Pathogen Prevalence Estimates and Diagnostic Methodology Trends in Laboratory Mice and Rats from 2003 to 2020.

Rodents used in biomedical research are maintained as specific pathogen-free (SPF) by employing biosecurity measures that eliminate and exclude adventitious infectious agents known to confound research. The efficacy of these practices is assessed by routine laboratory testing referred to as health monitoring (HM). This study summarizes the results of HM performed at Charles River Research Animal Diagnostic Services (CR-RADS) on samples submitted by external (non-Charles River) clients between 2003 and 2020. Summarizing this vast amount of data has been made practicable by the recent introduction of end-user business intelligence tools to Excel. HM summaries include the number of samples tested and the percent positive by diagnostic methodology, including direct examination for parasites, cultural isolation and identification for bacteria, serology for antibodies to viruses and fastidious microorganisms, and polymerase chain reaction (PCR) assays for pathogen-specific genomic sequences. Consistent with comparable studies, the percentages of pathogen-positive samples by diagnostic methodology and year interval are referred to as period prevalence estimates (%PE). These %PE substantiate the elimination of once common respiratory pathogens, such as Sendai virus, and reductions in the prevalence of other agents considered common, such as the rodent coronaviruses and parvoviruses. Conversely, the %PE of certain pathogens, for example, murine norovirus (MNV), Helicobacter, Rodentibacter, and parasites remain high, perhaps due to the increasing exchange of genetically engineered mutant (GEM) rodents among researchers and the challenges and high cost of eliminating these agents from rodent housing facilities. Study results also document the growing role of PCR in HM because of its applicability to all pathogen types and its high specificity and sensitivity; moreover, PCR can detect pathogens in samples collected antemortem directly from colony animals and from the environment, thereby improving the detection of host-adapted, environmentally unstable pathogens that are not efficiently transmitted to sentinels by soiled bedding.

Evaluation and Refinement of a Spot-change-only Cage Management System for Mice.

Maximizing operational efficiency while maintaining appropriate animal housing conditions is a continuous focus of research animal care programs. Our institution's longstanding approach to cage-change management included scheduled cage changes every 2 wk, with spot changes if cages met established visual criteria during the intervening period. This 2-wk plus spot changing (2WS) practice for mice housed in IVC was problematic during the COVID-19 pandemic when the need arose to minimize workload to reduce on-site staffing out of concern for employee health and possible absenteeism. With the approval of the IACUC, a spot-change-only (SCO) process was adopted, with the requirement to evaluate microenvironmental parameters under both practices to confirm acceptable equivalence. These parameters (humidity, temperature, and ammonia) were evaluated in a controlled study that found no significant difference between the 2 groups. Ammonia levels did not exceed 10 ppm in any group throughout the study. To assess operational differences between these 2 approaches, we collected cage-change data and employee feedback from facilities operating under these schemes. The SCO method required fewer cage changes than did the 2WS method (10.3% per day with 2WS and 8.4% per day with SCO). Despite this benefit, through a Plan-Do-Check-Act process that has been regularly employed at our institution, employee feedback identified important operational challenges associated with the SCO practice. The SCO approach was thus refined into a scheduled spot change (SSC) practice that builds on the SCO model by incorporating a scheduled focused cage evaluation period. Based on subsequent feedback, the SSC was found to retain the efficiency benefits afforded by the SCO model and simultaneously alleviated staff and operational concerns. This result underscores the importance of integrating staff feedback with a performance standard-based approach when assessing cage-change management.

Lack of social touch alters anxiety-like and social behaviors in male mice.

The importance of social interactions has been reported in a variety of animal species. In human and rodent models, social isolation is known to alter social behaviors and change anxiety or depression levels. During the coronavirus pandemic, although people could communicate with each other through other sensory cues, social touch was mostly prohibited under different levels of physical distancing policies. These social restrictions inspired us to explore the necessity of physical contact, which has rarely been investigated in previous studies on mouse social interactions. We first conducted a long-term observation to show that pair-housed mice in a standard laboratory cage spent nearly half the day in direct physical contact with each other. Furthermore, we designed a split-housing condition to demonstrate that even with free access to visual, auditory, and olfactory social signals, the lack of social touch significantly increased anxiety-like behaviors and changed social behaviors. There were correspondingly higher levels of the pro-inflammatory cytokine interleukin-6 in the hippocampus in mice with no access to physical contact. Our study demonstrated the necessity of social touch for the maintenance of mental health in mice and could have important implications for human social interactions.

Sex-Specific Effects of Plastic Caging in Murine Viral Myocarditis.

BACKGROUND: Myocarditis is an inflammatory heart disease caused by viral infections that can lead to heart failure, and occurs more often in men than women. Since animal studies have shown that myocarditis is influenced by sex hormones, we hypothesized that endocrine disruptors, which interfere with natural hormones, may play a role in the progression of the disease. The human population is exposed to the endocrine disruptor bisphenol A (BPA) from plastics, such as water bottles and plastic food containers. METHODS: Male and female adult BALB/c mice were housed in plastic versus glass caging, or exposed to BPA in drinking water versus control water. Myocarditis was induced with coxsackievirus B3 on day 0, and the endpoints were assessed on day 10 post infection. RESULTS: We found that male BALB/c mice that were exposed to plastic caging had increased myocarditis due to complement activation and elevated numbers of macrophages and neutrophils, whereas females had elevated mast cell activation and fibrosis. CONCLUSIONS: These findings show that housing mice in traditional plastic caging increases viral myocarditis in males and females, but using sex-specific immune mechanisms.

