General parity between trio and pairwise breeding of laboratory mice in static caging.

Changes made in the 8th edition of the Guide for the Care and Use of Laboratory Animals included new recommendations for the amount of space for breeding female mice. Adopting the new recommendations required, in essence, the elimination of trio breeding practices for all institutions. Both public opinion and published data did not readily support the new recommendations. In response, the National Jewish Health Institutional Animal Care and Use Committee established a program to directly compare the effects of breeding format on mouse pup survival and growth. Our study showed an overall parity between trio and pairwise breeding formats on the survival and growth of the litters, suggesting that the housing recommendations for breeding female mice as stated in the current Guide for the Care and Use of Laboratory Animals should be reconsidered.

Antibiotic Treatment of Corynebacterium bovis-associated Clinical Disease in NSG Mice.

Depending on the strain of immunodeficient mice, Corynebacterium bovis infection can be asymptomatic or cause transient or prolonged skin disease. C. bovis infection of NOD. Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice results in clinical skin disease that progresses in severity. Amoxicillin metaphylaxic and prophylaxic therapy prevents transmission and infection of mice after exposure to C. bovis and inhibits the growth of C. bovis isolates at therapeutic doses that are clinically achievable in mice. Amoxicillin is not efficacious for treatment of transient clinical skin disease in athymic nude mice, but the efficacy of amoxicillin treatment has not previously been characterized in C. bovis -infected NSG mice. In the current study, NSG mice were treated with amoxicillin beginning at 5 wk after exposure to C. bovis, at which time they had well-established clinical signs of disease. Clinical signs were scored to assess disease progression, regression, and reappearance. Our results showed that amoxicillin treatment for 3 or 6 wk reduced the clinical scores of NSG mice with C. bovis -associated clinical disease. In addition, withdrawal of treatment led to the recurrence of clinical signs. Collectively, our data suggest that amoxicillin treatment is effective in alleviating the clinical signs associated with C. bovis infection for the duration of treatment in NSG mice. Clinical intervention with antibiotics for C. bovis -infected NSG mice can be an option for management of C. bovis -related clinical disease either before or during facility-wide remediation efforts.

Effects of Extended Cage Component Sanitation Interval on the Microenvironment, Health, and Gastrointestinal Microbiome of Rats (Rattus norvegicus).

Washing and sanitizing rodent cage components requires costly equipment, significant personnel effort, and use of natural resources. The benchmark frequency for sanitation of individually ventilated caging (IVC) has traditionally been every 2 wk. In this study, we investigated the effects of extending this interval on the cage microenvironment, basic markers of health, and the gastrointestinal microbiota of rats. We compared our institutional standard of changing the sanitation interval for rat cage lids, box feeders, and enrichment devices from every 4 wk to an interval of 12 wk. The cage bottom and bedding continued to be changed every 2 wk for both groups. We hypothesized that we would find no significant difference between our current practice of 4 wks and continuous use for 12 wk. Our data showed that intracage ammonia levels remained below 5 ppm for most cages in both groups, with the exception of cages that experienced a cage flood. We found no significant difference between groups in bacterial colony forming units (CFU) on cage components. We used 3 novel methods of assessing cleanliness of enrichment devices and found no significant effect of continuous use for 12 wk on the number of CFU. In addition, we found no significant differences between groups for animal weight, routine blood work, or fecal and cecal microbiomes. These data indicate that a sanitation interval of up to 12 wk for components of rat IVC caging has no significant effects on the microenvironment or health of rats. Using the longer interval will improve efficiency, reduce the use of natural resources, and decrease costs while maintaining high-quality animal care.

A Clinical Scoring Systems for the Evaluation of Corynebacterium bovis -associated Disease in NSG Mice.

