prophylactic cholecystectomy, hepatic artery phine with Repeat Dosing in Rats collateral vessel ligation, and placement of a 2Fr. vascular access port (VAP) into the gastroduodenal artery under a surgical microscope. LE Neidig*1, M Haenisch1, SM Meeker1, AM Martinson2, C Murry2, The port head was secured subcutaneously to the rectus muscle. TL Brabb1 Correct placement of the VAP catheter end was verified during surgery by direct inspection using a surgical microscope and 1Comparative Medicine, University of Washington, Seattle, WA; 2Pa- palpation using microforceps, and was successful in all 4 animals. All thology, University of Washington, Seattle, WA 4 ports supported an experimental drug infusion of 3 mL/kg over 30 minutes without complications and were functional at the time of Sustained release formulation of buprenorphine (SR Bup) has become euthanasia in all animals (4-28 days post implantation). One of the widely used in laboratory animal medicine, particularly in rodents, four woodchucks suffered postoperative bleeding at the port head where reduced handling likely decreases stress while providing site, which was attributed to the animal’s degree of liver dysfunction, extended analgesic coverage. Current literature has documented and 4/4 woodchucks were partially anorexic for up to 7 days post blood plasma levels at or above 1 ng/ml for 72 hours following a op. In conclusion, we describe a new model of regional HAI single subcutaneous (SQ) dose in rats; however, there is a lack of chemotherapy using a VAP that provides a feasible and practical published data on repeated dosing for animals undergoing multiple treatment modality for evaluating novel regional cancer chemothera- survival surgeries. Side effects associated with higher levels of py agents in a large animal model of HCC. buprenorphine in rats such as rebound hyperalgesia, respiratory depression, gastrointestinal distress, and pica highlight the need for P244 Vertical Sleeve Gastrectomy in Mice: Validating a Mouse SR Bup to be administered at appropriate dosages and time intervals. Model for the Surgical Intervention Of Obesity In this study, the pharmacokinetics of SR Bup were examined when a second dose was administered 4 days after the initial dose. Adult LA Stewart*1,2, TA Knotts1,2, DA Kieffer1, K Lloyd1,2, KD Evans1,2, K athymic nude male rats (Hsd:RH-Foxn1rnu) received a dose of SR Grimsrud1,2

670 Abstracts of scientific papers 2016 AALAS National Meeting

1Mouse Biology Program, University of California—Davis, Davis, were no statistically significant differences, this pilot study suggest CA; 2Mouse Metabolic Phenotyping Center, Davis, CA that marble burying behavior may be a reliable and objective way to assess pain in rats. Obesity is rapidly becoming a global health epidemic. Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is currently the P246 Continuous Assessment of Marble Burying in Rats Is most effective long-term treatment for obesity and has shown to Inadequate for Assessing Postoperative Pain markedly improve glucose homeostasis. The VSG surgical procedure removes approximately 75% of the stomach creating a small remnant W McGee, LV Kendall* tube that limits food intake. In order to investigate the metabolic effects of these surgeries, it is critical to have appropriate translation- Laboratory Animal Resources, Colorado State, Fort Collins, CO al, clinically relevant animal models. We developed and validated the VSG surgical procedure in diet-induced obese (DIO) C57BL/6J mice Behavioral assessments of post-operative pain in rats can be (n = 160). During the developmental phase, we modified an existing subjective and prone to interobserver variability. We demonstrated procedure to improve survival rates and reduce the occurrence of that marble burying behavior could be used as an objective means to surgical site leakage, ischemia, and abscess formation. Our modified assess post-operative pain in a pilot study using vasectomized rats. technique improved the survival rate from approximately 50% to This study evaluated the use of marble burying behavior to assess 90%. The validation was comprised of mice fed a 60% high-fat diet post-operative pain in female rats following an ovariectomy. Twelve for 9 weeks prior to surgery. VSGs, pair-fed shams (PFS), ad-lib Long Evans rats were anesthetized with isoflurane and underwent a shams (ALS), and control mice (C) were measured for body weight, surgical ovariectomy, and 12 rats received anesthesia only. Six body composition, feed intake, and glucose tolerance over a 3-month ovariectomized rats received post-operative analgesia treatment with period. Significant differences in body composition of VSG mice were meloxicam (2 mg/kg s.c. bid) and six received saline (0.1 ml s.c. bid). observed. VSG male and female mice gained significantly less body Six rats in the anesthesia only group received meloxicam and 6 weight post surgery compared to the ALS mice; no difference received saline as controls. Pain was assessed at 1, 3, 6, 12, 24, and 48 between VSG and PFS mice was observed. Male VSG mice had hours post operatively. Rats were observed for 2 minutes to evaluate improved glucose tolerance compared to ALS mice. No difference behavioral indicators of pain including rearing, grooming, wound was observed among female mice. Our validation demonstrated that licking, posture, orbital tightening, and general activity. Marbles (1.5 the pattern of body weight change and the effect on food intake and cm diameter) were placed in the recovery cage and the number of body composition in our VSG mouse model paralleled the human marbles that were buried 2/3 of their diameter were counted at the prognosis following VSG surgery. In summary, the surgical mouse time of pain assessment. Ovariectomized rats had increased orbital model we created and validated is translational and can be used to tightening and wound licking, and reduced rearing compared to further investigate the effects of VSG as a surgical intervention of unsurgerized rats. There was no significant difference in the number obesity. of marbles buried over time between the 4 groups. This study demonstrates that marble burying behavior may not be an objective P245 Marble Burying Test as a Practical Pain Assessment in Rats means to assess post-operative pain if the marbles are left continuously in the home cage. NN Lee, LV Kendall* P247 The Cynomolgus Macaque (Macaca fascicularis) Model of Laboratory Animal Resources, Colorado State University, Fort Pneumonic Tularemia Collins, CO LL Lanning*1, P Sanz1, L Wolfraim1, C Houchens2, K Omland3, M Behavioral assessments using ethograms are frequently used as a Williams1, J Hewitt1, T Guina1 means to assess pain in rats. These measurements are often subjective and prone to inter-observer variability. As a prey species, rats may 1NIH/NIAID/DMID, Bethesda, MD; 2BARDA/HHS, Washing- hide behavioral signs during observations, which further complicates ton; 3Mergus Analytics, Jerricho, VT pain assessment. Rats typically bury novel objects as a natural defense mechanism to cope with stress or anxiety caused by novel The National Institute of Allergy and Infectious Diseases (NIAID) objects. Marble burying test are frequently used to assess anxieolytic and the Biomedical Advanced Research and Development Authority drugs and obsessive compulsive disorders. We evaluated the marble (BARDA) formed a Working Group for qualification of the cynomol- burying test in an effort to identify a more objective measurement of gus macaque (Macaca fascicularis) model of pneumonic tularemia as a pain in rats. Six male Sprague–Dawley rats were anesthetized with Drug Development Tool with the Food and Drug Administration. isoflurane and underwent a surgical vasectomy. Six rats received Qualification of an animal model through FDA’s Animal Model anesthesia only, no surgery. Three vasectomized rats received Qualification Program is limited to those proposed for use in product postoperative analgesia treatment with meloxicam (1 mg/kg s.c. bid) approval under the Animal Rule. This is a regulatory mechanism for and 3 received saline (0.1 ml s.c. bid). Three rats in the anesthesia- approval based on evaluation of the efficacy of new-generation only group received meloxicam and 3 received saline. Pain was therapeutics and vaccines in suitable animal models [21 CFR 314.600 assessed at 1, 3, 6, 12, 24, and 48 hours post operatively. Rats were for drugs; 21 CFR 601 Subpart H for biologic products]. The observed for 5 minutes to evaluate behavioral indicators of pain adequacy of an animal model for qualification is based on data from including rearing, grooming, wound licking, posture, orbital natural history studies and from the human disease or condition tightening, and general activity. Following the 5-minute observation showing that the specific animal species given a specific challenge period, rats were placed in a new cage with 12 marbles. Rats were agent by a specific route, produces a disease that corresponds to the removed after 30 minutes and the number of marbles that were human disease or condition of interest in multiple important aspects. buried 2/3 of their diameter were counted. Vasectomized rats had We intend to qualify the natural history model of pneumonic reduced rearing, increased wound licking, and increased orbital tularemia in the cynomolgus macaque based on results of 2 LD50 and tightening scores compared to unsurgerized control rats. Wound 5 natural history studies that have been completed. Mortality, licking was greatest in the saline treated group. Marble burying in average time to death, average time to fever onset, average interval saline treated vasectomized rats was reduced for the first 12 hours between fever and death, initial level of bacteremia post-challenge, post-operatively; whereas, meloxicam treatment increased the marble timing of bacteremia onset, clinical signs, necropsy findings, and burying behavior 3-6 hours post-operatively. This was similar to the histopathology from the studies were analyzed and form the basis for unsurgerized, saline treated rats. Meloxicam treatment in unsurger- the proposed cynomolgus macaque model parameters. Data collected ized rats had the greatest marble burying activity. Compared to the to date support the conclusion that symptoms and signs of pneu- ethogram observations, marble burying behavior showed the most monic tularemia in cynomolgus macaques exposed to a target dose of apparent differences between the 2 surgical groups. Although there 1,000 cfu (range 300 to 3000 cfu) aerosolized F. tularensis Schu S4,

671 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 under the conditions described herein, and human pneumonic groups that received nutritional supplement compared to mice that tularemia cases are highly similar. did not receive nutritional supplement. The results of this study suggest an altered response to tumor growth in mice that receive P248 Safety of Epidural Morphine with Preservative in Domestic nutritional supplement prior to but not during chemotherapy. The Goats (Capra aegagrus hircus) improvement on body weight recovery for mice receiving supplement during chemotherapy may improve the overall health of the MG Lin*1, JJ Elliott1, M Jones2, M Wight-Carter3, G Carroll2 animal subjects and extend the life of the research study. If nutritional support is used during chemotherapy studies with mice we 1Comparative Medicine Program, Texas A&M, College Station, recommend providing supplement to all cages during dosing to TX; 2College of Veterinary Medicine, Texas A&M, College Station, avoid variability in tumor growth or body weight loss between cages. TX; 3ARC Diagnostic Lab, University of Texas Southwestern Medical Center, Dallas, TX P250 Urine Cortisol:Creatinine Ratios as an Assessment of Stress and HPA-Axis Function in Rhesus Macaques (Macaca mulatta) The management of perioperative pain in livestock species using opioid epidurals has become increasingly popular. Preservative-free SC Adams*1, C Guyot1, K Berry1, A Loar2, M Leblanc1 morphine sulfate (PFM) is currently the standard for epidural use, as phenol and formaldehyde preservatives found in morphine with 1ARD, Salk Institute, La Jolla, CA; 2STAT Veterinary Lab, San Diego, preservative (MWP) have been shown to be neurotoxic. However, CA due to costs and national shortages of PFM, many veterinarians are being forced to use MWP instead. The objective of this study was to Monitoring biomarkers of stress in laboratory animals is critical to determine if epidurally (L6-S1) administered MWP produced evaluate the potential impact of research activities on animal welfare. behavioral or spinal cord changes that were different from those seen Cortisol is a sensitive marker of stress in many species, including with PFM, normal saline, or no epidural administered. One hundred macaques. Cortisol levels may be evaluated in several samples, young adult goats were randomized into 7 groups, and necropsied including serum, saliva, feces, hair, and urine. While serum and 2-4 weeks following no epidural (2 weeks, n = 15), saline epidural (2 saliva best reflect real-time cortisol levels, they require stressful weeks, n = 15; 3 weeks, n = 11), PFM epidural (2 weeks, n = 15; 4 restraint that may alter results. Fecal cortisol is non stressful, but weeks, n = 14), or MWP epidural (2 weeks, n = 15; 4 weeks, n = 15). results may be confounded by urine contamination, diet, and Behavior and clinical signs of toxicity were evaluated. At necropsy, intestinal transit. Hair cortisol is also noninvasive but provides spinal cords were removed from the lumbar region to the cauda retrospective values over weeks to months. Urine cortisol:creatinine equina and histology performed. All slides were examined by a ratios (UCCR) are commonly used in small animal and human veterinary pathologist (blinded to treatment groups) for evidence of medicine and reflect hypothalamic-pituitary-adrenal (HPA) activity neurotoxicity by the presence of gliosis, central chromatolysis of over a period of a few hours to a day; however, information is neurons, white blood cell infiltrates, thickening of dura mater, and lacking regarding its use in nonhuman primates (NHPs). We fibrosis. Histologic lesions of neurotoxicity were not found in any therefore evaluated the use of UCCR in macaques to assess animal treatment group. Behavior did not vary among treatment groups, welfare and HPA axis. We hypothesized that UCCR values would be and no clinical signs of toxicity were noted. Thus, a single dose of elevated in working cranially implanted macaques and would be MWP administered epidurally in goats does not cause histologic responsive to dexamethasone suppression. Urine was collected from evidence of neurotoxicity and may be considered a safe practice. singly housed animals that were separated due to veterinary or scientific exemptions or paired-housed macaques separated at the time of P249 Nutritional Supplement and Its Effect on Tumor Growth and feeding or during work. We first compared UCCR in working Body Weight in NSG Mice Undergoing Chemotherapy macaques with cranial implants (n = 4) and naïve non-working monkeys (n = 8). UCCR values were significantly higher (P < 0.001) M Tewodros*1, L Yao1, M Cheng1, M Creamer2 in working monkeys (58.2 ± 23.4) as compared to nonworking animals (12.5 ± 12.7) suggesting higher levels of stress associated 1In Vivo, The Jackson Laboratory, Sacramento, CA; 2Comparative with research activities. We also evaluated the use of UCCR to assess Medicine and Quality, The Jackson Laboratory, Sacramento, CA HPA function by performing a Low Dose Dexamethasone Suppres- sion (LDDS) test. Oral dexamethasone (DEX) (0.01 mg/kg) was given Chemotherapeutics are effective in the treatment of cancer; however at 6 am, 12 pm, and 6 pm on the same day and urine samples were body weight loss and subsequent malnutrition are potential collected before and at 6, 12, 24, 32, and 56, and 102 hours post first complications during therapy. The use of supplemental nutrition in DEX dose. UCCR levels decreased by 67% in the first 12 hours and cancer patients remains controversial. This study seeks to investigate reached nadir at 32 hours before gradually returning to normal at 102 whether nutritional supplement can alleviate body weight loss hours, thus suggesting that cortisol is predictably suppressed by low during chemotherapy and its effects of tumor growth using NSG doses of DEX and can be used to assess near-real time cortisol levels mice. Female NSG mice (n = 60) were subcutaneously engrafted with and HPA function in macaques. Overall, these results suggest that human lung patient derived xenograft (PDX) fragments. A subset of UCCR is a noninvasive method that can be used to evaluate near-real mice (n = 30) were used to evaluate tumor growth response with or time cortisol levels, stress, HPA axis, and welfare in research without nutritional supplement and the remaining engrafted mice (n macaques. = 30) received the chemotherapeutic Docetaxel with or without nutritional supplement. Tumor growth and body weight were P251 Antigen, Antibody, and Echocardiographic Results for Cats measured twice weekly for all groups. Nutritional supplement Experimentally Infected with L3 of Dirofilaria Immitis and consisted of ad libitum access to 2oz cups of a commercially available Implications for Future Studies nutrient-fortified water gel placed within the cage. Cups were replaced twice weekly. The study found that mice receiving M Krecic*1, J Lizer2, T McTier2 Docetaxel had an equal amount of body weight loss regardless of access to nutritional supplement. However, mice that received 1Zoetis, Florham Park, NJ; 2Zoetis VMRD, Kalamazoo, MI nutritional supplement saw a significantly faster (P < 0.05) increase in body weight once chemotherapy decreased or ended and were able Feline heartworm disease caused by Dirofilaria immitis remains to regain their body weight 5-7 days sooner than mice which did not difficult to diagnose. Serological tests do not always correlate well receive supplementation. There was no significant difference in with infection and little controlled data is available on the use of tumor growth of chemotherapeutic treated mice irrespective of echocardiography in determining infection and infection levels. The nutritional supplementation. Interestingly, tumor growth in mice not objective here, part of a wider feline heartworm diseases program, undergoing chemotherapy was significantly greater (P < 0.05) in was therefore twofold: to determine serological heartworm status

672 Abstracts of scientific papers 2016 AALAS National Meeting

through 8 months post experimental infection and to correlate University of Missouri, Columbia, MO; 2Veterinary Medicine and serological heartworm test results and echocardiographic findings to Surgery, University of Missouri, Columbia, MO; 3Mason Eye the known numbers of adult heartworms identified at necropsy 238 Institute, University of Missouri, Columbia, MO; 4Veterinary days postinfection. Twenty cats experimentally infected with 100 Medicine and Surgery, Veterinary Pathobiology, and Mason Eye third stage larvae (L3) of D. immitis (day 0) were tested for antigen on Institute, University of Missouri, Columbia, MO; 5Harry S. Truman and between days 28 through 238 and antibody on and between days Memorial Veterans’ Hospital, Columbia, MO 56 through 238, and examined echocardiographically on days 177 and 231. Sixteen of 20 (80%) cats developed adult heartworm Corneal scarring (fibrosis) is a leading cause of blindness worldwide infections; 13 of 16 (81%) cats had antigen on day 238 and 16/16 as a result of the normal corneal wound healing response. Rho-asso- (100%) cats had antibodies on days 182 and 238. Four of 20 (20%) cats ciated protein kinase (ROCK) pathways regulate cellular did not develop adult heartworm infections. These cats were also proliferation, migration, adhesion, wound healing, angiogenesis, and negative for antigen and antibody on days 182 and 238 and also fibrosis in vivo. We tested the postulate that topical application of negative for adult heartworms with echocardiography on days 177 ROCK inhibitor HA1077 to the eye following corneal insult will and 231. Results suggest that combined diagnostics are useful in a attenuate corneal fibrosis and corneal neovascularization (CNV) in research setting for accurately identifying adult heartworm infections vivo by mitigating exuberant wound healing. The study was in cats at 8 months post experimental infection with 100 L3 of D. approved by the IACUC. Corneal fibrosis and CNV were produced immitis without the need for necropsy. Veterinarians examining cats in 12 New Zealand White rabbits by a single topical alkali (1N suspected of adult heartworm-induced disease may equally be able NaOH) application for 1 minute to the central cornea. Following to exclude infection with negative serological test and corneal wounding, animals were divided into 2 groups: Group 1 echocardiographic results as early as 7 months post natural infection. served as controls and received 50μL balanced salt solution (BSS) topically twice daily for 3 days. Group 2 served as treatment cohort P252 Isolation of Lytic Bacteriophages against Multidrug-Resis- and received 50μL HA1077 (3nM) topically twice daily for 3 days. tant Enterococcus faecalis ST55 Isolated from Research Macaques Contralateral untreated eyes served as negative controls. Serial slit and stereomicroscopy evaluated the degree of ocular inflammation, MT Lieberman*1, RJ Citorik2, TK Lu3, JG Fox1 corneal edema, and corneal opacity. Corneas were harvested on day 14 with H&E and immunofluorescence staining employed to 1Division of Comparative Medicine, Massachusetts Institute of characterize levels of fibrosis, CNV, inflammation, and apoptosis. Technology, Cambridge, MA; 2Department of Biology, Massachusetts Biomicroscopy detected a significant decrease (~2.8 fold; P < 0.01) in Institute of Technology, Cambridge, MA;3Department of Biologic corneal fibrosis and CNV in eyes treated with HA1077 compared to Engineering, Massachusetts Institute of Technology, Cambridge, MA BSS-treated controls. Further, HA1077-treated corneas showed significant decreases in fibrosis markers (smooth muscle actin, Multidrug-resistant Enterococcus faecalis (MDR-EF) is a serious cause fibronectin, and F-actin; 55-60%; P < 0.0001), and did not exhibit of human nosocomial infections. We have identified 14 MDR-EF significantly increased numbers of CD11b+ (2-11%) or TUNEL+ isolates of sequence type (ST) 55 (n = 7) and ST4 (n = 7) from cephalic (0-5%) cells compared to BSS-treated controls during immunofluores- recording chambers of macaques used in neuroscience research. cence analyses. A substantial decrease in CNV was also noted in Whole genome sequencing identified genes encoding resistance to HA1077-treated eyes as compared to BSS-treated controls (quantifica- aminoglycosides, tetracycline, erythromycin, and macrolide tion pending). Clinical ocular examinations and histologic evaluation antibiotics. Due to the difficulties in treating infections caused by did not reveal evidence of acute toxicity from topical application of multidrug-resistant bacteria, new antimicrobial strategies are needed. HA1077. Topical application of ROCK inhibitor HA1077 is a viable Lytic bacteriophage therapy has historically been used to treat option for treating corneal fibrosis and neovascularization resulting bacterial infections in Eastern Europe since the early 1900s. We from corneal insult. Further in vivo analysis is warranted. hypothesized that we could isolate lytic bacteriophages with activity against MDR-EF. Bacteriophage enrichments were prepared by P254 Noonan Syndrome with Multiple Lentigines Associated adding sterile-filtered sewage samples to double-strength phage Hypertrophic Cardiomyopathy Treatment with Angiotensin II broth and individual inoculation with single MDR-EF isolates. Receptor Blocker Following overnight incubation, enriched samples were centrifuged and supernatants were spot tested on each MDR-EF isolate using a MA Bellrichard*1, M Krenz2,3 modified double-agar overlay method. Individual phages were purified by 3 serial passages of isolated plaques, then concentrated 1Veterinary Pathobiology, University of Missouri, Jonesburg, from lysed broth cultures via polyethylene glycol precipitation and MO; 2Medical Pharmacology and Physiology, University of Missouri, stained with uranyl actetate for transmission electron microscopy Columbia, MO; 3Dalton Cardiovascular Research Center, University (TEM). Lytic activity was observed against all 7 ST55 isolates with 3 of Missouri, Columbia, MO isolates showing the strongest lysis as indicated by plaque size. Initial TEM imaging identified the 3 phages as likely Siphoviruses of Noonan syndrome with multiple lentigines (NSML) is a rare genetic 2 distinct morphologies featuring long, flexible, apparently disease that leads to a variety of clinical manifestations. These noncontractile tails. Capsid morphologies included icosahedral (50.7 include skin, skeletal, and cardiovascular abnormalities. Importantly, ± 2.4 nm in diameter) and 2 sizes of prolate capsids (104.5 ± 5.7 nm x 80% of NSML patients have hypertrophic cardiomyopathy (HCM). 37.64 ± 1.1 nm and 92.8 ± 4.6 nm x 36.5 ± 1.3 nm). Lytic phage Genetic studies have shown that 90% of NSML cases are due to therapy offers an alternative to traditional antimicrobials and may be mutations in the nonreceptor protein tyrosine phosphatase Shp2. especially valuable against highly antimicrobial-resistant bacteria. Mechanistically, animal studies have shown that mutations in Shp2 Future studies include continued efforts to isolate lytic phage with result in hyperactivation of downstream signaling through Akt and activity against ST4 isolates, as well as phage sequencing and mammalian target of rapamycin, both of which are potent stimula- characterization of anti-biofilm activity by ST55 lytic bacteriophages. tors of cardiac growth. Myocardial hypertrophy and fibrosis are a major part of the pathology of HCM and both of these processes can P253 Pharmacologic Inhibition of Rho-Associated Protein Kinase be stimulated by angiotensin II. In addition, another study has Pathways as a Therapeutic Strategy for Attenuating Corneal revealed that angiotensin II directly activates Akt through angioten- Fibrosis and Neovascularization in Vivo sin II receptor type 1. This information lead to the hypothesis that blocking angiotensin II via losartan, an angiotensin II receptor MK Fink*1,5, S Gupta2,5, M Possin3,5, PR Sinha2,5, EA Giuliano2, RR antagonist, will improve cardiac morphology and function in NSML. Mohan4,5 We chose the Q510E-Shp2 mutation for our studies since this mutation is associated with a particularly severe form of NSML in 1Veterinary Pathobiology and Comparative Medicine Program, both humans and mice. Expression of Q510E-Shp2 in mice causes

673 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 severe early onset HCM with interstitial fibrosis, thickened ventricu- drinking water. It is questionable whether this is an effective protocol lar walls, and depressed contractile function. In our study, in minimizing clinical disease in strains that are susceptible to 8-week-old Q510E mutant mice and nontransgenic controls were specific bacterial agents. In these instances, supplementation with treated with losartan or a control for 8 weeks. The mice were antibiotics may be recommended. Normally antibiotics are provided evaluated with echocardiography at both the onset and conclusion of to the mice via the drinking water; however, acidification of the the treatment trial. Cardiac function, as measured by fractional drinking water may interfere with antibiotic efficacy. In this project, shortening, was decreased by 20% in the untreated NSML mice. we evaluatedStaphylococcus xylosus (S.xylosus) cultured from However, the NSML mice treated with losartan showed no decrease immunocompromised mice being treated with either acidified water fractional shortening in comparison to their nontransgenic counter- only or neomycin prepared with acidified water. Thirty female parts. At the conclusion of the study, heart to body weight ratios were B6.129S-Cybbtm1Din/J (Cybb) mice were received and housed 2/cage assessed as a measure of cardiac hypertrophy. Our data show a trend and placed on either acidified water (Group 1-5A) for the duration of with NSML mice having 20% heavier hearts than the nontransgenic the study or neomycin acidified water (Group 1-5B and 6-10). mice and the losartan treatment decreased this hypertrophy to only Animals in Group 1-5B alternated between acidified and neomycin 10%. Taken together, this information shows losartan has promise as acidified every 2 months. Group 6-10 were treated with neomycin a novel treatment modality for maintaining cardiac function in NSML acidified water for 5 months. At the end of the study both groups patients, but would have only minor effects on the degree of cardiac were removed from antibiotic treatment and placed back on acidified hypertrophy. water. Oropharangeal (OP) cultures were performed weekly until S. xylosus was recovered and antibiotic susceptibility testing could be P255 Characterization of the Fecal Microbiota of Mink (Neovison performed. Sensitivity disks were prepared with 0.25 inch blank vison): A Representative Carnivore disks saturated with 20 ul of each treated water neomycin acidified or acidified as well as neomycin in water deionized by reverse NR Compo*1, B Tapscott2, D Gomez1, J Weese1, PV Turner1 osmosis (RO-DI) to confirm the reduction in microbial inhibition was indeed due to the acidified water. Sensitivity disks were placed on 1Pathobiology, University of Guelph, Guelph, Canada; 2Ontario 5% Sheep Blood Agar plates streaked with S. xylosus and incubated Ministry of Agriculture, Food and Rural Affairs, Guelph, Canada at 37 degrees for 24 hours. Zones of inhibition were recorded. S. xylosus could not be cultured while on antibiotic water; however, S. The mammalian gut microbiota has received increasing attention xylosus was recovered 3 weeks after animals returned to acidified because of its role in health and disease. In particular, reports have water. Microbial sensitivity testing revealed neomycin had a emerged from mice, rabbits, and other species suggesting that significant reduction in microbial inhibition when prepared with differences in gut microbiota can influence research outcomes. To acidified water compared to neomycin prepared with RO-DI water. date, most microbiota work has been conducted in herbivores and Therefore the type of water used when preparing antibiotic treated omnivores, although some work has also been conducted in water should be considered. companion cats. The objective of this study was to characterize and compare the fecal microbiota of mink (Neovison vison), to contribute P257 Reference Growth Curves for Long-Tailed Macaques (Macaca to the understanding of comparative microbiota differences in fascicularis) from Mauritius carnivore models. In addition, we sought to determine whether temporal or age differences exist in the mink fecal microbiota, and S Naiken, P Honess* whether dietary changes, as occur in female mink prior to breeding, affect the gut microbiota. Pooled fecal samples (n = 344) were Animal Welfare, Bioculture Group, Riviere des Anguilles, Mauritius collected from healthy females and weaned kits from mink farms during 2 consecutive summers and from healthy females (n = 36) The long–tailed macaque (Macaca fascicularis) of Mauritian origin is before winter breeding. Bacterial DNA was extracted and character- widely used in biomedical research. However, there is little informa- ized using 16S rRNA gene PCR and next generation sequencing. tion on its growth pattern. The purpose of this study is to provide Following quality control filtering, approximately 7.2 million weight for age and sex reference standards for this important animal sequences have been identified. The predominant phyla in the feces model. Reference values were derived from retrospective data over a of females and kits are Firmicutes, with mean relative abundances of 10-year period for all live births of captive-bred monkeys from birth 54.7 and 57.3%, respectively, and Proteobacteria, with means of 42% to a maximum of 84 months. The animals were weaned at between 12 and 39.7%, respectively. The latter is greater than the that seen in and 24 months into peer groups housed outside under ambient other species, such as mice, rabbits, and humans. Other phyla were conditions. Housing and care meets or exceeds standards set in the identified but all were uncommon accounting for <2% of total NRC Guide (2011) and the EU Directive (2010/63/EU). The smooth sequences each. There were no significant differences in phyla curves of weight for age and sex were created using Generalised identified between females and kits. At the genus level, >900 genera Additive Models for Location Scale and Shape (GAMLSS) in R were identified, only 3 of which represented >4% of the total statistical software (v.3.1.2) and included 244,809 weight observations sequences: Atopostipes (~5%), previously only isolated from an from 37,105 males (mean = 6.6 observations/animal, SD=8.53) and underground swine manure storage pit; Lactobacillus (~9%), involved 223,432 from 35,455 females (mean: 6.3 observations /animal, in lactic acid production; and Ignatzschineria (~21%), the predominant SD=8.13). It was found that males not only grow faster than females genus in the gut of spotted flesh fly larvae. It is unclear whether this (regression coefficient, 0-84 months: male=0.09, P = 0.000; fe- last finding represents a fecal contaminant or true gut colonization in male=0.065, P = 0.000), but they also continue to grow well after mink, which receive a significant portion of their diet from livestock females have attained their peak body mass at around the age of 48 byproducts. Limited data are available on the microbiota of carni- months. Males appear to go through an adolescent growth spurt vores, so this data will be valuable for understanding comparative from around 30 months, whereas females continue to grow at a modeling. 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 P256 Antibiotic Drinking Water for Immunocompromised Strains: establish age-weight reference ranges which can be useful for Does the Type of Water Make a Difference? assessing growth in long-tailed macaques of Mauritian origin.

