Effects of Mouse Kidney Parvovirus on Pharmacokinetics of Chemotherapeutics and the Adenine Model of Chronic Kidney Disease.

Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy in severely immunocompromised mice and renal interstitial inflammation in immunocompetent mice. Here we sought to determine the effects of MKPV on pre-clinical murine models that depend on renal function. To assess the effects of MKPV infection on the pharmacokinetics of 2 renally excreted chemotherapeutic agents, methotrexate and lenalidomide, we measured drug concentrations in the blood and urine of MKPV-infected or uninfected immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and immunocompetent C57BL/6NCrl (B6) female mice. No differences in plasma pharmacokinetics were observed for lenalidomide. However, the AUC of methotrexate was 1.5-fold higher in uninfected NSG mice compared with infected NSG mice, 1.9-fold higher in infected B6 mice compared with uninfected B6 mice, and 4.3-fold higher in uninfected NSG mice compared with uninfected B6 mice. MKPV infection did not significantly affect the renal clearance of either drug. To assess effects of MKPV infection on the adenine diet model of chronic kidney disease, MKPV-infected and uninfected B6 female mice were fed a 0.2% adenine diet, and clinical and histopathologic features of disease were assessed over 8 wk. MKPV infection did not significantly alter urine chemistry results, hemogram findings, or serum concentrations of BUN, creatinine, or symmetric dimethylarginine. However, infection did influence histologic outcomes. As compared with uninfected mice, MKPV-infected mice had more interstitial lymphoplasmacytic infiltrates after 4 and 8 wk of diet consumption and less interstitial fibrosis at week 8. Macrophage infiltrates and renal tubular injury were similar between in infected and uninfected mice. These findings indicate that MKPV infection had minimal effects on the renal excretion of 2 chemotherapeutics and on serum biomarkers of renal function. However, infection significantly influenced two histologic features of the adenine diet model of chronic renal disease. MKPV-free mice are critically important in studies evaluating renal histology as an experimental outcome.

Evaluation of In-cage Filter Paper as a Replacement for Sentinel Mice in the Detection of Murine Pathogens.

Recent studies have evaluated alternatives to the use of live animals in colony health monitoring. Currently, an alternative method that is suitable for all rack types and that has been verified to detect the infectious agents most commonly excluded from mouse colonies is unavailable. We compared the use of filter paper placed on the inside floor of mouse cages to the traditional use of sentinel mice in the detection of several prevalent murine pathogens including mouse hepatitis virus (MHV), murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler murine encephalomyelitis virus (TMEV), Helicobacter spp., Syphacia obvelata, and Aspiculuris tetraptera. Experimental groups comprised 7 cages containing either 2 pieces of filter paper on the cage floor or 2 ICR sentinel mice. Soiled bedding from pet-store mice was transferred to the experimental cages weekly for 8 wk. At 1 and 2 mo after bedding transfer, the filter papers were evaluated by PCR and sentinel mice were tested by serology and fecal PCR. Filter papers detected all pathogens as effectively (MHV, MNV, MPV, MVM, TMEV S. obvelata, and A. tetraptera) or more effectively (Helicobacter spp.) than sentinel mice at both time points. Filter papers more readily detected pathogens with a high copy number per RT-PCR analysis than a low copy number. Helicobacter spp. were not detected by sentinel mice at either time point. These results indicate that the use of filter paper placed on the interior floor of empty mouse cages and exposed to soiled bedding is efficient in detecting bacteria, endoparasites, and most of the common mouse viruses included in an animal health monitoring program.

LEPTOSPIRA, PARVOVIRUS, AND TOXOPLASMA IN THE NORTH AMERICAN RIVER OTTER (LONTRA CANADENSIS) IN NORTH CAROLINA, USA.

