Optimal age at fostering for derivation of Helicobacter hepaticus-free mice

Helicobacter hepaticus is well established as an unwanted variable in laboratory rodent colonies. Historically, cesarean section and embryo transfer have been used to derive Helicobacter-free mouse colonies. Neonatal transfer of newborn mice onto Helicobacter-free foster dams was recently reported as an alternative method of deriving Helicobacter-free mice, but until now, the age by which pups must be fostered to remain Helicobacter-free was unknown. The purpose of the study reported here was to determine the age by which mouse pups must be fostered to remain free of H. hepaticus. Beginning on the day of birth, 20 C57BL/6 mice were fostered from H. hepaticus-positive parents onto Helicobacter-free BALB/c dams each day for 14 days for a total of 280 pups fostered. Fecal specimens collected at weaning, and fecal, liver, and cecal specimens collected at euthanasia were analyzed by use of polymerase chain reaction (PCR) analysis. No pup fostered within 24 h of birth became infected with H. hepaticus; however, many of those fostered after 24 h became infected. These results were supported by those of a large field trial, in which 201 litters representing 71 strains of mice were fostered within 24 h of birth. Follow-up fecal PCR analysis was performed on 52 mice or their progeny that were randomly sampled from the 201 fostered litters. All mice tested remained free of H. hepaticus approximately 100 days after fostering. The results indicate that mouse pups must be fostered within 24 h of birth to remain free of H. hepaticus. In addition, cecal and fecal PCR analyses detected more infections, than did liver PCR analysis, thus indicating that those specimens are preferred for detection of H. hepaticus infection.     

Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system

This study established the feasibility of rederiving numerous mouse hepatitis virus (MHV) and mouse encephalomyelitis virus (MEV) antibody positive strains of mice using cross fostering techniques and a new caging system, thus permitting introduction of virus antibody free mice into a barrier facility. Serologic status of dams within the nucleus breeding colony was determined, and all mice within the breeding colony were housed in individual Microisolator cages. Specific pathogen free (SPF) foster mothers purchased from a commercial source were determined to have no detectable serum antibody to 11 murine viruses including MEV and MHV. Pups delivered naturally from time pregnant dams were cross fostered onto the SPF foster dams. The procedure of cross fostering was conducted within a positive flow, HEPA-filtered, mass air displacement unit within 24 hours of parturition. The virus status of pups from 49 litters was monitored serologically at weaning and again at 6 weeks of age. All cross fostered litters were serologically negative for antibody to mouse hepatitis virus. Seven of 29 litters were negative for MEV antibody titer using this cross fostering technique. Those litters negative serologically to both MHV and MEV (at 3 and 6 weeks) were transferred to a barrier facility and held in isolation. All rederived mice transferred to the barrier facility remained negative for MHV and MEV when sampled at 12 weeks of age.     

Prevalence of natural virus infections in laboratory mice and rats used in Canada

Results of serological tests carried out over a period of 6 years to detect the presence of antibodies against 14 indigenous viruses in mice and rats used in 32 Canadian institutions are reported. Close to 20,000 individual sera were tested by the complement fixation or the hemagglutination inhibition technics. In order of mouse colony prevalence the six most common viruses present were pneumonia virus of mice, mouse hepatitis virus, rat virus, minute virus of mice, Sendai, and Theiler's mouse encephalomyelitis viruses. The most common viruses present in rat colonies were minute virus of mice, K virus, coronaviruses (rat coronavirus or sialodacryoadenitis virus), rat virus, H-1, pneumonia virus of mice, Theiler's mouse encephalomyelitis viruses, Sendai, and reovirus 3.     

Evaluation of Helicobacter hepaticus bacterial shedding in fostered and sex-segregated C57BL/6 mice

Neonatal fostering has been evaluated as a means of eliminating Helicobacter hepaticus infection in laboratory mouse colonies. The purpose of the present study was to evaluate cross-fostering of neonatal C57BL/6 pups from experimentally infected dams after male-absent parturition and to determine the effects of sex and housing strategy on H. hepaticus populations. Approximately 20 C57BL/6 mice (age, 1 to 4 days) were fostered daily. In all fostered mice, fecal samples collected at 21 and 42 days of age and cecal samples collected at 42 days of age tested negative for H. hepaticus by polymerase chain reaction analysis. Our results demonstrate that removal of the male prior to parturition extends the fostering period to yield Helicobacter-free mice. In a second experiment, the effects of time of infection, housing strategy, and sex on fecal H. hepaticus shedding and cecal colonization were evaluated. Neither time nor housing strategy affected bacterial shedding. In contrast, fecal and cecal bacterial loads were higher in male mice versus female mice. A novel predictive algorithm was developed to predict cecal bacterial colonization levels in light of fecal bacterial loads. Our findings likely will prove useful in Helicobacter eradication efforts and in studies designed to further elucidate the role of H. hepaticus in disease.     

Review of successful treatment for Helicobacter species in laboratory mice

Three variations of the amoxycillin-based triple therapy (amoxycillin, metronidazole and bismuth) were administered in the diet, by oral gavage or in the diet in conjunction with cross-fostering on to Helicobacter-free foster mothers to mice naturally infected with H. hepaticus and/or H. bilis. The presence of Helicobacter species was determined by polymerase chain reaction (PCR) analysis of faecal pellets. Helicobacter infection was eliminated in 50% of strains of mice treated by oral gavage; 57% of strains of mice treated by medicated diet alone and 100% of strains of mice treated with the medicated diet in conjunction with cross-fostering on to Helicobacter-free foster mothers. Eight strains of mice were successfully treated for Helicobacter infection over a two-year period. The mouse colony has been maintained Helicobacter free, as determined by PCR analysis and has remained off treatment from December 2002 to March 2005.     

Viral-induced neurodegenerative disease.

Viral etiology has been postulated in a variety of neurological diseases in humans, including multiple sclerosis. Several experimental animal models of viral-induced neurodegenerative disease provide insight into potential host- and pathogen-dependent mechanisms involved in the disease process. Two such mouse models are the Theiler's murine encephalomyelitis virus (TMEV) infection and mouse hepatitis virus (MHV) infection.     

Efficacy of three microbiological monitoring methods in a ventilated cage rack

The use of individually ventilated caging (IVC) to house mice presents new challenges for effective microbiological monitoring. Methods that exploit the characteristics of IVC have been developed, but to the authors' knowledge, their efficacy has not been systematically investigated. Air exhausted from the IVC rack can be monitored, using sentinels housed in cages that receive rack exhaust air as their supply air, or using filters placed on the exhaust air port. To aid laboratory animal personnel in making informed decisions about effective methods for microbiological monitoring of mice in IVC, the efficacy of air monitoring methods was compared with that of contact and soiled bedding sentinel monitoring. Mice were infected with mouse hepatitis virus (MHV), mouse parvovirus (MPV), murine rotavirus (agent of epizootic diarrhea of mice [EDIM]), Sendai virus (SV), or Helicobacter spp. All agents were detected using contact sentinels. Mouse hepatitis virus was effectively detected in air and soiled bedding sentinels, and SV was detected in air sentinels only. Mouse parvovirus and Helicobacter spp. were transmitted in soiled bedding, but the efficacy of transfer was dependent on the frequency and dilution of soiled bedding transferred. Results were similar when the IVC rack was operated under positive or negative air pressure. Filters were more effective at detecting MHV and SV than they were at detecting MPV. Exposure of sentinels or filters to exhaust air was effective at detecting several infectious agents, and use of these methods could increase the efficacy of microbiological monitoring programs, especially if used with soiled bedding sentinels. In contemporary mouse colonies, a multi-faceted approach to microbiological monitoring is recommended.     

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.     

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.     

Infectious microorganisms in mice (Mus musculus) purchased from commercial pet shops in Germany

In this study, we investigated the prevalence of infectious microorganisms (viruses, bacteria, fungi and eukaryotic parasites) in mice from different pet shops in Germany; such animals may compromise the hygienic integrity of laboratory animal vivaria if private pet holders act as unintended vectors of infections carried by them. House mice sold as pets or feed specimens were purchased from different pet shops and tested for a comprehensive panel of unwanted microorganisms. We found a number of microorganisms in these pet shop mice, the most prevalent of which were Helicobacter species (92.9%), mouse parvovirus (89.3%), mouse hepatitis virus (82.7%), Pasteurella pneumotropica (71.4%) and Syphacia species (57.1%). Several microorganisms (e.g. mouse parvovirus, Theiler's murine encephalomyelitis virus, pneumonia virus of mice, Encephalitozoon cuniculi, Clostridium piliforme) had considerably higher prevalences than those reported in similar studies on wild mice from North America, Europe or Australia. Our study shows that direct contact with pet shop mice may constitute a risk for laboratory animal vivaria if hygienic precautions are not taken. However, even relatively simple precautions seem effective enough to hold the risk at bay.     

