Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Chlamydia muridarum infection causes bronchointerstitial pneumonia in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice.

The murine bacterial pathogen Chlamydia muridarum (Cm) has been used to study human Chlamydia infections in various mouse models. CD4+ T-cells, natural killer cells, and interferon-gamma (IFN-γ)-mediated immunity are important to control experimentally induced Cm infections. Despite its experimental use, natural infection by Cm has not been documented in laboratory mice since the 1940s. In 2022, the authors reported the discovery of natural Cm infections in numerous academic institutional laboratory mouse colonies around the globe. To evaluate the impact of Cm infection in severely immunocompromised mice, 19 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were cohoused with Cm shedding, naturally infected immunocompetent mice and/or their soiled bedding for 4 weeks and subsequently euthanized. Clinical disease, characterized by lethargy, dyspnea, and weight loss, was observed in 11/19 NSG mice, and 16/18 NSG mice had neutrophilia. All mice exhibited multifocal to coalescing histiocytic and neutrophilic bronchointerstitial pneumonia (17/19) or bronchiolitis (2/19) with intraepithelial chlamydial inclusions (CIs). Immunofluorescence showed CIs were often associated with bronchiolar epithelium. CIs were frequently detected by immunohistochemistry in tracheal and bronchiolar epithelium (19/19), as well as throughout the small and large intestinal epithelium without lesions (19/19). In a subset of cases, Cm colonized the surface epithelium in the nasopharynx (16/19), nasal cavity (7/19), and middle ear canal (5/19). Endometritis and salpingitis with intraepithelial CI were identified in a single mouse. These findings demonstrate that Cm infection acquired through direct contact or soiled bedding causes significant pulmonary pathology and widespread intestinal colonization in NSG mice.

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.

Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Research Mouse Colonies.

Chlamydia muridarum (Cm) was detected in 2 colonies of mice with lymphoplasmacytic pulmonary infiltrates by using PCR and immunohistochemistry. This discovery was unexpected, as Cm infection had not been reported in laboratory mice since the 1940s. A Cm specific PCR assay was developed and testing implemented for the resident colonies of 8 vivaria from 3 academic institutions, 58 incoming mouse shipments from 39 academic institutions, and mice received from 55 commercial breeding colonies (4 vendors). To estimate Cm's global prevalence in research colonies, a database containing 11,387 metagenomic fecal microbiota samples from 120 institutions and a cohort of 900 diagnostic samples from 96 institutions were examined. Results indicate significant prevalence among academic institutions, with Cm detected in 63% of soiled bedding sentinels from 3 institutions; 33% of incoming mouse shipments from 39 academic institutions; 14% of 120 institutions submitting microbiota samples; and 16% of the diagnostic sample cohort. All samples from commercial breeding colonies were negative. In addition, naïve NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice exposed to Cm-shedding mice and/or their soiled bedding developed clinical disease at 21 to 28 d after exposure. These mice had a moderate-to-severe histiocytic and neutro- philic bronchointerstitial pneumonia, with their respiratory epithelium demonstrating inclusions, chlamydial major outer membrane protein immunostaining, and hybridization with a Cm reference sequence (GenBank accession no. U68436). Cm was isolated from lungs, cecum, and feces of a Cm-infected NSG mouse by using HeLa 229 cells. The considerable prevalence of Cm is likely due to widespread global interinstitutional distribution of unique mouse strains and failure to recognize that some of these mice were from enzootically infected colonies. Given that experimental Cm colonization of mice results in a robust immune response and, on occasion, pathology, natural infection may confound experimental results. Therefore, Cm should be excluded and eradicated from enzootically infected mouse colonies.

Using Filter Media and Soiled Bedding in Disposable Individually Ventilated Cages as a Refinement to Specific Pathogen-free Mouse Health Monitoring Programs.