PH Myers*, TE Whiteside, J Locklear, TL Blankenship P258 Evaluation of Anthelmintic Resistance and Exhaust Air Duct PCR as a Diagnostic Tool in Mice Endemically Infected with As- National Institutes of Health, Research Triangle Park, NC piculuris tetraptera

Immunocompromised mice are often placed on acidified water to P Kapoor*2, Y Hayes2, L Jerrell1, DA Bellinger2, R Thomas3, C reduce the spread of bacterial disease among animals through the Fletcher2, J Nielsen2

674 Abstracts of scientific papers 2016 AALAS National Meeting

1Sobran Inc, Dayton, OH; 2Division of Laboratory Animal Medicine, tions (day 0-35) were tested by ELISA and MFIA assays. Assays University of North Carolina at Chapel Hill, Chapel Hill, NC; 3De- developed using nMAV-2 antigen detected early seroconversion at partment of Psychology and Neuroscience, University of North the same time points (7 DPI) as seen by both conventional and Carolina at Chapel Hill, Chapel Hill, NC recombinant MAV-2 antigens. Antigen specificity was evaluated by testing sera from historically known negative guinea pig colonies. The influx of infectious agents in rodent colonies occurs despite a None of the 300 tested sera showed positive results thus demonstrat- robust sentinel monitoring program, strict quarantine measures, and ing a 100% specificity of both nMAV-2 ELISA and MFIA assays. superior biosecurity practices. Due to several outbreaks with Aspicu- Purity of the nMAV-2 antigen was tested by screening heterologous luris tetraptera in our facilities, we investigated the presence of sera positive for other infectious agents (i.e. LCMV, MPV, MTLV). anthelmintic resistance and the use of exhaust air dust (EAD) PCR Results showed no cross-reactivity with non-MAV antibodies. In for the early detection ofAspiculuris infection. To determine anthel- summary, ELISA and MFIA assays developed using nMAV-2 antigen mintic resistance, C57BL/6, DBA/2, and NCr nude strains of mice demonstrated species cross-reactivity for detection of GPAV sera were experimentally inoculated with embryonated Aspiculuris ova antibodies and can be used for routine screening of GP colonies. IFA harvested from endemically infected mice, followed by treatment test can continue to be used for confirmation of suspect ELISA and with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 MFIA positive GPAV sera samples. 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 P260 Development and Confirmation of a Novel Technique for demonstrated anthelmintic resistance as indicated by the absence of Orthotopic Engraftment of Colon Organoids in Mice worms at necropsy. In order to evaluate the effectiveness of EAD PCR, 69 cages of breeder mice endemically infected with Aspiculu- R Garcia-Gonzalez*1, EP Chua1, J Yamada1, C Sohn1, P Manzanillo2, J ris were housed on a sterilized IVC rack. Pooled fecal pellets and fur Lesch2, K Leong2 swabs were analyzed by PCR and fecal centrifugation floatation (FCF). At day 0, 56-58% of the cages on this rack tested positive 1Laboratory Animal Resources, Genentech, Inc., South San Francisco, for Aspiculuris by PCR and FCF. PCR from EAD swabs became CA; 2Research, Genentech, Inc., South San Francisco, CA positive forAspiculuris within 1 week of placing the cages on the rack. Following treatment with 150 ppm fenbendazole in feed, the EAD Inflammatory bowel diseases (IBD), including both Crohn’s disease PCR reverted to negative within 1 month of treatment and remained and ulcerative colitis, are disorders of chronic inflammation of the negative for the duration of the study (8 weeks). Additionally, gastrointestinal tract marked by episodes of relapse and remission. no Aspiculuris worms were observed in the colons of culled mice at These chronic, lifelong conditions can be treated symptomatically but necropsy. The sensitivity of EAD PCR was investigated by housing not cured. The incidence and prevalence of IBD has been increasing 6 Aspiculuris-infected mice on another sterilized IVC rack. The EAD worldwide across pediatric and adult populations. IBD can signifi- PCR from this rack was positive for Aspiculuris within 1 week of cantly affect a patient’s quality of life and may have a high financial placement. Aspiculurisinfection was confirmed by direct visualization burden. Researchers at our institution developed a new animal from cecum/colon samples. The IVC racks and air handling units model for studying IBD and other gastrointestinal diseases. We were sanitized prior to each study and confirmed negative by aimed to verify a recently published approach to transplant pinworm PCR, as a rack control. These results demonstrate that GFP-labeled colon organoids into a mouse’s intestinal tract via fenbendazole is still an effective anthelmintic and that EAD PCR is a enema infusion. Organoids are 3D structures that are generated from rapid, noninvasive, and reliable assay that can be an important stem cells or tissue progenitor cells. These small systems organize diagnostic tool for antemortem detection of Aspiculuris infection in themselves to recreate the organ’s architecture and demonstrate conjunction with fecal PCR and FCF. organ-specific functionality. We hypothesized that colon organoids could easily engraft or integrate into a DSS-damaged intestinal P259 Development of a New Adenovirus Antigen for Serological epithelium by infusing the intestinal tract via enema with these Detection of Adenovirus Infections in Guinea Pig Colonies organoids in suspension. Developing this model required several refinements to the original approach. We sought to ensure organoids RK Dhawan*, ML Wunderlich, R Brouillette remain in the mouse colon for several hours to settle and implant themselves into the damaged epithelium. The original published BioAssay Services, Charles River Laboratories, Wilmington, MA reference used tissue glue around the delicate skin and mucus membranes of the perianal region to allow for the infusion of Adenovirus has a moderate prevalence in lab guinea pigs and organoids to remain in the GI tract for up to 6 hours. We determined therefore it’s important to have a high throughput serological we could refine the technique with a more consistent and less screening assay for detection of antibodies against it. Failure to make invasive approach by applying common veterinary methods to treat conventional guinea pig adenovirus (GPAV) antigen in the past was rectal prolapses and administering suppositories or enemas. The due to difficulty in virus propagation and/or in making a recombi- major areas of refinement that was added was the introduction of nant antigen. Historically, highly purified conventional and sterile petrolatum/mineral oil-based ointment after intrarectal recombinant mouse adenovirus (MAV) antigens, both type-1 and injection of the labeled organoid infusion, followed by placement of a type-2, failed to demonstrate significant cross-reactivity with GPAV grain of Israeli couscous to function as an intestinal plug, coupled antibodies. We hypothesized that a new GPAV cross-reactive with a purse string suture around the perianal region. Shortening the conventional MAV-2 antigen (nMAV-2) could be prepared from time for intestinal infusion to 4-5 hours, suture was removed and CMT-93 mammalian cells infected with mouse adenovirus type-2 animals were allowed to normally defecate. The animals were (MAV-2, K87) virus. Reactive viral proteins were isolated from constantly monitored, and only 1 animal that presented hunched infected cells by using a combination of techniques including low posture before the final timepoint had the purse string suture speed centrifugation, ultra-centrifugation, and detergent extraction/ removed. Ten days post transplantation, proper engraftment of washing. Analysis by ELISA and MFIA® confirmed that purified organoids was a success, as determined by microscopic examination antigen was cross-reactive with GPAV seropositive antisera collected of the intestinal epithelium and viewing GFP-labeled gastrointestinal from naturally and experimentally infected animals. Sensitivity of stem cells. The potential for the ability of intestinal stem cells to this nMAV-2 antigen was evaluated by screening 20 GPAV antibody repair damaged epithelium brings great hope for all inflicted with positive field sera previously confirmed positive by IFA. All of these IBD. samples, 20/20 (100%) were found positive by both nMAV-2 ELISA and MFIA®. Early detection capability of nMAV-2 antigen was P261 Assessment of Sensitization and Pain Medication Treatment compared to previously purified conventional and recombinant in the Guinea Pig Maximization Test MAV-2 antigens. Antisera collected from experimentally MAV-1 and MAV-2 infected mice and rats at various time points post inocula- RM Morales*, PC Modine, T Smith, D Maillett

675 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Quality Biology Laboratory, Edwards Lifesciences, Irvine, CA associated with amino acid metabolism and the Krebs cycle. Transcriptomics using RNAseq showed significant changes in the The Guinea Pig Maximization Test (GPMT) is the most sensitive expression of genes involved in immunity, metabolism, and ion commonly employed skin sensitization assay used in preclinical transport. Altogether, our data suggests important roles for Fnip1 in testing of medical devices. The assay requires use of complete renal development, function, metabolism, and that alterations in Freund’s adjuvant, which raises concern for the associated pain and Fnip1 expression may predispose to polycystic kidney disease and distress that animals may experience throughout the test. As a renal cancers by altering amino acid metabolism and ion transport. potential refinement, 2 pain medications were evaluated for effect on GPMT in correctly identifying known sensitizers and alleviation of P263 Species Variation in Pinworm (Aspiculuris tetraptera) test-related discomfort. Testing followed ISO 10993-10 guidelines. Susceptibility in a Wild-Derived Colony of Mice of the genus Mus Pain medication was given subcutaneously twice daily on the initial induction phase, and on bandage removal of the topical induction RC Curtis*1, J Murray1, P Campbell2, Y Nagamori3, A Molnar4, TA and challenge phases. The pain medications were Buprenorphine Jackson1 (0.05 mg/kg), an opioid, and Flunixin Meglumine (2.5 mg/kg), a nonsteroidal antiinflammatory drug. Test materials included polar 1Animal Resources, Oklahoma State University, Stillwater, OK; 2Inte- and nonpolar non-sensitizers (0.9% sodium chloride (NaCl) and grative Biology, Oklahoma State University, Stillwater, sesame oil (S.Oil)), an organic strong sensitizer (0.2% dintrochloro- OK; 3Veterinary Pathobiology, Oklahoma State University, Stillwater, benzene (DNCB)), and an inorganic weak sensitizer (1-3% nickel OK; 4Statistics, Oklahoma State University, Stillwater, OK sulfate (NiSO4)). Each test material group had 36 guinea pigs divided into 2 test groups (test material + pain medication) and 1 control Pinworms are common parasites found in wild and laboratory group (test material only, no sham injection). Additionally, 2 groups rodents. Despite being relatively nonpathogenic in immunocompe- of 6 guinea pigs were included to control for test material vehicles. tent models, pinworm infections add an unwanted variable and may Guinea pigs were weighed and clinically observed periodically. play a role in compromising certain types of research. For this reason, Observations included behavioral signs such as movement, vocaliza- health monitoring programs and biosecurity measures aim to tion, and pruritus. Throughout all treatment groups, DNCB had minimize the spread of pinworm infections into colonies free from sensitization responses of intense erythema and edema while disease. Wild-derived and laboratory strains of mice infected

NiSO4 had only mild erythema. DNCB showed a higher proportion with Aspiculuris tetraptera have shown varied susceptibility in the (83% - 100%) and amplitude (scores 0-3) of sensitization than literature. Work with laboratory and wild-derived strains of Mus

NiSO4 (33% - 67%, scores 0-1). No sensitization (0%) was detected for musculus has illustrated higher susceptibility in wild-derived mice, in NaCl, S.Oil, and the two negative control groups. Buprenorphine younger animals, and in males. Routine surveillance at our groups were generally characterized by diminished vocalization, institution revealed pinworm infection (Aspiculuris tetraptera only) resistance, movement, interactivity, and weight gain as compared to within a colony of multiple, wild-derived species of Mus, although the no pain medication group. In contrast, Flunixin Meglumine only certain species showed positive results during initial sampling. groups had similar weight gain and clinical signs as the no pain It was our hypothesis that there were differences in susceptibility medication group. In all cases, the GPMT correctly distinguished between various species within the genus Mus. To examine this nonsensitizing and sensitizing materials, demonstrating further, fecal samples from every cage of the colony were analyzed noninterference of Buprenorphine and Flunixin Meglumine with the (using the Wisconsin egg-counting test) to assess fecal egg counts GPMT. of Aspiculuris tetraptera. Our results revealed significant differences in susceptibility between various species/subspecies of Mus. Egg P262 Modulation of Kidney Development and Polycystic Kidney counts in M. spicilegus were found to be significantly higher than Disease by Folliculin Interacting Protein 1 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 R Centini*, M Tsang, J Ramirez, H Gu, D Raftery, BM Iritani 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 Department of Comparative Medicine, University of Washington, find significant differences in regard to age, sex, and number of mice Seattle, WA per cage. As wild-derived mouse models continue to compliment research largely based on laboratory strains, it will be important to Folliculin Interacting Protein 1 (Fnip1) is cytoplasmic protein which understand host-parasite interactions and their effect on research. interacts with Folliculin, Fnip2, and the master metabolic regulator AMP kinase (AMPK). In response to low energy, AMPK stimulates P264 Comparison of Cross-Foster Rederivation and Antibiotic mitochondrial biogenesis to produce more ATP, while inhibiting Administration in the Drinking Water to Eradicate Bordetella energy consuming biosynthetic and growth pathways regulated by pseudohinzii mechanistic target of rapamycin (mTOR). Whereas the cellular and molecular functions of Fnip1 are unclear, the Fnip1 gene is amplified SE Clark*1, JE Purcell1, X Bi2, JD Fortman1 in up to 16% of renal cell carcinomas, suggesting an important role 1Biologic Resources Laboratory, University of Illinois at Chicago, for Fnip1 in kidney development and/or function. In addition, Chicago, IL; 2University of Illinois Urbana—Champaign, Champaign, Folliculin (encoded by the Bhd gene) is mutated in Birt-Hogg Dube’ IL Syndrome, an autosome recessive disorder in humans characterized by renal tumors, skin hamartomas, and lung cysts. We hypothesized Bordetella pseudohinzii is a microbial agent of potential importance in that Fnip1 has a normal role in the development and function of the mice and may confound pulmonary research. At our institution there mammalian kidney, in part by regulating AMPK and/or mTOR is an association between infection with B. pseudohinzii and increased activation. For this study, we used mice lacking Fnip1 (Fnip1-/- neutrophils (> 4%) in bronchoalveolar lavage fluid. We sought to C57BL6J), which we previously generated using ENU chemical evaluate cross-foster rederivation and antibiotic administration in mutagenesis, and wildtype controls (n = 4-8 mice per group), to drinking water as methods to eradicate B. pseudohinzii. To evaluate define how loss of Fnip1 alters AMPK and mTOR activation, kidney cross-foster rederivation 29 litters representing 16 strains of mice metabolism, gene expression, and renal function. By comparing were cross-fostered from B. pseudohinzii positive cages to B. pseudohin- kidney/brain weight ratios to assess kidney size, we found that ziinegative Crl:CD1-Elite surrogate dams. To evaluate antibiotic Fnip1-/- kidneys are significantly larger than wildtype kidneys (P = administration, sulfamethoxazole at a concentration of 0.66 mg/ml 0.018). Hematoxylin and Eosin stained histologic sections showed and trimethoprim at a concentration of 0.13 mg/ml (TMS), and increased numbers of small cysts in the renal cortex of the Fnip1- tetracycline at a concentration of 4.5 mg/ml were administered in /- relative to WT mice (P < 0.01). Metabolomics using mass drinking water. There were 3 antibiotic treatment groups with spectrometry revealed increased levels creatinine and metabolites twelve B. pseudohinzii positive cages per group (6 cages of CD1 and 6

676 Abstracts of scientific papers 2016 AALAS National Meeting

cages of C57BL/6 mice), TMS for 4 weeks, TMS for 6 weeks, and P266 Bone Marrow Transplantation with Autologous Lenti-GFP tetracycline for 6 weeks. Twenty-four of the 29 litters that underwent Transduced Cells in Nonhuman Primates for Assessment of Cell cross-foster rederivation were negative for B. pseudohinzii. Five of 12 Mobilization in Tissue Regeneration 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 SS Lankford*, A Dean, JK Williams TMS was negative 2 weeks post treatment. Three of 12 cages treated Regenerative Medicine, Wake Forest University, Winston-Salem, NC with tetracycline were negative for B. pseudohinzii 2 weeks post treatment. Pearson chi square test analysis revealed a significant A major question remaining in the development of tissue engineering association (p < .0001) between method of eradication (cross-foster medicine approaches to tissue and organ replacement is the role of rederivation or antibiotic administration) and B. pseudohin- injected/implanted versus native cells in tissue regeneration. The zii infection, and an odds ratio estimate from a logistic regression goal of this study was to address this gap in knowledge by develop- demonstrated that cross-foster rederivation was more successful. ing a method to pullulate the bone marrow with autologous cells Antibiotic administration in drinking water failed to eradicate B. carrying a lentivirus promotor for green fluorescent protein (GFP). pseudohinzii, however cross-foster rederivation was successful and Bone marrow samples were collected from the humerus of 10 female has been used to establish a B. pseudohinzii negative barrier that has cynomolgus monkeys (Macaca fascicularis). The buffy coat was remained negative for 11 months. isolated using gradient centrifugation and the sample was placed on a fibronectin coated plate and cultured in EGM-2. Adherent stromal P265 APE2 Couples SSB End Resection to Checkpoint Signaling in cells were expanded in culture and labeled with a lentivirus GFP Oxidative Stress Response in Xenopus laevis Egg Extracts promoter. Five million autologous cells (30±5% of those exhibiting positive fluorescence for GFP) were injected intravenously 4 days S Yan* following the monkeys receiving 3 intravenous injections (on 3 consecutive days) of busulfan (1 mg/kg/day). The recipient Biologic Sciences, University of North Carolina—Charlotte, Char- monkeys had surgically induced urinary sphincter deficiency lotte, NC produced 6 weeks post injection of the labeled cells. Tissue content of the labeled cells was assessed in the urinary sphincter complex. We Oxidative stress is defined as an imbalance between the production analyzed the BMCs by immunohistochemistry. We will present data of reactive oxygen species (ROS) and antioxidant defenses. ROS of successful transduction of BMCs by lenti-GFP (30% efficiency), include hydrogen peroxide and hydroxyl radicals, and are generated including the presence of lenti-GFP+ cells in the bone marrow (30% endogenously from normal cellular metabolism and exogenously positive) and the presence of lenti-GFP+ cells in the lamina propria of from chemotherapeutic and environmental agents. Oxidative stress the urinary sphincter complex. Labeled cells strongly expressed leads to DNA damage in genome, such as DNA single-stand breaks markers for endothelial and stromal progenitor cells (CD133, CD34, (SSBs) and AP (apurinic/apyrimidinic) sites. Oxidative stress is often CD117, and CD44) both in vitro and in vivo. It is concluded that this the underlying pathology in a variety of diseases including cancer is a safe and effective method to efficiently label bone marrow cells and neurodegenerative disorders. To eliminate oxidative DNA for assessment of their migratory/mobilization behavior. Safety was damage, base excision repair (BER) has evolved as a major DNA assessed by examining the urinary sphincter tissue histologically for damage repair mechanism. However, it remains unknown how evidence of infection, inflammation, atypical cells, and necrosis. unrepaired oxidative DNA damage is sensed and recognized by cells None were observed, nor were there any effects of treatment on to coordinate the cell cycle progression and DNA repair. APE2 (AP plasma white cell counts, red cell counts, kidney, or liver function endonuclease 2) has weak AP endonuclease activity and strong markers. 3’-phosphodiesterase and 3’-5’ exonuclease activities, playing essential role in PCNA-dependent repair of hydrogen peroxide- P267 Targeting tMUC1 for Treatment of Pancreatic Cancer Using induced oxidative DNA damage. We hypothesize that APE2 may Monoclonal Antibody TAB004 play an essential role in the DNA damage response (DDR) pathway. Xenopus laevis egg extract system is a cell-free biochemical S Wu*1, A Fowler2, C Ogle2, P Mukherjee1 system from extract derived from the eggs of African clawed frogs. Xenopus egg extract is supplemented with sperm chromatin 1Biological Sciences, University of North Carolina—Charlotte, (4000/μl) and hydrogen peroxide (100mM). After a 45-day Charlotte, NC; 2Chemistry, University of North Carolina—Charlotte, incubation, total extracts were examined via immunoblotting Charlotte, NC analysis. We found that hydrogen peroxide triggers Chk1 phosphorylation at the Serine 344 residue, indicating the activation of Pancreatic cancer (PC) is a lethal disease with a 5-year survival rate the ATR-Chk1 DDR pathway. Notably, the hydrogen peroxide- of 7% and an annual death rate of ~41,000. Poor prognosis is the induced Chk1 phosphorylation was compromised when endogenous result of being diagnosed late in the progression. More than 80% of APE2 was depleted in Xenopus egg extracts via immunodepletion PC cases are locally advanced or metastatic at diagnosis and less than with anti-APE2 antibodies. This observation suggests that APE2 20% of patients eligible for surgical resection. Pancreatic ductal plays a vital role in ATR-Chk1 checkpoint signaling in response t adenocarcinoma (PDA) accounts for more than 85% of PCs and over oxidative stress. We then added back wild type APE2 recombinant 80% of PDAs over-express tumor-associated Mucin-1 (tMUC1), a protein or mutant APE2 recombinant protein deficient for its membrane-tethered glycoprotein protein. Tumor-associated MUC1 exonuclease activity to the APE2-depleted egg extracts supplemented exhibits changes to its glycosylation pattern that expose its protein with sperm chromatin and hydrogen peroxide. Samples were further core, thus making it identifiable to the TAB004, a specific antibody analyzed via immunoblotting analysis after a 45-min incubation. We we developed and have shown to only recognize this form of MUC1. found that wild type APE2, but not mutant APE2, rescued the There is a need to diagnose pancreatic cancer efficiently and to target hydrogen peroxide-induced Chk1 phosphorylation in APE2-depleted the delivery of anti-cancer treatments to specific areas to avoid egg extracts, suggesting that APE2 resects the SSB in the 3’ to 5’ adverse side effects in patients. Thus, we hypothesize that TAB004 direction via its exonuclease activity to generate single-strand DNA antibody can be used as a targeting agent to accurately diagnose PDA for checkpoint activation. We have demonstrated distinct mecha- and to increase the accumulation and duration of anti-cancer nisms of how APE2 couples SSB end resection with checkpoint treatments, which will increase their overall therapeutic index. Con- signaling following oxidative stress. Our findings from Xenopus egg focal microscopy and high-resolution fluorescence microscopy were extracts system will help to better understand how cells trigger used to determine TAB004 specificity and internalization and oxidative stress response to prevent genomic instability, a hallmark nanoparticle (NP) internalization in several MUC1-expressing PDA of cancer. 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

677 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 incubation process, the cells were fixed and imaged. NuLink kit was is a suitable model to use for reagents for virus identification for used in TAB004-NP conjugation (T-NP). Nanoparticles were global influenza surveillance. The CDC is continuing to optimize suspended in sterile, ultrapure water and treated with the NuLink methodologies to increase the precision of these kits in detecting linking reagent and TAB004 antibody. Cell viability was determined emerging influenza viruses. Previously, sheep or ferret antisera were using the Dojindo CCK -8 Cell and MTT (3-(4, 5-dimethylthiazol- included in the kits. To improve sensitivity and specificity of the 2-yl)-2, 5-diphenyltetrazolium bromide) assay. The cells were plated WHO kits’ antisera, goats were used in this study. Animals were and treated with different treatments, ranging from controls, blank primed (SubQ with 100 µg) and boosted (SubQ with 15µg) 2 to 4 nanoparticles, drug loaded nanoparticles, and TAB004-conjugated weeks with bromelain purified hemagglutinins from representative nanoparticles. After 1.5 hours of treatments, the cells were washed seasonal influenza viruses, then apheresed at intervals following and allowed to incubate for 48 hours before results were read with a boosting. The methods chosen demonstrated that immunized goats plate reader. An IVIS Spectrum was used to image mice injected with provided larger volume of antisera (compared to ferrets) and TAB004 conjugated with ICG, NPs with indocyanine green (ICG), demonstrated less nonspecific cross-reactivity than that obtained and T-NPs with ICG. Mice were injected with TAB004-ICG, NP-ICG, from sheep. Therefore, goat antisera appears to be a valuable reagent and T-NP-ICG retroorbitally and intraperitoneally and imaged at in addressing global influenza virus identification. In addition, reduc- increasing time points starting at 0. Results show that TAB004 was tion in time required to generate large quantities of quality antisera specific for and internalized significantly more in tMUC1 expressing allows for refinement of antisera production. Furthermore, experi- cell lines, able to target tMUC1 expressing orthotopic tumors, able to mental animals are maintained and boosted with new variants each increase the duration, and accumulation of NPs in tMUC1 expressing year to broaden the coverage of responses to current influenza cells in vitro and in vivo upon conjugation. TAB004 is a promising viruses. These findings assure that high quality reagents are provided targeting agent for diagnosing and treating tMUC1 expressing PDA. each year, reducing the number of animals used and refining the length of time they are on study. P268 Postoperative Analgesia of Liposomal Bupivacaine in a Model of Incisional Pain in Rats (Rattus norvegicus) P270 Anesthesia Does Not Affect Development of Diabetes in ZDF Rats S Kang*1, K Jampachaisri2, TL Seymour1, S Felt1, C Pacharinsak1 V Karicheti*, Y Luo, R Velez, L Williams, GB Mulder, A Williams, JA 1Comparative Medicine, Stanford University, Stanford, CA; 2Nare- Cornicelli suan University, Phitsanulok, Thailand Charles River Laboratories, Wilmington, MA Bupivacaine is a local anesthetic that is valuable for multimodal analgesia. Here, we evaluated the application of liposomal bupiva- The obese ZDF rat develops diabetes over time and is an excellent caine for postoperative analgesia. The aim of this study was to assess model of late stage type 2 diabetes. The development of diabetes in whether liposomal bupivacaine effectively attenuates mechanical and that strain is diet dependent and can be influenced by many thermal hypersensitivity in the postoperative period in an incisional environmental factors. Anesthesia has been associated with transient pain model in rats. Rats (Rattus norvegicus) (n = 36) were randomly changes in blood glucose in rodents. However, long-term effects of assigned to 5 treatment groups: 1) saline 1 ml/kg SC BID for 2 days anesthesia on regulation of blood glucose levels and development of (C); 2) buprenorphine HCl 0.05 mg/kg SC BID for 2 days (Bup HCl); 3) the diabetic phenotype is not known in rodents. We sought to 0.5% bupivacaine 2 mg/kg SC local infiltration once (Bupi); 4) determine whether or not administration of anesthetic agents would liposomal bupivacaine 1 mg/kg SC local infiltration once (Exp-1); 5) alter the development of the diabetic phenotype in these animals. liposomal bupivacaine 6 mg/kg SC local infiltration once (Exp-6). Thirty, 8-week-old male ZDF obese rats were distributed to 3 groups Mechanical and thermal hypersensitivity were evaluated daily on days of 10 animals (ketamine/xylazine, isoflurane, and control). Diet, 1, 0, 1, 2, 3, and 4 post surgery. Bup HCl attenuated mechanical bedding, and water were provided ad libitum for this IACUC-ap- hypersensitivity for 2 days and thermal hypersensitivity for 1 day post proved study. Blood glucose (BG) was measured from a tail nick surgery. Bupi attenuated only thermal hypersensitivity for 4 days. blood sample using a handheld glucometer. Anesthesia was induced Exp-1 attenuated both mechanical and thermal hypersensitivity for 4 by intraperitoneal injection of ketamine (75 mg/kg) and xylazine (6 days post surgery. Exp-6 attenuated mechanical hypersensitivity on mg/kg), or inhalation of isoflurane using a chamber (2 to 4%) and day 0 and thermal hypersensitivity for 4 days post surgery. These data followed by a 15-minute maintenance period (0.5 to 2%). Control suggest that Exp-1 effectively attenuates both mechanical and thermal animals experienced identical cage changes as the other groups. hypersensitivity for up to 4 days in a rat incisional pain model. Immediately post anesthesia, food was returned to half the animals in each group, while the remaining animals were fasted through the P269 Use of Goat Antisera in Virus Identification for Global acute sampling period. Acute BG was measured every 30 minutes for Influenza Surveillance 4 hours. Chronic BG was measured once every week for 11 weeks (until 20 weeks of age). Fasted glucose was estimated 16 hours T Rowe*, E Blanchard, X Xu, JL Weed, G Langham following collection of the nonfasting sample. All the animals tolerated the study well and showed similar weight gains post Center for Disease Control and Prevention, Atlanta, GA anesthesia. Ketamine/xylazine increased BG immediately following anesthesia in both fasted and non fasted animals, followed by a Can goats be used in virus identification for global influenza decrease compared to controls. BG in isoflurane animals was lower in surveillance? Seasonal influenza outbreaks have the potential to fasted animals only relative to the control group. Long-term affect 15-60 million people in the US each year. The majority of the assessment of both fasting and nonfasting BG demonstrated no effect influenza A (H3N2) viruses that circulated in 2014-15 influenza on the conversion to the diabetic phenotype in the animals. Based on season were antigenic drift variants. This season was characterized these data, we conclude that ketamine/xylazine or isoflurane acute by increased hospitalizations rates and higher mortality among exposure does not affect the onset and development of diabetes in affected populations. Constantly evolving viruses make it difficult to male ZDF obese rats. select the ideal strains of influenza viruses that will best protect against multiple cocirculating drift variants in the upcoming P271 Prevalence of Rotavirus, Astrovirus, and Hepatitis E Viruses influenza season. Researchers are developing ways to rapidly deploy in Domestic Rabbits counter measures or test isolates to track these emerging pathogens. One approach is the use of the WHO Influenza Reagent Kit for W Xie*1, J Bil2, E Shantz2, J Hammermueller1, PV Turner1 Identification of Influenza viruses. These kits, sent worldwide each year, represent a simple and effective way to identify circulating 1Pathobiology, University of Guelph, Guelph, Canada; 2University of influenza viruses with relative ease. Our question is whether the goat Guelph, Guelph, Canada

678 Abstracts of scientific papers 2016 AALAS National Meeting

Gastrointestinal viruses acting alone or in concert with other gut flora P273 Alfaxalone as an Adjunct Constant Rate Infusion Provides play a significant role in causing animal disease. Often, diseases Safe and Stable General Anesthesia in Rats (Rattus norvegicus) caused by viral infections are difficult to characterize and differentiate from other infections, and there are few validated diagnostic tools K Heng*1, C Pacharinsak1, K Jampachaisri2 for rabbits. Astrovirus, rotavirus, and hepatitis E virus infections are common in many species, and have been associated with enteric 1Comparative Medicine, Stanford University, Menlo Park, CA; 2Nare- disease in both humans and rabbits, and rabbit hepatitis E virus is suan University, Phitsanulok, Thailand potentially zoonotic. None of these viruses are currently reported for rabbits in vendor quality assurance surveillance reports. We Because ketamine/xylazine provides an unreliable surgical plane of hypothesized that virus prevalence would differ between Canadian general anesthesia in rats, a constant rate infusion (CRI) of alfaxalone domestic rabbit populations, and further, that subclinical astrovirus was added as an adjunct, and the combination was evaluated. The infection would be more common in commercial meat rabbits, based aim of this pilot study was to investigate whether alfaxalone CRI in on reduced biosecurity and hygiene practices. The prevalence of combination with ketamine/xylazine safely and effectively extends a these 3 viruses was evaluated in pooled fecal samples collected from stable surgical plane of general anesthesia in rats. Rats (n = 8) were clinically healthy rabbits from 28 commercial rabbit farms (n = 101), randomly assigned to 2 treatment groups: 1) KX - ketamine/xylazine individual companion and shelter rabbits (n = 76), and pooled rabbit (80/8 mg/kg, SC), and 2) KXA - ketamine/xylazine (80/8 mg/kg, fecal samples from 7 research facilities (n = 13). Viral RNA was SC) and alfaxalone (10 mg/kg/hr, IV). In both groups, time 0 was extracted, reverse-transcribed into cDNA, and PCR amplified using assigned when the rats lost righting reflex (RF) after KX injection. In rabbit-specific primers. Agarose gels run using the PCR products the KXA group, tail vein catheter placement was initiated at time 0, confirmed viral infections. Astrovirus was found in 50% of commer- and alfaxalone CRI was started at time 5. All rats were monitored cial rabbit samples, with one rotavirus positive and no rabbit every 5 minutes for 45 minutes and provided 100% supplemental hepatitis E virus. Rabbit hepatitis E virus was isolated from 3 oxygen throughout. The surgical plane was evaluated by a surgical companion rabbit samples. None of the 3 viruses were detected in stimulation (ST, 0.5 cm incision on right lateral thigh) after loss of jaw pooled fecal samples from multiple specific pathogen-free research tone (JT) or paw withdrawal reflex (PWR). Measured anesthetic

rabbits or the vendor from which these rabbits were sourced. The parameters included oxygen saturation (%SpO2), heart rate (HR), presence of astrovirus and rotavirus in farmed rabbits is of concern, respiratory rate (RR), apnea, and body temperature (T). Recovery as conventional research rabbits may be purchased from these time was evident by the presence of RF after subcutaneous atipa- sources. Although the research rabbits used in this study were mezole at 45 minutes to reverse xylazine’s effects. After full recovery, negative for the tested viruses, continued monitoring of viral animals were monitored for 3 days. KX provided up to 15 minutes of infections is important to prevent confounding factors in research, a surgical plane with some animals responsive to JT or PWR. KXA and improve understanding of clinical disease in research animals. provided up to 45 minutes of a stable surgical plane with unrespon- siveness to JT, PWR, and ST. There were no significant differences in

P272 A Study of Investigating High-Fat Diet with Coronary the measured anesthetic parameters (%SpO2, HR, RR, or T). Apnea Balloon Compression Technique Modeling of Resting Electrocar- was not observed in either group. Animals receiving KX recovered diograph Effect faster than the KXA group after atipamezole. These findings suggest that alfaxalone CRI in combination with ketamine/xylazine safely X Gao*1, G Yang2, Z Zhe1, N Yin1, J Pan1, S Wang2, Y Feng2, Y Gao2, H and effectively extends the stable surgical plane of general anesthesia Zhou2 in rats.

1Department of Scientific Research, Affiliated Hospital of Liaoning Platform Sessions University of Traditional Chinese Medicine, Shenyang City, China; 2Li- aoning University of Traditional Chinese Medicine, Shenyang, China PS1 Comparison of Per Diem Rates in Europe and the USA

This study investigates the effect of high-fat diet on pigs treated with J Hau*, O Kalliokoski coronary balloon compression to induce myocardial ischemic injury. The study proceeded for 48 weeks, with the effect of the diet being Experimental Medicine, University of Copenhagen, Copenhagen, measured by resting electrocardiograph (ECG). Fiftt-five Bama Denmark minipigs were randomly divided into a control group of 5 and treatment group of 50. The 5 in control group received basic feed Mice are used in large numbers in present day’s high-profile without coronary vessel injury, whereas the treatment group was biomedical research, and housing large numbers of mice for a long given a high-calorie diet for 24 weeks. At the second week, coronary time is expensive. Per diem rates for mice vary between universities balloon compression was conducted at the left anterior descending and are a subject of much discussion between scientists. Institutions coronary artery to induce vascular injury in the treatment group. generally subsidize their laboratory animal care and use program ECG was recorded during the operation to generate a baseline with researchers paying per diem rates that only reflect a certain measurement of cardiac function. At 0 week, 4th week, 12th week, proportion of the real costs. It is often claimed by scientists that high 24th week, 36th week, and 48th week, 8-channel electrocardiogram per diem rates are inhibitory to their research, and render institutions was conducted and RR interval(s), heart rate (BPM), PR interval(s), P noncompetitive compared to institutions offering low per diem rates. duration(s), QRS interval(s), QT interval(s), QTc(s), JT interval(s), This is a statement with interesting far-reaching implications, which Tpeak Tend Interval(s), P amplitude (mV), QRS amplitude (mV), ST is why we decided to sample per diem rates at different institutions height (mV), and T Amplitude (mV) was recorded. At the 0, 4th, and, and to benchmark our own rates. We were particularly interested in 12th week, there was no statistical significance between the treatment investigating whether scientifically high-ranking institutions group and the control group in ST height (P > 0.05), but at the 24th deliberately kept their per diem rates low to attract top scientists and week, 36th week, and 48th week, ST height was significantly lower in stimulate scientific production. We analysed per diem rates at 37 the treatment group. Difference in T Amplitude between groups research institutions (95 different programs), 28 universities, and 9 reached statistical significance by the 12th week (P < 0.05), with the T other nonuniversity research institutions. All per diem rates were wave appearing flat and two-way or inverted in the minipigs that calculated as median costs per mouse and day. The data are had received the high fat diet. In animals given a high-fat diet with associated with some uncertainty because of the different caging coronary balloon injury, ST segment changes relatively slowly and T types used at the various institutions and differing pricing models wave changes relatively early. Eight-channel electrocardiogram (whether researchers are charged by day or week and by animal or measurement, together with coronary artery angiography was one of cage). As expected per diem rates were significantly lower in US the criteria for the evaluation of this animal model of chronic institutions compared with European institutions, which may be due myocardial ischemia. to overall higher costs in Europe than in the US. The per diem rates

679 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 varied from 2 cents to $3 per animal, and the European Max Planck staff conducted mock exercises on driving the truck, as well as the institutes were unique in not charging their scientists for animal process of large scale material handling. A failure mode analysis was accommodation. When plotting the per diem rates against the institu- also conducted to think through scenarios that could disrupt this tions’ ranking in the Times Higher Education World University process. A complete overhaul of our typical push/pull cage wash Rankings no correlation could be found. This suggests that the approach was needed during the project. Once the new machine was institutions do not use per diem rates and subsidization of their installed and functional, widespread testing and validation were animal care and use program as a tool to attract scientists reliant on completed while new wash techniques were found to increase mouse models in their research, or to stimulate the quality and operational efficiencies. The new machine has allowed us to quantity of their research performance. streamline our push/pull approach while also being far more environmentally friendly (less water) and employee friendly (far less PS2 Creation of a Risk Assessment and Operational Procedures noise, steam, and better ergonomics). By using a thorough and Matrix for the Use of Hazardous Chemical in Animal Studies systemic approach based on business improvement practices, our downtime and impact for such a large scale project was decreased M Zelivyanskaya*, A Hall, M Blayney significantly.