A serological survey on 15 murine pathogens in mice and rats

A serological survey for 15 murine pathogens was performed on 269 mouse sera collected from 21 conventional and 12 barrier colonies, and on 376 rat sera collected from 21 conventional and 23 barrier colonies. Animals having an antibody against at least one of the antigens were contained in 81.0% of conventional and 16.7% of barrier mouse colonies and also in 81.0% of conventional and 43.5% of barrier rat colonies. Main contaminants were mouse hepatitis virus and Sendai virus in mice, and Sendai virus and pneumonia virus of mice in rats. Results also indicated that antibodies to Toolan's H-1, minute virus of mice and PVM were positive in mice from a considerable number of colonies and those to Kilham rat virus, Mycoplasma pulmonis and Toolan's H-1 were sometimes detected in rats, suggesting prevalences of these pathogens in mice and rats in Japan.     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

Serologic study on the prevalence of murine viruses in a population of wild meadow voles (Microtus pennsylvanicus)

The demography and serology of a population of wild meadow voles (Microtus pennsylvanicus) were monitored from 1982 to 1984 near Pinawa, Manitoba, Canada. Serologic tests were performed on 486 samples to detect the presence of viral antibodies to 11 common murine viruses. Meadow voles showed evidence of infection with Theiler's encephalomyelitis, reovirus-type-3, ectromelia, lymphocytic choriomeningitis, adenovirus, and mouse hepatitis viruses. At times of good survival and breeding performance the population was nearly free of evidence of viral infection. During a period of severe mortality in the winter of 1982-1983, evidence of infection by Theiler's encephalomyelitis virus and reovirus-type-3 was obtained. A high prevalence of antibodies and high titers to these two viruses were characteristic of voles that were captured late in the decline in density in the spring of 1983. This association of mortality with a viral outbreak is consistent with the hypothesis that vole population declines are sometimes related to opportunistic pathogens present in the voles' biotic and social environment.     

Ten-year long monitoring of laboratory mouse and rat colonies in French facilities: a retrospective study

From 1988 to 1997, a total of 69 mouse colonies and 36 rat colonies were examined for the presence of antibodies to 14 indigenous viruses of mice and rats. Among mouse viruses, high positivity rates were observed with mouse hepatitis virus (MHV), Theiler's encephalomyelitis virus (THEMV), minute virus of mice (MVM), Sendai virus and pneumonia virus of mice (PVM); the prevalence rates were high in rats with Khilam's rat virus (KRV), THEMV, Toolan's H-1 virus, Sendai virus, Parker's rat coronavirus (RCV/SDA) and PVM. During the last decade, the prevalence of some agents such as MHV, Sendai virus, THEMV, PVM and MVM has apparently decreased although they were still present in 1997 (except for PVM). Another point is the constant increase of colonies found free of viruses through this decade, demonstrating the efforts of the French research community to increase the quality of hygiene in laboratory animals.     

B-lymphocyte requirement for vaccine-mediated protection from Theiler's murine encephalomyelitis virus-induced central nervous system disease.

The role of humoral immunity in the protection of vaccinated SJL/J mice from central nervous system disease induced by the DA strain (DAV) of Theiler's murine encephalomyelitis virus was investigated in B-cell-deficient mice. Mice were depleted of B cells by treatment with a mouse monoclonal antibody specific for immunoglobulin M. DAV-vaccinated, B-cell-deficient mice failed to clear viral infection and were no longer protected from Theiler's murine encephalomyelitis virus-mediated central nervous system disease. CD4+ T cells are required in this model of protection to provide help for the development of an antiviral antibody response in the central nervous system.     

Mode of spread to and within the central nervous system after oral infection of neonatal mice with the DA strain of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus is a neurotropic enterovirus known to cause biphasic neural disease after intracerebral inoculation into adult mice. The present study characterizes a neonatal mouse model with a high disease incidence for the study of the acute phase of the pathogenesis of the DA strain of Theiler's murine encephalomyelitis virus after oral infection. The route of viral spread to and within the central nervous system (CNS) was determined by examining the kinetics of viral replication in various organs and by performing histopathological analysis. Viral antigen was detected widely in the neonatal CNS, mainly in the gray matter, and it was asymmetrical and multifocal in its distribution, with considerable variation in lesion distribution from animal to animal. Necrotizing lesions appeared to expand by direct extension from infected cells to their close neighbors, with a general disregard of neuroanatomical boundaries. The diencephalon showed particular susceptibility to viral infection. Other areas of the CNS, including the cerebellum and dentate gyrus of the hippocampus, were consistently spared. Neurons with axons extending peripherally to other organs or receiving direct input from the peripheral nervous system were not preferentially affected. The kinetics of viral replication in the liver, spleen, and CNS and the histopathological findings indicate that viral entry to the CNS is via a direct hematogenous route in orally infected neonatal mice and that the disease then progresses within the CNS mainly by direct extension from initial foci.     

Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections

Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals.
Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus , and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns.
Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H . bilis , in 91% for H. hepaticus, and in 82% for H. ganmani . The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium .
Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.     

Specific infection and destruction of dopaminergic neurons in the substantia nigra by Theiler's virus.

Theiler's murine encephalomyelitis virus was stereotaxically inoculated unilaterally into the substantia nigra of the mouse brain. Virus specifically infected tyrosine hydroxylase-positive neurons and spread rostrocaudally throughout this subpopulation of neurons, resulting in impaired function and degeneration of the substantia nigra. The spread of the virus to other areas of the brain was minimal and rare.     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.