Clinical signs of Corynebacterium bovis infections are well-known in athymic nude mice. However, C. bovis can also infect and cause clinical signs in many hirsute, immunocompromised mouse strains such as NSG (NOD. Cg-Prkdcscid Il2rgtm1Wgl/SzJ). Typically, the clinical assessment of C. bovis-infected mice begins when overt clinical signs are initially observed and thus the early course of infection has not been thoroughly described. The goal of this study was to characterize the clinical progression of C. bovis infection in NSG mice under experimental conditions and develop a quantifiable clinical scoring system. For the development and application of this clinical scoring system, 54 naïve NSG mice were exposed to soiled bedding from clinically ill C. bovis-infected NSG mice and the emergence of clinical signs was monitored and scored weekly for 8 wk. Overall, we identified 6 benchmark changes associated with C. bovis clinical infection. Four changes were the appearance of the eyes, ears, hair coat, and posture. Two behavioral changes were increased grooming activity and rapid head shaking. All clinical signs appeared consistently and progressed temporally with increasing clinical severity. Characterization of clinical signs and scoring of clinical disease will aid veterinarians in the assessment of C. bovis-infected NSG mice and may help in the evaluation of current and future clinical interventions used to prevent or treat C. bovis-infected immunodeficient mice.

Characterization of a Jumping Stereotypy in Gerbils (Meriones unguiculatus) and Assessment of Opaque Tubing Enrichment on Stereotypies and Breeding.

Mongolian gerbils can develop stereotypic behaviors, including corner digging. At our institution, gerbils also engage in repetitive corner jumping, which we sought to characterize as a potentially novel stereotypy in gerbils. We then attempted to mitigate this behavior by mimicking the natural habitat by adding intracage environmental complexity. Seventeen gerbil breeding pairs were video recorded in their home cages during the light cycle. Repetitive corner jumping and digging were compared between different times of day to assess when the behaviors occurred and whether they were temporally associated. To determine whether we could reduce the incidence of stereotypic behaviors, we tested a straight tube or 1 of 3 angled opaque tubes in different orientations, which were fitted to the gerbils' preexisting opaque nesting box. Behavior was assessed at baseline and at 1, 4, 8, and 12 wk to evaluate opaque tube placement as an intervention. In addition, breeding efficiency, valuated as the number of gerbil pups born and weaned per breeder pair, was compared with pre- and poststudy data. The number of corner jumps was highest at the end of the light cycle and the majority were associated with corner digging. After placement of the enrichment tubes, an initial increase in corner digging behavior was observed and persisted throughout the study period. The opaque tubes were not associated with significant changes in corner jumping. After adjusting for age, the addition of opaque tubing to gerbil breeding cages was not associated with significant changes in breeding efficiency. The addition of opaque tubing did not effectively address concerns about stereotypic behaviors and was associated with a chronic increase in stereotypic corner digging among breeding gerbil pairs.

Effect of Antimicrobial Prophylaxis on Corynebacterium bovis Infection and the Skin Microbiome of Immunodeficient Mice.

Corynebacterium bovis is an opportunistic pathogen of the skin of immunodeficient mice and is sensitive to oral antibiotics that reach therapeutic blood concentrations. However, prophylactic antibiotics are considered to be ineffective at preventing C. bovis infection. In addition, the effect of C. bovis on the skin microbiome (SM) of common immunodeficient mouse strains has yet to be characterized. Consequently, we evaluated whether oral prophylactic antibiotics prevent C. bovis infection after inoculation. An infectious dose of C. bovis was applied to the skin of Hsd:Athymic Nude (nude) and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were then housed individually and assigned randomly to receive either untreated drinking water (Cb+Abx-group) or prophylactic amoxicillin-clavulanic acid in the drinking water (0.375 mg/mL) for 14 d (Cb+Abx+group). A third treatment group of each mouse strain was uninoculated and untreated (Cb-Abx-group). Mice from all groups were serially sampled by using dermal swabs to monitor C. bovis infection via quantitative real-time PCR and the SM via 16S rRNA sequence analysis. Fourteen days of prophylactic antibiotics prevented the perpetuation of C. bovis skin infection in both strains. Only the combination of C. bovis inoculation and oral antibiotics (Cb+Abx+) significantly affected the SM of NSG mice at day 14; this effect resolved by the end of the study (day 70). In mice that did not receive antibiotics, C. bovis significantly altered the SM of nude mice but not NSG mice at days 14 and 70. These findings demonstrate the potential benefit of prophylactic antibiotics for prevention of C. bovis infection. However, indirect effect of antibiotics on commensal bacteria and potential effects on xenograft models must be considered.