Office for Research Safety, Northwestern University, Chicago, IL PS4 Location-, Equipment-, and Task-Associated Assessment of Lab Animal Allergen Exposure in Animal Care and Use Spaces A vast amount of health science research is based on the use of hazardous chemical compounds in animal studies. There are known P Preisig*1, JD Macy3, C King2, D Dicks2, D Ormrod2, D Scavone2, J and unknown hazards and risks associated with this research. Not Merk3, R Kline4 surprisingly, there are potential hazards and risks to scientists and vivarium staff from these chemicals, as well. Despite decades of 1Medicine/Nephrology, Yale University, New Haven, CT; 2Environ- animal-based science, there is still no standardized risk assessment mental Health and Safety Office, Yale University, New Haven, process for commonly used hazardous chemicals in the animal care CT; 3Comparative Medicine, Yale University, New Haven, CT; 4Coast- facility. Of greater concern is that institutionally approved study al Safety, Clinton, CT precautions may not be practical or achievable within the operational limits of the vivarium. In cooperation with the veterinary staff, we Laboratory animal allergen (LAA) exposure is a well-established developed and implemented a risk assessment and operational occupational hazard of animal care staff and researchers, and an procedures matrix. For simplicity, we focused on the most commonly increasing concern for incidental exposure to others. Protecting staff encountered hazardous chemicals in our animal studies. The risk is critical, but has cost implications. Thus, evidenced-based perfor- assessment process included expected criteria such as animal species, mance standards are needed to ensure appropriate protection while dose, route of administration, and excretion. The information was minimizing cost and regulatory burden. A systematic approach was further organized, not by individual chemical, but as generalized taken using standard industrial hygiene exposure monitoring groups of chemicals with the same approximate degree of health methods to collect data across animal care and use locations, hazard. The risk assessment information was then distilled into a equipment, tasks, and procedures to establish performance-based table consisting of 9 operational procedures (and combinations of personal protection equipment (PPE) requirements. Using >5 ng/m3 procedures) matched to the day-to-day operational standards of the mouse urinary protein (MUP) as a threshold for requiring respiratory vivarium. Each of the 9 procedures contains safety guidelines for protection, the data show that there is high exposure variability for husbandry (disposal of bedding, animal housing, and cage changing tasks with the potential to generate larger amounts of allergens, e.g., locations, etc.), personal protective equipment, and warning signs. cage dumping and cage changing without use of a clean air device The table was designed as a tool for investigators to complete the (CAD). This variability is likely related to a combination of staff occupational health and safety section of the animal study proposal. technique and ventilation control efficiency. However, MUP levels Faculty report that they find this table helpful in preparing their were consistently <5 ng/m3 in housing rooms, regardless of cage proposals. The long-term effectiveness of this approach will be type or manufacturer, on the clean side of washroom, in animal evaluated within the vivarium by comparing the approved work facility hallways, and in staff breakrooms. Importantly, MUP levels practices of the study with the operational outcomes and under- were near or below detection in spaces outside the vivaria, e.g., in standing of the animal care staff. labs while surgical or other procedures were being performed and where mice were held for >24 hrs. These data suggest that: 1) PS3 Out with the Old, In with the New: How We Replaced a respiratory protection is likely required when dumping cages, even Tunnel Washer in a Fully Functioning Research Facility when a CAD is used because of variability in staff technique and equipment, and when changing cages without use of a CAD, 2) LAA JR Chipps* exposure is insignificant when entering vivaria hallways, animal housing, or procedure spaces and 3) the risk of incidental exposure is Animal Resources Core, Nationwide Children’s Hospital, Columbus, negligible when entering spaces where live animals are taken outside OH of the vivaria. A gown is required for handling live animals and/or soiled/used caging in any location, given the potential for direct Typically, a tunnel washer is one of the largest and most expensive exposure with clothing and the potential for subsequently re-expos- pieces of equipment in the vivarium. Like many large research ing oneself or exposing others (second-hand allergens) to low levels facilities that are 20+ years old, our tunnel washer was becoming of MUP until clothes are removed. Removing PPE requirements for unreliable and in dire need of replacement. Size, cost, and sheer vivarium hallways and animal room entries (no animal handling) importance in day-to-day operation make the thought of replacing it should realize a substantial cost savings. a very intimidating situation. Roughly 80% of our caging needs were being washed in that specific machine. In addition to cost concerns, PS5 Smartphone or Smartphomite?: Evaluation of 6 Bacterial there were numerous operational concerns. We used a systematic Sanitization Methods approach to our machine replacement that required interaction with a number of different departments, including budgeting, finance, MT Lieberman*, CM Madden, JG Fox strategic planning, business process improvement, engineering, and epidemiology. Focus groups containing people from many different Division of Comparative Medicine, Massachusetts Institute of areas of expertise were constructed to determine machine needs, Technology, Cambridge, MA what functions were important, removal details, and installation concerns, among others. Cage wash functions were shifted to a Smartphone usage has increased in popularity, with both smart- secondary vivarium through use of a leased box truck and cage wash phones and tablets used frequently in laboratory animal facilities.

680 Abstracts of scientific papers 2016 AALAS National Meeting

Portable devices present a risk for fomite transmission when devices facilities, their operational efficiencies, and the animal program are transferred between facilities. The goal was to evaluate different financial structure can lead to lower operating costs for their bacterial sanitization methods for smartphones. We hypothesized institution. that ultraviolet light (UV) would be an effective nonliquid method to decrease aerobic bacterial colonization of phones. Sterile swabs were PS7 Starting an AALAS Certification Mentorship Program through used to sample the smartphone face (PF), the phone-case junction Your Local AALAS Branch (PJ), and the back and sides of the smartphone case (PC) before and after different sanitization methods. Phones were sanitized using 2 LJ Hughes* commercially available UV devices lasting either 30 seconds (UV-30) or 5 minutes (UV-300) according to manufacturer instructions, a AHCS/NINDS, National Institutes of Health, Bethesda, MD bleachwipe (BW), quaternary ammonium disinfectant spray (QA), 70% ethanol spray (ET), or by cleaning with a sterile cleanroom wiper The AALAS National Capital Area Branch (NCAB) Education/ (KW). Swabs were plated on tryptic soy agar with 5% sheep blood Workshops Committee launched the NCAB AALAS Certification and incubated aerobically overnight at 37°C. Following incubation, Mentorship Program (NACMP) for NCAB members seeking to the number of colony-forming units (CFU) was enumerated for obtain AALAS certification The goal of this somewhat unorthodox plates pre and post disinfection and percent reduction of bacterial program was to foster AALAS certification among NCAB members. colonies calculated. The mean and standard deviation for CFU on PF, This program partnered a senior laboratory animal professional PJ, and PC prior to sanitization were 17.5±24.1, 29.7±30.1, and (manager/supervisor, trainer, senior technician with LATG certifica- 25.6±26.0, respectively. All sanitization methods were effective at tion, or a veterinarian) with a laboratory animal professional desiring decreasing bacterial colonization levels and there was no significant to obtain AALAS certification. Since this program was newly difference in percent reduction of bacterial colonization between launched, we solely focused on the Laboratory Animal Technologist UV-300, BW, QA, ET, or KW (Wilcoxon Signed-Rank Test). Percent (LATG) certification exam. This type of collaboration provided reduction of bacterial colonies was significantly increased using NCAB members the benefit of one-on-one training sessions with a UV-300 compared to UV-30 sanitization (P < 0.001, Mann-Whitney senior lab animal professional, attentiveness to different learning Test) and UV-300 was significantly more likely to reduce aerobic styles, and scheduling flexibility. This also gave the mentee a bacterial colonization to zero as compared to UV-30 (P < 0.001, personal cheerleader. So far, all of those who successfully completed Fisher’s Exact Test). Overall, “dry” sanitization methods using UV the program and took the exam have successfully obtained LATG light or cleaning with a sterile cleanroom wiper were as effective in certification. This presentation will share the behind-the-scenes percent reduction of bacterial colonization as “wet” disinfection development and coordination of the program, the pros and cons of methods using bleach, quaternary ammonium disinfectants, or 70% the program, selection process of mentors, mentees, and their ethanol. Further investigation is needed to evaluate optimal UV pairings, as well as the use of technology to aid in the delivery of exposure times to reliably minimize aerobic bacterial colonization of material and flexibility of one-on-one meetings. smartphones. PS8 Measuring Intracage Ammonia Concentrations: What’s All the PS6 Doing More with Less: Consoldiation of Small, Inefficient Stink About? Facilities R Morrow*, R Wiler J Percifield, MP Mottet* TgT, Genentech, Davis, CA HDR Architecture, Atlanta, GA The measurement of intracage ammonia is often used as a surrogate Many Institutions have seen small to significant reductions in their for quality of husbandry. The literature documents a variety of research funding and have been forced to operate existing facilities sensors and methods to measure intracage ammonia. However, and programs with less money. These instituions are looking for attempts to duplicate results are a challenge due to poorly defined ways to improve the efficiency of their programs with various methods, incomplete reporting of methods, and differences in facility strategies, including higher-density caging systems, faster through- operations. Adopting a standardized methodology for ammonia put washing equipment, and other means of making their animal measurement will benefit the lab animal research community by facilities more efficient. Several Institutions have or have begun to delivering reproducible results. Selecting the appropriate gas sensor, assess their existing vivarium facilities inventory to determine if and while understanding the limitations of a measurement device, is what opportunities exist to consoldiate older, inefficient facilities into critical to understand how these factors affect the microenvironment fewer, larger, and more efficient facilities. The first step in the and the generation of ammonia. We sought to develop defined approach to the consolidation of a group of facilities is to assess their methodology to measure intracage ammonia. A side-by-side condition and their functionality and to develop and document the comparison of 3 types of ammonia gas sensors: electrochemical (EC), baseline conditions. Older, inefficient facilities are highlighted, and photoionization (PID), and colorimetric was performed. The EC and the animal census and research programs are incorporated into a PID sensors have continuous real-time measurements, while the spreadsheet to track this data. Meetings with institutional leadership colorimetric sensor provides a single snapshot of the intracage are used to discuss and document areas of anticipated growth so that ammonia levels. Similar concentration results were achieved using the long-term needs of the institution can be charted against existing the EC and PID sensors, but reproducible results were unachievable capacities. Discussions with vivarium operations leadership will using the colorimetric sensor, a device that is commonly used for uncover operational inefficiencies and individual facility challenges assessing intracage ammonia in the lab animal setting. Furthermore, that will also impact consolidation opportunities. Finally, a thorough ammonia concentrations should reflect a core data set that includes review of the animal progam’s financial status is required to both macro- and microenvironmental ammonia, temperature, relative understand the total operating costs and the sources and amount of humidity, air changes per hour, bedding material and volume, animal income. The results of applying the outlined apppoach at several occupancy, and latrine locations. By adopting a standardized institutions vary, but many similar outcomes have evolved. First, reproducible measurement methodology, the lab animal research most institutions do not have spare vivarium capacity, which makes community will improve the reporting of intracage ammonia. the reduction of existing facilities a challange and often leads to the Moreover, it will enable discussion and comparison between recommendation for a need for a new facility. Consolidations through institutions. 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

681 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

PS9 An Economical Solution to Whole Room Scavenging of 1Division of Veterinary Resources, University of Miami, Miami, Chlorine Dioxide Gas FL; 2Pathology and Laboratory Medicine, University of Miami, Miami, FL; 3Gastroenterology, University of Miami, Miami, J Ludwig*, JM Criley FL; 4Physiology and Biophysics, University of Miami, Miami, FL

University of Illinois, Urbana, IL Colitis-associated cancer is a widespread disease that affects 130,000 people annually in the US alone. Colitis-associated cancer can be Chlorine dioxide gas generated with a portable gas generator is an modeled in mice by treating them with a combination of the geno- effective method of sterilizing many pieces of equipment and animal toxic agent azoxymethane (AOM) and the colitis inducer dextran rooms. The use of the generator requires multiple steps: sealing off a sodium sulfate (DSS). We aimed to see if environmental enrichment, room or chamber, increasing the humidity in the room, introduction social housing, and sex have an effect on intestinal tumorigenesis us- and exposure of room/chamber to the gas to set parameters, and ing the AOM-DSS murine model. We used aged-matched C57BL/6J removal of the gas. Room exhaust or active scavenging can both be (n = 64) mice that were randomly assigned to groups, each having used to remove gas from the room. The majority of animal rooms an equal number of males and females. Mice were housed either have dedicated exhaust without recirculation of air so room exhaust singly or in groups (4 mice/cage) as a model for social housing. is typically used in our animal facilities. We have recently had Nesting substrates were provided to half of the mice as environmen- requests to sterilize areas outside of the animal facility and without a tal enrichment. All mice were given AOM (14.6mg/kg, i.p.) on day portable active scavenging unit our decontamination services were 1 of the study. On day 14, mice were administered a cycle (7 days) limited to rooms with nonrecirculating room exhaust. The largest of 3% DSS. Following this, mice were given 2 weeks of recovery obstacle in obtaining a portable whole room scavenging system was time and subsequently treated with a second cycle of 3% DSS for an financial. A commercial scavenging unit can cost between $1,800 and additional 7 days. On day 56, we measured tumor number, tumor $2,000. With an understanding of the mechanics of a scavenger we size, calculated tumor load, and characterized the inflammatory and were able to build a unit ourselves with materials available from neoplastic changes in the colon by histopathology. We also exam- local home improvement stores for less than $650. By adding a whole ined the welfare of the animals by recording body weights and nest room portable scavenger we were able to increase our decontamina- scores over the course of the treatment. We found that singly housed tion services to accommodate more areas of our institution and animals (n = 24) get higher numbers of tumors (P < 0.05), and had provide service to our larger campus community. an increased tumor load (P < 0.05) compared to their group housed counterparts (n = 32). Tumor number and load were also significantly PS10 Fine Tuning Steam Sterilization: One Cycle Does Not Fit All higher in female than in male mice, especially when singly housed (P < 0.05). Environmental enrichment had no effect on tumorigenesis. K Lux*, K New, J Vollmer, R Santos Although environmental enrichment had no effect in tumorigenesis, sociality and sex both had significant impacts. These results provide The Jackson Laboratory, Sacramento, CA performance-based data that can be used when providing recommendations for the use of social housing, environmental enrichment, The use of steam sterilization is common practice in laboratory and when selecting sex of study subjects. Ultimately, this study has animal operations to prevent the introduction of pathogenic or shown that both social housing and sex can influence rodent welfare, opportunistic microorganisms. Controlling sterilization variables is revealing the importance of selecting proper environmental and critical to maintaining biosecurity. Although traditionally steam housing conditions, specifically in murine models of colitis-associat- autoclaves are regularly evaluated and monitored to ensure their ed neoplasia. effectiveness via steam tape, biologic indicators (BIs), and/or chemical integrators, we suggest that additional variables are taken PS12 Animal Room Light Phase LED Light Enhances Nighttime into account. As part of a steam autoclave optimization project, the Circadian Melatonin Inhibition of Rodent Hepatoma Metabolism following were evaluated for the 16 autoclaves that we have between and Growth our 2 sites: cycle parameters, load contents, load configuration, indicators, documentation, operator training, and machine differences. We first performed temperature distribution studies in an MA Wren-Dail*1, RT Dauchy1, AE Hoffman2, JP Hanifin3, B Warfield3, empty chamber to identify cold spots and create a baseline for GC Brainard3, SM Hill1, VP Belancio1, EM Dauchy1, DE Blask1 autoclave operation. Materials were classified into families and material(s) within each family identified as the most challenging to 1School of Medicine, Department of Structural and Cellular Biology, sterilize within a cycle. The biologic indicator selection (product and Tulane University, New Orleans, LA; 2Epidemiology, Tulane Uni- concentration) was made after evaluating the bioburden of certain versity School of Medicine, New Orleans, LA; 3Neurology, Thomas materials, ensuring that the BIs were more difficult to “kill” than our Jefferson University, Philadelphia, PA materials. BIs were placed in the center of the materials and strategically placed throughout the load during fractional studies (a Melatonin, produced by the pineal gland in mammals primarily process of reducing exposure time until the failure point is deter- at night, regulates linoleic acid (LA)-dependent metabolism and mined). Differences in load configuration were noted to impact proliferation in human xenograft tumors in vivo.Melatonin inhibits sterility of materials, which lead us to establish minimum/maximum LA-uptake and conversion to the mitogenic agent 13-hydroxyocta- loads and load configuration guides for each cycle and autoclave. decadienoic acid (13-HODE), a lipoxygenase product that enhances Several surprising lessons were learned regarding challenging epidermal growth factor and insulin-like growth factor-I-induced mi- materials and mechanical components that impact cycle effective- togenesis. Previously, our laboratory demonstrated how transmission ness. Each unit must be evaluated with special consideration to the of light in the blue-appearing portion of the visible spectrum (465-485 contents and their configuration, and it is critical to ensure that your nm) through standard rodent caging during light phase results in quality control check is more challenging than your most challenging amplification of the nighttime melatonin signal in rats and leads to material, paying careful attention to the selection and placement of suppression of human prostate tumor metabolism and growth. Here BIs. With the current trends towards maximum barrier and gnotobi- we tested whether light phase exposure of hepatoma-bearing rats to otic housing, sterility is key to our research results. white light-emitting diode (LED) lighting, enriched in blue emissions, results in amplified nighttime melatonin levels and influences PS11 It’s Not Where You Live but with Whom You Live: Housing tumor metabolism and growth. Male Buffalo rats (BUF/CrCrl; 250 Effects on Tumorigenesis in Murine Colon Cancer g; n = 12/group) bearing “tissue-isolated” Morris hepatoma 7288ctc xenografts were maintained on a lighting regimen 12L(172.0 ± 12.3 lx; J Zaias*1,2, JK Lang3, R Dheer3, R Santaolalla3, M Phillips3, J Grant4, M 70.5 ± 5.0 mW/cm2 [inside cage]; lights on 0600):12D under control Abreu3 broad spectrum cool white fluorescent (CWF) or experimental LED

682 Abstracts of scientific papers 2016 AALAS National Meeting

lighting. Tissue-isolated hepatoma latency-to-onset of growth and ficient models is a factor of age, as well as housing environment growth rates were significantly delayed (P < 0.001) in the LED group and the background strain of the model. Here, serum IgG was used (21 days; 0.36 ± 03 g/d) versus CWF group (11 days; 0.71 ± 0.03 to determine the extent of leakiness in various immunodeficient g/d), respectively. Arteriovenous measurements were taken when mouse models; survival and lymphoma incidence was also analyzed. tumors reached an estimated weight of 6-8g. Results showed that Male and female (n = 23/26) C.B-17-SCID, NOD SCID, SCID Beige, arterial plasma melatonin levels (mean ± 1 SD) were low in the light SHrN®, Beige Nude XID, and Nude mice were housed in flexible film phase (1200 h) in both groups (1.0 ± 0.2 pg/mL), but significantly isolators. Blood was collected and analyzed using dried blood spot higher (P < 0.001) in LED (1065.0 ± 38.0 pg/mL) compared to CWF technology at 12, 16, 20, and 24 weeks of age for IgG concentration (154.0 ± 16.5 pg/mL) at peak, mid-dark phase (2400 h). Hepatoma analysis using multiplex fluorescent immunoassay. The leakiness xenograft cAMP levels, uptake-metabolism of LA to 13-HODE, threshold was set at 5 µg/mL of IgG for analysis. At 24 weeks of age, aerobic glycolysis (Warburg effect) and growth signaling activities necropsies were performed to determine incidence of lymphoma. At were significantly reduced in rats maintained in LED versus CWF 24 weeks of age, the SHrN® model had the smallest percentage of lighting environments (P < 0.001). These findings are the first to show leaky animals followed closely by the NOD SCID. Beige Nude XID in vivo that amplification of nighttime melatonin levels by exposing mice display an age associated increase in serum IgG levels, as well rats to daytime blue-enriched LED light significantly reduces rodent as an increasing percentage of animals above the 5 µg/mL thresh- hepatoma metabolic and proliferative activities. old 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 PS13 Ophthalmologic Assessment of the Common Marmoset (Cal- lowest survival rate at 24 weeks; however, survivability in all models lithrix Jacchus) Involved in Visual Neuroscience was greater than or equal to 79%. There is a positive correlation between the percentage of leaky animals and survival in males and C Boisvert2, J Mitchell3, SC Adams1, J Reynolds4, M Leblanc*1 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 1ARD, Salk Institute, La Jolla, CA; 2UC Irvine Gavin Herbert Eye highest incidence of lymphoma. But when the hairless mutation is Institute, Irvine, CA; 3Brain and Cognitive Sciences, University of crossed onto the NOD SCID mouse to create the SHrN®, incidence Rochester, Rochester, NY; 4Systems Neurobiology Laboratories, The of lymphoma is decreased. This is the first study reporting IgG con- Salk Institute for Biologic Studies, La Jolla, CA centration in multiple immunodeficient models to examine leakiness as well as 24-week survival and lymphoma incidence. Additional The common marmoset (Callithrix jacchus) is gaining popularity as studies measuring immune cell activity and function will be useful a leading nonhuman primate (NHP) model for visual neuroscience. to further characterize immunodeficient models; however, these As with the macaque, marmosets can easily be trained to perform data will be valuable information in determining the best model for complex behavioral tasks, have natural gaze behavior, and highly xenograft research. developed oculomotor systems that enable smooth-pursuit and saccades, as well as brains with organizational features very similar PS15 Daytime Blue-Enriched LED Light Enhances Nighttime Circa- to humans. Marmosets have the added advantage of a nearly lis- dian Melatonin Regulation of Rodent Metabolism and Physiology sencephalic 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 RT Dauchy*1, MA Wren-Dail1, AE Hoffman1, JP Hanifin2, B Warfield2, development of transgenic marmosets is also a major scientific break- GC Brainard2, SM Hill1, VP Belancio1, DE Blask1 through that will allow the exploration of complex brain functions in NHP using genome-editing tools. As is true in humans, marmosets 1Tulane University School of Medicine, New Orleans, LA; 2Depart- are susceptible to refractive errors that may affect research outcome. ment of Neurology, Thomas Jefferson University, Philadelphia, PA Here, we report ophthalmology and visual acuity examinations in a colony of marmosets used in visual neuroscience. Across the Light entrains the master biologic clock within the suprachiasmatic colony, the mean refractive error was -0.35±1.48D for the right eye nucleus (SCN), which tightly controls the nocturnal pineal melatonin and -0.20±1.55D for the left eye. A high correlation coefficient (R2= signal, a critical contributor to the temporal coordination of circadian 0.93, P<0.001) was found between the spherical equivalents of both rhythms in metabolism and physiology of all mammals. Previous eyes in individual animals. Refraction was correlated with age with studies from our laboratory demonstrated that the spectral transmit- mild hyperopia to emmetropia at a younger age evolving to myopia tance (color) of light passing through standard laboratory rodent after 3 years of age. Myopia (as defined as a spherical equivalent caging impacts these responses in rats. Here, we examined the refraction less than or equal to −0.50D) was found in 13 (36.1%) eyes, hypothesis that white light-emitting diode lighting (LED) during with 2 adult animals being severely nearsighted, with refractions daytime, high in emission of light in the blue-appearing portion exceeding -3.25D in both eyes. In those animals, vision would be (460-480 nm) of the visible spectrum, compared to standard cool blurry over a 30 cm working distance and would greatly limit per- white fluorescent lighting (CWF), alters the nocturnal circadian formance on standard behavioral and visual tasks unless corrected. melatonin signal, thereby altering normal metabolic and physiologic There was also a tendency for adult females (-1.06±0.84D) to be more functions in laboratory rodents. Male Buffalo rats (BUF/CrCrl); n = nearsighted than males (-0.28±0.63D) (NS). There were no other 12/group) were maintained on a common lighting regimen 12L(172 pathologies observed on ocular examination. This study stresses the ± 12.3 lx; 70.5 ± 5.0 mW/cm2[within cage]; lights on 0600):12D in importance of screening marmosets involved in visual science for translucent clear polycarbonate rodent cages in an AAALAC-accred- refractive errors and ocular pathologies on a regular basis throughout ited facility. After 1 week, animals were subjected to a series of six their experimental life and especially after 3 years of age to ensure low-volume blood draws via cardiocentesis (0400, 0800, 1200, 1600, the quality of research. 2000, and 2400) over a 4-week period to assess arterial blood

melatonin, total fatty acid (TFA), glucose, lactic acid, pO2, pCO2, PS14 Characterization of Immunodeficient Mouse Models insulin, leptin and corticosterone concentrations. Results showed lower dietary and water intake, and body growth rates in LED versus J McClellan*1, R Macasocal2, C Buckner2, T Hare2, MJ Horn1 CWF (P < 0.05). Plasma melatonin levels in pg/mL (mean ± 1 SD) 1Veterinary Science, Research and Support, Envigo, Indianapolis, were low (>2.0 ± 0.3) in the light phase (1200 h) in both groups, IN; 2Envigo, Indianapolis, IN 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).

Leakiness in SCID mouse models refers to the occasionally produc- Arterial blood diurnal rhythms of TFA, glucose, lactic acid, pO2, tive VDJ rearrangement leading to clonal expansion of these limited pCO2, leptin, insulin, and corticosterone levels were significantly B and T cell clones. The rate and extent of leakiness in immunode- altered in LED, compared to CWF (P < 0.05). Together with our

683 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 previous results, the present findings suggest that daytime broad weeks, n = 22) and G B6 (C57BL/6J.129S7- Ifngr1tm1Agt, 4-8 weeks, n = spectrum LED light with high blue emissions, compared to CWF 14), to challenge from ZIKV (PF/13 strain). AG 129, AG B6, and A B6 light, has a profound positive impact on the circadian regulation of were inoculated at 103 and 102 pfu/mouse in a volume of 0.1 ml in neuroendocrine, metabolic, and physiologic parameters that Leibovitz (L-15) medium by either the retro-orbital (RO) or influence laboratory animal health and wellbeing, and ultimately the subcutaneous routes. Age-matched controls (AG 129, n = 3; AG B6, n outcome of scientific investigations. = 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, PS16 A Nonhuman Primate Model for Prediction of Clinical AG B6, and A B6 strains were susceptible to ZIKV infections Outcomes in Regenerative Medicine Approaches to Tissue and resulting in death or euthanasia, the latter based on the criteria of Organ Repair 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 JK Williams*1, A Dean1, SS Lankford1, K Andersson1,2 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 1Regenerative Medicine, Wake Forest University, Winston-Salem, (18%) of A B6 mice, inoculated SC with either 103 or 102 pfu/mouse, NC; 2Urology, Aarhus University, Aarhus, Denmark survived. By characterizing this ZIKV model in adult and pregnant mice, this research will elucidate how ZIKV affects the developing It is not unusual for animal models of disease to inaccurately predict fetus and the peripheral nervous system and will transform how we clinical outcome of clinical studies. One such example is stem cell treat ZIKV infections by generating a desperately needed small therapy for stress urinary incontinence (SUI) where preclinical animal model to test antiviral compounds and vaccines. studies report almost complete remission of symptoms, whereas clinical studies report only around 50% remission in 50% of patients. PS18 Murine Norovirus Alters B Cell Development in the Bone The answer is most likely because animal models (which create acute Marrow of Stat1 Knockout Mice SUI in relatively young animals) do not represent the most common clinical scenario where SUI is most common as a chronic disease in CC Hsu*, SM Meeker, JA Ramirez, S Escobar, B Iritani, TL Brabb, L peri/post-menopausal women with coexisting risk factors such as Maggio-Price obesity and Type 2 diabetes. To better predict the effects of cell therapy for UI, we developed a cynomolgus monkey model of Comparative Medicine, University of Washington, Seattle, WA urinary incontinence (surgical nerve and muscle damage to the urinary sphincter complex) that reproduces the functional and Murine norovirus (MNV) is known to infect macrophages and structural changes in the urinary sphincter complex seen in women dendritic cells. Recently, it was reported that MNV also infects B with clinical SUI. In these studies, we modeled both acute and cells. Therefore, we hypothesized that MNV would alter the chronic SUI in younger and older female NHPs with varying degrees proportions of B cell subsets during development in the bone of estrogen deficiencies and impaired glucose/insulin metabolism. marrow of infected mice, and that this effect would be more With an n = 6 experimental group, autologous skeletal muscle pronounced in Stat1 knockout mice that have impaired responses to precursor cells (skMPCs) were isolated from a muscle biopsy, viral infections. Female, 7- to 8-week-old wild-type 129 mice (129S6/ expanded to 5 million cells, and injected directly into the urinary SvEvTac) and Stat1 knockout mice (129S6/SvEv-Stat1tm1Rds) were sphincter complex of NHPs with SUI. skMPCs almost completely infected with MNV-4 by oral gavage and B cells from the bone restored sphincter muscle content and urethral pressures in younger marrow were stained with markers to identify subsets of developing (5-8 years) NHPs (P < 0.05 vs SUI/no treatment), but not older (15-28 B cells including B220, CD19, CD43, IgM, and IgD, and stained cells years) NHPs (P > 0.05 versus SUI). This same pattern of efficacy was were evaluated by flow cytometric analysis. Stat1 knockout mice observed in NHPs with acute versus chronic SUI, in intact versus infected with MNV had significantly (P < 0.05) decreased developing ovariectomized NHPs, in normal cycling dominant NHPs versus B cell populations in the bone marrow including pro-B/pre-B, dysmenorrheic subordinate NHPs, and in normal weight/normal immature, and mature B cells compared to mock-infected control glucose metabolism versus heavier impaired glucose/insulin ratio mice. This effect was observed as early as 7 days postinfection (PI) as NHPs. Thus, there are multiple determinants of cell therapy efficacy well as at 3 weeks PI. No differences in any B cell subsets were noted that can be modeled in NHPs and are critical to translational in MNV infected 129 mice versus. mock-infected control mice at 3 applicability of regenerative medicine approaches to tissue repair. weeks PI. MNV was detected by RT-PCR in the bone marrow of all infected mice. Additionally, splenic B cells isolated from Stat1 PS17 A Mouse Model to Study Zika Virus Pathogenesis 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 CM Nagamine*1, K Majzoub2, Y Ooi2, R Mateo3, KA Kirkegaard4, J hours PI, respectively, when compared to MNV-4 genome copies at 2 Carette2 hours PI. This increase in viral genome copies was not seen in similarly infected splenic B cells isolated from wild-type 129 mice. 1Comparative Medicine, Stanford University, Stanford, CA; 2Microbi- These results suggest that MNV-4 infection can alter B cell develop- ology and Immunology, Stanford University School of Medicine, ment in the bone marrow and can replicate in splenic B cells, and Stanford, CA; 4Genetics, Stanford University School of Medicine, these changes are dependent on Stat1 deficiency. Stanford, CA PS19 Drug-Induced Neurotoxicity Assessments: Validation of a Zika virus (ZIKV), a mosquito-borne virus in the family Flaviviridae, Neurologic and Functional Observation Battery in the Sinclair is an emerging pathogen that is associated with congenital micro- Miniature Swine cephaly and Guillain-Barré syndrome, an autoimmune disorder where the immune system attacks the peripheral nervous system, A Stricker-Krongrad*, D Brocksmith, M Zhong, GF Bouchard, J Liu leading in severe cases to life-threating paralysis. Well-characterized animal models are critical to generating insights into ZIKV pathogen- Sinclair BioResources, LLC, Auxvasse, MO esis and to test antiviral compounds and vaccines. The AG 129 mouse strain (129/SvEv-Ifnar1tm1Agt Ifngr1tm1Agt) has knockouts for the The nervous system is one of the most complex organs in the body, receptors for both Type I (IFN-a/b) and Type II (IFN-g) interferons on and has its unique vulnerabilities to toxic compounds. The central the 129 genetic background. We placed the double KO on the nervous system, along with cardiovascular and respiratory systems, C57BL/6 genetic background generating the AG B6 strain has been listed in the safety pharmacology core battery of the (C57BL/6J.129-Ifnar1tm1Agt Ifngr1tm1Agt). We compared the DKO harmonized test guidelines for preclinical testing of human pharma- strains, AG 129 (8-9 weeks of age, n = 14) and AG B6 (8-14 weeks, n = ceuticals. A functional observational battery (FOB) has been 20), and the single KO strains, A B6 (C57BL/6J.129S2-Ifnar1tm1Agt; 8 recommended in the guideline as the first tier neurotoxicity screening

