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.     

Alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus

Pre-infection with mouse hepatitis virus (MHV) strains S, 3, or JHM reduced the ability of mice to seroconvert to PVM. Geometric mean antibody titers to PVM among MHV pre-infected mice were lower than those for control mice given only PVM, and dually infected mice seroconverted to PVM later than mice given PVM alone. PVM was not recovered from normally permissive respiratory tract tissues of MHV-S pre-infected mice. Pre-infection of DBA/2 mice with MHV-S compromised the susceptibility of these mice to lethal Sendai virus infection but did not substantially reduce the titers of infectious Sendai virus recovered from the lungs. Serologic responses to Sendai virus and lung Sendai virus titers were similar in Sendai virus-resistant C57BL/6 mice pre-infected or not with MHV-S.     

Differences in antibody production against mouse hepatitis virus (MHV) among mouse strains

Several strains of mice were examined for antibody production after intranasal inoculation with a low virulence strain of mouse hepatitis virus (MHV), MHV-NuU. C57BL/6N mice were shown to be high responders in the production of complement fixing (CF) antibody as compared to C3H/HeN, BALB/c-AnN, DBA/2N mice. F1 hybrids B6C3 and BDF1 from C57BL/6N mice, showed CF antibody responses as high as C57BL/6N, suggesting that high responsiveness is genetically controlled. All these mouse strains were able to produce high titred neutralizing antibody to MHV.     

Studies on the development of an ELISA kit for microbiological monitoring. 1. Evaluation of the reliability of the prototype kit by field tests

The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV)/sialodacryoadenitis virus (SDAV)/Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2 micrograms/ml and MHV Nu-67 strain, 5 micrograms/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10,094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.     

A comparison of two antigen strains of each of mouse hepatitis virus and mouse adenovirus for detection of complement fixation antibody in mouse and rat sera

Detection rates of complement fixation antibodies in mice and rats were compared between two antigen strains of each of mouse hepatitis virus (MHV) and mouse adenovirus (MAV). Among 66 and 47 naturally infected MHV-positive sera of mice (18 facilities) and rats (16 facilities) respectively, 17 mouse and 21 rat sera reacted with both Nu-67 and MHV-2 strains, but 49 mouse and 25 rat sera were positive to Nu-67 strain alone. Only one rat serum reacted with MHV-2 strain alone. In comparison with K87 and FL strains of MAV, all 8 positive mouse sera (3 facilities) reacted with K87 strain alone whereas out of 53 positive rat sera (20 facilities), 43, 6 and 4 sera reacted with K87 strain alone, with FL strain alone and with both the two strains, respectively.     

Adverse effects of mouse hepatitis virus on ascites myeloma passage in the BALB/eJ mouse.

During experimental serial passage of ascites myelomas through BALB/cJ mice, unexpected illness and premature deaths occurred. Postmortem examination of affected mice revealed focal or diffuse discolored depressed areas in the liver and, in some cases, splenomegaly. Histopathologic findings consisted of focal to diffuse areas of necrosis with minimal leukocytic infiltration. Aerobic and anaerobic bacterial cultures of livers and spleens from affected mice were negative. Mouse hepatitis virus (MHV) was isolated from livers of clinically ill mice and from the ascites myeloma lines. An MHV contaminated ascites myeloma line, when passed into nude (nu/nu) mice, killed the animals in 6 days; the virus was isolated from livers of inoculated mice. Attempts to determine the source of the infection were unsuccessful. Serologic survey of newly acquired mice indicated no evidence of antibodies to MHV while mice in holding rooms had titers that ranged from 1:10 to 1:40. Two solid myeloma lines (being maintained by subcutaneous passage) were negative for MHV when tested by virus isolation techniques, and nine lines were negative to 11 murine viruses when tested by mouse antibody production assay. Attempts to demonstrate Eperythrozoon coccoides in control BALB/cJ mice were unsuccessful. Because of the outbreak, changes were made in animal handling procedures. A colony of BALB/cAn mice negative to MHV antibodies was established to provide animals for experimental passage of tumors, and animals in both the breeding and transfer room were placed under filter tops. The results were encouraging. In the four newly established tumor lines, one having been passed 46 times, no illness or unexplained deaths were observed.     