Antimicrobial Susceptibility of Corynebacterium bovis Isolates from Immunodeficient Rodents.

Corynebacterium bovis, the causative agent of hyperkeratotic dermatitis in immunodeficient mice, is a significant problem in preclinical oncology research. Infection results in lifelong skin colonization and a decrease in successful engraftment of patient-derived xenograft tumor models. The use of antimicrobial agents for C. bovis is controversial in light of reports of poor efficacy and the possibility of selection for resistant strains. The purpose of this study was to describe the antimicrobial susceptibilities of C. bovis isolates obtained exclusively from immunodeficient rodents in order to aid in antimicrobial dose determination. Between 1995 and 2018, 15 isolates were collected from 11 research institutions across the United States. Antimicrobial susceptibility testing was performed for 24 antimicrobials commonly used against gram-positive bacteria. Our results provide an updated understanding of the susceptibility profiles of rodent C. bovis isolates, indicating little variability between geographically and temporally distant isolates. These results will facilitate appropriate antimicrobial use to prevent and treat C. bovis infections in immunodeficient rodents.

Metaphylactic Antibiotic Treatment to Prevent the Transmission of Corynebacterium bovis to Immunocompromised Mouse Offspring.

Current methods for eradicating Corynebacterium bovis, such as depopulation, embryo transfer, and cesarean rederivation followed by cross fostering, are expensive, complex, and time-consuming. We investigated a novel method to produce immunocompromised offspring free of C. bovis from infected NOD. Cg-PrkdcscidIl2rgtm1Wgl/SzJ (NSG) breeding pairs. Adult NSG mice were infected with C. bovis, paired, and randomly assigned to either a no-antibiotic control group (NAB, n = 8) or a group that received amoxicillin-clavulanic acid (0.375 mg/mL) in their drinking water for a mean duration of 7 wk (AB group, n = 7), spanning the time from pairing of breeders to weaning of litters. The AB group also underwent weekly cage changes for 3 wk after pairing to decrease intracage C. bovis contamination, whereas the NAB mice received bi-weekly cage changes. Antibiotics were withdrawn at the time of weaning. All litters (n = 7) in the AB group were culture- and qPCR-negative for C. bovis and remained negative for the duration of the study, whereas all litters in the NAB group (n = 6) remained C. bovis positive. A single adult from each breeding pair was sampled at weaning and at 5 and 10 wk after weaning to confirm the maintenance of (NAB) or to diagnose the reemergence (AB) of C. bovis infection. By the end of the study, C. bovis infection had returned in 3 of the 7 (43%) tested AB adults. Our data suggest that metaphylactic antibiotic use can decrease viable C. bovis organisms from adult breeder mice and protect offspring from infection. However, using antibiotics with frequent cage changing negatively affected breeding performance. Nevertheless, this technique can be used to produce C. bovis-free NSG offspring from infected adults and may be an option for salvaging infected immunocompromised strains of mice that are not easily replaced.

Diagnosis, Surveillance and Management of Streptococcus equi subspecies zooepidemicus Infections in Chinchillas (Chinchilla lanigera).