684 Abstracts of scientific papers 2016 AALAS National Meeting

that encompasses motor activity, behavioral changes, coordination, of 3-day-old mice and 8 CD1 dams with litters of 13-day-old mice sensor/motor reflex responses, and body temperature. The miniature were inoculated orally with filtered colon homogenates containing swine is an appropriate species for neurologic and behavioral MuAstV. No clinical signs were observed in the dams or pups and studies. Its central nervous system and specifically the brain are MuAstV had no effect on weight gain in pups. MuAstV RNA was similar to those of human with respect to tissue composition, detected in feces from 15/16 dams for 18 days postinfection (dpi) gyrencephalic structure, and developmental growth and myelination and 9 dams were shedding MuAstV at dpi 29 or 39. MuAstV RNA patterns. In addition, behavioral studies, as well as modeling of was highest in intestines of mice infected at postpartum day (ppd) 3 neurologic human diseases, have been increasingly performed in at 12 dpi and highest in intestines of mice infected at ppd 13 at 4 dpi. minipigs. Because the miniature swine has increasingly been used as Low levels of MuAstV were sporadically detected in spleen, liver, an alternative to dog or nonhuman primate in regulatory toxicology and kidney of mice infected at 13 dpi. MuAstV was detected in the studies, we validated a FOB for the safety pharmacology assessment feces of all 3 to 6 week-old weaned mice and in 94% of feces from of pharmaceutical products intended for human use. A swine FOB 7-9 week-old mice. Many of the dams became pregnant immediately protocol was developed with multiple observation parameters to after delivering their first litters and intestines from their second litter monitor effects on 6 majors categories related to sensory and motor pups were MuAstV negative at ppd 0-5, indicating that fostering may functions, autonomic and voluntary functions, and behavior. Known be feasible for generating MuAstV negative mice. Weekly swabs of CNS acting agents were administered to the Sinclair minipigs via cages housing MuAstV infected mice were MuAstV positive through intramuscular route. Amphetamine, ketamine, and diazepam were 6 weeks. Swabs of the rear plenum of the ventilated rack were demonstrated to positively or negatively dose-dependently affect MuAstV negative at 6 and 13 dpi and positive at 20 dpi and beyond. different parameters related to autonomic functions, excitability, Ninety-seven percent of sentinels that received soiled bedding from behavior, motor activities, gait, and reflexes. 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 PS20 Successful Group Formation of Mature Male Long-tailed sentinels exposed to soiled bedding stored for 1, 2, or 3 weeks prior Macaques (Macaca fascicularis): Can Minimizing Coalitions Help to addition of the sentinels were MuAstV positive indicating that Reduce Aggression? MuAstV is stable in the environment for several weeks. Collectively, these results show that MuAstV is shed for extended periods, from T Andrianjazalahatra, P Honess* 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 Animal Welfare, Bioculture Group, Riviere des Anguilles, Mauritius effective at 2 wpi; soiled bedding sentinels and cage swabs were effective from 1 wpi; and MuAstV remained infective in soiled bedding Forming groups of mature male macaques in captivity without for at least 2 weeks. potentially fatal aggression has long been a challenge. With roughly equal birth sex ratios in breeding facilities where breeding groups are PS22 Laboratory Mice Are Colonized with Colibactin-Encod- single-/two-male, bachelor groups are a necessity for efficient use of ing Escherichia coli caging. We aimed to reduce aggression and improve welfare when creating groups of long-tailed macaque males over 4 years old, by A Garcia*1,2, A Mannion2, CM Madden2, V Bakthavatchalu2, Y Feng2, reducing the number of potential existing coalitions (cooperative Z Shen2, Z Ge2, JG Fox2 relationships based on prior acquaintance) between introduced males through minimizing previous experience of each other. It was 1Molecular Sciences Research Center, University of Puerto Rico, San hypothesised that minimizing coalitions would reduce aggression Juan; 2Division of Comparative Medicine, Massachusetts Institute of recorded in animal records as fight injuries requiring veterinary Technology, Cambridge, MA attention. In a retrospective analysis of colony records (January 2010–November 2015) we compared aggression levels for 30 days Colibactin (Clb) is a genotoxin encoded by Escherichia coli strains following mergers using the new reduced coalition system (43 belonging to phylogenetic group B2 that have been isolated groups) and the old system (36 groups) (overall mean = 11.03/ from humans with intestinal diseases, including cancer as well group). Study animals were group-housed outside under ambient as from urogenital disease, meningitis, and bacteremia cases. E. conditions. Housing and care meets or exceeds standards set in the coli NC101 is a prototype murine Clb-encoding E. coli that has NRC Guide for the Care and Use of Laboratory Animals and the EU been used to experimentally infect mouse models and investigate Directive on the Protection of Animals used for Scientific Purposes cancer pathogenesis. However, the relative prevalence of naturally (2010/63/EU).There was no difference between methods in occurring Clb-encoding E. coli in laboratory mice is unknown. aggression (events/merger) (P = 0.771). However further analyses Furthermore, E. coli, historically considered a commensal, has not showed the new system produced 47% lower aggression (P = 0.048) been excluded from laboratory rodent colonies. We obtained rectal among wild-caught males and 86% higher aggression (P = 0.031) in swabs or feces, and/or extraintestinal samples from surveillance captive-bred males. In conclusion, success of the refined system (sentinel) mice [Crl:CD1(ICR)], a breeding colony (which supplies depends on the animals’ origin highlights the importance of tailoring mice for our surveillance program), 2 commercial vendors (1 the management system to the characteristics of the animals. These vendor supplied mice for our breeding colony), and nonsurveillance findings have relevance for those working in a range of captive (nonsentinel) mice (various strains). The extraintestinal samples contexts including breeding facilities, laboratories, zoos, and included nare/lung wash samples from surveillance mice (collected sanctuaries with potential significant improvement in animal welfare as part of our health monitoring program) and uterine samples during mergers. from clinically affected genetically modified mice (collected as part of necropsy procedures). Samples were cultured and E. coli isolates PS21 Murine Astrovirus Infection and Detection 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%) SR Compton*, JD Macy isolates were PCR positive for Clb genes (clbA and clbQ). Clb-encoding E. coli belonged to phylogenetic group B2. Selected E. coli isolates Yale University School of Medicine, New Haven, CT were serotyped as O2:H6, OM:H+, O22:H-, and O-:H-. Clb- encoding E. coli induced megalocytosis, a cytotoxicity phenotype, in Murine astrovirus (MuAstV) is a recently identified, widespread HeLa cells. A Clb-encoding E. coli was isolated from a uterine sample infection among laboratory mice, yet little is known about its of a clinically affected genetically modified mouse diagnosed with pathogenesis. Our goal was to determine the duration of MuAstV cystic endometrial hyperplasia. Our study suggests that laboratory infection, susceptibility of pups, and the efficacy of soiled bedding mice are colonized with Clb-encoding E. coli that may be associated sentinels and environmental monitoring. Eight CD1 dams with litters with disease and potentially confound research studies. This is the

685 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 first report documenting the presence of Clb-encoding E. coli in PS35 A Robust and Mandatory Training Program Promotes laboratory mice and suggests the monitoring and exclusion of these High-Quality Science Clb-encoding E. coli strains from rodent colonies. NA Rogers*, E Horrigan, JG Fox PS33 Reduce, Replace, and Refine the Due Diligence Process for Vendors, Contract Research Organizations, and Academic Research Division of Comparative Medicine, Massachusetts Institute of Organizations Technology, Cambridge, MA

CM Charlebois* In casual conversations with training coordinators at other academic institutions, there often seems to be a concern to not burden MRL QA Animal Welfare, Merck, West Point, PA researchers with mandatory training, instead presenting a minimal slate of required introductory training and offering further training Site visits to vendors, contract research organizations (CROs), and as optional or merely recommended. In our experience in a large and academic research organizations (AROs) are a necessary part of the diverse academic research program (with over 1,500 active animal due diligence process. Universities, government, and pharma spend users), we have found that a comprehensive mandatory training significant funds sending staff to service providers to evaluate their program for the investigative community (students, postdocs, staff, quality and compliance. If resources are not available to conduct site and faculty) is not only achievable, but has reaped rewards in visits, compliance may be at risk. Service providers are also impacted compliance, competency, and fostering a spirit of cooperation and due to the commitment of multiple staff members for visits that may collaboration with the research community. Over the course of the last several days. Site visits can be inconsistent due to the auditors’ past decade, we have dramatically expanded our training program qualifications and experience, questions posed, time allotted, and while simultaneously streamlining and simplifying the onboarding available access to the facility. Additionally, these site visits are process for investigators. Mandatory introductory training for all duplicative as each university, government agency, and pharma animal users now includes online e-courses, hands-on species-specif- conducts a site visit to each service provider. Alternatively, the lab ic training, and animal facility orientations. Supplemental training in animal industry can use the 3Rs to decrease the resources needed to surgery, hazmats, imaging, irradiation, and food/water regulation is ensure the animal care and use program at any service provider required, as necessary. Continuing education for all mouse and rat meets the same high standards of your own institution. Options may users comes in the form of an annual “rodent refresher”; an online include sharing site visit reports, using third party auditors, and continuing education program for nonhuman primate users is to be standardizing the site visit process. We will initiate a conversation launched in the fall of 2016. Compliance is achieved by gating about these options and the way they can reduce the industry’s certification to work with animals, protocol approval, and access to resources for site visits while still ensuring animal welfare compli- animal facilities through completion of the requisite training. Efforts ance at CROs, AROs, and vendors. have been made to minimize growing pains; new mandatory training is phased in where feasible and, when warranted, can be waived for PS34 The New R: How an Animal Welfare and Ethical Review long-standing investigators. All training can now be completed or Body in the UK Is Tackling Reproducibility scheduled online. While the requirements can appear formidable on first glance, feedback from researchers has been positive; comments JC Gebbie*, B Whitelaw commending our staff both for our efforts and the scope of our training program are common. The rewards of instituting a robust The Roslin Institute, Easter Bush, United Kingdom training program extend beyond our own animal care program to the broader scientific community—more thorough training fosters better The remit of an Animal Welfare and Ethical Review Body (AWERB) informed and skilled researchers, well cared for animals, and, in the UK includes encouraging implementation of the classic 3Rs of ultimately, higher-quality research. humane animal science. Of late, there have been several new Rs appearing in science publications and media, lead by “reproducibil- PS36 Spreading the Word: Creating a Veterinary Technician ity.” Our AWERB recognised that reproducibility of scientific studies Student Outreach Program is fundamentally an issue of science quality and is critically important in animal studies. Studies that cannot be reproduced because of TT Mufford1,2, JA Reisinger*2 poor quality work represent use of animals that has not achieved a useful objective and therefore the animals’ adverse experiences and 1Office of Animal Resources, University of Colorado—Boulder, lives have been wasted. Also public trust in animal science will be Boulder, CO; 2Division of Medical Oncology, University of Colorado, eroded if it is not perceived to meet high standards.We established a Aurora, CO reproducibility subgroup to consider issues related to reproducibility and identify means to improve the reproducibility of animal work Veterinary technicians play a unique and vital role on the laboratory conducted at the institute. To achieve this aim we have shared and animal professional team. They are an essential bridge for various discussed relevant publications and guidelines, introduced the species, specialties, and procedures for which traditional clinical subject to academic development seminars, and incorporated veterinary technicians generally are not prepared. Most vet tech reproducibility issues into a new “ethics day” for PhD students and degree programs offer little more than basic introduction to labora- postdoctorate staff. We have also trialled a process of following the tory animal medicine, as required for AVMA for accreditation. This paper trail of a study from planning to publication to see how amounts to the equivalent of 3 credit hours of lecture, minimal reproducible the study has been and what we can learn about hands-on experience in technical procedures, and virtually no practices in our Institute which affect reproducibility. The AWERB exposure to research vivaria. Oftentimes this leads to misunderstand- also has a statistician member who raises general issues regarding ings of the mission of the lab animal field, and fosters most to seek experimental design and statistical analysis and is also involved in positions in companion animal clinics. We sought to change the the local review of detailed study request forms.The number of publi- negative bias towards the field by starting an outreach program cations on reproducibility is growing and our scientists are geared towards veterinary technician students. Through networking increasingly aware of these issues. The AWERB has taken the lead in with instructors in local technician programs, we integrated standard our Institute because of the importance of reproducibility in animal lab animal classes to generate exposure and interest, as well as to studies, but it is a wider issue for all our science. Our conclusion is educate students on the wide variety of related topics. Lectures were that this is an issue that is not going to go away. The AWERB needs to geared towards the importance of the veterinary technician in be actively involved in identifying relevant issues in our Institute, research, relevance to their lives, and educational and advancement gathering sources of advice and tools, and promoting communication opportunities with employment. A central theme focused on how a and education through creative means. background in animal science is precisely what the field needs for

686 Abstracts of scientific papers 2016 AALAS National Meeting

healthy animals and valid science. Instructors surveyed the students PS38 Making Matters Worse: A Snapshot how Outside Influences pre and post lecture with questions regarding student feelings Can Impact Model Endpoints and Animal Welfare 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 JK Lang*1,2, J Zaias3 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 1Department of Medicine/Gastroenterology, University of Miami, tech schools. Although discussing a career in laboratory animal Miami, FL; 2Department of Immunology and Infectious Diseases, research can be a sensitive subject, this program has demonstrated Harvard T.H. Chan School of Public Health, Boston, MA; 3Divison of that with the proper information, enthusiasm, and personalization on Veterinary Resources, University of Miami, Miami, FL how animal research has affected their lives, outreach programs should help persuade this essential cohort to embrace their role in Suggestions for study design often come from outside sources. For supporting animal research. example, IACUC directives can change animal protocols, especially when related to welfare matters. The Guide has been updated with PS37 Real versus Perceived Costs of Self-Imposed Regulatory recommendations for enrichment and social housing. Recently, the Burden in Different Administrative Models of Animal Welfare NIH has strongly suggested using both sexes in animal experiments. Assurance It is difficult to understand how these potentially positive directives can impact longstanding predictable model endpoints. We JM Wallace*1, VK Bergdall6, JF Bradfield10, LA Conour4, LJ DeTolla7, demonstrate how outside influences suggesting alterations to study AW Grady11, DL Hickman9, SM Niemi5, JN Norton3, JD Thulin2, BJ variables can impact model outcomes and animal welfare. Our lab Weigler8 studies colitis using DSS and colitis-associated cancer (CAC) using AOM-DSS. Both are widely accepted models in the GI field. Using 2 1Pathology, Microbiology and Immunology/Comparative Medicine, separate study designs, we tested the effects of several variables Vanderbilt University Medical Center, Nashville, TN; 2Biomedical which were recently addressed by the Guide, NHI, and our IACUC. Resource Center, Medical College of Wisconsin, Milwaukee, These variable were environmental enrichment, social housing, using WI; 3Division of Laboratory Animal Resources, Duke University, both sexes, and fluid therapy as a medical refinement. We performed Durham, NC; 4Laboratory Animal Resources, Princeton University, a CAC study using various housing variables, enrichment, grouping, Princeton, NJ; 5Office of Animal Resources, Harvard University, and sex to determine how strongly these might affect our model Cambridge, MA; 6University Laboratory Animal Resources, The outcome. Our results demonstrate these variables had an effect on Ohio State University, Columbus, OH; 7Program of Comparative the tumorigenicity of our model, as nonenriched single-housed Medicine, University of Maryland, Baltimore, MD; 8Office of the Vice females were prone to higher tumor numbers and loads than the President for Research and Innovation, University of Nevada, Reno, other groups.Our IACUC suggested we hydrate our DSS colitis mice. Reno, NV; 9Laboratory Animal Resource Center, Indiana University We provided fluid hydration and examined the mice at several time School of Medicine, Indianapolis, IN; 10AAALAC International, points during the study. Interestingly, mice had dramatically lower Frederick, MD; 11Laboratory Animal Facilities, University of inflammation when provided with fluids. We were no longer able to Mississippi Medical Center, Jackson, MS use our protocol as a robust model of colitis. Therefore, the outside proposal from IACUC, which was suggested to refine our model, A growing number of biomedical research institutions, particularly in ultimately would have caused an increase in sample size, as many of academia, have organizationally separated administrative support the animals were not inflamed enough for our studies.Both study for their IACUC from their animal resources program (ARP), with results highlight the need for strong standardization of experimental each entity reporting separately to their institutional official (IO). design. Seemingly minor deviations in the environment can cause There are a variety of valid management rationales for adopting this significant differences in experimental outcomes. This can be difficult particular organizational structure, including the limited veterinary and limiting due to animal availability, cost factors and convenience. management resources in small programs. However, a common Hence prior to accepting a suggestion from an outside source, it is driver has been the fear of a purported conflict of interest for the important to consider what sort of impact minor changes could have attending veterinarian (AV), who typically directs the ARP. The on your model. reasoning is that in also directing the IACUC administrative staff, the AV could unduly influence IACUC activities and decisions, espe- PS39 Engaging Investigators Using a Diversified Approach to cially as they pertain the the IACUC’s regulatory responsibility for Postapproval Monitoring (PAM) Process oversight of the ARP. Yet by the same reasoning, any individual charged with directing the IACUC administrative support could be E Koncza*, E Kapsalis, K Sklavounos, J Zaias similarly conflicted because each could inappropriately influence IACUC activities and decisions for personal advantage unrelated to Office of the Vice Provost, University of Miami, Miami, FL welfare concerns. In spite of this largely theoretical conflict, the specific separation of IACUC administrative support from ARP The purpose of our postapproval Mmnitoring (PAM) processi is to management is repeatedly portrayed as a best practice, a character- engage with animal users in constructive exchange, to assist ization too often misinterpreted as favored or required rather than researchers in identifying and correcting any deviations, and to merely one of several acceptable options. In addition to claimed verify research is being conducted as approved. Our initial PAM advantages, separation of IACUC and ARP administrative support process included semiannual inspections, monitoring by veterinary has drawbacks, including duplication of effort, inefficiencies, and personnel, and limited one-on-one meetings with the PI and IACUC unnecessary discord. These outcomes in turn can result in increased Director. In 2011, a new PAM process was launched revealing that of costs and regulatory burden without appreciable benefit to animal those investigators that underwent the process, a significant number welfare or research quality. We considers the merits of an alternative had medical recordingkeeping deficiencies. It soon became evident organizational arrangement in which both ARP and IACUC support that a typical PAM process took much longer than we originally staff report to the AV, preserving the tripartite approach for trustwor- expected and that we needed to develop a more systemic and thy oversight involving the IO, IACUC, and AV. Effective time-efficient approach. In 2012, a process called self-PAM was communication, full transparency, constructive conflict resolution, initiated in which investigators perform their own internal protocol and mutual respect are essential and ensure a healthy system of monitoring and report their outcomes to the IACUC. In 2014, a checks and balances. 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

687 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 prior to any animal work by new investigators on newly approved ogy study requirements. Provided that the solution is selected protocols. During the first year, of 22 investigators that participated according to the specific features of each option, to the outcome of a in PIM, 19 opted to revise their protocols subsequent to the feedback comprehensive risk analysis, and that, as illustrated by examples, they received during the PIM process. In 2015, a new cross-PAM appropriate training and practices are used, IVC-based solution process was piloted. In this process, we focus on a particular represent a reliable and practical alternative for biocontainment and compliance concern such as rodent enrichment or tumor burden and gnotobiology. review its implementation across several investigators’ protocols. In first 5 months, we were able to work with 69 investigators and 115 PS44 Combined Use of Isolators and IVC System for Production, protocols. Diversifying our approach to PAM increased our efficiency Maintenance, and Experimentation with Axenic Animals in reaching animal users and provided us with an expanded platform for positive educational and preventive interaction with investigators. J Bom*1, K Berman2, A Ribeiro1, M Pereira1, AS Leocádio1, M Rebelo1, J Demengeot1 PS40 Rodent Survival Surgery: Where Are Your Investigators Learning Aseptic Technique? 1Animal House Facility, Instituto Gulbenkian de Ciência, Lisboa, Portugal; 2Baylor College of Medicine, Houston, TX JL Haupt*, JG Fox In 2005 we started with rigid walls isolators for axenization and Massachusetts Institute of Technology, Cambridge, MA maintenance of mutant and wild-type mouse strains of different genetic backgrounds. Gnotobiology techniques allow maintaining Aseptic surgical technique for rodent survival surgery is mandated animals in axenic environment, with the possibility to restore specific by the Guide, as well as being required in many institutional components of the microflora. In 2012, the need to implement gnoto standards. Proper training of sterile surgical techniques and specific experiments led us to reorganize space, optimizing human and surgical procedures is vital for institutional educational programs financial resources. The IsoCage P system was designed to achieve and for the welfare of laboratory animal species. With advances in the safety and the protection features of an isolator, with the web-based training, many investigators are seeking outside sources advantages in terms of ergonomics, flexibility, and density of an IVC. for procedure specific training. Concerns regarding these resources Each cage in an IsoCage P system is a microisolation cage, allowing include the demonstration of inappropriate sterile surgical technique, multiple studies on the same rack, providing full bioexclusion for as well as inadequate anesthetic and analgesic regimens. To maximum protection, excluding cage-to-cage contamination. This investigate potential deficits in these areas, 100 randomly selected IVC system has been in use since 2013 for experiments with GF video-based articles on the topic of rodent survival surgery were animals, in combination with the IsoCage Biosafety Station (IBS). The assessed for aseptic surgical principles, appropriate tissue handling IBS was specifically conceived to enable the manipulation of cages and wound closure, analgesic and anesthetic use, and postoperative and animals in a fully sterilized environment. The use of the IsoCage monitoring. All videos were viewed and scored by a boarded P system for gnoto experiments allowed freeing space in isolators veterinary surgeon. Assessment of the video articles revealed that and expanding the number of GF animals produced. From 2013 to only 44% of the articles described or demonstrated appropriate 2015, 44 different experiments were performed using this system. preoperative skin disinfection. In addition, surgical asepsis was Standard operational procedures have been implemented and deemed to be maintained in only 10% of the videos with 51% and improved, registering a decrease of contaminated experiments from 35% having minor and major breaks in aseptic technique, respec- 45% to 5-10%. To validate our working system, GF sentinel animals tively. Appropriate tissue handling and wound closure were present were implemented in the IsoCage P system. These animals are in only 67% of the videos and 13% of videos had no mention or use manipulated exactly the same way experimental animals are, and of analgesics. This study highlights many of the potential deficits in their health status checked every month for quality control. The video-based articles for rodent surgery and alerts the laboratory ongoing results demonstrate that sentinels have been maintaining animal community regarding the use of these video articles by their axenic status for more than 1 year, indicating that our system is investigators for purposes of surgical training. working efficiently. In summary, the combined use of isolators for production and maintenance of GF animals stocks and IsoCage P PS43 Comparative Review of Key Issues for Biocontainment and system for experimentation with axenic animals has been running for Gnotobiology IVC System Use versus Glove-Box Isolators in the last 3 years. Based on the low fraction of contaminated experi- Rodent-Based Research ments and our sentinel program we conclude that this system was successful, optimizing human, financial, and space resources. PR Hardy*1, PC Willan2 PS45 Effects of Bedding Material and Water Treatment on the Gut 1Veterinary & Professional Services, Allentown Inc., Bussy Saint Microbiota Georges, France; 2Allentown Europe, Reading, United Kingdom WA Bidot*1, A Ericsson2,3, CL Franklin2,3 Biosafety requirements include the use of biosafety cabinets or other physical containment devices (primary containment) and suitable 1Veterinary Pathobiology and Comparative Medicine Program, working practices. They must respond to a risk assessment and a University of Missouri, Columbia, MO; 2Metagenomics Center, related mitigation plan, be validated accordingly, properly main- University of Missouri, Columbia, MO; 3Mutant Mouse Resource and tained, and used by experienced and qualified personnel. Both for Research Center, University of Missouri, Columbia, MO animal care and use, practical constraints and risk assessment outcome related to the use of BSC-III/glove-box isolators (sensitive Rodent models are invaluable to understanding health and disease in procedures, space requirements, ergonomics, use of sharp items, many areas of biomedical research. Unfortunately, many models etc.) led to the development of innovative biocontainment IVC suffer from lack of phenotype reproducibility. Our laboratory has systems, associated to the use of suitable biosafety cabinets. Such recently shown that differences in gut microbiota (GM) can modulate alternatives must respond to the specific and integrated needs of phenotypes of models of colon cancer and inflammatory bowel scientists and technicians carrying rodent-based research, as well as disease. We and others have also shown that a number of factors to biocontainment regulations and best practices and biosafety associated with rodent research, including vendor, housing ventila- officers expectations. A comparative review of the design, validation, tion, and bedding can alter GM. The objective of this study was to and use of IVC-based systems and associated working cabinets expand the latter studies to examine the effect of additional bedding allows comparing their benefits and drawbacks when compared to materials and methods of water decontamination on GM diversity isolator use. A similar reverse approach was used to develop, and composition. To this end, Crl:CD1 (ICR) mice were housed on validate, and use bioexclusion IVC systems designed for gnotobiol- corncob or compressed paper chip bedding and provided water that

688 Abstracts of scientific papers 2016 AALAS National Meeting

was decontaminated by reverse osmosis, autoclaving, sulfuric acid because of the large difference in weight as they grow and lack of any treatment, or hydrochloric acid treatment. Feces was collected at days visible markers for various stages of adult life. Current cage space 0, 7, and 14. At day 28, animals were euthanized and both fecal and recommendations are based on weight. Considering the potential for cecal material samples were collected. DNA was extracted from cross-contamination of different colonies, it is not advisable to use a samples, amplified by PCR using conserved bacterial primer sets and single scale from room to room, and it is cost prohibitive to have a subjected to next generation sequencing. Raw sequence data was dedicated scale for each rat colony. An alternate technique to analyzed using Qiime and resulting metagenomics data compared determine the proper housing density of rats is needed. Skeletal size among groups using principal component analysis and multivariate has been shown to be proportional to body weight in previous ANOVA. Principal component analysis of data revealed differences studies. However, the studies were not done on live animals and in microbiota composition in the following comparisons: corncob were not correlated with size of the tail, therefore this study explored versus paperchip in mice given either sulfuric acid-treated or the feasibility of using tail diameter instead of weight as a surrogate autoclaved water, and sulfuric acid-treated water versus autoclaved marker of weight. A digital caliper was fitted with polyoxymethylene water in mice on corncob bedding. These findings expand the list of blocks, 25 x 38mm, to create a standardized surface for the measure- husbandry factors that can modulate GM to water decontamination ment. Rats were then weighed and their tails measured at the methods and bedding/water interactions and further emphasize the boundary of fur to no fur near the tail base. A series of 3 measure- need to consider microbiota changes as possible sources of pheno- ments were collected and averaged. Average data from no less than type variability. 50 adult male and 50 adult female animals from several different colonies resulted in a positive correlation of 0.85 between tail PS46 Static and Individually Ventilated Caging Acceptable for diameter and body weight. Using that information, it was calculated Mouse Housing Density Exceeding Guide Recommendations 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, E Helman*, D Duvall, JM Cadillac, E Dohm, T Schoeb, SC Cartner and 9.5mm for a 400g animal. We will use these measurements to create a standardized rat tail gauge for verification of proper housing Animal Resources Program, University of Alabama—Birmingham, density. It was determined that the diameter of a rat’s tail at the fur to Birmingham, AL 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 Many references have determined that the mouse housing density a scale, which will save money and time of the husbandry staff. recommendations of the Guide can be exceeded when IVC is used. However, having different requirements for animals housed in IVC PS48 Cross-Fostering within the Same Animal Housing Room to or under static conditions is not practical for the mouse housing Eliminate Murine Norovirus and Helicobacter spp. in Mice density policy at our institution. Therefore, a study was undertaken to both validate published IVC housing density recommendations CA Stevens* and to confirm that those same higher housing densities are also acceptable for cages kept under static conditions. The experiment Central Michigan University, Mount Pleasant, MI consisted of a breeding and a nonbreeding group housed in standard cages of approximately 70 inches2 of floor space with hardwood chip In efforts to preserve the SPF status of our facility with mice arriving bedding. Each group contained 24 cages of inhouse bred C57BL/6 from other institutions, cross-fostering to eliminate pathogens in the mice equally divided into control and test densities and housed on space housing room was initiated. Our goal was to minimize static or IVC racks. The breeder control density consisted of 8-week potential unexpected changes in transgenic mice which had old mice set up in a trio breeding scheme, 1 male and 2 females, with previously occurred with rederivation. A cost and time effective no more than 9 pups total per cage, strict weaning at 21 days, and no protocol was implemented to cross-foster incoming mice with known more than 14 days separation in age of the pups. The breeder test MNV and Helicobacter spp. status. The procedure was put in place density was also a trio breeding scheme, but with no limit on pups involving cross-fostering litters in the same housing room due to and no restriction in litter age difference. The nonbreeder groups limited housing space. The pathogen-positive colony and the included a control density of 5 and test density of 7 weaning age surrogate cross-foster CD1-Elite females were Mouse Surveillance males. Measurements of intracage conditions included ammonia at Plus Pria tested prior to being housed on separate ventilated racks in cage change, which occurred once weekly for IVC and twice weekly quarantine. Clear-cut, extensive steps were implemented to prevent for static cages. Evaluation of the soiled bedding, water, food, nest breaches in cross-contamination. Mouse Surveillance Plus Pria material, pups born, and health assessment were recorded daily. At testing was repeated several times up to 12 weeks of age to ensure weaning the litter weight was recorded, and number of pups negative MNV and Helicobacter spp. status of cross-fostered pups and surviving from birth through weaning was calculated. Initially, ATP surrogate mothers prior to transferring the offspring from quarantine luciferase test swabs were used at cage change as an objective into regular housing. Quarterly health monitoring has continued to assessment of cage hygiene, but it was quickly determined that food confirm the efficacy of our cross-fostering protocol. Our results dust confounded results, so testing was changed to CONTACT indicated that our protocol was 100% successful in eliminating both PLATES cultures. Observations continued for at least 2 months. MNV and H. ganmani,H. hepaticus, H. mastomyrinus, and H. typhlo- There were no significant differences between any of the groups for nius. This protocol is recommended to eliminate pathogens in ammonia, CONTACT PLATES growth, soiled bedding, nest cross-foster programs within the same housing room. evaluations, pup birth rate, and survival. Housing of mice at densities exceeding those recommended in the Guide is acceptable at PS49 An Assessment of Recycling Crinkled Paper Nesting Material our institution regardless of static or IVC status and in agreement for 2 Cage Changes in Individually Ventilated Cages with other published data on the subject. ML Kain*1, TL Martin2, P Velleman1, C Hedrick2, N Jurcak2, S Lewis2 PS47 Quick and Easy Determination of Rat Housing Density by Husbandry Staff Using Tail Diameter as a Surrogate for Body 1Cornell University, Ithaca, NY; 2The Ohio State University, Colum- Weight bus, OH

K Browning*, JM Cadillac, E Dohm, SC Cartner Nesting material as environmental enrichment has been shown to reduce stress, anxiety, pup mortality, and aggression. Recent studies Animal Resources Program, University of Alabama at Birmingham, indicate that mice prefer to build nests with naturalistic bedding, Birmingham, AL such as commercially available brown crinkled paper. Literature has also demonstrated that mice prefer dirtier cages to clean ones, with Determining the appropriate housing density for rats is difficult an increase in adverse behaviors such as aggression and stereotypies