Molecular-based methods have shown potential for improving pathogen detection and reducing animal use. While increasing evidence supports rodent-free environmental health PCR pathogen detection, limited information is available regarding efficacy for disposable individually ventilated caging systems. In such systems, testing of plenum exhaust air dust is ineffective, and the use of collection media is optimal. We performed a series of studies to compare PCR infectious agent detection with dust collected on media placed in a mouse-free soiled bedding cage, the cage exhaust filter of an occupied sentinel cage, and direct sampling from colony and sentinel mice with traditional soiled bedding mouse sentinels. We hypothesized that after a 3-mo period, testing of filter media agitated in a soiled bedding cage would be equal to or more sensitive than more traditional methods. Agitated media detected Astrovirus-1, segmented filamentous bacteria and Helicobacter ganmani to a degree comparable to testing lid exhaust filter PCR from a sentinel mouse cage, but opportunists such as Staphylococcus aureus and Proteus mirabilis were not detected consistently, and H. hepaticus was not detected at all. Direct sampling of pooled fecal pellets and body swabs from sentinel mice and testing using PCR also failed to reliably detect opportunists and Helicobacter spp. While further work is needed to refine use of filter media in soiled bedding for detection of lower prevalence opportunists, this report provides evidence that a rodent-free method of reliably detecting murine agents in a disposable individually ventilated cage system with cage-level filtration outperforms direct sampling of soiled bedding sentinel mice.

Assessing Elimination of Mouse Kidney Parvovirus from Cages by Mechanical Washing.

Mouse kidney parvovirus (MKPV), a newly identified parvovirus of the genus Chaphamaparvovirus, causes inclusion body nephropathy in severely immunocompromised mice and is prevalent in research mouse colonies. As nonenveloped viruses, mammalian parvoviruses are stable and generally resist thermal inactivation; however, as a novel and highly divergent parvovirus, the thermal stability of MKPV is undefined. This study aimed to evaluate the ability of cage sanitization in a mechanical washer to eliminate MKPV. Cages contaminated by MKPV-infected mice were assigned to 1 of 3 treatment groups: 1) control (bedding change only); 2) sanitization in a tunnel washer (88°C final rinse for 20 s); or 3) sanitization in a tunnel washer followed by autoclave sterilization (121 °C for 20 min). The presence of MKPV on the cage's interior surface was assessed by PCR of cage swab extracts collected before and after cage treatment. After treatment and swabbing, each cage housed 4 MKPV-negative CD1 mice. Each group of naive CD1 mice was assigned to one of the treatment groups and was housed in a cage from this group for two, 1 wk periods. At 12, 17, and 20 wk after the first exposure, renal tissue was collected from 1 test mouse per cage and assessed for MKPV by PCR. MKPV was detected by PCR on the surface of 63% of the pretreatment cages. All cages sanitized in a tunnel washer with or without sterilization were PCR negative after treatment. Seven of 10 mice housed in untreated cages contained a mouse positive for MKPV by 20 wk after exposure. None of the mice housed in cages sanitized in a tunnel washer with or without sterilization tested positive for MKPV at any time point. This study indicates that MKPV contaminated caging can result in MKPV infection of mice, and the use of a tunnel washer at the temperature and duration evaluated was sufficient to remove MKPV nucleic acid and prevent MKPV transmission.

Infectivity and Shedding of Mouse Kidney Parvovirus After Oronasal Inoculation of C57BL/6, CD1, and NSG Mice.

Mouse kidney parvovirus (MKPV), the etiology of murine inclusion body nephropathy, has been identified globally in mice used for research, with an estimated prevalence of 10% in academic colonies. In immunodeficient strains, MKPV causes significant morbidity and mortality, and severe renal pathology. In contrast, in immunocompetent mice, the infection is subclinical and causes minimal pathology. We investigated viral infectivity and shedding in inbred C57BL/6NCrl (B6), outbred Crl:CD1(ICR) (CD1), and highly immunocompromised NOD. Cg - Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four doses, ranging from 1.16 × 10 3 to 1.16 × 10 6 viral copies per microliter, of an MKPV inoculum were administered oronasally to 3 mice per dose per mouse type. All 3 types (B6, CD1, and NSG) had persistent infection with prolonged shedding in urine and feces. Viral copy number in the urine generally increased over time, while shedding in the feces was more variable. Among the 3 populations, CD1 mice developed viral shedding in urine earliest (4 wk after inoculation) and at higher levels (greater than 1 × 10 7 viral copies per microliter). B6 mice become viruric later (7 wk after inoculation), with lesser virus shed (1 × 10 6 viral copies per microliter or less). In CD1 and B6 mice, peak urine shedding occurred at 11 to 14 wk after inoculation, after which levels gradually declined until 35 wk after inoculation (study endpoint). In contrast, NSG mice did not become viruric until 10 wk after inoculation and continued to shed large amounts of virus (greater than 1 × 107 viral copies per microliter) in urine until the study endpoint. Two commercial immunofluorescent serologic assays failed to detect serum antibodies to MKPV nonstructural protein 1 as late as 58 wk after inoculation, whereas immunohistochemistry of infected renal tissue successfully detected anti-MKPV serum antibodies. These results increase our knowledge of the biology of MKPV and have practical application for development of effective screening programs for this pathogen.

Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2.

Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains-a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD- Prkdcem26Cd52Il2rgem26Cd22 /NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

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.

Mite Burden and Immunophenotypic Response to Demodex musculi in Swiss Webster, BALB/c, C57BL/6, and NSG Mice.

Detection methods for Demodex musculi were historically unreliable, and testing was rarely performed because its prevalence in laboratory mice was underestimated. Although infestations are unapparent in most mouse strains, D. musculi burdens are higher and clinical signs detected in various immunodeficient strains. The parasite's influence on the immune system of immunocompetent mice is unknown. We characterized mite burden (immunocompetent and immunodeficient strains) and immunologic changes (immunocompetent strains only) in naïve Swiss Webster (SW; outbred), C57BL/6NCrl (B6; Th1 responder), BALB/cAnNCrl (BALB/c; Th2 responder) and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; immunodeficient) mice after exposure to Demodex-infested NSG mice. Infested and uninfested age-matched mice of each strain (n = 5) were euthanized 14, 28, 56, and 112 d after exposure. Mite burden was determined through PCR analysis and skin histopathology; B-cell and CD4+ and CD8+ T-cell counts and activation states (CD25 and CD69) were evaluated by using flow cytometry; CBC counts were performed; and serum IgE levels were measured by ELISA. Mite burden and PCR copy number correlated in NSG mice, which had the highest mite burden, but not in immunocompetent strains. Infested immunocompetent animals developed diffuse alopecia by day 112, and both BALB/c and C57BL/6 mice had significantly increased IgE levels. These findings aligned with the skewed Th1 or Th2 immunophenotype of each strain. BALB/c mice mounted the most effective host response, resulting in the lowest mite burden of all immunocompetent strains at 112 d after infestation without treatment. Clinically significant hematologic abnormalities were absent and immunophenotype was unaltered in immunocompetent animals. Topical treat- ment with imidacloprid-moxidectin (weekly for 8 wk) was effective at eradicating mites by early as 7 d after treatment. IgE levels decreased substantially in infested BALB/c mice after treatment. These findings demonstrate a need for D. musculi surveillance in mouse colonies, because the infestation may influence the use of infested mice in select studies.

Serendipitous Discovery of a Novel Murine Astrovirus Contaminating a Murine Helper T-cell Line and Incapable of Infecting Highly Immunodeficient Mice.

The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying <30% capsid identity, yet were closely related to murine astrovirus 2 (85% capsid identity), which had recently been isolated from feral mice in New York City. A MuAstV-2 specific PCR assay was developed and used to eradicate MuAstV-2 from the infected colony using a test and cull strategy. The newly identified MuAstV2 readily transmits to immunocompetent mouse strains by fecal-oral exposure, but fails to infect NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.

Comparison of two prevalent individually ventilated caging systems for detection of murine infectious agents via exhaust air particles.

We compared the efficacies of two different individually ventilated cage systems (Allentown and Tecniplast) for health monitoring (HM) of murine infectious agents using exhaust air particle (EAP) capture and real-time PCR. After three months of monitoring, both EAP capture media allowed detection of Helicobacter, Pasteurella and Entamoeba. Use of the EAP real-time PCR for HM reduces the number of mice used.

Using Hysterectomy Rederivation to Produce Guinea Pigs (Cavia porcellus) Free of Guinea Pig Cytomegalovirus.

Due to similarities in placentation, guinea pigs can be used as models of human cytomegalovirus infection, but they mustbe free of guinea pig cytomegalovirus. Many commercial guinea pig colonies are enzootically infected with guinea pigcytomegalovirus, which can be transmitted vertically as well as horizontally through saliva, vaginal secretions, and milk.These characteristics make its eradication in a commercial setting challenging. Because embryo transfer technology in guineapigs is in its infancy, it is not generally a viable option for obtaining animals free of guinea pig cytomegalovirus. However,a combination of hysterectomy rederivation and testing by PCR assay and serology of both dams and offspring from anenzootically infected colony produced offspring free of guinea pig cytomegalovirus.

Contaminated Shipping Materials Identified as the Source of Rotaviral Infection of Exported Mice.