During a 6-mo period, two 5-6 mo old female chinchillas (Chinchilla lanigera) were examined at the University of Colorado Anschutz Medical Campus after the discovery of firm, nonmobile masses in the left ventral cervical and left axillary region. Other than these findings and mild weight loss, both chinchillas' physical exams were normal. Bloodwork revealed an inflammatory leukogram characterized by leukocytosis, toxic neutrophils, lymphopenia, and monocytosis with mild, nonregenerative anemia. At necropsy, both masses were identified as abscesses. Streptococcus equi, subspecies zooepidemicus (S. zooepidemicus) was isolated in pure culture. Histology of the lungs, liver, spleen, and kidneys showed a marked increase in the numbers of both polymorphonuclear leukocytes and lymphocytes. Both animals were deemed unsuitable for research and were euthanized under isoflurane anesthesia by an intracardiac injection of pentobarbital sodium solution. S. zooepidemicus is an opportunistic, commensal organism found in the upper respiratory tract of horses. This organism has been documented to cause disease in other species and is zoonotic. Infections in humans have been reported, resulting in glomerulonephritis, endocarditis, septic arthritis, osteomyelitis, meningitis, and death. To aid in diagnosis and prospective surveillance of this bacteria, oral and nasal swabs were collected from the remaining cohort of chinchillas, and a qPCR screening assay was implemented. Within 12 mo, 4 of 41 additional females tested positive by culture or qPCR, resulting in a disease prevalence of 14% (6 of 43). However, only 2 of the additional 4 S. zooepidemicus positive animals developed clinical signs. The potential for the spread of infection, zoonosis, and adverse effects on research demonstrate that surveillance for S. zooepidemicus should be considered in a biomedical research environment.

Effects of an Extended Cage-change Interval on Ammonia Levels and Reproduction in Mongolian Gerbils (Meriones unguiculatus).

Prompted by the cage cleanliness of Mongolian gerbils (Meriones unguiculatus), we evaluated a prolonged cage-change interval. We compared the effects of a 2-wk and 6-wk cage-change schedule on ammonia levels, temperature, humidity, and reproductive performance in breeding pairs housed in IVC. We hypothesized that ammonia levels would remain below our threshold for cage changing and that reproductive performance would not be affected. Although ammonia levels increased over time, they remained low (less than 5 ppm) over the 6-wk period. In addition, the 6-wk cage-change interval did not significantly influence reproductive parameters, such as average pup weaning weight, number of litters, and number of pups per litter. We conclude that an extended cage-change interval (6-wk) can be used for gerbils without significant increases in intracage ammonia levels or effects on reproduction.

Transmission of Chagas disease via blood transfusions in 2 immunosuppressed pigtailed macaques (Macaca nemestrina).

A 2.25-y-old male pigtailed macaque (Macaca nemestrina) was experimentally irradiated and received a bone marrow transplant. After transplantation and engraftment, the macaque had unexpected recurring pancytopenia and dependent edema of the prepuce, scrotum, and legs. The diagnostic work-up included a blood smear, which revealed a trypomastigote consistent with Trypanosoma cruzi, the causative agent of Chagas disease (CD). We initially hypothesized that the macaque had acquired the infection when it lived in Georgia. However, because the animal had received multiple blood transfusions, all blood donors were screened for CD. One male pigtailed macaque blood donor, which was previously housed in Louisiana, was positive for T. cruzi antibodies via serology. Due to the low prevalence of infection in Georgia, the blood transfusion was hypothesized to be the source of T. cruzi infection. The transfusion was confirmed as the mechanism of transmission when screening of archived serum revealed seroconversion after blood transfusion from the seropositive blood donor. The macaque made a full clinical recovery, and further follow-up including thoracic radiography, echocardiography, and gross necropsy did not show any abnormalities associated with CD. Other animals that received blood transfusions from the positive blood donor were tested, and one additional pigtailed macaque on the same research protocol was positive for T. cruzi. Although CD has been reported to occur in many nonhuman primate species, especially pigtailed macaques, the transmission of CD via blood transfusion in nonhuman primates has not been reported previously.

Oral squamous cell carcinoma in a pigtailed macaque (Macaca nemestrina).