689 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 observed following cage changes. We hypothesized that recycling 1Veterinary Science, Research, and Support, Envigo, Indianapolis, crinkled paper for 2 cage changes (28 days total) would result in IN; 2Envigo, Indianapolis, IN reduced adverse behaviors in mice without affecting nest quality. We proposed that the crinkled paper would preserve its strength and Enrichment is important for laboratory rodent models as it can cleanliness throughout the 28-day period. To test this, we gave 20 reduce stress and abnormal/aggressive behaviors. Proper enrich- individually ventilated cages of mice 8g of crinkled paper and ment can lead to a reduction in variability in research outcomes and monitored behavior, nest scores (as a measure of paper strength), and reducing the number of animals needed for a study. Two studies intracage ammonia levels (as a measure of paper cleanliness) for 28 were performed to evaluate the use of several enrichment types in days. Half of the cages (n = 10) received new crinkled paper at cage various rat and mouse models. In the initial study, a cotton fiber pad change (day 14), while half (n = 10) retained the original crinkled (DN) was evaluated. Usage of this enrichment was examined in paper. Strength testing and visual assessments of paper cleanliness breeding pairs of the Brown Norway rat, C57BL/6N, ICR, Athymic were also conducted at each cage change (days 14 and 28). We Nude, SHrN®, and FVB/N mouse models for 6 weeks starting at 8-10 observed no significant behavioral changes in mice receiving clean weeks of age. DN usage was also examined in C57BL/6N, ICR, versus recycled paper. We found no significant difference in intracage Athymic Nude, SHrN®, and FVB mouse models for from weaning to ammonia levels in cages receiving clean versus recycled crinkled 6 weeks of age. The number and size of pups per litter was recorded paper, although the recycled paper did subjectively appear dirtier in the breeding cages. Nests were scored weekly according to the upon visual examination. We found reduced paper strength via following system: 0-5 where 0=undisturbed, 1=disturbed, 2=flat nest, strength testing, and significantly lower nesting scores in cages 3=cup, 4=incomplete dome, and 5=complete dome. The second study receiving recycled paper (p = .0049). We did observe strain differ- evaluated usage and preference of various enrichment types in ences in nest score and structure, indicating that uniform scoring breeding pairs: DN, a compressed cotton pad (IB), rolled tissue paper systems in the literature should be tailored to the strain being (DT), or crinkle paper (SN) for 13 weeks in Athymic Nude, BALB/c, examined to be most useful. Overall, we concluded that the crinkled DBA/1, C57BL/6N mice, and Sprague–Dawley® rats. Production paper did not retain sufficient strength and visual cleanliness to be efficiency index, aberrant findings, and enrichment usage were recycled for 2 cage changes. recorded weekly. There were no aberrant findings in either study with the use of enrichment. All models built nests with the DN PS50 Prevalence of Murine Adventitial Infections in Biomedical enrichment. The number of pups per litter was significantly higher in Research Facilities ICR mice with DN compared to no enrichment. The percent of pups weaned was also significantly higher in the Athymic Nude mice with JO Marx*, AL Smith, D Gaertner DN compared to those with no enrichment. The SHrN® breeders built better nests with SN compared to DN. BALB/c mice built nests Pathobiology, School of Veterinary Medicine, University of Pennsyl- more readily with DN but didn’t build nests with IB until 6 weeks vania, Philadelphia, PA 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. Adventitious infections in mice used in biomedical research may Brown Norway breeding pairs built nests with DN and the Sprague– have a profound impact on study outcomes. Effects are not limited to Dawley® rats efficiently used the DT. Studies such as these will deaths and clinically apparent illness in mice, since undetected enable us to determine best practices for nesting enrichment in infections may have subtle effects on the immune system, hormone laboratory rodent models to better care for our animals and promote production, and other physiologic systems that can unknowingly the 3Rs. alter collected data. Many immunosuppressed mouse strains are highly vulnerable to these normally benign infections and any viral, PS52 Methods Development for Cohousing Male Crl:CD1(ICR) bacterial, or parasitic infection can profoundly impact study results Mice from mice used in immunology research. Additionally, genetically modified mice may have subtle, undetected alterations in immune RR Hukkanen, L McFadden, J Murray*, J Reinart, J Wachner, J Lacher function. Because of this, research institutions spend a great deal of time and resources to control these infections in their colonies. We Dow Chemical Company, Midland, MI recently conducted a survey querying 62 of the top 100 NIH-funded biomedical research institutions regarding the prevalence of all Mice readily seek conspecific contact and are capable of forming common infections in their colonies during the previous 2-year social relationships. However, males have strain-dependent territo- period. These results were compared with those from 2 previous rial tendencies that often prohibit pairing of adults. Group housing published surveys conducted in 1996 and 2006. We found that the of mice is advantageous and encouraged as enrichment to promote frequency of most viral, bacterial, and parasitic infections decreased species-specific behavior and deter stereotypy. Nonetheless, indi- over time, despite improved sensitivity of diagnostic techniques. The vidual housing is the current practice on toxicological studies here most commonly identified agents included fur mites (56% of due to the potential for male aggression and the need to normalize institutions reporting infections during the 2-year period), pinworms experimental environments across sexes/subjects. Despite histori- (44%), and mouse parvovirus (37%). The prevalence of mouse cal challenges, we hypothesized that male Crl:CD1(ICR) mice could hepatitis virus (13%), epizootic diarrhea of mice virus (15%) and be successfully paired through reducing factors associated with minute virus of mice (3%) were dramatically decreased compared to aggression. We explored the effect of pheromone continuity, age at the previous surveys. Twenty-two percent of institutions completely first pairing, partner permanence, and delayed intervention within excluded norovirus, while 48% of institutions had select suites or a 28-day repeat-dose toxicity test study design. We further explored facilities which were maintained free of norovirus. In both situations, the presence of novel enrichment within new and sustained-pairings 21% of institutions experienced norovirus outbreaks. Management of over a 1-5 month period. Four groups of 10 mice aged 5 or 6 weeks viral and fur mite outbreaks varied widely among institutions. were paired once or twice using husbandry techniques designed to Ultimately, our ability to completely eradicate these infections will transfer pheromones at cage change-out (marking clean cages with depend on the use of strict barrier housing practices, the prevalence urine-soiled gauze, transfer gauze from dirty to clean cages, and of the organisms in wild mouse populations, the biology of the agent, transfer feed on separate days from cages). A control group of 10 and the perceived impact of the agents on research. mice was paired using standard husbandry techniques. Under veterinary supervision, animal care staff did not separate pairs exhibiting PS51 Preferential Treatment: Enrichment Options for Mice and nontraumatic interactions including vocalization and fighting. After 4 Rats months, the first 4 groups of mice were transitioned to standard husbandry and offered novel enrichment. All pairings were successfully J McClellan*1, M Strine2, M Gallimore2, J Bowman2, L Figueroa2, MJ maintained throughout the study. A single injury (minor laceration) Horn1 was observed within the first 28 days of pairing; however, the pair

690 Abstracts of scientific papers 2016 AALAS National Meeting

was maintained and the laceration resolved. These data indicate that Pittsburgh compound-β (PiB) to measure amyloid plaque accumula- pheromone continuity, age at first pairing, and partner permanence tion. MRI revealed decreased cerebral blood flow and PET revealed do not significantly impact pairing success. We therefore conclude decreased cerebral metabolism and increased levels of amyloid that male Crl:CD1(ICR) mice may be successfully paired on repeat- plaques. These findings correlated with the histologic findings, which dose toxicity studies up to 90 days of duration. employed novel 3-dimensional immunofluorescence imaging. We propose these techniques as sensitive methods to evaluate the PS53 The Placental Estrogen, Estriol, Protects Female Mice against progression of AD in mice, allowing longitudinal evaluation in lieu Influenza A Virus by Reducing Pulmonary Inflammation of postmortem analysis at specific time points.

MS Vermillion*1,2, SL Klein2 PS55 Host Genetic Drift and Gut Microbiota Variability between C57BL/6- ApcMin Mouse Colonies Affects the Intestinal Tumor 1Molecular and Comparative Pathobiology, Johns Hopkins Univer- Phenotype sity, Baltimore, MD; 2Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, Baltimore, MD JE Moskowitz*, S Busi, ML Hart, CL Franklin, J Amos-Landgraf

Estriol (E3) is a pregnancy-specific estrogen in humans and mice that Comparative Medicine Program, University of Missouri, Columbia, is produced by the placenta and found in highest concentrations MO during late pregnancy. In humans and mice, E3 has potent immunomodulatory functions and can ameliorate diseases caused by Reproducibility in animal studies has emerged as a significant inflammation, including multiple sclerosis (MS). Like MS, pulmonary concern across a breadth of research fields, yet the underlying causes disease associated with influenza A virus (IAV) infection is predomi- of phenotypic variability among animal models often remains nantly caused by aberrant inflammation and immunopathology, unclear. Host changes such as genetic drift between different colonies rather than virus replication. We hypothesized that exogenous E3 are likely to influence these phenotypes, but other poorly character- may protect female mice against IAV-associated disease by reducing ized factors such as the gut microbiota (GM) may have a considerable pulmonary inflammation and tissue damage. Adult gonadally intact impact as well. The ApcMin mouse model of colorectal cancer is a female C57BL/6 mice received a subcutaneous implant of either highly used model that has demonstrated phenotypic variability 21-day release 5mg E3 or placebo (n = 20 animals/treatment). This among colonies. Consistent with these reports, our laboratory has dose of E3 increased concentrations to levels found in pregnant found significant differences in both small intestinal (SI) and colonic female mice as measured by mass spectrometry. Females were tumor numbers between two ApcMin mouse colonies; the C57BL/6J- intranasally infected with a sublethal dose of mouse-adapted 2009 ApcMin (B6-Min/J) from the Jackson Laboratory, and the H1N1 IAV or vehicle, and body mass was recorded daily as a C57BL/6JD-ApcMin (B6-Min/D) closed colony that has been main- measure of morbidity. Pulmonary inflammation and immune cell tained at the University of Wisconsin. To determine if phenotypic populations were evaluated by histology and flow cytometry at 9 variability between these 2 colonies is due to host genetic changes or and 14 days postinfection (dpi), corresponding to peak disease and the environmental impact of the GM, we used complex microbiota recovery, respectively. Following infection with IAV, E3-treated targeted rederivation (CMTR) to rederive embryos of the 2 females lost significantly less body mass compared with placebo- Apc mutant colonies onto CD1 surrogate dams that possessed GM treated females. Clinical protection against IAV was associated with from either The Jackson Laboratory (Crl:CD1GMJAX) or Envigo Min significantly less pulmonary inflammation and tissue damage, which (Hsd:CD1GMHSD). We generated a total of 4 Apc groups: B6-Min/ was characterized by reduced perivascular and peribronchiolar JGMJAX, B6-Min/JGMHSD, B6-Min/DGMJAX, and B6-Min/DGMHSD. inflammation at 9 dpi. Characterization of the T cell response by flow Tumor counts revealed significant increases in both SI (P = 0.03) and cytometry showed that E3-treated females had a significantly greater colonic tumors (P = 0.006) in the B6-Min/J group with a GM from ratio of CD4+ to CD8+ T cells at both 9 and 14 dpi, and skewing of HSD compared to the B6-Min/J group with a GM from JAX, CD4+ T cell responses toward Th2 and away from Th1 and Th17 suggesting that the GM is critical in the SI and colonic tumor responses at 9 dpi. These data suggest that pregnancy levels of E3 are phenotype. Additionally, B6-Min/D animals had more SI tumors protective against IAV infection and indicate that the estrogen-driven than B6-Min/J animals within each GM group. However, there were immunomodulation during pregnancy is not the cause of pregnancy- no differences between these groups in colonic tumor numbers, associated IAV disease severity. These data also provide evidence suggesting that host genetic differences play a role in the SI pheno- that E3 has broad immunomodulatory effects and may be beneficial type, but not the colonic phenotype. We conclude that both the GM in the context of inflammation-driven disease. and host-genetic factors within the C57BL/6J-ApcMin population contribute to the SI tumor phenotype, while the GM primarily PS54 In Vivo Methods for Monitoring Disease Progression in defines the colonic tumor phenotype. These results demonstrate how Mouse Models of Alzheimer’s Disease interactions between host and environment can shape a given phenotype within our animal models, and thus significantly impacts NM Tataryn*1,2, H Ahn2, S Strickland2 reproducibility in animal studies.

1Veterinary Services, Memorial Sloan Kettering Cancer Center, New PS56 Comparison of the Vaginal Environment in Rhesus Macaques York, NY; 2The Rockefeller University, New York, NY (Macaca mulatta) and Cynomologus Macaques (Macaca fascicularis) Several mouse models of Alzheimer’s disease (AD) employ the integration of human transgenes into the mouse’s genome that GJ Daggett*1, C Zhao3, F Connor-Stroud1, F Villinger2 upregulate the production of amyloid-β peptides leading to the deposition of amyloid plaques in the brain. Traditionally, methods to 1Department of Animal Resources, Emory University, Atlanta, monitor disease progression in these models have included behav- GA; 2New Iberia Research Center, New Iberia, LA; 3Centers for ioral testing and postmortem histologic evaluation. These methods Disease Control and Prevention, Atlanta, GA are often insensitive and preclude longitudinal analysis of individual mice. We evaluated multiple in vivo imaging modalities to monitor Rhesus (Macaca mulatta) and cynomologus macaques (Macaca disease progression and to equate these findings with innovative fascicularis) are valuable animal models for the development and 3-dimensional immunohistochemical techniques in 2 mouse models study of Human Immunodeficiency Virus (HIV) prevention of AD. The MRI method of arterial spin labeling (ASL) was used to strategies. Prevention of vaginal acquisition of HIV is highly relevant measure cerebral blood flow. Additionally, positron emission as it represents a major portal of viral entry that is heavily influenced tomography (PET) was performed using 2 different radiotrac- by local infections, inflammation, hormonal changes, and the ers: 18Fluordeoxyglucose (FDG) to measure cerebral metabolism, and resident microbiota. To this end we tested the vaginal microbiota and

691 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 pH of rhesus and cynomolgus macaques to characterize the normal increased over time, suggesting a chemical was eluting from some microflora present in these models of HIV infection. In women, Lacto- component of the plastic syringe. bacillus spp dominate a healthy vaginal microbiota, leading to the production of lactic acid, a lower pH, and the inhibition of other PS58 Characterization of Multi-Drug Resistant Enterococcus bacterial species, as well as viruses. Higher Nugent scores, referred to faecalis Isolated from Research Macaques as bacterial vaginosis, and higher vaginal pH have been shown to increase susceptibility to mucosal transmission of HIV in humans. In MT Lieberman*1, SE Woods1, F Lebreton2, J Dzink-Fox1, MS Gilmore2, this study we compared the Nugent scores and vaginal pH of rhesus JG Fox1 and cynomologus macaques to determine if a significant species difference exists, which could suggest that the vaginal environment 1Division of Comparative Medicine, Massachusetts Institute of of one is a closer analogue to humans than the other. This experiment Technology, Cambridge, MA; 2Department of Ophthalmology, has shown that both parameters are significantly different between Massachusetts Eye and Ear Infirmary, Harvard Medical School, the 2 species. The natural vaginal environment in both species was Boston, MA equivalent to bacterial vaginosis in humans. We therefore attempted to colonize the macaque vagina with Lactobacillusrhamnosus to create Multi-drug resistant (MDR) Enterococcus faecalis is a common and an induced model of a healthy vaginal environment. Successful serious cause of nosocomial infections. Previous characterization of colonization indicated that both macaque species are able to 14 E. faecalis isolates from cephalic recording chambers of macaques host Lactobacillus spp vaginally. The colonization was sustained for a used in neuroscience research revealed two lineages with marked longer period of time in rhesus macaques with significant decreases multi-drug resistance. Lineage A (LA) isolates (n = 7) showed in Nugent scores. The Nugent scores in cynomologus macaques did differing susceptibilities to gentamicin, with 4/7 isolates displaying not change significantly. Vaginal pH following colonization with L. high-level gentamicin resistance while Lineage B (LB) isolates (n = 7) rhamnosus decreased significantly in both species. These results displayed resistance to neomycin. All isolates from Lineages A and B suggest that normal rhesus and cynomologus macaque vaginal displayed marked streptomycin resistance. Resistance to tetracycline, environments more closely represent humans with bacterial chloramphenicol, erythromycin and trimethoprim-sulfamethoxazole vaginosis. Future studies of vaginal HIV transmission should was also noted. DNA was extracted from 2 LA and 1 LB isolate and consider the normal vaginal environment when selecting an animal sequenced on a single SMRT cell on a Pacific Biosciences RS2 model. Induction of a healthy vaginal environment via colonization sequencer. Genomes were assembled and FASTA sequences were with Lactobacillus spp should also play an important role in future analyzed by PubMLST, ResFinder and VirulenceFinder to confirm investigations. sequence type (ST) and identify genes of interest. Two isolates from LA were confirmed to be ST4 and the isolate from LB was confirmed PS57 Diluted Buprenorphine Degrades over Time in Several to be ST55. All isolates had a unique antimicrobial resistance gene Common Storage Conditions profile with the lsa(A) gene encoding intrinsic resistance to lincosamides and streptogramins A as the only common gene JM DenHerder*1,4, RL Reed2,3, JL Sargent1,4, BT Bowers6,5, JF Ste- between all 3 isolates. Additional macrolide resistance encoded vens2,3, HE Diggs1,4 by erm(B) was identified in 2 isolates. Four genes encoding aminoglycoside resistance were identified: str and 3 aminoglycoside-modifying 1Department of Biomedical Sciences, Oregon State University, enzymes: aph(3’)-III, aac(6’)-aph(2”) and ant(6)-Ia. Genes encoding Corvallis, OR; 2Department of Pharmaceutical Sciences, Oregon State tetracycline resistance included mechanisms for both efflux pumps University, Corvallis, OR; 3Linus Pauling Institute, Oregon State (tetL) and ribosomal protection (tetSand tetM). Phenicol resistance University, Corvallis, OR; 4Laboratory Animal Resources Center, was conferred by the cat gene and was present in ST4 isolates, but not Oregon State University, Corvallis, OR; 5College of Veterinary the ST55 isolate. Trimethoprim resistance encoded by dfrG was noted Medicine, Oregon State University, Corvallis, OR; 6College of in 1 ST4 isolate. Other virulence factors identified included cytolysin, Pharmacy, Oregon State University, Corvallis, OR enterococcal surface protein, aggregation substance, gelatinase, collagen adhesion precursor and endocarditis antigen. E. faecalis iso- Buprenorphine is a partial mu opiate agonist used for analgesia. Due lates from cephalically-implanted macaques display genetic to the small size of rodents, buprenorphine HCl is typically diluted similarities to isolates associated with human nosocomial infections. 10- or 20-fold with a sterile diluent such as saline to aid in accurate Macaques represent a unique research model to study nosocomial dosing. Protocols for preparing and storing diluted buprenorphine infections due to their long-term residence following cephalic vary by institution, and there is little information in the literature implantation, and intermittent antimicrobial exposure. regarding beyond-use dating of specific preparations. Buprenorphine HCl is packaged without a preservative in 1 ml glass ampules, vials, PS59 Intraperitoneal Administration of Ethanol as a Means of or syringes intended for immediate use as a single human dose. Euthanasia in Neonatal Mice (Mus musculus) Inadvertent microbial contamination when diluting buprenorphine poses a significant risk to the patient and degradation of the CE de Souza-Dyer*, JO Marx compound as a result of dilution and storage could decrease efficacy. The purpose of this study was to determine the chemical and Unit for Laboratory Animal Resources, University of Pennsylvania, microbiological stability of diluted buprenorphine stored for up to 21 Philadelphia, PA days. Our hypothesis was that the concentration of buprenorphine would decrease over time and that the rate of degradation would be The euthanasia of animals in research is of utmost importance to our affected by container type and storage temperature. Buprenorphine field. Regrettably, euthanasia of neonatal mice can be challenging was diluted 1:10 with sterile saline using aseptic technique in a Class because of differences in physiology compared to adults. In 2013, the II, type A2 biologic safety cabinet. Diluted samples were stored in AVMA’s Guidelines for the Euthanasia of Animals included glass vials or plastic syringes protected from light at refrigerated or intraperitoneal (IP) injection of ethanol as “acceptable with room temperature. Samples were stored for several time points from conditions,” and a recently published study confirmed that this 0 to 21 days and then analyzed with high performance liquid method is adequate for euthanasia of adult mice; however, neonatal chromatography (HPLC) to determine relative concentration of mice have not been tested. This study examined whether IP ethanol buprenorphine. Samples were also submitted to the Oregon results in humane euthanasia in neonatal mice. Mouse pups Veterinary Diagnostic Laboratory for sterility testing. All samples (C57BL/6 and CD1 (n = 162)) were injected with 100% ethanol, a stored for 0 to 21 days were negative for bacterial and fungal growth. pentobarbital/phenytoin combination, or saline at 7, 14, 21, 28, or 35 HPLC results suggest that buprenorphine concentrations decreased days of age. Electrocardiogram, respiratory rate, and times to loss of under all storage conditions. In addition, there was another peak on righting reflex and death were recorded. No mice recovered after the chromatograms of all samples stored in plastic syringes which injection with pentobarbital or ethanol. The average time to death

692 Abstracts of scientific papers 2016 AALAS National Meeting

(TTD) was not significantly different between strains so results were outbreak of preputial gland abscesses (PGA) in C57BL/6 mice in an pooled. However, there was a significant difference in TTD between exclusion barrier room in a transgenic mouse facility, housing only ethanol and pentobarbital at 7, 14, and 21 days, as well as between commercially sourced mice or mice generated within the facility. The ethanol groups at 7, 14 and 21 days compared to 35 days. The PGA in the index case, a founder mouse, was lanced and treated with average TTD (time±SD) in minutes at 7, 14, 21, 28 and 35 days old antibiotics. Within the next 14 days, 22 additional mice developed using ethanol was 70.3±39.8, 51.7±30.5, 32.3±20.8, 13.1±14.1, and PGA in the same room. Males had been rotated through breeding 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 cages, which may have contributed to the additional cases. Offspring for pentobarbital, respectively. Although there was no statistically subsequently born to the infected sires also developed PGA at 6-8 significant difference in TTD between ethanol and pentobarbital at 28 weeks of age, despite having euthanized the clinically apparent sires. days of age, the TTD in 2 of 12 mice was greater than 30 minutes While identifying the source of this PGA problem, further cases of following ethanol administration. Therefore, 100% ethanol IP should PGA occurred in commercially sourced mice in a separate mouse not be used as a method of euthanasia in mice younger than 35 days, room within 1-2 weeks of arriving from the commercial vendor. The as the criteria for humane euthanasia (rapid and minimally distress- only observable sign of infection in all affected mice was nodular ful) were only met in mice 35 days or older. swelling at the base of the penis. Histologically, lesions consisted of extensive preputial gland suppurative inflammation with large PS60 Effects of Low-Level Brodifacoum Exposure on the Feline numbers of intralesional coccoid bacteria. Methicillin-susceptible S. Immune Response aureus (MSSA) was isolated in pure culture from samples of affected mice. Molecular characterization including pulsed-field gel JH Kopanke*1, K Horak2, E Musselman1, K Bennett1, S VandeWoude1, electrophoresis, Staphylococcus protein A typing and multilocus S Bevins2 sequence typing were further performed on the isolates involved in the outbreak. Of the 6 isolates genotyped, 3 were identified as 1Microbiology, Immunology, and Pathology, Colorado State Univer- ST1-t127-MSSA, 2 as ST88-t2649-MSSA and 1 as ST88-t7464-MSSA. sity, Fort Collins, CO; 2USDA APHIS, Fort Collins, CO ST88 has been previously reported to be associated with PGA. To avoid potential spread to other parts of the barrier, we salvaged Anticoagulant rodenticides have recently been implicated as a valuable strains through embryo rederivation and decontaminated potential inciting factor in the development of notoedric mange in the room and equipment. For the past 15 months, no further PGA bobcats and other wild felids that may regularly consume rodents. To cases have been observed. In cases of persistent PGA, we recommend date, however, these studies have failed to provide direct evidence of the causative agents be identified and typed as some bacterial strains a causative association between anticoagulant rodenticide exposure may be commonly involved. Control strategies may require and immune suppression in non-target species. Therefore, the depopulation of affected animals. present study sought to determine whether chronic, low-level exposure to brodifacoum resulted in alterations in the direct and PS62 Estimated Prevalence of Demodex spp. in a 1600-Cage Mouse recall immune response in felines. Age-matched, specific pathogen- Colony and in Imported Mice from Other Academic Institutions 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, RJ Ricart Arbona*1, MA Nashat2, NS Lipman1, FR Wolf1 14, 21, 28, 35) to model environmentally realistic exposure scenarios. Complete blood counts (CBC) and prothrombin time (PT) were moni- 1Center of Comparative Medicine and Pathology, Memorial Sloan tored biweekly as measures of immune function and coagulation. Kettering Cancer Center and Weill Cornell Medical College, New York Cats were vaccinated and boosted with 50 μg of two irrelevant City, NY; 2Tri-Insitutional Training Program in Laboratory Animal antigens (ovalbumin and keyhole limpet hemocyanin (KLH)) at Medicine and Science, New York, NY different points during the course of the experiment to assess the recall and direct immune responses, respectively. Measures of We attempted to estimate the prevalence of Demodex spp. in a colony immune response included delayed-type hypersensitivity (DTH) of mice (~ 1600 cages) of various strains used by 14 laboratories, tests and cell proliferation assays in the presence of ovalbumin and which were imported en masse, using a PCR assay offered by a KLH. Ovalbumin- and KLH-specific antibodies were detected and commercial diagnostic laboratory. Two rounds of testing were quantified via ELISA assays. Cytokine induction following exposure performed 3 weeks apart. For each round, cages and mice were to vaccine antigens was also analyzed. The mean blood brodifacoum randomly selected. Fur swabs from 1 mouse per cage from 10 cages level in exposed cats was 4.76 ± 0.577 ng/mL on Day 70, and the were pooled into a single sample. Twenty four samples (representing mean liver brodifacoum level was 1806 ± 106 ng/g on Day 77. 240 cages) were submitted. Thirteen of 24 samples were positive Remarkably, no coagulopathies developed during the course of the for Demodex spp.In the second round of testing, 5 additional samples study, and there was only one time point (Day 42) where the KLH of 27 (representing 270 cages) were found to be positive. All cages DTH response and KLH-specific antibody titers were lower in the tested in the second round were different than those tested in the first brodifacoum-treated group than the control group. This study round. This data suggests the prevalence of this ectoparasite in this indicates that cats maybe more resistant to clinical effects of group may be between 3.5% and 35%. Additionally, from February to brodifacoum exposure than other species and suggests that gross May 2016, the same PCR assay was used to test smaller imported impacts of environmentally realistic brodifacoum exposure on groups of mice (1- 10 cages) from different academic institutions humoral and cell mediated immunity against foreign antigen while in quarantine. Five of 38 groups (point prevalence = 13.2%) exposures in domestic cats are minimal. tested positive for Demodex spp. Skin testing (fur pluck and deep skin scrape evaluated by microscopy) was also performed on these groups PS61 Staphylococcus aureus Strains Isolated from an Outbreak of simultaneously. Only one group was confirmed positive by micros- Preputial Gland Abscesses in C57BL/6 Mice under Exclusion copy. All groups tested negative by PCR after a 4-week antiparasitic Barrier treatment protocol. These data may indicate that the Demodex spp. PCR is significantly more sensitive than fur pluck and deep skin UM Jonnalagadda*1, L Ward2, K Zhang2, W Pan2, C Pantigoso2, C scrape and the prevalence of Demodex spp. may be greater than Fielding2, C Geary-Joo2, D Morck2, M Ayroud2 suspected. Additional characterization and comparison of the PCR assay is warranted. 1Laboratory Animal Medical Services, University of Cincinnati, Cincinnati, OH; 2Cummings School of Medicine University of PS63 Pharmacokinetics and Pharmacodynamics Fail to Support the Calgary, Calgary, Canada Use of 3 Commonly Considered Delivery Methods for Postopera- tive Analgesia Using Meloxicam in Mice Colonization of laboratory mice maintained in barrier conditions by Staphylococcus aureus is relatively common. We describe an JA Herrod*1, AM Mexas1, C Doane2

693 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

1Veterinary Medicine, Midwestern University, Glendale, AZ; 2Univer- posture (>5 min/hr) with concomitant hypercortisolemia. The animal sity of Arizona, Phoenix, AZ was placed on Fluoxetine and provided increased environmental enrichment. To evaluate the effect of treatment, we performed serial Meloxicam, a commonly used NSAID, is routinely given to mice UCCR and behavioral measurements and observed a return of UCCR following minor surgical procedures. Researchers have explored the values within normal range and improvement of behavioral scores use of NSAID’s such as Meloxicam administered in sustained-re- after 6 weeks of treatment. This case highlights the importance of leased formulations, solid feed pellets, or gel as a stress-free way to evaluating chronically implanted animals for depression and the deliver analgesia postoperatively. These delivery methods allow for utility UCCR at baseline and following LDDS for screening behavioral less frequent handling following a surgical procedure and therefore and HPA abnormalities. should reduce pain and stress. Anecdotal evidence supports the use of these delivery methods, but objective data comparing the different PS65 Use of a Murine Sepsis Scoring System (MSS) for a Mouse formulations is lacking in the literature. In this study the pharmaco- Model of Hemolytic Uremic Syndrome kinetics and pharmacodynamics of Meloxicam-containing feed (dosed at 3.3mg/kg), Meloxicam-containing gel (dosed at 4mg/kg), PA Castro, LV Gigliello* and injectable sustained-release Meloxicam (dosed at 5mg/kg) were compared. An osmotic pump was placed in the subcutaneous region Research, VA Palo Alto, Palo Alto, CA between scapulae of each mouse to demonstrate a routine, minor surgical procedure. We measured Meloxicam drug plasma levels Seven C57BL/6 (WT) and 7 Carboxypeptidase N knockout (CPN -/- using liquid chromatography/mass spectrometry and evaluated “KO”) adult male mice were each given a single intraperitoneal common appearance parameters such as the mouse grimace scale injection of Shiga toxin 2 (6ug/kg) and E. coli LPS (1.5mg/kg) mixed (MGS), hair coat, nest building, activity, and subjective pain scoring in 500uL of saline to induce Shiga toxin associated kidney disease as to evaluate pre and postoperative pain. All mice displayed signs of a mouse model of Hemolytic Uremic Syndrome (HUS). These mice pain postoperatively; mice showed a decrease in activity and were monitored and scored using the Murine Sepsis Scoring (MSS) grooming and an increase in pain based on the MGS on postopera- system at 6 hours intervals for up to 132 hours. The MSS evaluated tive days 0, 1, and 2. Meloxicam blood plasma levels in all groups mice based on 7 clinical variables: appearance, level of consciousness, were below therapeutic plasma concentrations. Although the highest activity, response to stimuli, eyes, respiration rate, and respiration concentration of meloxicam in the blood plasma was noted in mice quality. Each of the 7 variables were scored from 0 (normal) to 4 that received subcutaneous Meloxicam SR, these values were below (significant clinical abnormalities) with a composite score range of 0 reported therapeutic levels, consistent with the subjective pain scores. to 28. Mice were additionally given subcutaneous injections of We concluded based on low Meloxicam blood plasma levels and sustained release buprenorphine (1mg/kg) every 72 hrs. The mean observations of pain postoperatively in all groups, that these three survival time for all experimental mice was 106.3 hours with a MSS approaches are not as effective in treating postoperative pain as mean composite score of 20.7 measured 6 hours prior to being found desired at the given doses. Higher doses of Meloxicam may need to dead. The mean survival time for WT mice was 116.6 hours with a be considered when formulating medicated pellets or gel in order to MSS mean composite score of 22.9 measured 6 hours prior to death achieve therapeutic concentrations. 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 PS64 Hypercortisolemia and Depression in a Cranially Implanted reached a MSS composite score of at least 18. Six hours prior to being Rhesus Macaque (Macaca mulatta) found dead, 13 of the study mice (92.8%) had a score of at least 12 when combining MSS variables appearance, level of consciousness, SC Adams*1, C Guyot1, K Berry1, S Wallack2, A Loar3, M Leblanc1 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 1ARD, Salk Institute, La Jolla, CA; 2Veterinary Imaging Center of San experimental mice had a score of at least 3 or more in appearance Diego, San Diego, CA; 3STAT Veterinary Lab, San Diego, CA 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 A 10-year-old cranially implanted rhesus macaque (Macaca mulatta) the next 6 to 24 hrs. The data from this study also highlights the with a history of weight loss and alopecia was presented for change in possible need for different endpoint criteria for the different mouse mentation described as dull in the cage. Initial physical examination strains used. Furthermore, this work demonstrates the ability to was normal while bloodwork revealed marked hypercortisolemia (23 measure clinical conditions based on a clinical observational scoring μg/dL). Other CBC and biochemistry values including fT4 and ACTH system (e.g. Murine Sepsis Score) that can be used with other disease were normal. Differential diagnosis for hypercortisolemia, weight models, such as HUS in this study, to more precisely determine loss, and alopecia included Cushing’s and Pseudo-Cushing’s suitable criteria for study endpoints. Syndrome. As an initial screening, we compared Urine Cortisol:Creatinine Ratio (UCCR) in the principal animal and naïve PS66 Use of PCR for Posttreatment Testing of Murine Fur Mites animals. UCCR was 10x higher in the presenting animal suggesting a possible Cushing. We then performed a low-dose dexamethasone DM Molk*, S Shainker suppression (LDDS) test by administering oral dexamethasone (DEX) to the presenting animal and 3 clinically normal nonworking animals Massachusetts General Hospital, Charlestown, MA 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, Pooled PCR testing is used to identify the extent of a fur mite 56, and 102 hours post first dose of DEX. All animals showed outbreak at our institution; fur pluck testing is the standard post- suppression and release of suppression at a similar time, which was Ivermectin treatment follow-up. We wanted to determine if PCR interpreted as a nonpathologic response. Abdominal ultrasound and testing could be used as an alternative to fur plucks for follow-up radiographs of the presenting animal revealed marked bilateral testing. However, dead mites can lead to false positive PCR findings. adrenal mineralization, which is a common finding in macaques, but Based on the work of other institutions that have shown that live no overt adrenal tumor. Overall, these results excluded Endogenous mites are readily passed to offspring and that young mice are likely Cushing’s Syndrome and prompted us to evaluate different causes of to have larger, detectable mite loads, we devised a new follow-up Pseudo-Cushing. Obvious differentials such as obesity or diabetes PCR-based testing plan focused on the testing of weanling mice to were immediately excluded based on previous results. Chronic stress minimize the likelihood of false positive findings. PCR was com- and depression were then evaluated by recording video and pared to fur pluck testing. We had 2 outbreaks of fur mites: the first quantifying behaviors in the principal animal and naïve nonworking consisted of 185 cages consisting of multiple transgenic lines on colony animals. A presumptive diagnosis of depression was made various backgrounds quarantined for Radfordia. Breeding continued, based on published criteria, which includes time spent in huddling and at the end of the treatment period, 8 cages contained mice of

694 Abstracts of scientific papers 2016 AALAS National Meeting

weanling age; PCR and fur plucks were performed on dams and women with absolute uterine factor infertility who hope to achieve weanlings, and all results were negative. Outcome from this sample full-term gestation. In Sweden, this quite anticipated option has now group suggested that PCR was at least as accurate as fur pluck resulted in several full-term pregnancies with successful births. As in testing. The second outbreak consisted of 256 cages positive any field of ongoing research, the beginning and continuity are never for Myobia. Seven cages contained mice of weanling age at the end of definitive and almost invariably the work needs to improve over the treatment period, and PCR and fur plucks were performed on the what has begun. Therefore, continued research seeks to make the weanlings and dams. One dam was PCR positive, while all offspring surgical approach safe and effective for donors (live donor) and were negative. This suggests that the breeder had been infected, recipients alike. Since 2011, we been a part of these investigations treatment was effective, and that live mites did not transfer to the where our Hamadryas baboons (Papio hamadryas) have played an offspring. Fur plucks were negative on this same dam. Sentinel invaluable role and have taken part in moving toward the first uterus testing resulted in positive PCR results but negative results on direct transplantation in the United States. For most of the phases of this animal testing. These results support the claim that PCR is more project, nulliparous females in reproductive age were used as sensitive for fur mite detection than fur plucks, and supports the recipient subjects whereas multiparous females in reproductive age possibility that targeting offspring of weanling age for testing via were used as donors. A critical component of the study was the use PCR after treatment avoids false positive findings. These scenarios of immunosuppressive drugs such as Tacrolimus, Mycophenolate foster consideration of how PCR can be used for follow-up testing in Mofetil, Anti-thymocyte globulin, Solu-Medrol, Ganciclovir, and a outbreak situations. few more, which presented a unique challenge to administer and balance when compare to other laboratory animals. Our investigators PS67 Novel Cageside Methods for Recognizing Unalleviated keep on working arduously to refine their surgical technique and Postoperative Pain in Mice reduce the operation time. An ambitious project nowadays is to retrieve the uterus from living donors through laparoscopy. This JL Lofgren*1, Y Kang1, C Luke2, D Myers2 involves dissecting and re-connecting the main blood vessels that irrigate this important organ once is transplanted. Based on 1Unit for Laboratory Animal Medicine, University of Michigan preliminary results, and the experience gained thus far, we can Medical School, Ann Arbor, MA; 2Conrad Jobst Vascular Research conclude that baboons may represent an ideal model for this type of Laboratory, University of Michigan, Ann Arbor, MI transplantation work.