Effects of long-term social isolation on central, behavioural and metabolic parameters in middle-aged mice.

Social isolation gained discussion momentum due to the COVID-19 pandemic. Whereas many studies address the effects of long-term social isolation in post-weaning and adolescence and for periods ranging from 4 to 12 weeks, little is known about the repercussions of adult long-term social isolation in middle age. Thus, our aim was to investigate how long-term social isolation can influence metabolic, behavioural, and central nervous system-related areas in middle-aged mice. Adult male C57Bl/6 mice (4 months-old) were randomly divided into Social (2 cages, n = 5/cage) and Isolated (10 cages, n = 1/cage) housing groups, totalizing 30 weeks of social isolation, which ended concomitantly with the onset of middle age of mice. At the end of the trial, metabolic parameters, short-term memory, anxiety-like behaviour, and physical activity were assessed. Immunohistochemistry in the hippocampus (ΔFosB, BDNF, and 8OHDG) and hypothalamus (ΔFosB) was also performed. The Isolated group showed impaired memory along with a decrease in hippocampal ΔFosB at dentate gyrus and in BDNF at CA3. Food intake was also affected, but the direction depended on how it was measured in the Social group (individually or in the group) with no alteration in ΔFosB at the hypothalamus. Physical activity parameters increased with chronic isolation, but in the light cycle (inactive phase), with some evidence of anxiety-like behaviour. Future studies should better explore the timepoint at which the alterations found begin. In conclusion, long-term social isolation in adult mice contributes to alterations in feeding, physical activity pattern, and anxiety-like behaviour. Moreover, short-term memory deficit was associated with lower levels of hippocampal ΔFosB and BDNF in middle age.

Serological Screening for Antibodies against SARS-CoV-2 in Dutch Shelter Cats.

The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%; CI 95%: 0.1-3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.

Reuse of Disposable Isolation Gowns in Rodent Facilities during a Pandemic.

Reuse of disposable personal protective equipment is traditionally discouraged, yet in times of heightened medical applications such as the SARS CoV-2 pandemic, it can be difficult to obtain. In this article we examine the reuse of disposable gowns with respect to still providing personnel protection. XR7, a fluorescent powder, was used to track contamination of gowns after manipulation of rodent cages. Mouse cages were treated with XR7 prior to manipulations. Disposable gowns were labeled for single person use and hung in common procedure spaces within the vivarium between usages. A simulated rack change of 140 cages was completed using XR7-treated cages. One individual changed all cages with a break occurring after the first 70 cages, requiring the gown to be removed and reused once. To simulate research activities, 5 individuals accessed 3 XR7-treated cages daily for 5 d. Each mouse in the XR7-treated cages was manipulated at least once before returning cages to the housing room. Disposable gowns were reused 5 times per individual. Gowns, gloves, clothing, bare arms, and hands were scanned for fluorescence before and after removing PPE. Fluorescence was localized to gloves and gown sleeves in closest contact with animals and caging. No fluorescence was detected on underlying clothing, or bare arms and hands after removing PPE. Fluorescence was not detected in procedure spaces where gowns were hung. The lack of fluorescence on personnel or surfaces indicate that gowns can be reused 1 time for routine husbandry tasks and up to 5 times for research personnel. A method for decontamination of used gowns using Vaporized Hydrogen Peroxide (VHP) was also validated for use in areas where animals are considered high risk such as quarantine, or for fragile immunocompromised rodent colonies.

Effects of different mating strategies in broiler breeder during peak and postpeak phase on subsequent broiler performance.

Two experimental trials on commercial broiler (Ross-308) were conducted to evaluate the carryover effect of artificial insemination (AI) in parent flock (PF) kept in cages (C), and on floor (F) in comparison to natural mating (NM) in floored PF. A total of 900 broiler chicks were obtained from 38-week-old PF (peak production), representing C, F, and NM evenly during first trial, whereas in second trial, similar number of chicks were obtained from same PF during postpeak phase (55 wk of age). Subsequent effects of AI and NM in PF were evaluated by bacteriology, posthatch mortality, growth performance, immune response, and carcass traits on experimental birds (broiler). Chicks being produced through NM exhibited significantly (P ≤ 0.05) improved growth performance (feed conversion ratio, weight gain, European efficiency factor) along with the least (P ≤ 0.05) posthatch mortality and prevalence of Escherichia coli, Salmonella Pullorum, and Mycoplasma gallisepticum. Moreover, the experimental chicks obtained from floored PF subjected to AI particularly during postpeak phase expressed the highest (P ≤ 0.05) contamination of the said pathogens along with posthatch mortality. However, immune response against New Castle disease and infectious bronchitis vaccines and slaughtering parameters remained nonsignificant (P > 0.05) among the 3 treatments under both trials. It is concluded that the best growth performance along with the least depletion and microbial load of concerned pathogens were being pertained by the experimental birds representing NM.