Over a 4-wk period in 2017, we received notification from 7 different institutions that mice exported from our SPF barrier facilities had tested positive for mouse rotavirus (MRV). The exports originated from several different buildings across multiple campuses. Our institution excludes MRV in all of our barrier facilities and has historically been free of this virus. Extensive testing of our rooms from which the exported mice originated did not detect the presence of rotavirus. The single commonality among the 7 shipments was the use of shipping boxes acquired from one vendor. These shipping boxes arrived at our institution prepackaged with unsterilized feed and bedding, which we hypothesized was the source of the rotavirus. To test this hypothesis, we housed naïve sentinel mice in clean cages with feed and bedding transferred from 29 unopened, unused shipping boxes. Sentinel mice were exposed to this bedding and feed for 14 d and then evaluated through MRV serology and PCR assay. Of the 29 sentinels, 24 were seropositive for MRV, and 14 of the 29 were PCR positive. These results provided direct evidence that MRV detected by recipient institutions originated from the contaminated feed or bedding within the shipping boxes. To our knowledge, this report is the first description of contaminated materials in shipping boxes resulting in rotaviral infection of mice during export.

PCR Testing of IVC Filter Tops as a Method for Detecting Murine Pinworms and Fur Mites.

We evaluated PCR testing of filter tops from cages maintained on an IVC system through which exhaust air is filtered at the cage level as a method for detecting parasite-infected and -infested cages. Cages containing 4 naïve Swiss Webster mice received 360 mL of uncontaminated aspen chip or α-cellulose bedding (n = 18 cages each) and 60 mL of the same type of bedding weekly from each of the following 4 groups of cages housing mice infected or infested with Syphacia obvelata (SO), Aspiculuris tetraptera (AT), Myocoptes musculinus (MC), or Myobia musculi (MB) and Radfordia affinis (RA; 240 mL bedding total). Detection rates were compared at 30, 60, and 90 d after initiating bedding exposure, by using PCR analysis of filter tops (media extract and swabs) and testing of mouse samples (fur swab [direct] PCR testing, fecal flotation, anal tape test, direct examination of intestinal contents, and skin scrape). PCR testing of filter media extract detected 100% of all parasites at 30 d (both bedding types) except for AT (α-cellulose bedding, 67% detection rate); identified more cages with fur mites (MB and MC) than direct PCR when cellulose bedding was used; and was better at detecting parasites than all nonmolecular methods evaluated. PCR analysis of filter media extract was superior to swab and direct PCR for all parasites cumulatively for each bedding type. Direct PCR more effectively detected MC and all parasites combined for aspen chip compared with cellulose bedding. PCR analysis of filter media extract for IVC systems in which exhaust air is filtered at the cage level was shown to be a highly effective environmental testing method.

Pre-Natal Exposure to Mouse Parvovirus at Day 5 and 12 Gestation Does Not Induce Immune Tolerance.

Parvoviruses have a predilection for rapidly dividing cells such as occurs during embryonic development. Potentially, in utero exposure could lead to immune tolerance in progeny mice. To determine if MPV infection in utero results in immune tolerance, pregnant mice were inoculated by oral gavage with 50 ID50 MPV1e or sham inoculated with phosphate buffered saline at day 5 and 12 gestation. Offspring were fostered to MPV-negative recipient dams prior to development of a milk spot. After confirming the offspring were seronegative for MPV by serology and not shedding by fecal PCR, they were challenged with 50 ID50 MPV1e by oral gavage at weaning or sham inoculated. At 4 weeks post inoculation, all weanlings exposed in utero developed antibodies to MPV, and MPV was detected by fecal PCR. Similarly, all weanlings from sham-inoculated dams challenged with MPV developed antibodies and MPV was detected by fecal PCR. None of the sham inoculated weanling mice from MPV infected dams or sham infected dams developed antibodies to MPV nor was MPV detected by fecal PCR. These results demonstrate that in utero exposure to MPV1e via oral gavage was insufficient to induce immune tolerance and provides greater confidence that rederivation techniques may successfully eliminate colonies of MPV. Furthermore, our findings do not provide evidence that MPV tolerance may contribute to hidden infections in mouse colonies.

A comparison of mouse parvovirus 1 infection in BALB/c and C57BL/6 mice: susceptibility, replication, shedding, and seroconversion.