Here we describe 2 novel cageside methods to quickly and objec- PS69 Comparison of 2 Saliva Collection Methods and Saliva-Serum tively identify unalleviated postoperative pain in mice. With normal Cortisol Correlation in Rhesus Macaques, Cynomolgus Macaques, grooming, a nontoxic material that glows under black light is and African Green Monkeys transferred from the top of the head to the paws, face, body, nest, and cage environment in a reproducible pattern. Additionally, mice K Rapp-Santos*1, CC Hofer1, L Altamura2, L Lugo-Roman1 readily consolidate nesting material, which can be quantified in any location of the home cage. Adult C57BL/6 male mice were evaluated 1Veterinary Medicine Division, The United States Army Medical by a blinded-observer postisoflurane anesthesia +/- analgesia and Research Institute for Infectious Diseases, Fort Detrick, MD; 2CTR postlaparotomy with inferior vena cava (IVC) ligation +/- analgesia. Diagnostic Systems Division, The United States Army Medical Buprenorphine (0.05mg/kg) (n = 9) or saline (n = 9) were provided Research Institute of Infectious Diseases, Fort Detrick, MD 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 Serum cortisol measurement has been widely used as a stress head; a black light was then used to identify transfer to body and indicator due to activation of the hypothalamic-pituitary-adrenal axis cage areas according to a novel scoring system ranging from 1 in nonhuman primates. Salivary cortisol measurement may be (original application site only) to 10 (completely groomed away). To superior because it allows for repeated sampling in a relatively short assess nesting consolidation behavior, one quarter of a cotton nesting period, represents relative acute stress levels, and is less subject to square was placed in each corner of the home cage. Consolidation degradation or contamination than other biosamples. The purpose of was scored from 0 (no pieces) to 4 (all pieces) consolidated within a this study was to evaluate a human clinical saliva collection device 2.5 x 3.5 inch area of one another. Nest consolidation was significant- for measurement of salivary cortisol in 3 nonhuman primate species. ly depressed after surgery compared to anesthesia for the first 24 Serum and saliva samples were collected from 20 nonhuman hours post surgery. Grooming behavior was significantly depressed primates of each species. Saliva was collected using the human after surgery compared to anesthesia throughout the 48 hour post clinical collection device and compared to passive drool. Both serum operative period. Taken together, laparotomy with IVC ligation and saliva samples were analyzed using a commercial ELISA kit to resulted in postoperative pain for at least 48 hours. At 1-hour determine cortisol levels. Mean serum cortisol was 370.2 ± 23.8 ng/ postsurgery, buprenorphine treated mice had significantly fewer mL in African green monkeys, 374.4 ± 17.9 ng/ml in cynomolgus differences in nest consolidation behavior and grooming behavior macaques, and 492.5 ± 25.3 ng/mL in rhesus macaques. Mean saliva between the postanesthesia and postsurgery conditions compared to cortisol was 33.4 ±4.3 ng/mL in African green monkeys, 24.37 ± 4.2 saline treated mice, indicating buprenorphine likely provided some ng/mL in cynomolgus macaques, and 34.3 ± 3.3 ng/mL in rhesus analgesia. However, by 8 hours postsurgery buprenorphine-treated macaques. The mean saliva cortisol was greater in both African green mice did not significantly differ from saline controls, indicating a monkeys (P = 0.03) and cynomolgus macaques (P < 0.001) when higher dose or additional analgesia was needed to effectively collected by passive drool compared to the clinical collection device. alleviate postoperative pain. This pilot study provided proof of In the rhesus macaque group, the mean saliva cortisol was indepen- concept that the Nest Consolidation Test and Grooming Transfer Test dent of collection technique. In both the African green and the rhesus are effective, objective, titratable cageside methods for assessing macaque group, the clinical saliva collection technique yielded unalleviated postlaparotomy pain in mice. greater volume than the passive drool technique (P < 0.001, P = 0.02). Serum and saliva cortisol demonstrated adequate correlation in all PS68 Baboon (Papio hamadryas) as Animal Model For Uterus species. This study represents the first evaluation of this human Transplantation clinical collection device for salivary cortisol determination in nonhuman primates. It also provides the first known published K Rivas-Wagner* ranges of salivary free cortisol in cynomolgus macaques and African green monkeys, as well as serum total cortisol in African green The Mannheimer Foundation Inc, Homestead, FL monkeys. Saliva cortisol demonstrated adequate correlation with serum cortisol, and the clinical saliva collection device was validated There are several key factors and recent developments that shed new for use in 3 nonhuman primate species. interest on uterus transplantation (UTx) as a medical treatment for

695 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

PS70 Albumin Is an Overlooked but Highly Relevant Allergen tank system and developing further enrichment ideas to more closely Potentially Affecting Laboratory Animal Professionals mimic this species’ wild habitat. We predict these measures will improve frog health and breeding success by lowering stress, O Kalliokoski*, TM Kousted, J Koch, J Hau increasing appetite, and improving water quality.

Experimental Medicine, University of Copenhagen, Copenhagen, PS72 Litter Box Training of Rabbits Housed in Pens Denmark C Merrill*, JA Ordile Although long recognized as a prevalent allergen, mouse serum albumin has taken the back seat to the urinary protein mus m1 in Comparative Medicine, Pfizer, Pearl River, NY investigations of mouse-derived allergen levels in laboratory animal facilities. Two implicit assumptions have been made: that albumin The Guide has recommended social housing of social animals as the functions mainly as a contact allergen, which is less of a workplace default housing system. AAALAC also supports this initiative. With health concern, and that quantification of the more volatile mus m1 these standards in place, the industry has begun to social house gives a more accurate indication of the presence of airborne rabbits. Group housing rabbits in a laboratory facility has shown to (inhalable) allergens in a laboratory animal facility. To assess the be an excellent source of enrichment and a way to improve animal prevalence of airborne allergens in an under-ventilated animal unit, welfare. Here, rabbits are socially housed in pairs or triples in dog we collected air samples using an IOM sampler and quantified mus runs that have been converted into rabbit pens. This group-housing m1 and albumin collected on the filters using ELISAs (a commercial program resulted in increased social contact and improved animal and an inhouse assay, respectively). We found, somewhat surpris- welfare. The daily husbandry and maintenance of these pens, ingly, that albumin was present in quantities comparable to mus m1, however, proved to be laborious and time consuming. Therefore, it but that the levels of the 2 allergens in the same sample were not was decided to attempt to litter box train rabbits with the expectation correlated. The findings likely reflect the allergens’ different means of of reducing manual labor, reducing chemical usage, increasing time spread in a laboratory animal facility. Mus m1 is a smaller protein, efficiency, and improving animal welfare. Two litter boxes containing thus an easily dispersed carrier of pheromones, whereas albumin is irradiated corncob bedding were introduced in the corners of the likely to spread adsorbed onto larger particulates like dust and pens where rabbits were housed and a sample of their excrement was dander. Consistent with this theory, the room-level ventilation was added to each box. Over time, the less-used box was removed from able to reduce the levels of airborne mus m1 following cage the pen. Boxes were emptied a minimum of every other day, and a changing, whereas the levels of airborne albumin were stable over sample of soiled bedding was taken from the dirty box and placed time in the animal rooms. Even though improved cage-level into the clean box. This helped the rabbits identify the area they ventilation and aerated cage-changing stations are becoming the marked previously for consistent use of the box. Used boxes were norm in laboratory animal facilities, higher stocking densities and old replaced with clean boxes a minimum of twice a week to prevent buildings with inefficacious room-level ventilation may outbalance urine scale build up. Litter box training of pen-housed rabbits was the benefits of the last few decades’ technical innovations. Continu- successful despite encountering several challenges that allowed for ously monitoring and addressing sources of airborne allergens in the opportunity for brainstorming and innovation. Overall, the animal facilities is important, in particular to reduce the sensitization success of this program has led to increased husbandry efficiency, risk for those who visit the facilities infrequently. In doing so, we sug- overall cleaner pens, and improved animal welfare. gest that controlling exposure to the albumin family of proteins is as important as the much-focused-on lipocalin family (e.g., mus m1 and PS73 Evaluation of 4 Commercially Available Applications as its rat counterpart rat n1). Assessing only lipocalin levels when Environmental Enrichment for Domestic Cats (Felis catus) in a determining the airborne allergen status of a laboratory animal Laboratory Animal Setting environment paints, in our experience, an incomplete picture. RG Hairston* PS71 Husbandry on a Budgett: Caring for Carnivorous Frogs (Lepidobratrachus laevis) and the Challenges of a Novel Model Unit for Laboratory Animal Resources, The Ohio State University , Organism Columbus, OH

I Bukovnik*, A Huffine Institutions seek to find creative and innovative ways to encourage species-specific behavior via their enrichment program in order to Laboratory Animal Resources, North Carolina State University promote higher-quality welfare for the animals in their care. The College of Veterinary Medicine, Raleigh, NC institution must meet this goal while also dealing with the reality of budget constraints, necessitating creative and cost-effective methods Lepidobatrachus laevis, commonly known as the Budgett’s Frog, is a to create enrichment whenever possible. Most programs have tablet carnivorous aquatic anuran from the Chaco region of South America. and/or smart phones available through the institution. These This animal has historically been kept as an exotic pet, but has also technologies offer a myriad of quick and useful tools that can be used recently begun to gain popularity as a developmental research model for a multitude of species. This purpose of this study was to due to its unusually large embryo size (over twice the size of X. determine if smart technology applications (apps) can be used as an laevis) and rapid speed of development. Current research efforts span enrichment device for domestic cats (Felis catus) housed in a the fields of development, evolution, morphogenesis, and tissue laboratory setting. Using a smart tablet, 4 commercially available cat regeneration. Our research facility maintains over 50 L. laevis of applications were chosen to be evaluated. All consisted of different various ages and origins, and we are currently working to establish animations that were developed to entice cats into interacting with an F3 generation as a known research strain. Because ours is the only the screen through visual engagement and/or touch. The applica- known L. laevis research colony in the United States, husbandry tions evaluated consisted of 1 of the following: a laser light simulator, protocols have been developed from the ground up and demand coy fish, insects, or mice in order to elicit the cats natural prey drive. constant innovation. L. laevis husbandry differs from normal aquatic A colony of 5 gang-housed cats were provided access to each app amphibian husbandry in several key ways, most noticeably in twice a day for 10-minute periods for 4 days. During each 10-minute feeding habits and tank design requirements. As a pioneer in L. time frame, ethogram data for each cat was collected at 1-minute laevis husbandry, we have made many improvements to our colony, scans by the same individual observing the animals through a including designing an individualized feeding schedule, additional window. It was observed that the cats were most engaged in the enrichment in the form of aquatic plants, and implementing a regular applications that featured single animations that moved in quick, weighing schedule to monitor health and development. In the future, random patterns. These are features present in both the laser and the we plan to make further improvements by building an integrated mouse animation applications. Variation in individual interest was

696 Abstracts of scientific papers 2016 AALAS National Meeting

also observed, with some cats consistently engaged while others panels would disrupt this pattern. Sleep parameters of 5 naïve adult demonstrated little to no interest in any of the applications. Most cats rhesus monkeys (1 single-housed socialized monkey and 2 pairs of showed decreased interest in the apps within the first 2 days. While pair-housed monkeys) were evaluated for 15 consecutive days using not a substitution for human interaction, smart tablet applications are Actiwatch monitors. Sleep measures of pair-housed monkeys were a relatively simple tool that can be easily used as a component of an significantly inferior compared to the single-housed subject, with the enrichment program. single-housed monkey showing higher sleep efficiency and lower sleep latency and fragmentation. Correlational analysis indicated a PS74 Implementation of a Supplemental Feeding Program positive correlation between sleep measures of 1 paired monkey and Improves Survival in Neonatal Common Marmosets (Callithrix its home-cage partner, suggesting that pair-housing influences sleep jacchus) quality. Paired animals were then separated through socialization panels and sleep-like measures were evaluated for 15 days. After MA Burns*1, M Siddalls1, J Hyman2, S Sancricca1, RP Marini1, L separation, sleep patterns of previously paired monkeys did not Wachtman1, JG Fox1 differ from that of the single-housed monkey. Further correlational analysis indicated a lack of correlation for all sleep measures between 1Division of Comparative Medicine, Massachusetts Institute of previously paired monkeys, suggesting that when separated the Technology, Somerville, MA; 2Brain and Cognitive Sciences, home-cage partner’s sleep does not influence the other monkeys’ Massachusetts Institute of Technology, Cambridge, MA sleep. In conclusion, our results suggest that social housing has a strong impact on home-cage partner’s sleep and that this pattern can The common marmoset (Callithrix jacchus) is a nonhuman primate be disrupted by separation through socialization panels. Studies species used in research as an attractive alternative to larger NHP evaluating sleep in pair-housed monkeys should take into consider- species due to their small size, high fecundity, and relative lack of ation the effects that the partner’s sleep may have on the subjects’ zoonoses. Marmoset mothers typically give birth to twins in the wild, sleep parameters. but often produce triplets in captivity. A number of studies have evaluated factors that are predictive for infant survival, including birth PS76 Artificially Intelligent Weight Management System for weight, maternal weight, and number of infants per birth, among Nonhuman Primate Research others. Triplet births have been associated with higher infant mortality. There is considerable variation in how institutions address the problem GD Lawrence*1, TR Hobbs2 of failing infants, ranging from providing no support to daily supplemental feeding. While studies describing supplemental feeding 1Director’s Office, Oregon National Primate Research Center, guidelines have been published, there remains a need to validate and Portland, OR; 2Surgery Unit, Oregon National Primate Research document increased survival after instituting supplemental feeding Center, Beaverton, OR programs. Initially, no infant support was provided to neonates born in our colony. During 2015, 44 infants were born, with 28 born to triplet Excessive variation in weight can in uncertain ways adversely effect (n = 24) or quad (n = 4) litters. Only 24 survived the neonatal period the suitability of an animal for its assigned research purpose, in (50% infant mortality). In an effort to improve infant survival, a addition to impacting on the animal’s overall health and wellbeing. supplemental feeding plan was instituted. Feeding began 24 hours We are challenged with managing the weight of nearly 600 nonhu- after birth, starting with frequent feedings of a dilute dextrose/infant man primates (NHPs). To do so we have developed a software formula mixture that gradually changed to infrequent feedings of application that allows us to review each case thoroughly yet quickly, infant formula mixed with rice cereal by 28 days post birth. Supple- modifying diet regimens and feeding methods when necessary. mental feeding was rotated among all infants in the litter. Each infant Weight is automatically tracked with respect to targets, with alerts was fed no more than once every 3 days unless weight loss was noted. being issued when the difference between target and actual weight Early on in supplementation, infants were kept in an incubator for exceeds a predefined threshold, or when body condition score approximately 8 hours per day, and this was decreased to <1hr by the deviates from ideals. The system puts the key components of the end of the 28-day feeding period. A total of 32 infants have been born animal record in easy reach of the clinician, and a structured in our colony this year, and 28 infants have undergone the supplemen- case-entry interface ensures that the weight management (WM) tal feeding and incubator support program. Of these 28 infants, 23 record is machine readable. This is essential to a rigorous determina- have survived the neonatal period (18% infant mortality). Institution of tion of efficacy for individual NHP weight-management regimens, as a supplemental infant feeding program significantly reduced infant well as evaluating performance of the WM program as a whole. In mortality (P < 0.01; Fisher exact test). This daily feeding program also particular, we wanted to know whether particular dietary interven- allowed for establishment of neonatal growth and diet consumption tions, changes in feed, ration, and/or feeding methods, were actually volume curves, which aid in assessment of neonates. effective in producing the desired weight change (gain, loss, or stability). Having established this relationship, we would be better PS75 Effects of Pair Housing on Sleep Parameters Evaluated with placed to automate additional aspects of the WM program, including Actigraphy in a Rhesus Monkey (Macaca mulatta) diet adjustment, allowing the system to identify cases where a modified regimen might be called for, and what sort of ration change LF Berro*1,2, ML Andersen1,2, LL Howell2 would be appropriate. This would further optimize time allocation, presenting clinicians with recommendations for review and 1Psychobiology, Universidade Federal de São Paulo, São Paulo, approval, or to be adjusted as appropriate. We proceeded by Brazil; 2Yerkes National Primate Research Center, Emory University, introducing new functionality in the WM software application that Atlanta, GA allowed us to graphically visualize the sequence of WM interventions (feed, ration, feeding methods) superposed on animal weight According to the Animal Welfare Act, institutions that house plots. We were able to assess time required for an intervention to nonhuman primates must develop a plan to provide for the animals’ have effect, and identify instances in which interventions were not psychologic wellbeing and address their social needs. Rhesus timed well. By embedding clinical expertise in our tools, making monkeys (Macaca mulatta) are known to live in social groups in them more “intelligent,” we found that we are able to both refine that nature and behavioral management strategies have been focusing on expertise and deliver a better weight management outcome. promoting pair-housing in order to decrease distress. Although social housing shows countless benefits, pair-housing may have effects on PS77 Challenges and Successes of Implementing Automated research outcomes. In humans, it is well known that co-sleeping can Feeders in an Outdoor Nonhuman Primate Facility have a major impact on bed partner’s sleep. In the present study we investigated if pair-housing would influence home-cage partner’s B Hughes*, MT Sharpless, J Moran, T Meeker, R Stavisky, J Cohen, KF sleep in rhesus monkeys and if separation through socialization Ethun

697 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Division of Animal Resources, Yerkes National Primate Research 1Medicine/Nephrology, Yale University, New Haven, CT; 2Compara- Center, Emory University, Lawrenceville, GA tive Medicine, Yale University, New Haven, CT

Over the past 3 years, 32 commercially available automated feeding IACUCs, attending veterinarians, animal resource centers (ARCs), stations have successfully been deployed in 8 outdoor compounds, and investigators are facing financial, operational, and regulatory housing approximately 42% of the rhesus macaque breeding colony. challenges that can impact the sustainability of animal-based As animals obtain food pellets, the computer-controlled system research. Such challenges likely require reengineering and/or records grams obtained in real-time by detecting unique radio reprioritization of current practices to provide the bandwidth and frequency identification (RFID) microchips implanted subcutane- resources to resolve the challenges and implement sustainable ously in each hand of individual monkeys. Because this novel system solutions. Analysis of discrete and longitudinal data from archived is a significant departure from standard husbandry practices, the and recent Yale Animal Resource Cost and Benchmarking survey animal care staff has been faced with various challenges related to data (reflecting ARCs in US academic Institutions) quantifies, network and infrastructure installation, equipment maintenance, qualifies, and/or contextualizes contemporary issues facing animal feeder sanitation, diet storage and dispensement, database manage- programs. The analysis profiles the highest-ranked challenges in the ment, record keeping, and animal care technician staffing and context of trends in the funding environment, consumer price index training. We took steps to address each of these challenges including (CPI), and federal regulatory requirements. The highest-ranked the establishment of internal written procedures, validation of an financial challenges had the theme of reducing operating costs and automated feeder sanitation protocol, formulation of new pelleted managing the budget, consistent with expectations as a result of the diets, employment of an operating system manager, development of plateauing of the NIH budget, steady increase in the CPI, and finding effective staffing strategies, and establishment of an effective animal that 40-70% of participating institutions report the inability to fully care technician training program. To ensure the successful implemen- recover mouse care costs, the latter possibly due to limits on per diem tation of these automated feeding stations on multiple outdoor rate affordability. The highest-ranked operational challenges were compounds, a team of animal care, colony management, and managing human resources, increased compliance requirements, and veterinary medicine personnel met routinely to review and evaluate inefficient facility size and layout. The highest-ranked technical new automated feeder operating procedures. Information from this challenges were tracking census and service charges. Despite these report will be of interest to the management staff of captive nonhu- challenges, participants felt that the noncompliance-related program man primate colonies considering the utilization of commercially changes increased researcher productivity, but also increased available automated feeding stations in either their indoor or outdoor operational costs, possibly contributing to the cost under-recovery of facilities. Successful implementation of automated feeding stations ARCs. Overall, compliance-related program changes more negatively has led to enhanced husbandry practices of socially housed rhesus effected researcher productivity. The most effective ARC program macaques. improvements focused on animal care and welfare, outcomes that require financial support but can be incorporated into the per diem PS78 Physiologic and Pharmacokinetic Effects of Multilevel rates; ~60% of participants reported per diem rate increases associ- Caging on Sprague–Dawley Rats under Ketamine-Xylazine ated with these improvements. In contrast, the most effective IACUC Anesthesia improvements focused on making the lives of the Investigators easier. While these improvements also require financial support, they A Dodelet-Devillers*1,2, C Zullian1, F Beaudry1, J Gourdon2, J are often not included in the per diem rate. Chevrette2, P Helie1, PJ Vachon1 PS79 Eradication Of Demodex musculi Using a Topical Parasiticide 1Medecine Veterinaire, Universite de Montreal, Montreal, Cana- in an Infested Transgenic Mouse Strain with Defective Adaptive da; 2Comparative Medecine and Animal Ressources Centre, McGill Immunity University, Montreal, Canada MA Nashat*1,2, NS Lipman3, FR Wolf3, RJ Ricart Arbona3, SF While cage refinement is a necessary step towards improving the Santagostino3 welfare of research rats, increasing the complexity and surface area of the living space of an animal may have physiologic impacts that need 1Comparative Bioscience Center, The Rockefeller University, New to be taken into consideration. In this study, ketamine (80 mg/kg) York, NY; 2 Center for Comparative Medicine and Science, Tri- and xylazine (10 mg/kg) caused a short duration anesthesia that was Institutional Training Program of Laboratory Animal Medicine and significantly decreased in Sprague–Dawley rats housed in multilevel Science, New York, NY; 3Center for Comparative Medicine and cages (MLC; n = 6), compared to rats housed in standard cages (SDC; Pathology, Memorial Sloan Kettering Cancer Center and Weill n = 6). The withdrawal reflex, the palpebral reflexes, and the Cornell Medical Center, New York, NY 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 A unique transgenic mouse strain (B6.Cg-Rag1tm1Mom Tyrp1B- addition, during anesthesia, cardiac frequencies were increased in w Tg(Tcra,Tcrb)9Rest/M, TRP-1/TCR mice) with defective adaptive animals housed in the smaller SDC (P < 0.05). Respiratory frequen- immunity presented with ulcerative dermatitis and suppurative cies, the blood oxygen saturation, and rectal temperatures during ocular and skin infections. Corynebacterium bovis and Pasteurella anesthesia did not vary between conditions during the anesthesia. pneumotropica were isolated from the lesions and Demodex muscu- While xylazine pharmacokinetics were unchanged with caging li mites were observed in skin sections from affected regions. As conditions, the clearance and half-lives of ketamine and its metabo- rederivation would be time consuming and costly, we assessed lite, norketamine, were altered in the rats housed in MLC. Finally, whether a parasiticide could be used to eradicate D. musculi from the while no difference was ultimately seen in rat body weights, isolated colony. Thirty mice were confirmed mite-positive by fur plucks and liver and adrenal gland weights were significantly lighter in rats deep skin scrapes. Infested mice were randomly assigned to 3 housed in the MLC (P < 0.05). Increasing cage sizes, while having a groups: untreated control, ivermectin (12 ppm) diet ad libitum for 8 positive impact on wellbeing in rats, can alter anesthetic drug weeks, or moxidectin 3.3 mg/kg and imidacloprid 13 mg/kg weekly metabolism and thus modify anesthesia parameters and associated for 8 weeks. Following treatment, half of the mice from each group physiologic processes. were euthanized and necropsied, or they were euthanized 3 months after cessation of treatment. Fur plucks, deep skin scrapes, and PS129 Using Trends to Understand Animal Resource Center histologic skin sections revealed large numbers of mites in untreated Challenges controls while ivermectin-treated mice had a reduced mite population and moxidectin/imidacloprid-treated animals were mite P Preisig*1, R McDonald2, JD Macy2 free. Subsequently, a shorter 4-week treatment trial revealed that 3/3 (100%) control and 1/7 (14.3%) moxidectin/imidacloprid-treated

698 Abstracts of scientific papers 2016 AALAS National Meeting

mice remained infested. These results indicate that an 8-week BLAST analysis indicates that the target sequence is ~90% treatment regimen of moxidectin/imidacloprid is necessary to homologous to Aspiculuris spp. To further investigate detection eradicate D. musculi mites in an infested immune-compromised methods, a diagnostic comparison of the colony was performed mouse strain and that ivermectin at 12 ppm in feed is not sufficient. using PCR targeting the 28s rRNA gene, fecal float, and tape test Additional studies evaluating other drug combinations, routes of methods. Ten male and 10 female New Zealand White rabbits were administration, and newer parasiticides are warranted. 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 PS80 Exhaust Air Particle PCR: A Highly Reliable Alternative the colony facility and perianal hair swabs were collected for qPCR Microbiological Monitoring Method for the Detection of Excluded screening and submitted to a diagnostic laboratory. Pinworm Pathogens in IVC-Housed Rodent Colonies detection rates were 68% via PCR, 38% for tape test, and 8% for fecal float. Although overall PCR was more sensitive, there were some M Brielmeier* instances where eggs were detected by tape test and not by PCR. Infestation rates, and the estimated copy number detected by qPCR, Research Unit Comparative Medicine, Helmholtz Zentrum München, are both higher in younger animals and decrease with age. There Neuherberg, Germany does not appear to be a notable gender bias in infestation rate. Environmental swab samples were also collected from the ceiling Recently, alternative health monitoring strategies for IVC-housed exhaust vents and floor of a newly populated room and were tested rodent colonies have been proposed to improve the detection of using the qPCR screening assay. P. ambiguus was detected in the excluded pathogens. One novel method uses exhaust air particles environment swabs after 6 weeks of exposure. As most rabbit polymerase chain reaction (EAP-PCR) to detect pathogen nucleic colonies are housed in open cages, environmental sampling provides acids associated with particles in the exhaust air of IVC cage rack a simple way to monitor for P. ambiguus by PCR testing. systems. Although the idea of environmental samples for microbiological monitoring is not new, only recently, suppliers of IVC systems PS82 Development and Qualification of a Probe-Based Fluorogenic have presented alterations to their equipment that provide user- Rt-qpcr for the Detection of Zika Virus In Nonhuman Primates friendly technical solutions. Yet, there is only limited peer-reviewed information on the reliability and sensitivity of EAP-PCR. Intensive C Wang*, P Momtsios, KS Henderson research was conducted to provide evidence that the method of Charles River Laboratories, Wilmington, MA EAP-PCR is superior to soiled bedding sentinel serology, a method flawed by serious drawbacks. EAP real-time PCR was systematically Zika virus (ZIKV) is a single-stranded RNA virus that is an emerging tested for Mouse Norovirus (MNV),Pasteurella pneumotropica, agent related to other Flaviviruses such as West Nile virus, yellow and Helicobacter hepaticus with multiple repetitions and at different fever virus, and dengue virus. ZIKV was initially isolated from a pathogen prevalence. Sentinels were exposed to soiled bedding rhesus macaque (Macaca mulatta) in 1947 in Uganda. Like other during multiple 3-month monitoring periods and gauze pieces Flaviviruses, it can infect multiple host species and can be transmit- pinned on the prefilter of an air handling unit of an IVC system were ted by mosquitoes (Aedes spp.) which is the major vector for exposed to particles in the IVC racks exhaust air. Under field transmission to humans. In humans, infection of ZIKV often have conditions, in animal rooms with medium to high prevalence, EAP influenza-like signs and symptoms and could be misdiagnosed PCR detected MNV in 57/59, SBS serology in only 28/59 3-month during the acute phase. Recently, outbreaks of ZIKV infection have periods, respectively. EAP-PCR detected Pasteurella pneumotropica in occurred in Brazil and distributed to USA and several other areas in 26/26 quarterly monitoring periods whereas SBS serology failed South America. There is no valid vaccine available to protect hosts completely (0/26). At low prevalence, EAP-PCR detected MNV- from ZIKV infection. Because nonhuman primates (NHPs) in and Pasteurella pneumotropica-infected animals in 6/6 monitoring southern United States are sometimes housed in facilities with access periods, while SBS detected MNV in only 1/6 periods and failed to to outdoor air, ZIKV-infected mosquitos could pose a risk for detect Pasteurella pneumotropica in all instances (0/6). EAP PCR infection of NHPs and to caretakers. To date, rapid laboratory proved highly sensitive and the minimum prevalence of MNV, Pas- detection of the presence of ZIKV in nonhuman primates and other teurella pneumotropica, and Helicobacter hepaticus for a positive PCR research animals, particularly for those housed outside in research result was repeatedly 1 cage with 5 infected mice in a 63-cage IVC facilities, has not been reported. Based on ~90 ZIKV sequences in the system. In addition, EAP-PCR was able to detect infections within 1 NCBI database, we developed a fluorogenic real-time RT-qPCR assay to 2 weeks after onset. The use of EAP-PCR is highly recommended targeting a conserved region of the nonstructure protein gene. The as an additional method to exclude unwanted organisms. 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 PS81 Detection of Passalurus ambiguus in a Laboratory Rabbit multiple host species and other pathogens. Inactivated ZIKV was Colony acquired and tested to confirm that the process from RNA isolation to PCR testing was functional. We obtained 45 NHP blood samples CL Perkins*1, KS Henderson1, LA Burlingame2, P Lester2 from 3 different institutions in the southern USA. These NHPs were housed outdoor and may be exposed to ZIKV infected mosquitos. 1Charles River, Wilmington, MA; 2University of Michigan, Ann When compared with the inactivated ZIKV template, the results from Arbor, MI all 45 samples were determined to be negative. ZIKV serology assay may not distinguish between different flavivirus NHP seroconver- Passalurus ambiguus is an oxyurid pinworm of rabbits (Oryctolagus cu- sion antibodies, such as antibodies from Dengue virus (DENV) and niculus) which has typically been reported in domestic and wild West Nile virus (WNV). Therefore, individual flavivirus infections rabbits, but has historically thought to have been uncommon in can only be confirmed by agent specific PCR assays. Samples from laboratory rabbits. In this study, we investigated a colony of ZIKV infected animal or infected colony will be further validated by laboratory rabbits from an academic institution as a result of repeated this RT-qPCR assay. In summary, we developed a ZIKV RT-qPCR low-positive findings in fecal samples by qPCR pinworm screening assay that may be used to routinely monitor for active infection of over a 1-year period. An investigative transition to using environ- ZIKV in NHP research colonies, and may also be considered as a tool mental swab samples and perianal hair swabs for PCR testing in ZIKV research studies. provided substantially higher and reproducible detection of a pinworm infestation. To confirm the presence of P. ambiguus, a 750bp PS83 Detection and DNA Sequencing of Pinworms and Novel Fur segment of the 28S rRNA gene was amplified and sequenced. Mites in a Laboratory Vole Colony (Microtus ochrogaster) Obtained sequence was consistent with P. ambiguus sequence previously obtained from individually isolated rabbit pinworms. CL Perkins*1, WR Shek1, SM Sullivan2, SE Perkins3 There is no P. ambiguus sequence currently available in GenBank, but

699 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

1Charles River, Wilmington, MA; 2Northeastern University, Boston, 0.0551). Post-surgical maternal cortisol and post-surgical fetal MA; 3Tufts University, Boston, MA hematocrit were significantly correlated (P = 0.0424, R=0.6836). Interestingly, fetal surgical pH was not correlated with post-surgical