The North American river otter (Lontra canadensis) is the largest mustelid in North Carolina, US, and was once extirpated from the central and western portions of the state. Over time and after a successful reintroduction project, otters are now abundant and occur throughout North Carolina. However, there is a concern that diseases may have an impact on the otter population, as well as on other aquatic mammals, either through exposure to emerging diseases, contact with domestic animals such as domestic cats (Felis catus), or less robust condition of individuals through declines in water quality. We tested brain and kidney tissue from harvested otters for the pathogens that cause leptospirosis, parvovirus, and toxoplasmosis. Leptospirosis and toxoplasmosis are priority zoonoses and are maintained by domestic and wild mammals. Although parvovirus is not zoonotic, it does affect pets, causing mild to fatal symptoms. Across the 2014-15 and 2015-16 trapping seasons, we tested 220 otters (76 females, 144 males) using real-time PCR for Leptospira interrogans, parvovirus, and Toxoplasma gondii. Of the otters tested, 1% (3/220) were positive for L. interrogans, 19% (41/220) were positive for parvovirus, and 24% (53/220) were positive for T. gondii. Although the pathogens for parvovirus and toxoplasmosis are relatively common in North Carolina otters, the otter harvest has remained steady and the population appears to be abundant and self-sustaining. Therefore, parvovirus and toxoplasmosis do not currently appear to be negatively impacting the population. However, subsequent research should examine transmission parameters between domestic and wild species and the sublethal effects of infection.

Seroconversion of 1-year-old Mice to Murine Norovirus.

Rodent sentinel screening for adventitious pathogens is an integral part of many biomedical research institutes and universities that use rodents in research. Typical screening programs involving live sentinel animals typically purchase young SPF sentinel animals that are sampled and replaced quarterly. Previous reports suggest that mice as old as 6 mo are effective sentinels for various agents. In efforts to reduce the number of animals used in our sentinel program, we wanted to investigate the possibility of keeping sentinel animals inhouse for 12 mo at a time. We exposed mice (age, 40 to 48 wk) to murine norovirus (MNV) to test whether they could reliably produce detectable levels of antibodies (similar to younger mice) to this adventitious pathogen. Mice first exposed to MNV at 40 to 48 wk of age seroconverted to MNV after both direct inoculation (through gavage) and indirect exposure (from soiled-bedding transfer) at the same or greater frequency than mice first exposed at 8 to 12 wk of age. These findings indicate that, at least for MNV, sentinel residence time can be extended from 3 to 12 mo without compromising the reliability of seroconversion, thus ultimately reducing sentinel animal numbers. This practice, combined with nonanimal testing modalities (for example, exhaust duct sampling), can increase the sensitivity and specificity of rodent surveillance programs and minimize the use of live animals.

Effects of Pelleting, Irradiation, and Autoclaving of Rodent Feed on MPV and MNV Infectivity.

Murine norovirus (MNV) and mouse parvovirus (MPV) are among the most common adventitial viruses seen in laboratory mice, and infections arise in barrier facilities despite rigorous biosecurity programs. Some authors have implicated nonsterilized feed as a source of MPV in rodent facilities, but none have conclusively documented viral particles in the feed. In this study, we hypothesized that both viruses can resist the pelleting process but not subsequent irradiation or autoclaving, thus revealing a potential source of outbreaks in rodent facilities. To test this hypothesis, we contaminated powdered feed with 10-fold concentrations of MNV and MPV and fed it to both Swiss Webster (SW) and C57BL/6NTac (B6) mice to determine a 'powdered ID50' according to seroconversion over a 28-d period. We repeated the experiment by using powdered feed that we contaminated with increasing viral doses (as no. of powdered ID50) and subsequently pelleted; from these results, we determined a 'pelleted ID50.' Finally we assessed the effect of irradiation and autoclaving on contaminated pellets by using the same experimental design. The powdered ID50 was relatively low and identical in both mouse strains (2.51 × 10² pfu) for MNV but higher in B6 (copy number, 3.20 × 106) than SW (3.98 × 104 copies) for MPV. As hypothesized, mice were infected by contaminated rodent feed despite the pelleting process. Indeed, pelleting resulted in a 1- to 2-log increase in ID50 in both strains for MNV and MPV. Irradiation and autoclaving of infected pellets effectively prevented seroconversion of mice exposed to all doses of MNV, whereas a single mouse seroconverted at the highest dose of MPV (1.35 × 107 copies). These data suggest that both MNV and MPV remain infectious after conditions reproducing the rodent chow pelleting process and that nonsterilized rodent chow might be a source of viral outbreaks.

Differential susceptibility of SD and CD rats to a novel rat theilovirus.

Antibodies to rat theilovirus (RTV) have been detected in rats for many years because of their serologic crossreactivity with strains of Theiler murine encephalomyelitis virus (TMEV) of mice. Little information exists regarding this pathogen, yet it is among the most common viruses detected in serologic surveys of rats used in research. In the study reported here, a novel isolate of RTV, designated RTV1, was cultured from the feces of infected rats. The RTV1 genome contained 8094 nucleotides and had approximately 95% identity with another rat theilovirus, NSG910, and 73% identity with TMEV strains. In addition, the genome size of RTV1 was similar to those of TMEV strains but larger than that reported for NSG910. Oral inoculation of Sprague-Dawley (SD) and CD male rats (n = 10 each group) with RTV1 revealed that SD rats were more susceptible than CD rats to RTV1 infection. At 14 d postinoculation, 100% of SD rats shed virus in the feces, and 70% were positive for RTV serum antibodies. By 56 d postinoculation 30% of SD rats continued to have detectable virus in the feces, and 90% had seroconverted. In contrast, in inoculated CD rats RTV was detected only in the feces at 14 d postinoculation, at which time 40% of CD rats were fecal positive. By 56 d postinoculation only 20% of CD rats had detectable RTV serum antibodies. Our data provide additional sequence information regarding a rat-specific Cardiovirus and indicate that SD rats are more susceptible than CD rats to RTV1 infection.

PCR Testing of Filter Material from IVC Lids for Microbial Monitoring of Mouse Colonies.

Testing sentinel animals exposed to soiled bedding from colony animals is the most common method used for health monitoring in rodent facilities. Although environmental sampling is being explored-and, in many cases, has been implemented-as an alternative, exhaust plenum sampling is not effective for all ventilated rack designs. This study evaluated PCR testing of filter paper from sentinel cages on ventilated racks. We hypothesized that testing filter paper from cages containing soiled bedding would be as effective as testing sentinel mice and that periodic shaking of cages would generate sufficient particulate movement to substitute for the presence of live animals. Three cages containing soiled bedding were maintained in each of 8 rooms; one cage contained 2 Cr:NIH(S) mice, one had no mice and was shaken twice weekly, and the remaining one had no mice and was left undisturbed. For 3 consecutive months, a piece of filter paper from the undersurface of the cage lid was tested monthly for adventitial agents and then replaced. A second piece remained on the cage undersurface for 3 mo. Fecal pellets and oral and fur swabs were collected from sentinel mice at months 1 and 3 and tested for the same agents. At month 3, serology was performed on the sentinel mice; feces and oral and fur swabs from colony animals were tested concurrently for comparison. Filter paper from cages without mice and shaken were at least as effective than all other methods in detecting the presence of endemic agents, including mouse norovirus, Helicobacter spp., Pasteurella pneumotropica, Entamoeba muris, and Spironucleus muris. For IVC systems where exhaust plenum testing is ineffective, PCR testing of IVC filter tops should be considered as an alternative to soiled bedding sentinels. Environmental sampling may provide increased reliability and reduce the number of rodents used for routine health surveillance.

Ivermectin-compounded Feed Compared with Topical Moxidectin-Imidacloprid for Eradication of Demodex musculi in Laboratory Mice.

Demodex musculi is a prostigmatid follicular mite that has rarely been reported in laboratory mice. Although prevalence of this species has not been assessed formally, we have found that many imported mouse strains from noncommercial sources harbor Demodex mites. To assess whether an acaricide can be used to eradicate this mite, infested immunocompromised mice were provided ivermectin-compounded (12 ppm) feed without restriction for 8 wk (n = 10), were treated topically with moxidectin and imidacloprid (MI; 3 and 13 mg/kg, respectively) weekly for 8 wk (n = 10), or remained untreated (n = 10). Mice were confirmed to be mite-infested before treatment and were tested after treatment by using fur plucks (FP), deep skin scrapes (DSS), and PCR analysis of fur swabs. In addition, the presence of mites was confirmed through skin biopsies at 2 study endpoints (1 wk [n = 5] and 12 wk [ n = 5] after treatment). Samples collected before treatment and from untreated mice were positive for D. musculi at all time points and by all test modalities. After treatment, all ivermectin-treated animals remained infested, whereas mice treated with MI were repeatedly negative by all test modalities. An additional shortened treatment trial revealed that 4 wk of MI (n = 7) was insufficient to eradicate mites. Neither treatment produced any evidence of adverse effects according to hematology, serum chemistry parameters, behavior, body weight, and histopathology. Of the 70 PCR assays performed in treated mice, 14 were positive when FP+DSS was negative. In 6 cases where PCR results were negative, 5 were positive by FP+DSS and a single sample was positive on skin biopsy. Although PCR analysis has a high detection rate for D. musculi, FP+DSS can further enhance the detection rate. In conclusion, topical MI administered for 8 consecutive weeks can safely eradicate D. musculi from an immunocompromised mouse strain.