This study characterized the effects of challenge with a field isolate of mouse parvovirus 1 (MPV1e) in C57BL/6NCrl (B6) and BALB/cAnNCrl (C) mice. We found that C mice were more susceptible to MPV1e infection than were B6 mice; ID50 were 50 to 100 times higher after gavage and 10-fold higher after intraperitoneal injection in B6 as compared with C mice. To evaluate the host strain effect on the pathogenesis of MPV1e, B6 and C mice were inoculated by gavage. Feces and tissues, including mesenteric lymph nodes (MLN), ileum, spleen and blood, were collected for analysis by quantitative PCR (qPCR) to assess infection and fecal shedding and by RT-qPCR to evaluate replication. Peak levels of MPV1e shedding, infection, and replication were on average 3.4, 4.3, and 6.2 times higher, respectively, in C than in B6 mice. Peaks occurred between 3 and 10 d after inoculation in C mice but between 5 and 14 d in B6 mice. Multiplexed fluorometric immunoassays detected seroconversion in 2 of 3 C mice at 7 d after inoculation and in all 3 B6 mice at 10 d. By 56 d after inoculation, viral replication was no longer detectable, and fecal shedding was very low; infection persisted in ileum, spleen, and MLN, with levels higher in C than B6 mice and highest in MLN. Therefore, the lower susceptibility of B6 mice, as compared with C mice, to MPV1e infection was associated with lower levels of infection, replication, and shedding and delayed seroconversion.

Efficacy of direct detection of pathogens in naturally infected mice by using a high-density PCR array.

We used a high-density array of real-time PCR assays for commonly reported rodent infectious agents (PRIA) to test naturally infected index mice and sentinel mice exposed by contact and soiled-bedding transfer. PRIA detected 14 pathogens--including viruses, bacteria, fur mites, pinworms, and enteric protozoa--in 97.2% of 28 pooled fecal samples, fur-perianal swabs, and oral swabs from 4 cages containing a total of 10 index mice. Among these pathogens, PRIA (like conventional health monitoring methods) failed to detect Mycoplasma pulmonis, Pasteurella pneumotropica, and Giardia spp. in all of the 9 contact and 9 soiled-bedding sentinels. PRIA demonstrated murine adenovirus and Cryptosporidium and Spironucleus spp. in contact but not soiled-bedding sentinels and detected Helicobacter and pinworms in fewer than half of the soiled-bedding sentinels. Of the 4 species of Helicobacter that species-specific PCR assays identified in index mice, only H. ganmani was found in soiled-bedding and contact sentinels. PRIA detected all of the pathogens in sentinels that were identified by conventional methods. Myobia musculi was detected by PCR in index and sentinel mice but missed by conventional parasitologic examinations. In summary, PRIA reproducibly detected diverse pathogens in heavily pooled specimens collected noninvasively from infected index mice antemortem. The inability of PRIA and conventional health monitoring methods (that is, parasitology, micro-biology, and serology) to demonstrate transmission of some pathogens to contact sentinels and the inefficient transmission of others to soiled-bedding sentinels underscores the importance of direct PCR testing to determine the pathogen status of rodents in quarantine and during routine colony surveillance.

Evaluation of diagnostic methods for Myocoptes musculinus according to age and treatment status of mice (Mus musculus).

Detecting and controlling murine fur mites continues to be challenging. Here we compared the efficacy of fur-pluck, cage PCR, and fur PCR testing of mice naturally infested with Myocoptes musculinus and make recommendations regarding the application of these diagnostic strategies in aged or treated mice. We compared all 3 diagnostic methods in groups of infested and noninfested control mice over time. For fur plucks, we used a scoring system to quantitatively compare mite infestations across ages. Mice that were 4 wk old had higher egg and mite scores than did older mice, with average scores at 4 wk corresponding to 40 to 100 individual fur mites and eggs per sample. Furthermore, 15% and 20% of samples from infested mice at 24 and 28 wk of age, respectively, lacked all fur mites and eggs. Cage PCR results varied as mice grew older. Fur PCR testing was the most sensitive and specific assay in untreated infested mice, particularly when mite densities were low. In addition, we compared fur-pluck and fur PCR tests for evaluating the efficacy of selamectin treatment. Two treatments with selamectin eliminated Myocoptes fur-mite infestations. At 8 wk after treatment, all fur-pluck samples were negative, but one-third of treated infested cages remained positive by fur PCR assay; at 16 wk after treatment, all cages were negative by fur PCR assay. Because offspring of infested mice were invariably heavily infested, breeding of suspected infested mice with subsequent testing of offspring was the definitive testing strategy when fur-pluck and PCR results conflicted.

Pathogenicity and genetic variation of 3 strains of Corynebacterium bovis in immunodeficient mice.

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice.