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 Cross-sectional Survey on Rodent Environmental Health Monitoring Practices: Benchmarking, Associations, and Barriers.

Tens of thousands of rodents are used each year in Rodent Health Monitoring programs. However, Environment Health Monitoring (EHM) could replace sentinel rodent use while maintaining or even improving diagnostic quality. Despite its advantages, widespread implementation of EHM appears to be relatively low. To better understand EHM's prevalence and factors influencing its use, we surveyed research animal professionals. Our hypotheses were (1) EHM prevalence would be low and (2) EHM use would be associated with beliefs and knowledge about EHM. Participants were recruited via online promotion. A total of 158 individuals completed a mixed-methods survey about current practices, beliefs, and knowledge about EHM. Qualitative data were coded using thematic analysis and analyzed using generalized linear models. Results showed that current EHM implementation was low; only 11% of institutions used EHM exclusively. Across the 111 institutions surveyed, over 20,000 soiled bedding sentinels were used each year. However, most participants believed EHM to be advantageous in replacing sentinel animals (78% of participants). Some participants believed EHM could save time (31%), cost less (27%), and be highly accurate (15%). Conversely, some participants believed EHM would be difficult to use due to their current caging type (40%), higher costs (21%), lower accuracy (16%), and personnel attitudes/expertise (14%). Overall, respondents with higher planned EHM use also had more positive attitudes, norms, and control of EHM. We also identified several factors that could promote the implementation of EHM. Communication efforts should emphasize that EHM is compatible with various types of caging, can provide cost savings, has high accuracy, and is consistent with the 3Rs as a replacement. Efforts should also focus on improving attitudes, encouraging peers, and providing resources to facilitate implementation. Implementation in just the surveyed institutions could eliminate the need for well over 20,000 rodents each year, consistent with 3Rs goals.

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.

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.

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.

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.

Low-cost, Small-scale Decontamination of Laboratory Equipment by Using Chlorine Dioxide Gas.

A significant concern in laboratory animal medicine is contamination due to pathogen outbreaks and how to adequately decontaminate small equipment. Many factors play a role in the selection of the decontamination method including cost, efficacy, personnel time and safety. Chlorine dioxide (ClO2) gas is an effective method, but decontamination often requires a ClO2 gas generator with a specialized air-tight exposure chamber. Although this method works well for large-scale decon- tamination, the use of a gas generator may be impractical and too costly for smaller-scale decontamination. The goal of this study was to create and validate an effective, small-scale decontamination method that uses ClO2 gas and which is an affordable, efficient, safe, and reproducible. First, we identified a product that generates ClO2 gas after the combination of 2 dry reagents. To find an affordable exposure chamber, we evaluated the ability of 4 household totes with gasket-seal lid systems to retain ClO2 gas and relative humidity (RH). The efficacy of decontamination was validated by concurrently using 2 different biologic indicators (BI), Bacillus atrophaeus (B.a.) and Geobacillus stearothermophilus (G.s.). All household totes evaluated held sufficient gas and RH for a 15-h cycle, providing adequate contact time to inactivate both BI evaluated. Our results suggest that a total exposure dose of 71 ± 42 ppm-h of ClO2 gas over 15 h at 90% or greater RH is adequate to inactivate both B.a. and G.s. There was no statistical significance between the 2 BI as indicators for decontamination; 65 of 230 (28.3%) B.a. and 75 of 230 (32.6%) G.s spore strips were positive for growth (P = 0.36). In conclusion, we successfully combined a variety of low-cost materials to establish an effective, small-scale method to decontaminate laboratory equipment. Depending on the size of the tote and whether BI are used, the cost of our method is roughly 1% that of large-scale ClO2 gas generators used with specialized air-tight exposure chambers.

Procedure for Horizontal Transfer of Patient-Derived Xenograft Tumors to Eliminate Corynebacterium bovis.

Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

Detection and Elimination of Corynebacterium bovis from Barrier Rooms by Using an Environmental Sampling Surveillance Program.

Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and even replaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance method for the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EAD surveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressive quarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages of infected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficient mice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVC rack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to our ability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into our barrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated, cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used to propagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, test-and-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.

Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds.

Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.

Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.

Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER+ luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg(-/-) mice under estrogen supplementation. Five transplantable patient-derived ER+ breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5-99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a naïve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

Soiled-bedding sentinel detection of murine norovirus 4.

According to serologic surveys, murine norovirus (MNV) is the most prevalent viral pathogen infecting mice used in biomedical research. However, the use of sentinel mice to detect MNV-infected mouse populations has not been evaluated thoroughly. To this end, an experimental method of soiled bedding transfer was created to mimic a quarterly sentinel monitoring program. Soiled bedding (15 or 30 cm3) from ICR mice experimentally infected with MNV4 was transferred weekly to cages of pair-housed 4-wk-old ICR mice. After 12 wk, both mice in 80% (4 of 5) of cages receiving either 15 or 30 cm3 of soiled bedding were seropositive for MNV and were shedding virus in feces. To evaluate the stability of MNV RNA in mouse feces, fecal pellets from MNV-infected sentinel mice were stored at room temperature for as long as 14 d. After storage, all fecal samples tested positive for MNV by RT-PCR. To determine whether fecal samples could be pooled for MNV detection, 1 MNV-positive fecal pellet was combined with either 9 or 19 MNV-negative fecal pellets. All pooled fecal samples were positive for MNV by RT-PCR at both dilutions. These data indicate that although MNV-infected mouse populations can be detected by exposing sentinel mice to MNV-contaminated bedding, detection failures can occur. In addition, there was high agreement in the MNV infection status of cohoused sentinel mice. These data also demonstrate that MNV is readily detectable in pooled fecal samples and in mouse feces stored at room temperature for 2 wk.