Response of healthy dogs to infusions of human serum albumin.

OBJECTIVE: To evaluate the clinical and immunologic response in healthy dogs to infusions of human serum albumin (HSA). ANIMALS: 9 healthy purpose-bred mixed-breed dogs. PROCEDURES: Each dog was administered a 25% HSA solution once or twice. Various physical examination and laboratory variables were serially evaluated. Antibody against HSA was assayed before and after infusion by use of an ELISA. Intradermal testing was also conducted. A repeated-measures ANOVA or Friedman repeated-measures ANOVA on ranks was used to compare results for the variables. RESULTS: Adverse clinical reactions were observed after the first or second infusion in 3 dogs. Anaphylactoid reactions were observed in 1 of 9 dogs during the first infusion and in 2 of 2 dogs administered a second infusion. Two dogs developed severe edema and urticaria 6 or 7 days after an initial infusion. All dogs developed anti-HSA antibodies. Positive responses for ID tests were observed in 8 of 9 dogs. Short-term increases were detected in blood protein, total bilirubin, and calcium concentrations after HSA infusion. Serum cholesterol concentrations and platelet counts decreased after HSA infusion. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of HSA resulted in profound reactions in 2 of 9 dogs administered a single infusion and in 2 of 2 dogs administered a second infusion. This indicates that there is risk of life-threatening adverse reactions to HSA infusion in healthy dogs.

Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine.

Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish.

Persistent infection with and serologic cross-reactivity of three novel murine noroviruses.

According to serologic testing, murine norovirus is the most prevalent viral pathogen in contemporary laboratory mice. Previously, murine norovirus 1 (MNV-1) was the only norovirus reported to infect research mice. In this study, 3 novel murine norovirus strains--MNV-2, MNV-3, and MNV-4--were isolated from geographically separate mouse research colonies. All 4 murine norovirus strains used as individual antigens in microsphere fluorescent immunoassays displayed serologic cross-reactivity to serum from mice inoculated with MNV-1, MNV-2, MNV-3, or MNV-4. In addition, reverse transcriptase-polymerase chain reaction analysis at 8 wk postinoculation detected virus in the feces and tissues of all mice experimentally inoculated with MNV-2, MNV-3, or MNV-4. This finding suggests that mice can have prolonged fecal shedding of and can become persistently infected with murine noroviruses.

Bordetella pseudohinzii as a Confounding Organism in Murine Models of Pulmonary Disease.

A group studying acute lung injury observed an increased percentage of neutrophils in the bronchoalveolar lavage (BAL) fluid of mice. BAL was performed, and lung samples were collected sterilely from 5 C57BL/6 mice that had been bred inhouse. Pure colonies of bacteria, initially identified as Bordetella hinzii were cultured from 2 of the 5 mice which had the highest percentages of neutrophils (21% and 26%) in the BAL fluid. Subsequent sequencing of a portion of the ompA gene from this isolate demonstrated 100% homology with the published B. pseudohinzii sequence. We then selected 10 mice from the investigator's colony to determine the best test to screen for B. pseudohinzii in the facility. BAL was performed, the left lung lobe was collected for culture and PCR analysis, the right lung lobe and nasal passages were collected for histopathology, an oral swab was collected for culture, and an oral swab and fecal pellets were collected for PCR analysis. B. pseudohinzii was cultured from the oral cavity, lung, or both in 8 of the 10 mice analyzed. All 8 of these mice were fecal PCR positive for B. pseudohinzii; 7 had increased neutrophils (5% to 20%) in the BAL fluid, whereas the 8th mouse had a normal neutrophil percentage (2%). Active bronchopneumonia was not observed, but some infected mice had mild to moderate rhinitis. B. pseudohinzii appears to be a microbial agent of importance in mouse colonies that can confound pulmonary research. Commercial vendors and institutions should consider colony screening, routine reporting, and exclusion of B. pseudohinzii.