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.     

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.     

Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-processing function of the Kupffer cells of both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis

Abstract The genetic sensitivity of mouse strains to mouse hepatitis virus 3 (MHV 3) has been related in vitro to a delay of virus replication in liver sinusoidal cells. In vivo immuno-histochemical studies of the liver from infected mice have demonstrated that mechanisms other than direct viral injury are in operation. To examine potential mechanisms, the interaction of lipopolysaccharide (LPS)-stimulated Kupffer cells with MHV 3 was studied. We first observed a dramatic inhibition in viral replication in LPS-treated Kupffer cells explanted from A/J resistant mice. Second, we demonstrated that MHV 3 induced a dose-dependent interleukin 1 (IL-1) activity in the supernatants of infected Kupffer cells of both strains. These results led us finally to examine the antigen-proceesing function of the Kupffer cellsof both strains of mice. No striking differences were observed in the ability of Kupffer cells from resistant or sensitive mice to collaborate with immunocompetent lymphocytes. Our data suggest that Kupffer cells play a double role which is crucial in the pathogenesis of MHV 3-induced hepatitis. First, they act directly as the genetically determined sensitivity of mice to MHV 3 infection is correlated with the efficiency of the antiviral activity induced in Kupffer cells by LPS. Second, they act indirectly through the synthesis of different amounts of IL-1 induced by MHV 3. This hypothesis is further borne out by the effects of indomethacin treatment on the course of MHV 3 infection in A/J resistant mice in vivo.     

Studies on the development of an ELISA kit for microbiological monitoring. 2. Improvement of the prototype ELISA kit with special references to mouse hepatitis virus antigen

Improvement of the mouse hepatitis virus (MHV) antigen in a prototype ELISA kit was performed. Equivalent divalent antigens of MHV Nu-67 and S strains with a protein concentration of 10 micrograms/ml showed the best sensitivity and specificity for the detection of MHV and sialodacryoadenitis/Parker's rat coronavirus antibodies in mice and rats, respectively. An increase in the reliability of macroscopic evaluation of both antibody tests is expected by using the newly manufactured kit with the improved antigen.     

Difference in response to mouse hepatitis virus among susceptible mouse strains.

After intraperitoneal inoculation with a high-virulent mouse hepatitis virus (MHV) a significant difference was seen in survival time between DDD and CDF1 (BALB/c X DDD) mice, while 50% lethal doses were not significantly different. With 3 X 10(3) PFU of the virus CDF1 and DDD mice died in 45 and 120 hr, respectively, on the average. This difference of susceptibility between DDD and CDF1 mice was first demonstrable at the age of 1 week and was more pronounced at the age of 4 weeks but showed no dependence of the sex. Virus titers ran 2 to 3 log higher in the liver and blood of CDF1 than in those of DDD mice, while being only 1 log higher in the spleen. At an early stage of infection viral antigen was demonstrable by immunofluorescence in sinusoidal lining cells of the liver more prominently in VDF1 than in DDD mice. Interferon production occurring in parallel with virus growth was significantly higher in CDF1 than in DDD mice. In DDD mice, liver lesions were rather focal with some accumulation of round cells, while they were confluent with poor cellular response in CDF1 mice. Viral growth in cultured peritoneal macrophages from CDF1 mice was 1 log higher than in those from DDD mice. The results suggest that the divergence in response to MHV among susceptible mice greatly depends upon the susceptibility of macrophages and reticuloendothelial cells which constitute primary targets of the virus.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

In vivo RNA-RNA recombination of coronavirus in mouse brain.

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.     