An adult, gravid, female pigtailed macaque (Macaca nemestrina) presented for facial swelling centered on the left mandible that was approximately 5 cm wide. Differential diagnoses included infectious, inflammatory, and neoplastic origins. Definitive antemortem diagnosis was not possible, and the macaque's condition worsened despite supportive care. Necropsy findings included a mandibular mass that was locally invasive and expansile, encompassing approximately 80% of the left mandibular bone. The mass replaced portions of the soft palate, hard palate, sinuses, ear canal, and the caudal-rostral calvarium and masseter muscle. Histologically, the mass was a neoplasm that was poorly circumscribed, unencapsulated, and infiltrative invading regional bone and soft tissue. The mass consisted of polygonal squamous epithelial cells with intercellular bridging that breached the epithelial basement membrane and formed invasive nests, cords, and trabeculae. The mitotic rate averaged 3 per 400× field of view, with occasional bizarre mitotic figures. Epithelial cells often exhibited dyskeratosis, and the nests often contained compact lamellated keratin (keratin pearls). The neoplasm was positive via immunohistochemistry for pancytokeratin, variably positive for S100, and negative for vimentin, smooth muscle actin, and desmin. The gross, histologic, and immunohistochemical findings were consistent with an aggressive oral squamous cell carcinoma. The neoplasm was negative via PCR for papilloma virus. In general, neoplasia in macaques is rare. Although squamous cell carcinomas are one of the most common oral neoplasia in many species, to our knowledge this case represents the first reported oral squamous cell carcinoma in a pigtailed macaque.

Thromboelastography values from pigtail macaques ( Macaca nemestrina): effects of age and sex.

Thromboelastography is a clinical laboratory test used to assess global hemostasis. With technologic advances and the test's reemergence in human medicine, its utility in veterinary medicine is being explored. Because assays for PT, aPTT, and d-dimers require platelet-poor plasma, whereas thromboelastography is performed on whole blood, thromboelastography provides a more accurate representation of coagulation and allows the identification of hypocoagulable, hypercoagulable, and hyperfibrinolytic states. Conflicting information has been reported about the effects of age and sex on thromboelastog- raphy in humans and animals. Human studies have reported significant effects of age and sex on thromboelastography more often than have animal studies, but few publications are available about thromboelastography in the nonhuman primate and laboratory animal literature. We used a sample of 50 pigtail macaques (Macaca nemestrina) to determine whether age or sex influence thromboelastography values. Of 5 measured and 2 calculated variables produced by thromboelastography, sex had a significant effect only on the lysis-30 parameter, which also showed significant interaction between age and sex; values increased with age in male macaques but decreased with age in female macaques. In addition, we used the data to define reference intervals for thromboelastography parameters in pigtail macaques.

Pulmonary embolization of fat and bone marrow in cynomolgus Macaques (Macaca fascicularis).

Fat embolization (FE), the introduction of bone marrow elements into circulation, is a known complication of bone fractures. Although FE has been described in other animal models, this study represents the first reported cases of FE and bone marrow embolism in nonhuman primates. Histopathologic findings from cynomolgus macaques (Macaca fascicularis) indicated that in all 5 cases, fat and bone marrow embolization occurred subsequent to multiple bone marrow biopsies. In the most severe case, extensive embolization was associated pulmonary damage consistent with acute respiratory distress syndrome. Fat embolism syndrome (FES) is an infrequent clinical outcome of FE and is triggered by systemic biochemical and mechanical responses to fat in circulation. Although clinical criteria diagnostic of FES were not investigated at the time of death, this severe case may represent the fulminant form of FES, which occurs within 12 h after trauma. Bone marrow biopsy as an etiology of FES has been reported only once in humans. In addition, the association of embolization with bone marrow biopsies suggests that nonhuman primates may be a useful animal model of FE. FE and FES represent important research confounders and FES should be considered as a differential diagnosis for clinical complications subsequent to skeletal trauma.