Influence of Rack Design and Disease Prevalence on Detection of Rodent Pathogens in Exhaust Debris Samples from Individually Ventilated Caging Systems.

Sampling of bedding debris within the exhaust systems of ventilated racks may be a mechanism for detecting murine pathogens in colony animals. This study examined the effectiveness of detecting pathogens by PCR analysis of exhaust debris samples collected from ventilated racks of 2 different rack designs, one with unfiltered air flow from within the cage to the air-exhaust pathway, and the other had a filter between the cage and the air-exhaust pathway. For 12 wk, racks were populated with either 1 or 5 cages of mice (3 mice per cage) infected with one of the following pathogens: mouse norovirus (MNV), mouse parvovirus (MPV), mouse hepatitis virus (MHV), Helicobacter spp., Pasteurella pneumotropica, pinworms, Entamoeba muris, Tritrichomonas muris, and fur mites. Pathogen shedding by infected mice was monitored throughout the study. In the filter-containing rack, PCR testing of exhaust plenums yielded negative results for all pathogens at all time points of the study. In the rack with open air flow, pathogens detected by PCR analysis of exhaust debris included MHV, Helicobacter spp., P. pneumotropica, pinworms, enteric protozoa, and fur mites; these pathogens were detected in racks housing either 1 or 5 cages of infected mice. Neither MPV nor MNV was detected in exhaust debris, even though prolonged viral shedding was confirmed. These results demonstrate that testing rack exhaust debris from racks with unfiltered air flow detected MHV, enteric bacteria and parasites, and fur mites. However, this method failed to reliably detect MNV or MPV infection of colony animals.

Delayed and Aberrant Presentation of VX2 Carcinoma in a Rabbit Model of Hepatic Neoplasia.

A socially-housed New Zealand white rabbit presented with a large subcutaneous mass on the ventral thorax approximately 11 mo after the intrahepatic delivery of a suspension of VX2 carcinoma cells to induce hepatocellular carcinoma as part of a nanoparticle study. The mass and closely associated axillary lymph node were removed en bloc. Immunohistochemical staining identified the mass as an undifferentiated carcinoma. The rabbit demonstrated no appreciable pathology at the study end point at 16 mo after VX2 inoculation. An additional rabbit from the same VX2 injection cohort was found at necropsy to have an unanticipated intraabdominal mass, also identified as an undifferentiated carcinoma. This case report summarizes the molecular analysis of both tumors through a novel PCR assay, which identified the delayed and aberrant onset of VX2 carcinoma in an extended timeframe not previously reported.

Pathogenicity of Helicobacter rodentium in A/JCr and SCID mice.

Helicobacter rodentium was first recognized as a potential pathogen when it was isolated, along with Helicobacter bilis, from a colony of scid/Trp53 knockout mice with diarrhea. Clinical disease in these mice was more severe than that previously reported in mice infected with H. bilis alone, thus suggesting that H. rodentium contributed to the pathogenesis of enteritis. The purpose of the study reported here was to address two questions: is H. rodentium pathogenic in mice, and when co-infection with a pathogenic helicobacter occurs, does H. rodentium augment disease? To this end, A/JCr and C.B-17/IcrCrl-scidBr mice were inoculated with H. rodentium and/or H. hepaticus. Twelve weeks after inoculation, mice were euthanized. The cecum and liver were evaluated microscopically for evidence of disease. Cecal interferon-inducible protein 10 (IP-10), macrophage inflammatory protein 1alpha (MIP-1alpha), interleukin 10 (IL-10), and interferon gamma (IFN-gamma) mRNA values were measured as an indicator of mucosal immune response. Hepatic lesions were not identified in mice mono-infected with H. rodentium; likewise, cecal lesion scores were not significantly different from those of uninfected controls. With the exception of an increased IL-10 mRNA value in SCID mice, mean immune-related gene expression in H. rodentium mono-infected and uninfected control mice was not significantly different. In contrast, all mice infected with H. hepaticus developed moderate to severe hepatitis, significant increase in cecal lesion scores, and increased immune-related gene expression. The C.B-17/IcrCrl-scidBr mice co-infected with H. hepaticus and H. rodentium had liquid cecal contents and low terminal body weight. Further, compared with mice infected with H. hepaticus alone, co-infection was associated with significant increases of IL-10, MIP-1alpha, and IP-10 mRNA values in C.B-17/IcrCrl-scidBr and IFN-gamma and MIP-1alpha mRNA values in A/JCr mice. These results suggested that H. rodentium alone does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, co-infection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical manifestation of disease in immunodeficient mice.