Voles (Microtus spp.) are occasionally used in laboratory research for hematocrit or PaO2. These findings suggest that fetal surgical neurobiology, genetics, and social behavior studies. In this study, we hematocrit, which reflects the physiologic status over days or weeks compared methods of detection for pinworms and fur mites infesting (versus pH, which changes rapidly), and maternal postsurgical an academic colony of prairie voles (Microtus ochrogaster). In addition, cortisol values, reflective of maternal stress, may be useful to predict we speciated the parasites by DNA sequencing. Comparative testing postsurgical fetal health. was performed on 8 male and 12 female voles of various ages. Using a dissecting microscope, fur was examined for fur mites and PS85 Cardiovascular Phenotyping in Mice through the Measure- macerated GI tract for adult pinworms. Also, qPCR assays targeting ment of Pulsed Doppler Ultrasound Blood Flow Velocity conserved regions of the 18S rRNA gene in the fur mite genera Myo- bia and Radfordia, and the 28S rRNA gene of the pinworm AK Reddy*1,2, S Madala1,2 genera Aspicularis and Syphacia were performed on body swab and fecal pellet pools. Direct examinations detected fur mites in 100% and 1Indus Instruments, Webster, TX; 2Medicine, Baylor College of pinworms in 55% of the animals. PCR detected fur mites in 80%, and Medicine, Houston, TX pinworms in 50% of the animals. The lower rate of detection of fur mites by PCR may be due to sampling limitations. The estimated Evaluation of cardiovascular (CV) function in genetically altered pinworm copy number detected was higher in younger versus older mouse models that mimic human CV diseases requires reliable animals whereas the fur mite DNA copy number remained measurements with minimal effects from experimental conditions. consistent. There were no apparent differences observed based on Invasive measurement of blood pressure or flow are reliable but can gender. DNA sequencing was performed on a 572bp segment of the be traumatic and/or terminal. Therefore, a noninvasive method to fur mite 18S rRNA gene and an 719bp segment of the pinworm 28S evaluate CV function in mice and one that meets “reduction” and rRNA gene amplified from PCR positive samples. Using Vector NTI, “refine” aspects of the 3Rs of animal welfare is desirable. Pulsed the consensus sequence for the vole fur mite 18S rRNA gene was Doppler ultrasound is such a method which allows for single or compared to sequences generated in our laboratory for Myobia mus- serial noninvasive measurements in living animals. Noninvasive culi, Radfordia affinis, Radfordia ensifera, and Myocoptes musculinus; it blood velocity measurements can be made from the heart and the was found to be 99% homologous to both M. musculi and R. peripheral vessels in a less cumbersome and relatively shorter time affinis, but with base pair differences suggesting a novel fur mite using pulsed Doppler ultrasound versus echocardiography. Blood species. It is unclear whether this mite species may infect mice, rats flow velocity measurements, along with electrocardiogram, are made or other rodent species. Using BLAST analysis, the consensus in isoflurane anesthetized mice. Measurement times for a given site pinworm 28S rRNA sequences was compared to pinworm sequences can range from 5-15 min. The following measurements can be made: in GenBank; it was found to 99% homologous with Syphacia montana. (1) left ventricular filling and ejection velocities to determine diastolic It is unknown whether S. montana may cross-contaminate other and systolic function; (2) coronary velocity to determine coronary rodent species, although literature categorizes this species as specific flow reserve; (3) pulse transit time in an arterial segment to deter- to the subfamily Microtinae. mine pulse-wave velocity (arterial stiffness index); (4) flow velocity waveforms from peripheral vessels to determine indices of arterial PS84 Using One-Time Fetal Surgery Measurements to Predict Fetal compliance, resistance, and wave reflections; and (5) stenotic Health and Growth velocities for estimation of pressure gradient; and 6. tail artery velocity for estimating blood pressure using a tail-cuff. This JS Streeter*1,2, S Louey2, S Jonker2 convenient and easy method can be used by investigators to evaluate cardiovascular function in various genetically, surgical, and/or 1Comparative Medicine, Oregon Health and Science University, pharmacologically altered mouse or other small animal models. Portland, OR; 2Knight Cardiovascular Institute, Oregon Health and Because flow velocity is independent of body size due to the scaling Science University, Portland, OR of lengths and time constants of vascular systems, the magnitude and shape of velocity waveforms in mice are similar to the waveforms Pregnant sheep are the gold standard model for studying cardiovas- obtained in humans. Thus, much of what we learn from mice is cular and pulmonary development because of close parallels to readily translatable to humans for the diagnosis, evaluation, and human development. Large animal studies are labor intensive and treatment of cardiovascular diseases. expensive, making selection of healthy, normally growing fetuses essential for acquisition of quality data. Surgical indicators of fetal PS86 Effects of Buprenorphine, Methylnaltrexone, and Their wellbeing would be a critical refinement, reduce wasted cost and Combination on Gastrointestinal Transit in Rabbits (Oryctolagus effort invested in suboptimal fetuses, improve data quality, and cuniculus) 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 M Martin-Flores*1, BS Singh2, CA Walsh3, EP Brooks3, LC Taylor3, LM time of fetal surgery that can be used to establish whether a fetus is Mitchell1 within the normal, or suboptimal ranges for further study. To date, no systemic studies have used measures at the time of fetal surgery 1Department of Clinical Sciences, College of Veterinary Medicine, to assess fetal wellbeing, growth trajectories, and suitability to Cornell University, Ithaca, NY; 2Center for Animal Resources and continue to experimental protocols. To test whether a single blood Education, College of Veterinary Medicine, Cornell University, Ithaca, sample predicts fetal outcomes, we obtained fetal arterial samples at NY; 3College of Agriculture and Life Sciences, Cornell University, surgery and 3-7 days post surgery; blood gases were analyzed on a Ithica, NY Radiometer ABL825 and hematocrit was determined by microcapil- lary tube centrifugation. Maternal samples were collected for Buprenorphine (BUP) is likely the most commonly used opioid measurement of cortisol by ovine-validated ELISA. Data were analgesic in rabbits (Oryctolagus cuniculus). However, like most compared by Pearson Correlation and Student’s T Test. Surgical and opioids, it can decrease gastrointestinal (GI) motility and contribute postsurgical fetal hematocrit were correlated (P < .0001, R=0.6120). to ileus. The peripheral opioid antagonist methylnaltrexone (MTX) Given known relationships between hematocrit with long-term might be useful in relieving the GI effects of BUP without affecting trends in fetal arterial oxygen pressure (PaO2), postsurgical analgesia. We evaluated the effect of buprenorphine and methylnal- PaO2 values were compared for fetuses with a surgical hematocrit trexone on GI transit time in 8 healthy male rabbits. The rabbits were <40% or ≥40%; this relationship is confirmed in our dataset (P = administered 4 treatments using a randomized crossover design (at

700 Abstracts of scientific papers 2016 AALAS National Meeting

least 1 week apart): BUP 0.05 mg/kg, MTX 1 mg/kg, both agents 1Department of Primate Medicine, California National Primate combined (B/M), or normal saline (control). All treatments were Research Center, University of California—Davis, Davis, CA; 2K.L. administered subcutaneously every 12 hours for 2 days. Prior to each Maddy Equine Analytical Chemistry Laboratory, School of Veterinary treatment, 20 barium-filled plastic spheres were administered via Medicine, University of California—Davis, Davis, CA; 3California orogastric tube to be later identified by radiography of feces. Feces Animal Health and Food Safety Laboratory, School of Veterinary were collected every 6 hours, and body weight, food, and water Medicine, University of California—Davis, Davis, CA; 4Department consumption were measured once daily, for 5 days. The time to first of Medicine and Epidemiology, School of Veterinary Medicine, detection of spheres in feces was compared between the groups using University of California—Davis, Davis, CA Kruskall-Wallis test. Changes in body weight, daily consumption of food and water, and daily fecal weight were compared between the Rhesus macaques (Macaca mulatta) are the most commonly used groups with mixed effects models. Time to first passage of spheres nonhuman primate biomedical model, consisting of both research was significantly longer for BUP [median (minimum - maximum)] and clinical procedures requiring analgesia. Providing analgesia is [30 (24-48) hours] than for control [18 (18-18)] and for MTX [18 imperative and opioids are a mainstay of analgesic therapy. A novel, (18-24)]. The weight of collected feces per day was significantly lower transdermal fentanyl solution (TFS) was developed to be a long- for BUP and BM than for the other 2 groups. Water and food acting, single-administration, topical opioid that delivers ≥4 d of consumption was lowest for groups BUP and B/M than for control therapeutic plasma concentrations as previously published in beagles and MTX, however, there was no difference between the groups in (Canis familiaris). The TFS dose was applied to clipped dorsal skin daily change in body weight. BUP delayed GI transit time, and under ketamine (10 mg/kg IM) sedation. We describe the pharmaco- decreased daily fecal output and food and water consumption. MTX kinetic profile of TFS in healthy, adult rhesus macaques (n = 6; 3M, (1 mg/kg) administered concurrently was insufficient to prevent 3F) in this 2-period, 2-treatment crossover study of a single topical these effects. 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 PS87 Quantification of Induced Hypothermia from Aseptic Scrub therapeutic plasma concentrations (≥0.2 ng/mL) as compared to during Rodent Surgery Preparation beagles. Plasma fentanyl concentrations were determined by tandem liquid chromatography-mass spectrometry prior to drug administra- FC Hankenson*3, A Skorupski2, D Ferguson3, J Zhang1, F Lawrence1 tion 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). Noncom- 1Center for Statistical Training and Consulting, Michigan State partmental pharmacokinetic analysis was performed. For each dose University, East Lansing, MI; 2College of Veterinary Medicine, (1.3 and 2.6 mg/kg), respectively, maximum plasma concentration Michigan State University, East Lansing, MI; 3Campus Animal was 1.95 ± 0.40 and 4.17 ± 0.70 ng/mL, occurring at 21.33 ± 4.09 and Resources, Michigan State University, East Lansing, MI 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 Laboratory mice, Mus musculus, are common models in biomedical 93.65 ± 7.06 and 98.75 ± 5.40 h. No adverse effects were noted at research, often which involve surgical procedures as part of either dose. Macaques maintained plasma fentanyl concentrations approved biomedical research objectives. Mice may be prone to ≥0.2 ng/mL as compared to beagles for at least 7 days after 1.3 mg/ develop undesirable levels of hypothermia while under anesthesia kg and at least 10 days after 2.6 mg/kg topical administration of TFS. for surgery; hypothermia may impede anesthetic recovery, wound A single TFS dose may provide efficacious therapy with less stress, healing, and future health. Previously, whole body temperatures of discomfort, and risk to animals and personnel. mice were shown to be influenced by a variety of supplemental heat sources while under isoflurane anesthesia. This related study was PS89 The Use of Ear Pressure Pain Analysis to Assess Pain and designed to quantify the whole body cooling effect, particularly by Analgesic Efficacy in the Laboratory Rabbit Oryctolagus( cuniculus) ethanol (EtOH) with its evaporative properties, that scrub applications may have on the progression of hypothermia in anesthetized K LaVallee*1, A Yorsh1, A Hess2, LV Kendall1 mice (n = 47) and to determine if a certain scrub application should be preferentially recommended. EtOH, room-temperature saline, and 1Laboratory Animal Resources, Colorado State, Fort Collins, warmed saline (37°C), coupled with the iodophore betadine, were CO; 2Statistics, Colorado State University, Fort Collins, CO assessed as to their impact on both core temperature, recorded by rectal probe, and surface temperature, recorded by infrared scanner. The ability to assess pain in laboratory animals is critical in order to Scrub options were applied in triplicate to a 2cm x 2cm shaved provide appropriate analgesia, while ensuring optimal animal care abdominal area on mice that were maintained on a warm-water and welfare. New Zealand White rabbits are common animal models recirculating blanket (at 38°C) in a surgical suite (20-23°C room temp for surgical implant biomedical research due to their large size, average) and given isoflurane anesthesia (1.5-2.0% @ 0.6 L/min) for a which enables relatively easy surgical operations. These procedures 30-minute period. Interestingly, all applications that included are predictably painful, but methods to quantitatively assess betadine resulted in animals with the coolest body temperatures, analgesic efficacy in rabbits are poorly described. We evaluated ear significantly different from control animals (P < 0.05) at the time of pressure pain analysis (EPP) using the Randall-Siletto anlgesiometer application and for the remainder of the surveillance phase of 30 as a means to assess analgesic efficacy in rabbits. Ear pressure pain minutes. Betadine plus saline options resulted in body temperatures analysis was conducted once per day for 3 days to acquire baseline that lowered throughout the 30-minute period, with no differences pain tolerance for 3 rabbits. Rabbits were treated with either topical observed despite the fluid warming. EtOH alone demonstrated the EMLA cream (Lidocaine and Prilocaine), Meloxicam (0.5 mg/kg SC), most rapid cooling after application, both for surface and core temp or Buprenorphine (0.05 mg/kg SC) in a cross-over design with a readings, followed by an unanticipated warming (rebound) phase 3-day washout period between treatments. EPP was conducted 30 that equilibrated with the control animals’ baseline temperature minutes after EMLA application, and 2 and 6 hours after administra- within 12 minutes of application. These findings demonstrate that tion of Meloxicam and Buprenorphine. Baseline EPP measurements EtOH can be part of an acceptable scrub protocol to prepare an were 209 g. Rabbits receiving ELMA had a significant increase in EPP incision site, as it resulted in near normothermia in mice that were to 550 g (P < 0.001). There was an increase in the EPP of Meloxicam maintained on an appropriate heat source while under isoflurane treated rabbits (240 g) and Buprenorphine (277 g) at 2 h (P = 0.9 and anesthesia. 0.4, respectively). There were no differences noted at 6 hours. These results suggest that EPP has the potential to quantitatively assess PS88 Pharmacokinetics of a Novel, Transdermal Fentanyl Solution pain in laboratory rabbits. in Rhesus Macaques (Macaca mulatta)

GW Salyards*1, HK Knych2, AE Hill3,4, KL Christe1,4

701 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

PS90 Prophylactic Antibiotics Prevent Corynebacterium bo- (6) using the term “ethology” is inherently confusing. We propose vis Infection Following Acute Exposure that the term “functionally appropriate primate environments” be used instead, as it emphasizes how environments work for nonhu- CA Manuel*1,2, MJ Schurr3, CJ Henry3,4 man primates, it better describes current activities underway to improve nonhuman primate welfare, and the balance that is achieved 1Office of Laboratory Animal Resources, University of Colorado— between meeting the needs of the animals and the requirements of Denver, Aurora, CO; 2Department of Pathology, University of the research in which they are involved. We propose a process to Colorado—Denver, Aurora, CO; 3Department of Immunology and further refine this term through literature reviews, interviews with Microbiology, University of Colorado—Denver, Aurora, CO; 4Depart- experts, identifying areas with convergence of opinions on major ment of Biochemistry and Molecular Genetics, University of features needed to support welfare, conducting a gap analysis to Colorado—Denver, Aurora, CO ascertain needs for future research, and developing a mechanism to integrate research findings into animal care standards. We believe Corynebacterium bovis is sensitive to commonly used antibiotics that this approach will be fruitful as we work to further refine environ- are able to reach therapeutic blood concentrations in mice when ments for nonhuman primates in biomedical research. administered orally. However, it is believed that prophylactic antibiotics are ineffective at preventing C. bovis infections. An PS92 Intermittent versus Continuous Pair-Housing in Laboratory experiment was performed to evaluate if prophylactic antibiotics can Rhesus Macaques (Macaca mulatta) and Activation of the prevent a C. bovis infection following acute exposure and to HPA-Axis investigate the innate immune response to infection in athymic nude mice (n = 32). Amoxicillin and clavulanic acid were administered in D Hannibal*2,1, L Cassidy5, A Barnard1, J Vandeleest1, K Chun3, S the water to naïve mice for 14 days following C. bovis exposure. Semple4, B McCowan2,1 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 1California National Primate Research Center, University of negative controls (n = 8 each) and antibiotic treatment groups were California—Davis, Davis, CA; 2Population Health and Reproduction, serially sampled by oral and dermal swabs on days 0, 5, 10, 15, 21, 35, University of California—Davis, Davis, CA; 3Semel Institute for 49, and 63 for C. bovis DNA by qPCR. Half of mice were euthanized Neuroscience and Human Behavior, University of California—Los at day 15, with the remaining mice euthanized 63 days post Angeles, Los Angeles, CA; 4Life Sciences, Roehampton University, exposure. At harvest, splenocytes were isolated and evaluated for London, United Kingdom; 5The German Primate Centre, Göttingen, activated macrophages (Mac-1+, IFNg+) and NK cells (DX5+, IFNg+) Germany by flow cytometry. Our results show that all mice given prophylactic antibiotics starting on -3 day and 0 day of acute exposure were C. A social environment provides optimal captive primate welfare. At bovis negative by qPCR at both endpoints. At 15 and 63 day post indoor research facilities, laboratory rhesus macaques are often exposure, the number of activated splenic macrophages was housed in pairs and may be temporarily or permanently separated significantly lower in both antibiotic treated groups as compared for study procedures, concerns over compatibility, medical surveil- to C. bovisinfected controls (P < 0.05). However, at 15 day post lance, or other reasons. While long-term separations, as well as exposure, the number of activated macrophages was significantly introductions, can stimulate the stress response systems to the point higher in the 0 d treatment group as compared to the -3 day of impacting inflammation and immune function, it is unknown treatment group (P < 0.05). There were no significant differences in whether short-term separations for less than a day can have an effect. activated NK cells between positive and negative controls at either In this study, we investigated differences in urinary cortisol among time point. Our results demonstrate that prophylactic antibiotics can 24 adult female rhesus macaques over a 10-week period. The sample prevent C. bovis infections in athymic nude mice when started -3 day consisted of 12 females in continuous pair-housing prior the start of and 0 day of an acute exposure. In addition, our data also shows that the study, then switched to intermittent pair-housing half-way macrophages of the innate immune response are activated when through the study (CI subjects), and 12 females in intermittent antibiotics are started on the day of C. bovis exposure, but are not pair-housing for management reasons prior to the start of the study, activated when antibiotics are started 3 days prior to exposure. then switched to continuous pair-housing half-way through the study (IC subjects). Creatinine and cortisol were assayed from urine PS91 “Functionally Appropriate Primate Environments” as an samples collected 2 times per week. Behavioral data was collected on Alternative to the Term “Ethologically Appropriate Environments” each pair using 8-minute focal observations twice each morning and to Describe Environments for Research Primates afternoon for measures of affiliative, aggressive, anxious, abnormal, and activity-state behaviors. At the end of the study, subjects were MA Bloomsmith*1, J Hasenau3, R Bohm2 scored for personality and pair-rating attributes. Cortisol data were analyzed using a generalized linear model with gamma distribution 1Yerkes National Primate Research Center, Atlanta, GA; 2Tulane and an information theoretical approach to determine the predictors National Primate Research Center, Covington, LA; 3Lab Animal for a best-fit model. The final model (X2=49.98; df=6; P < 0.001) Consultants, Reno, NV 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 The term “ethologically appropriate environments” was first (beta=-0.65; P < 0.001) have lower cortisol. There is also an interaction published in the report of the Institute of Medicine Committee on the of intermittent-housing with “tense” pair rating, showing that Use of Chimpanzees in Biomedical and Behavioral Research in 2011. subjects in the intermittent condition are likely to have increased In 2015, the United States Department of Agriculture requested urinary cortisol if they are in a “tense” pair (beta=0.24; P = 0.002). comments on a petition which called for amending the Animal This suggests that to avoid chronic activation of the HPA-axis, rhesus Welfare Act so that all research primates would be housed in macaque females should be housed in continuous contact in “ethologically appropriate physical and social environments.” We are compatible pairs rather than maintained through intermittent critical of this term because (1) it does not provide clarification housing in less compatible pairs. beyond that in current regulatory language, (2) it does not provide for balance between animal welfare goals and the reasons why the PS93 Use of Temperament in Behavioral Management Practices for primates are housed in research facilities, (3) it discounts the Captive Nonhuman Primates adaptability that is inherent in the behavior of primates, (4) it conveys that duplication of features of the natural environment are K Coleman* required for suitable holding environments, (5) objective studies reveal that environments that appear to be more ethologically Oregon National Primate Research Center, Beaverton, OR appropriate do not necessarily better meet the needs of animals, and

702 Abstracts of scientific papers 2016 AALAS National Meeting

Effective behavioral management programs are designed to address PS95 Primate Behavior Management Certification Course for the behavioral needs of the animals. However, there is often a one Animal Care Staff size fits all approach, in which techniques that have been found to be beneficial for some are assumed to be good for all. However, JE Perlman, S Hastings, J Cowan-Brown*, K Neu, MA Bloomsmith individuals vary with respect to a wide range of traits, such as temperament or personality, that may affect how they respond to Yerkes National Primate Reseach Center, Emory University, Atlanta, these practices. Temperament influences how individuals cope with GA novel or stressful situations. Thus, it stands to reason that it may affect how individuals react to various management procedures, Animal care technician responsibilities include identification of including positive reinforcement training, socialization, and response adverse or abnormal behaviors in the species with which they to environmental enrichment. Over the past 5 years, an increasing work. The Guide states animal care personnel should receive training number of studies have examined how measuring temperament can in the behavioral biology of the species. Our Behavioral Management help guide behavioral management decisions. For example, knowing Unit developed a certification course for the purpose of teaching the the temperament of macaques can assist in social introductions. behavioral biology of nonhuman primates and to provide a frame- Further, an individual’s propensity to use environmental enrichment work, practical tools, and a system of communication with which to can be influenced by its temperament. Shy, inhibited individuals may address behavioral issues. Five areas of behavioral management are not use or get the same benefits from some common environmental presented. These topics are primate behavior, environmental enrichment options compared to more exploratory individuals. enrichment, animal training, social housing, and welfare. The course Temperament can also affect an individual’s trainability. Bold includes instruction on the behavior of laboratory primate species in monkeys are easier to train using positive reinforcement techniques the wild and captivity (chimpanzee, macaque, squirrel monkey, and than their shy conspecifics. This presentation will examine how managbeys); environmental enrichment includes applications of the individual differences in temperament might affect behavioral 4 inanimate enrichments; animal training covers terminology, management practices for captive nonhuman primates. techniques, and application that promote positive reinforcement techniques and how to apply alternative methods; and social housing PS94 An International Survey of Approaches to Monkey Chair includes the impact of different housing options on behavior and Restraint daily management. Captive wellbeing research is discussed with an emphasis on practical applications such as identifying and reporting JL McMillan*1, M Prescott2, MA Bloomsmith1 behaviors of concern. Information is presented via lecture, discussion, demonstrations, and group exercises. Testing is conducted at 1Yerkes National Primate Research Center, Atlanta, GA; 2National the end of each unit to demonstrate information is synthesized and Centre for the Replacement, Refinement and Reduction of Animals in can be applied to everyday care. Students receive certificates for each Research, London, United Kingdom unit successfully completed and receive full certification in behavioral management after all of the units are passed. The use of specifically designed restraint chairs is the preferred method of restraint for studies that require nonhuman primates to sit PS96 Size Doesn’t Matter: Behavior Management of a Small in place for sustained periods of time. Restraint chairs have been Population of Nonhuman Primates in a Research Setting used for decades within the research environment and there is a lot of variation in the equipment employed and the methods used for AN Rehrig* preparing animals for these procedures. With increasing emphasis on refinement of restraint procedures to promote animal welfare, it’s University of Rochester, Rochester, NY important to have a better understanding of this potentially stressful procedure. Chair restraint is used all over the world, yet there is little Implementing and managing a behavior program for a small published information available for researchers on the subject. We population of nonhuman primates may seem like a simple endeavor developed a survey to obtain a better overview of chair restraint at first glance; however, program implementation in a close knit equipment, procedures, and plans for improvement. We received 101 research community poses numerous challenges. Regardless of size, responses representing academic, pharmaceutical, and government successful programs depend on securing funding for behavioral staff facilities within the United States, Canada, Europe, and Asia. and resources, staff training specific to behavior management in a Findings indicated that the majority of researchers using restraint research setting, and fostering program acceptance with the IACUC, chairs work with macaque species and conduct a wide range of principal investigators, and husbandry staff. At our institution, the procedures including cognitive testing, recording neuronal activity, animal behaviorist oversees the behavior management for the entire fMRI, intravenous infusion, and blood sampling. Two-thirds of research collection, including everything from rodents to primates laboratories reported using an enclosed chair, where the animal and is involved in all aspects of the program including hands-on enters and is trained to extend his/her head through an opening on animal work and administrative duties. Prior to start up, an outside top of the chair, and 39% use an open chair which typically requires consultant was brought in to assess the existing program and the the use of the pole and collar system to transfer and secure the behaviorist visited sister institutions to gain insight on how to animal into the chair. Researchers reported considering a variety of improve and grow the program using best practices in the field. factors when choosing the type of chair for their studies including Acceptance was achieved through open and effective communication comfort for the monkey, ease of use, and ability to adjust fit between with all involved parties, demonstration of improved animal welfare, different-sized monkeys. We were encouraged with the number of and increased compliance and recognition from regulatory institu- facilities incorporating positive reinforcement training methods into tions (e.g., USDA). Effective behavior management programs, even in their approaches. The most commonly reported period of training small-scale operations, can have a significant and long lasting, time before research procedures begin was 1-2 weeks (38%) for users positive impact on primate welfare, as well as the culture of the of open chairs and 1-2 months (38%) for users of enclosed chairs. research institution overall. Similar proportions of open (69%) and enclosed chair users (71%) felt additional preparation time would be beneficial. As a research PS97 Taking Behavioral Management to the Next Level: Providing community using restraint procedures, there is a need to work Chimpanzees with Opportunities to Participate in Their Own Care together to define methods of best practice and this survey can help movement in that direction. 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

703 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016

Behavioral management programs have advanced considerably primates (NHPs) housed in laboratories reveal notable progress during the 5 years since the publication of the most recent version of between 2003 and 2014, particularly with respect to social housing, the Guide. We have focused our efforts on behavioral management positive reinforcement training (PRT), the use of enrichment devices, techniques that allow the animals to make meaningful choices and to and personnel resources. The aim herein is to evaluate whether this control some aspects of their care. Many of the chimpanzees are progress is manifest irrespective of facility size, and to identify how greater than 30 years of age, and some are experiencing aging-related current practices vary with facility size. Among 2014 survey maladies, particularly arthritis. In addition to the administration of responses, facility size was dichotomized per the 2003 ratio of caged typical arthritis medications, we provide our chimpanzees with animals to behavioral management technicians (358:1), resulting in 23 opportunities to voluntarily seek a variety of other treatments for large facilities (LFs; mean population 1,952 NHPs) and 18 small their arthritis symptoms, including acupuncture, low-level laser facilities (SFs; mean population 82 NHPs). In comparison to the use therapy, and a medication choice procedure. Many chimpanzees of social housing for caged NHPs in 2003 (41% of NHPs across all voluntarily participate in these treatment regimens, and a number of facilities surveyed), it is significant that gains are evident both in LFs them have demonstrated behavioral improvements (including (67%) and SFs (66%). Increases in the use of enrichment devices since increased mobility, decreased inactivity, and increased sociality; some 2003 are also seen in both. In both LFs and SFs, animal care techni- of the changes are statistically significant) as a function of acupunc- cians (ACTs) participate more in 2014 than 2003, particularly in ture, laser therapy, and/or choosing their medication. We are human interaction as enrichment, PRT, and implementation of continuing to 1) expand the number of treatment options that intervention for abnormal behavior. Currently, SF programs are chimpanzees can choose to receive and 2) refine our techniques for primarily overseen by veterinarians (67% of facilities) as opposed to assessing the benefits of such treatments. The ultimate goal of the behavioral scientists (6%). In contrast, LFs are overseen more often by behavioral management program at the NCCC is to establish a behavioral scientists (44%) than veterinarians (31%). While 87% of communication system that allows the chimpanzees to ‘tell’ us how LFs employ behavioral management technicians, a third of SFs do so. they want to be managed and treated. This appears to be compensated for by some increase in ACT participation, but to a greater degree by veterinary technician PS98 The Critical Role of Animal Care Staff in Executing Behav- participation in social housing, manipulanda, devices, and new ioral Management Programs employee training. While long-term program progress is not limited to large facilities with dedicated behavioral management scientists MT Sharpless*, JE Perlman, J Cowan-Brown and technicians, SFs report fewer recent program changes as well as fewer future plans to alter programs, underscoring the utility of Field Station, Emory University/Yerkes Primate Center, Lawrencev- pertinent literature, conferences, and other mechanisms for cross- ille, GA facility communication.

Primate behavioral management programs encompass animal enrich- PS100 Procedural and Socialization Similarities and Differences ment activities, animal training procedures, and socialization efforts. between Pair Housing Macaques (Macaca mulatta) and African The size of the facility, the number and type of species housed, and Greens the nature of the research being conducted are some of many factors in the design, structure, and staffing of an organization’s behavioral J Weed*1, M Jorgensen2 management program. Behavioral management programs, especially in facilities housing nonhuman primates, are often dynamic and can 1Comparative Medicine Branch, Centers for Disease Control, Atlanta, be complex. These programs need to be flexible and adaptable in GA; 2Pathology, Wake Forest School Of Medicine, Winston-Salem, order to accommodate a range of approved activities and options and NC to implement any necessary changes or modifications. Animal care technicians are key components to the behavioral management Vervets or African green monkeys are a nonhuman primate species program. They are often responsible for some or all aspects pertain- widely used in biomedical research. At present there are relatively ing to the preparation, distribution, implementation, and record few references available on how to effectively social house this keeping associated with enrichment activities. Care technicians are species in a captive environment. Social housing is currently the also on the front lines throughout the socialization process during the default value promoted in the Guide for captive-housed nonhuman course of their daily husbandry and care duties and responsibilities. primates unless there is a valid scientific justification for individual Enrichment and socialization efforts continue to increase as we gain housing. Herein we describe the subtle similarities and differences better understanding and as we increase our knowledge of the between techniques for social housing African green monkeys versus impact of behavioral management on the physical health and macaques under laboratory conditions. We describe procedures psychologic wellbeing of captive animals as well as the impact on implemented for social housing African greens in several laboratories research data and data collection. Laboratory animal care technicians and provide an overview of the methods used in one captive are part of this positive forward movement and work culture. Care population with over 6 years of experience of pair housing. Compat- technicians enter the field with a compassion for animals and they ibility was measured at 14, 30, and 60 days post introduction. In 1 are seeking guidance, information, and mentorship from supervisors cohort (vervet research colony), successful pairing rates at 14 days for and managers on ways to improve and enhance the lives of the males was 100%: females 96% (n = 72 female pairs, n = 52 male pairs). animals for which they provide care. Discussion topics will include By 30 days post introduction, rates for both male and female pairs challenges to behavioral management programs and possible remained high, 98% and 94%, respectively. At 60 days post pairing, resolutions, methods used in the development of a cooperative work 75% of males and 82% of female pairs remained intact. Some pairs culture, strategies employed to motivate and engage staff, and ways were separated for practical purposes, such as research needs rather to provide support and additional educational resources to care than due to antagonistic behavior or distress. We emphasize that technicians. African greens are not macaques and should not necessarily be treated the same way. Gradual introduction techniques may not be PS99 Changes in Practices over Time and Variations Associated beneficial or suitable for this species. Variables such as age, sex, and with Facility Size prior familiarity with new partners influenced successful socialization rates. The discussion will focus on behavioral differences KC Baker* between African green monkeys and macaques as well as future directions for research on pair housing this species. Veterinary Medicine, Tulane National Primate Research Center, Covington, LA

Surveys of behavioral management programs for nonhuman

704 Abstracts of scientific papers 2016 AALAS National Meeting

PS111 Preparing a Research Facility for Work on Emerging Ovarian cancer is diagnosed in approximately 225,000 women per Infectious Diseases year accounting for 4% of all cancer cases worldwide on an annual basis. There is an estimated 140,000 deaths per year that are BL Kick*2,1, S Jean1, M Thompson1 attributed to ovarian cancer. Diagnosis at advanced stages and resistance to treatments contribute to the high 5-year mortality rate. 1Yerkes National Primate Research Center, Atlanta, GA; 2Division of Domestic laying hens (Gallus gallus domesticus) have been identified Animal Resources, Emory University, Atlanta, GA as a spontaneous model for ovarian cancer with the potential, once fully characterized, to be the first predictive model and model for In early February the WHO declared Zika virus a Public Health therapeutic trials. The chicken develops spontaneous and progressive Emergency of International Concern. In response, several institutions ovarian cancer that have epidemiologic, morphologic, and molecular have begun working with Zika virus, both in vitro and in vivo. We will traits that are similar to humans. The incidence of spontaneous cover the steps and challenges associated with preparing a research ovarian cancer in chickens ranges somewhere between 40-70% in institution for work on Zika virus in both rodents and nonhuman chickens over 2 years of age. This is a consequence of constant primates. We will also emphasize the collaborative efforts needed ovulation resulting in DNA damage and repair of ovarian epithelial between biosafety professionals, occupational health providers, cells leading to ovarian cancer, similar to postmenopausal women researchers, institutional administrators, animal care resources, and the which account for over half the diagnosed cases of ovarian cancer in veterinary care team. The first step to developing a good team and women. Chickens make an ideal model due to their limited genetic functional facility for work on emerging infectious disease agents will diversity and predictable age of onset of disease. The development of be to develop an agent specific risk assessment. Once the comprehen- this model will allow for development of predictive assays and allow sive risk assessment is completed and the appropriate risk level for new therapeutics to be tested on spontaneously occurring ovarian determined, the core team moves forward with developing a facility cancer. Currently, the only way to diagnose chickens with ovarian plan and work flow. When working with emerging infectious disease cancer is to do invasive endoscopy. Development of surgically agents that may require higher levels of containment several questions implanted ports created with a 3D printer allows for a minimally have to be addressed. For example, where will this research occur? invasive repeated visualization of the ovary, following the progres- Who will conduct the animal work with this agent? What quantity of sion of disease, repeated sampling, and treatment monitoring. This animals are required and what training will be needed (both humans allows for the endoscopic visualization and biopsy of the ovary to and animals)? How will diagnostic samples be processed and how will change from requiring a surgical procedure to only being an occupational health requirements be met? Based on a risk assessment, anesthetic event. The original design was a hard plastic port that was work on Zika Virus at our facility is managed at an animal biosafety easily implanted but suffered the complication of rejection. The level 2+. We will lay the framework for how we developed our material was then switched to a soft, flexible silicone and the shape policies, procedures, facility work flow, and the challenges we faced. was adjusted to accommodate the location. The new material and Hopefully this information will assist other organizations to develop design has shown no evidence of port rejection, allowed endoscopic their risk assessment to aid in preparing their facilities for work on visualization of the ovary, and ports have remained implanted for emerging infectious disease agents. over 9 months.