Sex influence on chronic intestinal inflammation in Helicobacter hepaticus-infected A/JCr mice.

Helicobacter hepaticus is a bacterial pathogen of mice that has been reported to cause chronic intestinal inflammation in A/JCr, germfree Swiss Webster, and immunodeficient mice. To the authors' knowledge, the influence of sex on development of chronic intestinal inflammation in H. hepaticus-infected mice has not been investigated. The purposes of the study reported here were to determine whether severity of intestinal inflammation differs between male and female A/JCr mice chronically infected with H. hepaticus and to characterize the mucosal immune response in these mice. The cecum of male and female A/JCr mice infected with H. hepaticus for 1 month and 3 months was objectively evaluated histologically for intestinal disease. Also, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was done to measure interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 4 (IL-4), IL-10, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-inducible protein of 10 kDa (IP-10), and monokine induced by gamma interferon (MIG) mRNA values in the cecal tissue of these mice. Significant differences in cecal lesion scores were not present at 1 month after infection. However, infected female mice had significantly up-regulated expression of cecal IL-10, MIP-1alpha, IP-10, and MIG mRNA compared with that in uninfected females, and expression of IL-10 and MIP-1alpha was significantly greater than that detected in infected male mice (P < or = 0.05). At 3 months after infection, cecal lesion scores were significantly (P < or = 0.05) increased in female and male mice compared with uninfected controls, and infected female mice had significantly (P < or = 0.05) higher cecal lesion scores than did infected male mice. In addition, infected females had significant (P < or = 0.05) increases in cecal IFN-gamma, TNF-alpha, IL-10, MIP-1alpha, IP-10, and MIG mRNA values compared with values in uninfected females and infected males, and male mice had significant (P < or = 0.05) increases in cecal TNF-alpha and IL-10 mRNA values compared with those for male control mice. These data indicate that, in H. hepaticus-infected A/JCr mice, females develop more severe intestinal inflammation than do males, and the chronic mucosal inflammation is polarized toward a Th1 response that is not down-regulated by increased activity of IL-10. We propose that H. hepaticus-infected A/JCr mice will serve as a good animal model with which to study the influence of sex on bacterial-induced mucosal inflammation.

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays.

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.

Diagnostic testing of mouse and rat colonies for infectious agents.

Rodent health monitoring programs make an essential contribution to biomedical research by identifying the presence of infectious agents that might confound animal research. The authors discuss the types of diagnostic tests available, which agents deserve monitoring, and the appropriate frequency for such interventions.

Development of a PCR assay for the detection of Spironucleus muris.

Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests.

Pneumocystis carinii infection causes lung lesions historically attributed to rat respiratory virus.

Idiopathic lung lesions characterized by dense perivascular cuffs of lymphocytes and a lymphohistiocytic interstitial pneumonia have been noted in research rats since the 1990s. Although the etiology of this disease has remained elusive, a putative viral etiology was suspected and the term 'rat respiratory virus' (RRV) has been used in reference to this disease agent. The purpose of this study was to determine whether Pneumocystis carinii infection in immunocompetent rats can cause idiopathic lung lesions previously attributed to RRV. In archived paraffin-embedded lungs (n = 43), a significant association was seen between idiopathic lung lesions and Pneumocystis DNA detected by PCR. In experimental studies, lung lesions of RRV developed in 9 of 10 CD rats 5 wk after intratracheal inoculation with P. carinii. No lung lesions developed in CD rats (n = 10) dosed with a 0.22-µm filtrate of the P. carinii inoculum, thus ruling out viral etiologies, or in sham-inoculated rats (n = 6). Moreover, 13 of 16 CD rats cohoused with immunosuppressed rats inoculated with P. carinii developed characteristic lung lesions from 3 to 7 wk after cohousing, whereas no lesions developed in rats cohoused with immunosuppressed sham-inoculated rats (n = 7). Both experimental infection studies revealed a statistically significant association between lung lesion development and exposure to P. carinii. These data strongly support the conclusion that P. carinii infection in rats causes lung lesions that previously have been attributed to RRV.