PS112 New NIH Requirements for Cell Line Authentication: What PS114 The Grooming Sleeve: Encouraging Conspecific-Like You Need to Know Behavior and Increasing the Human-Animal Bond

BA Bauer*1, LK Riley2 CM Sands*, C Evans, CA Buckmaster

1Genetic Services, IDEXX Bioresearch, Columbia, MO; 2IDEXX CCM, Baylor College of Medicine, Houston, TX Bioresearch, Columbia, MO Nonhuman primates are naturally social animals that live in large The National Center for Biotechnology Information estimates that family groups called troops. In the wild, monkeys forage and build between 18–36% of cell lines are contaminated contaminated with bonds with one another through grooming and other social activities. cells from other cell lines, with the International Cell Line Authenti- The act of grooming has been found to greatly decrease stress, heart cation Committee (ICLAC) listing 475 known cross-contaminated or rate, and cortisol levels in nonhuman primates. This decrease in misidentified cell lines in existence today. There is more attention stress and cortisol is seen in the animal being groomed as well as the than ever on this topic with the recent publication of NIH Notifica- groomer. In the research setting, singly housed nonhuman primates tion (NOT-OD-16-011) calling for grant applicants to include an do not have the opportunity to groom another individual. In order to authentication plan for key biologic materials with new and offer our singly housed macaques the option of grooming, we competitive renewal grant applications. This presentation will designed a grooming sleeve. This device is worn over the protective discuss the new requirements for cell line authentication in NIH gear, on the arm of the caregivers, and gives our singly housed grants and how to implement cell line authentication monitoring pro- macaques the opportunity to groom. We had a high success rate, with grams. Participants will learn what is involved in cell line 80% of our macaques grooming the device, and 50% presenting to be authentication and different strategies for implementing monitoring groomed. Due to the overwhelmingly positive response to the programs. In addition, participants will learn how cell line authenti- grooming sleeve we recommend that it be used in other facilities cation fits into a more comprehensive biologics monitoring program housing nonhuman primates. to ensure materials used in animal studies to improve reproducibility and minimize risk to animal facilities. The target audience includes PS115 Adding Fuel to the Fire: Performance-Based Evaluation of veterinarians, facility managers, and technical personnel who work Welfare Interventions in the DSS Murine Model of Colitis with animal models using cell lines or human patient derived xenograft (pdx) models. JK Lang*1,4, J Zaias2, R Santaolalla1, R Dheer1, M Phillips1, S Diaz1, J PS113 Development of a Flexible Port for Repeated Biopsy of Grant3, M Abreu1 Spontaneous Ovarian Cancer in Domestic Laying Hen (Gallus gallus domesticus) 1Department of Medicine/Gastroenterology, University of Miami, Miami, FL; 2Divison of Veterinary Resources, University of Miami, S Galaviz2, AT Richert*1, BD Smith1 Miami, FL; 3Physiology and Biophysics, University of Miami, Miami, FL; 4Department of Immunology and Infectious Diseases, Harvard 1Comparative Medicine, Texas A&M, College Station, TX; 2Texas T.H. Chan School of Public Health, Boston, MA A&M, College Station, TX Ulcerative colitis (UC) affects a large percentage of the population by 705 Vol 55, No 5 Journal of the American Association for Laboratory Animal Science September 2016 causing inflammation mainly in the colonic mucosa. The DSS-in- for a short survival period (typically ≤ 24 hours) for physical and duced UC model in mice can cause debilitating consequences (e.g., behavioral evaluation, then euthanized. Results of pilot experiments diarrhea, weight loss, bloody stool, hunched posture, and difficulty are submitted to the IACUC and veterinary staff for evaluation. By walking). These clinical signs are often viewed as painful by the setting standard expectations for device construction and animal IACUC, thus possibly conflicting with researcher goals, as adding welfare, the IACUC may ensure consistency in the evaluation of medical treatments to mitigate symptoms may interfere with devices used in mTBI studies. experimental endpoints. To directly assess this, we examined the effect of fluid supplementation on animal welfare and experimental PS117 Successful Outcomes of a Behavioral Response Plan (BRP): endpoints in DSS-treated mice. Nine mice were assigned to 4 A Standardized Approach to Respond to Stereotypic Behaviors in treatment groups, with or without fluid supplementation and at 2 Nonhuman Primates 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 JN Camacho*, DM Jarrell supplementation was started on day 3 of DSS treatment and continued for 4 days. Welfare associated behaviors were measured Massachusetts General Hospital, Charlestown, MA (nest building and time to nest integration (TNT)). Clinical parameters included body weight/condition, stool characterization, and Our standardized approach to manage stereotypic behavior is intestinal inflammation. We found that acutely, there were no documented using a Behavioral Response Plan (BRP) combined with differences in clinical signs or parameters measured; however, TNT an Intervention Enrichment Assessment (IEA) and an Individual and nest scores acutely were greater in fluid supplemented mice. Behavior Assessment (IBA). The BRP categorizes 4 stereotypic During recovery after DSS cessation, mice who received fluids behaviors: repetitions, alopecia, feces Smearing, and self-directed regained weight faster, had higher serum protein and albumin, and behaviors. Additionally, the plan recognizes 4 other NHP behaviors improved health than those who did not receive fluids. Colonic that indicate concern: fear, aggression, higher needs, and social inflammation did not differ acutely, but was significantly reduced 6 conflict. Dependent on the frequency and/or severity of each days after DSS cessation in mice given fluid supplementation. behavior, a grading system is used and a specific enrichment Overall, fluids given to DSS-treated mice did not have a significant approach prescribed, followed by the use of a quantitative scale to impact on welfare acutely, but significantly improved their health determine the effectiveness of the enrichment to match the animal’s and welfare during recovery. Fluid supplementation does impact behavioral needs and eliminate the stereotypic behavior. An example colonic inflammation and serum protein levels during recovery. of this approach is an NHP that presents as having overgrooming Therefore, use of fluid supplementation as a welfare tool is not clear alopecia, categorized as displaying moderate severity (classified by cut acutely and it affects (decreases) our model endpoint (induction 2-4 patches). The NHP would have an initial intervention of of inflammation) which could ultimately result in an increase in total destructible enrichment daily, followed by a quantitative assessment numbers of animals used. These data can be applied henceforth by used by husbandry staff. An IEA score of 1 indicates enrichment is IACUC and investigators to provide performance-based decision- completely used, while a score of 3 indicates the enrichment is not making for therapy options and humane endpoints for this model. 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 PS116 Establishment of Novel IACUC Guidelines for Evaluation of result in worsening the stereotypic behavior. After analyzing a group Mild Traumatic Brain Injury Devices for Mice and Rats of 80 NHPs identified to have stereotypic behaviors and were subsequently treated with the BRP, 55 (60%) of the NHPs responded KL Cavanaugh*1, S Pritt1, R Hammer2 successfully to the BRP with stereotypic behavior resolved (without reoccurrence for 4+ months) within an average of 156 days (15-323 1IACUC, UT Southwestern, Dallas, TX; 2Biochemistry, UT Southwest- day range). While tracking prior to the use of the BRP plan was not ern, Dallas, TX as standard, it would not be ununusual for a NHP identified as having a stereotypic behavior to be receiving some sort of treatment Mild traumatic brain injury (mTBI), such as a concussion, comprises for years with little change in behavior. The BRP plan has been highly 70-80% of all traumatic brain injuries. To mimic these injuries, investi- successful at treating stereotypic behaviors observed by primates at gators create mTBI within an experimental animal model using our institution, using animal care staff making daily observations various methods of impact. These methods, such as weight drop, and responding based on individual animal needs. may use linear or rotational accelerated force to strike an animal’s head at a desired region. These methods produce a mild, noninvasive PS41 Withholding of Analgesics: When Is Justification Simply closed head injury that simulates injuries commonly sustained Enough? through sports, motor accidents, or other events. As commercially available devices are not viable for the experimental needs of close DA Pellecchia* head injuries, the published literature references the use of noncom- mercial, individually created devices. Therefore, investigators Comparative Medicine, Pfizer, Pearl River, NY wishing to reproduce these methods of injury must create their own devices, which then come under IACUC oversight for approval. As A researcher’s decision to not administer analgesics should always investigators embark on creating their own equipment, it becomes be accompanied by adequate justification in the animal use protocol. increasingly important for the IACUC to set standards for these Many times though, this rationale could be lacking appropriate scien- mechanisms. To ensure consistent consideration of animal welfare tific evidence. An older article may be cited from a renowned medical and device evaluation, novel standard criteria for mTBI studies using journal describing a reliable study which indicated that a particular mice and rats were established by the IACUC at an academic class of analgesics is “very likely” to interfere with scientific objec- research institution. This standard criteria is encompassed within a tives. For example, an analgesic’s mechanism of action may inhibit developed IACUC policy on traumatic brain injury to communicate tumor growth in an oncology study. Or, an analgesic might interfere to investigators the baseline expectations of the IACUC. Once initial with an immune-modulated response on a vaccination study. But do device construction is inspected by an ad hoc of the committee and we truly verify whether this tried and true “evidence” is still scientifi- injury parameters are addressed, PIs are then required to complete cally valid? Has the prior science been disproven with the discovery pilot studies to evaluate device performance. The IACUC requires of other innovative pain relievers that use a different mechanism of that pilot studies be conducted in 3 phases, each of which is action which consequently may not interfere with the study objec- immediately followed by gross pathology analysis: (1) carcass trials tives? IACUCs are charged with ensuring that principal investigators with veterinary supervision, (2) anesthetized animals that are have indeed conducted through literature searches to support their euthanized immediately after injury, but prior to anesthesia recovery, stance to withhold analgesics. But, how far do we expect the com- and (3) live animals allowed to recover from anesthesia after injury mittee to delve into whether alternative options are available to

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mitigate potential pain and distress? We aim to provide guidance to points, whereas carprofen-treated animals displayed behaviors IACUC members, regulatory personnel, veterinarians, and research- indicative of insufficient analgesia at 8 hours post surgery. After both ers regarding how to determine whether the justification to withhold anesthesia and surgery conditions, the multimodal and buprenor- analgesics is scientifically valid and capable of withstanding the phine treated animals lost at least 10% of their baseline weights (P < scrutiny of regulatory and accreditation agencies. Some methods 0.05) and were unable to return to baseline weights within 96 hours. will be described to help ensure that IACUCs have conducted due In contrast, carprofen treated animals animals lost significantly less diligence when deciding to approve a protocol in which analgesics weight (~3%, P < 0.05) and returned to baseline weights within 72 will be withheld. hours. Plasma levels of extended-release buprenorphine were above 0.9ng/ml from 8 to 96 hours post-injection with peak levels (1.2ng/ PS42 IACUC Meeting Minutes: Defining the Balance between ml) at 48 hours. Of the 3 analgesia regimens evaluated, multimodal Adequate and Too Much Information analgesia appeared to provide the greatest analgesia efficacy, however the significant weight loss and mild sedation observed in DA Pellecchia* the extended-release buprenorphine-treated groups may need to be considered when selecting an analgesia regimen. Comparative Medicine, Pfizer, Pearl River, NY PS119 Spontaneous Group B Streptococcal (GBS) Septicemia in IACUC meeting minutes are frequently a bone of contention among Gravid Long-Evans Rats the regulatory community in regards to the level of detail they should be. Ideally, minutes would be an accurate reflection of the CM Bodi*1, V Bakthavatchalu1, M Esmail2, Y Feng1, L Richey2, J discussions that transpired during the course of convened meeting. Dzink-Fox1, B Beall3, SE Perkins2, JG Fox1 However, some IACUC deliberations can become extremely tangen- tial and spiral into more of a political debate than a productive and 1Division of Comparative Medicine, Massachusetts Institute of efficient dialogue. IACUCs must abide by the regulations governing Technology, Cambridge, MA; 2Division of Laboratory Animal their institution and industry expectations regarding compilation Medicine, Tufts University, Boston, MA; 3Streptococcus Laboratory, of meeting minutes. Unfortunately, that particular guidance is not CDC/NCIRD/DBD/RDB, Atlanta, GA provided anywhere in an enumerated or concise description for the research community to follow. Most institutions tend to convey Group B Streptococcus (S. agalactiae, GBS) is a gram-positive way too much information about the events that occurred within a opportunistic pathogen that inhabits the respiratory, urogenital, 2-hour meeting. What probably could have been captured succinctly and gastrointestinal tracts of humans and animals. Maternal in a simple page or 2 ends up instead looking like a multipage colonization of GBS is a risk factor for infectious disease in humans transcription. How do you avoid this pitfall? Although there is no and a principle cause of neonatal meningitis and septicemia. It also straightforward answer, there is a method to tackle the problem. By is an emerging pathogen in the elderly, immunosuppressed, and studying several years’ worth of our IACUC’s meeting minutes along pregnant women. We describe the presentation of polymicrobial with queries from our regulatory authorities and accreditation orga- sepsis including GBS in 3 gravid adult female Long-Evans rats nizations, our institution decided to pare down our meeting records experiencing acute mortality (septicemia, meningitis, endocarditis, to just enough to cover the basics while avoiding the minutiae and nephritis) from a colony of long-term breeding pairs. Fluorescent in not burying all of the details. It definitely helps to have folks on your situ hybridization was performed to confirm the organism IACUC who have an affinity toward concise editing. This session will associated with the pathology in the affected tissues. Antibiotic help to provide guidance on how to trim the fat without losing too susceptibility testing indicated similar resistance and susceptibility much of the core of your IACUC deliberations. Your meeting minutes patterns to that of human strains of GBS. Seven GBS isolates can fulfill regulatory requirements without being overly verbose. obtained from affected and nonaffected animals identified by 16S rRNA sequencing were submitted to the CDC for whole genome PS118 Evaluation of Analgesia Efficacy in a Guinea Pig Cavia( sequencing and comparison of the rat GBS genomes to human porcellus) Surgical Pain Model 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 pathoge- V Oliver*1, K Simon1, S Athavale1, LV Kendall2, JA Nemzek1, J nicity, and shared serotypes (Ib, V) with invasive isolates collected Lofgren1 from human cases. SNP analysis revealed similar genetic relationships among isolates, strengthening the notion that zoonotic 1University of Michigan, Ann Arbor, MI; 2Colorado State University, transmission is possible. To our knowledge, this is the first report of Fort Collins, CO GBS associated with spontaneous maternal septicemia in gravid female rats. GBS is zoonotic, but the factors contributing to Approximately 50,000 guinea pigs (Cavia porcellus) are used in intraspecies and zoonotic transmission remain unknown. Due to research studies in which more than momentary or slight pain is the potential for significant GBS-induced disease, particularly in expected. As a prey species, they do not readily display overt signs of gravid and neonatal animals, prevention and consideration of elimi- pain, thus challenging our ability to accurately assess efficacy of nation of GBS in the colony are advised. Preventative measures current analgesic recommendations. Our laboratory examined including stringent PPE practices and repopulation have decreased analgesia efficacy for treating postoperative pain. Briefly, 24 female the incidence of clinical disease in the rat colony. In the interest of Hartley guinea pigs were placed into analgesic treatment groups: both public health and assisting in ensuring healthy rats, consider- extended-release buprenorphine, carprofen, or multimodal treatment ation of excluding GBS as a potential pathogen for with both analgesics. Assessments included daily body weight immunocompromised or stressed animals is recommended. measurements, electronic vonFrey testing, and video recording for ethogram analysis at 2, 8, 24, 32, 48, 72, and 96 hours in the following PS120 Mycoplasma hyorhinis: Resident Flora and Opportunistic time-matched conditions: baseline, anesthesia and analgesia only, Pathogen of Swine Conjunctiva and hysterectomy surgery. An additional 8 guinea pigs were used for pharmacokinetic evaluation of the extended-release buprenorphine. MP Bradley*, MJ Hoenerhoff, JA Nemzek Animals treated with a single analgesic experienced increased mechanical sensitivity for at least 96 hours post surgery. In contrast, Unit for Laboratory Animal Medicine, The University of Michigan, animals receiving multimodal analgesia only demonstrated increased Ann Arbor, MI mechanical sensitivity at 2 hours post surgery. Ethogram data indicated the extended release buprenorphine had a sedating but In the fall of 2015, 5 adult pigs (35-55kg), representing approximately analgesic effect during all anesthesia/analgesia and surgery time 16% (5/32) of those acquired from one SPF agricultural farm over a

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3-month period, were reported at intake exam for mild bilateral PS122 Spontaneous Mutation with Limb Deformity in a Colony of conjunctivitis. The condition became severe over the next 3 days Swiss Webster Mice following arrival. The conjunctivitis was typified by conjunctival hyperemia, chemosis, elevated nictitans, and purulent discharge. A Dickerson*1, H Sandy1, J Blaisdell2, CA McGee1, R Cannon2, TL Diagnostic workup ncluded conjunctival cytology, biopsy, and Blankenship1 aerobic bacterial culture. PCR analysis was performed for several pathogens, including Porcine Reproductive and Respiratory 1Comparative Medicine Branch, NIEHS/NIH/DHHS, Research Syndrome virus (serum), Swine Influenza Virus (oropharyngeal Triangle Park, NC; 2Signal Transduction Laboratory, NIEHS/NIH/ fluids), Chlamydia, and Mycoplasma (conjunctival swabs). PCR DHHS, Research Triangle Park, NC analyses were negative for viruses and Chlamydia, but positively identified Mycoplasma hyorhinis in all samples. Cytology revealed We report a spontaneous mutation that was observed in a Swiss neutrophils, lymphocytes, and basophilic inclusions within epithelial Webster breeding colony. The colony is used to generate sentinel cells. Biopsy samples revealed widespread submucosal lymphoplas- mice. The mice are bred in an isolator and removed from the isolator macytic infiltration with marked lymphoid follicle formation. at weaning. Although outbred, the colony is closed and inbreeding is Aerobic cultures grew various bacteria including Staphylococcus spp., likely due to the small numbers of breeding mice. The mice with the Streptococcus spp., and multiple coliforms. Prophylactic oxytetracy- mutation demonstrated prominently shortened front limbs with cline (2mg/kg IM SID) for 4 days prior to shipment reduced the missing digits. The footpads were deformed and found on the dorsal incidence of severe conjunctivitis to 2% (1/57) over the following 6 surface of the front limbs (versus the normal ventral location). months. PCR analysis of conjunctival swabs from 9 clinically normal Radiographs demonstrated absence of an ulna and missing phalan- pigs from the same farm, as well as two other farms revealed M. geal bones. Affected males and females were infertile. Breeding hyorhinis in all samples analyzed. These findings definitively experiments were set up with normal siblings to affected animals to document that M. hyorhinis, a known component of normal upper determine heritability and heritability pattern. Normal appearing respiratory tract flora in pigs, is also a common inhabitant of the siblings were intercrossed. Once a pair yielded mutant mice, they conjunctiva. Under certain conditions, M. hyorhinis is an were identified as potential heterozygotes or carriers of the trait. opportunistic pathogen, typically associated with polyserositis, These were then test mated with other normal siblings. Punnet arthritis, and otitis. The diagnostics and response to oxytetracycline squares of expected phenotypes were examined; and, assuming basic in this report strongly suggest M. hyorhinis may also cause severe Mendelian genetics of dominant and recessive phenotypes, the trait conjunctivitis. This is the first definitive report of M. hyorhinis as appeared to be completely recessive. Tail biopsies were collected normal flora of the swine conjunctiva, and these data suggest that M. from all offspring and identified as either carrier, affected, or hyorhinis may act as an opportunistic pathogen in the conjunctiva wildtype (based on breeding results and phenotype). Literature under stressful conditions such as transport and acclimation. reviews lead us to believe that the Wnt 7a pathway was involved as Importantly, our data shows that this severe conjunctivitis can be this mutation has been reported in outbred ICR mice. A PCR was largely prevented with prophylactic antibiotic therapy. optimized for the exons of Wnt 7a and the 4 exons were examined. Analyses revealed that there were no exon size differences among the PS121 Evaluation of Firocoxib in Sprague–Dawley Rats Using a 3 different phenotypes; however, this does not rule out a single nucle- Plantar Incision Model of Pain otide 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 TE Noe*, TJ Pavek, WS Schwark, BS Singh undergone or whether it is a spontaneous mutation. Spontaneous mutations do occur and can contribute to our understanding of Cornell University, Ithaca, NY disease processes. They also serve as a valuable educational tool on basic genetics for those in laboratory animal science. For rodent analgesia, NSAIDs are preferable over opioids due to reduced frequency of required dosing and ease of availability. We PS123 Focal Naso-Maxillary Swelling and Hemorrhage from an Ear sought to evaluate the relative efficacy of firocoxib, a highly COX-2 Canal in a Cynomologus Macaque (Macaca fascicularis) selective NSAID, versus meloxicam and buprenorphine for reducing KE Scott*, V Bakthavatchalu, JG Fox, RP Marini allodynia and hyperalgesia in rats in the plantar incision model of surgical pain. Sprague–Dawley rats (n = 12 per group, 6 per group of Division of Comparative Medicine, Massachusetts Institute of each gender) were divided into 6 groups: sham (anesthesia, no Technology, Cambridge, MA surgery), control (no analgesia), buprenorphine (0.05 mg/kg SC q8hr), meloxicam (2 mg/kg SC q24hr), firocoxib 10 (10 mg/kg SC A 15-yr-old, intact, singly housed, male cynomolgus macaque q24hr), and firocoxib 20 (20 mg/kg, SC q24hr). An incision was made (Macaca fascicularis) with a head-post implant presented with an into the plantar aspect of the right hind foot, the plantaris muscle was acute focal swelling infraorbital, overlying the maxillary bone (1 cm elevated, and the incision closed with 6-0 nylon suture. Nociceptive diameter), which ulcerated and had serous discharge within 48 hours assays were performed using von Frey and Hargreaves methodology post presentation. Two and a half years prior to presentation, the to test mechanical allodynia and thermal hyperalgesia, respectively. animal had been treated for purulent otitis externa and an oral These assays were performed at 24, 20, 28, 44, 52, 68, and 76 hours abscess from which Staphylococcus aureus and Streptococcus pyo- after surgery. Data were analyzed using mixed-model ANOVA, and a genes were cultured. Multiple radiographic studies revealed no oral Bonferroni correction for multiple comparisons was applied. The von or otic pathology. Biopsies of the facial swelling were nondiagnostic Frey assay revealed increased allodynia with no evidence of and aerobic, anaerobic, and fungal cultures were negative. An MRI significant alleviation of nociception after firocoxib or meloxicam was performed and during the imaging session, the right ear bar was injection, and minimal evidence of alleviation of nociception after difficult to place due to hyperplastic tissue in the ear canal. Blood buprenorphine. The Hargreaves assay revealed no statistically drained from the canal after the procedure. MRI revealed that the significant difference between any of the analgesic groups and the rostral aspect of the facial swelling had a focal hyperintense linear control group at any timepoint. Additionally, females appeared to tract that extended 5mm into the underlying tissues and a less experience greater hyperalgesia in the Hargreaves assay than males, intense soft tissue material extending through a small focal defect in though this trend did not reach statistical significance. These findings the maxillary bone to the level of the root of the right maxillary suggest that commonly recommended dosages of analgesics do not canine tooth. MRI also revealed heterogenous T2W hyperintensity provide sufficient analgesia in rats in the plantar incisional model of filling the right tympanic bulla with extension of strongly T2W pain. 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

708 Abstracts of scientific papers 2016 AALAS National Meeting

on MRI findings, a diagnosis of otitis media and periodontitis was = 43) and 4 out of 5 were of subordinate status in their social groups, confirmed. Myringotomy and tympanocentesis was attempted but suggesting a predisposition toward this phenotype. This is the first unsuccessful due to the depth and narrow diameter of the external report of nongluten, adverse food reactions in laboratory macaques. ear canal. The right maxillary canine was extracted, and histology of Our findings indicate that social stress (subordinate status) and a associated soft tissue showed an infiltration of a low number of preexisting, proinflammatory phenotype (high serum globulins) may neutrophils, macrophages, lymphocytes, and plasma cells. Due to the have predisposed these animals to an adverse food reaction. small size of the tympanic bulla of cynomolgus macaques, Additionally, the findings of elevated serum eosinophils and radiographic examination for bulla-associated pathology is not as resolution of clinical symptoms following walnut-elimination diet sensitive as in other species. In this animal, the otitis media remained suggest a potential intolerance of some individuals to tree nuts. subclinical, without signs of discomfort or neurologic signs. Otitis media can remain subclinical chronically and is difficult to confirm PS126 Review and Enhancement of Rodent Body Condition and culture in cynomolgus primates. Monitoring Program

PS124 Urinary System Lesions Associated with a Rat Model of S Shainker*, DM Molk Spinal Cord Injury Center for Comparative Medicine, Massachusetts General Hospital, RL Munden*, JL Booth, TK Cooper Charlestown, MA

Comparative Medicine, Penn State University, Hershey, PA We empower our front line husbandry staff, or research animal specialists (RAS), to use our veterinary-derived rodent health poster Each year approximately 12,000 individuals suffer a traumatic spinal to perform daily health observations. The poster is composed of a cord injury. Rats are the most widely used animal model to study designated treatment plan for 23 common rodent health concerns. spinal cord injury. Recently at our facility, necropsy evaluation of 5 Our most commonly reported health concern is poor body condition male Sprague–Dawley rats following experimental traumatic spinal (PBC) (30-40% of all health concerns) with an average treatment cord injury revealed that all cases had significant disease of the timeline of ~6 weeks. Monitoring of animals during treatment varied urinary system. Cystitis, pyelonephritis, lower urinary tract among staff who shared their confusion regarding when to close PBC obstruction, and urinary bladder rupture were observed. Severe cases as resolved. Veterinary validation of resolution was common. dermatitis and cellulitis secondary to urine scald were observed in Using Plan-Do-Check-Act (PDCA), a lean management problem- one of the cases. Evaluation of the lesions for these 5 rats prompted solving tool, an assessment of 25 current PBC cases in 1 facility was exploration of treatments and supportive care options for these performed. Each cage was followed from initial health alert to studies going forward. A literature search revealed treatment and resolution. Data on supply cost, staff time, and animal body weight management options such as indwelling (suprapubic) catheters, was collected. This assessment revealed that 84 min and $56.60 were intermittent urinary catheterization, and the use of a hand-held spent per case. It also revealed that body weights were stable or digital ultrasound imaging system to monitor urinary retention in the improving long before staff stopped performing daily monitoring. bladder. Pharmacologic treatment with prazosin, dantrolene, or light Weights were tracked on newly reported cases until they stabilized. induction with isoflurane may be additional options to facilitate Within 2 weeks of daily supplementation, mice maintained, gained, effective and less traumatic emptying of the bladder. Additionally, or lost weight; treating mice longer than 2 weeks did not alter the adjusting the amount of subcutaneous fluids and/or the amount of outcome of the case. A new standard approach for treating PBC cases, times the bladder is expressed per day may be other considerations including a prescribed 2-week monitoring period was created. Cases to aid in bladder maintenance for future studies. Awareness of are treated daily with supplements (Napa Nectar) for 2 weeks, after potential complications associated with this model allows better which the case is closed. This saved 60 mins and $36.40 per case and recognition of clinical concerns, guides treatment recommendations addressed staff hesitation to close a case. It is important to regularly and minimizes potential animal distress. review standard processes in our facilities to assess if workflow efficiency can be improved and to ensure that staff have clear PS125 Adverse Food Reaction to a Novel Diet in Adult Cynomol- guidance on veterinary care expectations. gus Macaques (Macaca fascicularis) PS127 Putative Virus Identified during Colony Health Monitoring MS Metzler*, E Floyd, B Uberseder, T Register, S Appt, K Michalson, with a Novel MTLV Antigen A LeGrande, M Gee, C Shively RJ Ricart Arbona*, NS Lipman, FR Wolf Pathology/Comparative Medicine, Wake Forest University, Winston-Salem, NC CCMP, Memorial Sloan Kettering Cancer Center, New York City, NY

Five out of 21 socially housed (3-4/group), adult female cynomolgus During routine sentinel health screening, soiled bedding sentinels macaques (Macaca fascicularis) presented with diarrhea and weight (SBS) from 2 cages in room A (RA) from a 4-room vivarium (FB) tested loss within 1 year of being switched from commercial monkey chow positive for Mouse T Lymphotrophic Virus (MTLV) by multiplex to a novel diet, designed to mimic healthy human diets with most of fluorescent immunoassay (MFIA) in house (IH) and confirmed at the protein and fat from plant sources. Physical exam findings Diagnostic Laboratory A (DLA) by MFIA (using the same antigen [Ag] revealed poor body condition, dehydration, and gas-filled intestines. used IH) and IFA. After discussions with DLA, the positive samples Laboratory findings included hypoalbuminemia (≤ 2.6 g/dL; 5 out of were retested and the IFA results were subsequently classified as 5 cases), leukocytosis (WBC > 16,000 cells/µL; 4 out of 5 cases), and nonspecific (TC), and the samples were reclassified as MTLV negative. eosinophilia (> 6%, 4 out of 5 cases). Abdominal ultrasound revealed Concurrently, 7 of the remaining 8 SBS cages, in the other 3 FB rooms, gas- and fluid-filled intestines with mildly increased mucosal were confirmed MTLV MFIA positive and IFA TC. The only other SBS distinction and wall thickening. Clinical and laboratory abnormali- cage having the same serologic profile on our campus of >360 SBS ties improved with cessation of the novel diet and returned when cages received soiled bedding from mice belonging to Research Group diet was reinitiated. Clinical improvement persisted after termination A (RGA), which had most of their mice in FB and routinely transferred of the novel diet. One animal was switched to a Western-like diet animals between these sites. It was determined that DLA had switched (protein from casein and whey), 2 were switched to commercial to a novel in vitro method (permissive lymphocyte cell line supple- monkey chow, and 2 were switched to a walnut-free version of the mented with several rodent-origin biologics) to generate the Ag used novel diet. Evaluation of laboratory values obtained prior to in their MFIA and also the MTLV-infected cells for IFA ~ 1 year prior. introduction of novel diet revealed that all cases had serum globulin Testing of a select number of the MTLV + samples by Diagnostic Lab concentrations in the top 10% (> 3.6 g/dL) of the total study cohort (n B, which uses the classic in vivo produced MTLV Ag, were negative.

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Further analysis revealed that the MTLV+ sera reacted, although less accidentally found while the dam was assisted through Cesarean intensely, to the uninfected cells suggesting that the serologic activity section. Major findings showed abdominal hemorrhage from a rup- was directed to an unknown virus carried by the cell line. Results from tured, grossly distended left uterus displaying 1 single fetus having additional tests including mouse antibody production, anatomic pa- a bilobed placenta. Subsequent obstetric manipulation for extraction thology, electron microscopy, and genomic sequencing of the cell line resulted in 9 fetuses among 11 pups. It was estimated that the dysto- will be presented and important issues to consider when interpreting cia was the result of the presence of 2 larger, malposed dead fetuses diagnostic assays will be reviewed. observed through the translucent uterine wall in caudal position blocking the birth canal. All fetuses and their placental remmants PS128 Gross and Morphologic Characterization of an Unusual were retrieved and weighed and the crown-rump index recorded. Bilobed Placenta in a Sprague–Dawley Rat 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 EF Tena Betancourt*2,1, S Jimenez3, G Carrillo-Marhx1, V Graullera3, the birth canal 6.44 g. Total body lengths (TBL) measured 4.88, 4.55, AM Zúñiga-Muñoz3, C Jiménez-Gutiérrez1, P Hernández-Jáuregui4 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, 1Animal Facility Services and Experimental Surgery, Universidad 0.57 (0.559 ± 0.176) respectively (fetuses 1-9). Microscopically, we La Salle, A.C., Mexico City, Mexico; 2Ethology, Wildlife and Labo- studied placentas belonging to animal 7 naming them primary (PP) ratory Animals, Universidad Nacional Autónonoma de México, and secondary (SP). Histologically, both placentas showed remark- Mexico City, Mexico; 3Pathology, INC Ignacio Chávez, Mexico City, able comparative analogies including the labyrinth zone, basal zone, Mexico; 4Dirección, Cyta Labs, AC, Puebla, Pue, Mexico and decidua. Morphologic features of PP demonstrated the labyrinth and basal zones congested with profuse erythrocytes delimitated by Placental development constitutes a key element in pregnancy. Exten- 1 hematoma. The SP exhibited a smaller hematoma among the basal sive research in eutherian mammals has reported numerous placental and labyrinth zones and scarce trophoblastic giant cells. The com- types depending on their gross shape and histologic structure of the municating umbilical cord showed a typical morphology including 2 materno-fetal interface, among other structures. However, no studies arteries and 1 vein buried within Wharton’s jelly. No anomalies were have reported an unusual twin placenta form in laboratory rats. This discerned among both placentas. This study highlights a striking report characterizes a bilobed placenta in a multiparous, 8-month- reproductive rarity never reported in an extensively studied mammal old Sprague–Dawley rat suffering maternal dystocia. This rarity was as the laboratory rat.

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