Microbiological monitoring in inbred mouse foundation stocks in Japan

Microbiological monitoring on 128 inbred mouse foundation stocks consisted of common 10 inbred strains and inbred strains originated from outbred dd mice was performed by cooperation of 24 organizations. A total of 881 mice were divided into 647 conventional animals from 95 colonies and 234 barrier-sustained animals from 33 colonies. Three viral, one mycoplasmal, 6 bacterial, one fungal and 3 parasitic agents selected as monitoring microbes according to the proposed selection standards. Among conventional colonies, 84.2% were positive for at least one agent. The highest detection rate was 44.2% for S. obvelata, followed by P. pneumotropica and S. muris, P. aeruginosa, G. muris, Sendai virus, M. pulmonis, MHV and E. coli O115a, c: K (B). Of these agents, only one microbe, P. aeruginosa, was detected in barrier-sustained colonies (36.4%), thus the efficacy of barrier system for the microbiological quality control of the inbred mouse foundation stocks was actually demonstrated. The positive rates of MHV (6.3%) and Sendai v. (16.8%) were significantly low compared with those in experimental mouse colonies. Positivity for parasites was rather high and they were infested together with other pathogens in many cases. Thus parasites including G. muris, S. muris and S. obvelata were regarded as useful indicators to see microbiological contaminations in conventional mice. There observed no strain difference in susceptibility to pathogens except for C57BL/6 and AKR mice which seemed to be high antibody responders to MHV.     

Rederivation of inbred strains of mice by means of embryo transfer

Embryo transfers were performed to rederive six inbred strains of mice, A/He, BALB/cByJ, BALB/c Lac, B10.BR/SgSnJ, C57BL/6J and DBA/2J. The aim was to determine whether it is possible to eliminate pathogens like mouse hepatitis virus (MHV) and Pasteurella pneumotropica (P.p.). The embryos were collected, handled and transferred into the oviduct of day one pseudopregnant SPF surrogate mothers under aseptic conditions. In 40.5% of the transfers, embryos developed to term. With respect to surrogate mothers delivering viable litters, 47.9% of the transferred embryos were born alive. Out of these 93.5% were reared. Virological and bacteriological examination of embryo donors verified the presence of P.p. and of antibodies against MHV in all strains. In some embryo donors P.p. could be isolated even from the uterine mucosa. However, neither in the surrogate mothers nor in the offspring could P.p. and antibodies against MHV be detected. Further bacteriological examination revealed that the offspring carried only the microbial flora received from the surrogate mother. The results indicate that embryo transfer is an appropriate tool to rederive mouse strains. In contrast to hysterectomy rederivation, embryo transfer has the advantage of avoiding postimplantational vertical transmissions of infections.     

Enterotropic mouse hepatitis virus infection in nude mice

The cause of emaciation and diarrhea in athymic nude mice was found to be hyperplastic typhlocolitis resulting from infection with enterotropic mouse hepatitis virus (MHV). The disease was reproduced in experimentally-inoculated nude mice using intestinal homogenates from affected mice and cell culture-derived virus. Material derived from an experimental mouse was passed into neonatal Swiss mice and caused acute typhlocolitis. Virus failed to grow in NCTC-1469 cells and 17Cl-1 cells, which are normally permissive for MHV, but grew to low titer in a mouse rectal carcinoma cell line, CMT 93. These results show that an enterotropic strain of MHV can cause chronic enteric disease in athymic nude mice. The pattern of infection differs markedly from the more common MHV wasting syndrome in nude mice caused by non-enteric strains of MHV.     

Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus.

BACKGROUND AND OBJECTIVES: Two serotypes of autonomously replicating parvoviruses infect laboratory mice. Genome regions coding for the nonstructural proteins of minute virus of mice [MVM] and mouse parvovirus [MPV] are almost identical, whereas capsid-coding sequences are divergent. We addressed these questions: Does humoral immunity confer protection from acute infection after challenge with homotypic or heterotypic parvovirus, and if it confers protection against acute MPV infection, does it also protect against persistent MPV infection? METHODS: Infant mice without maternal antibody or antibody to MVM or MPV and young adult mice given normal mouse serum or antibody to MVM or MPV were challenged with homotypic or heterotypic virus. In situ hybridization with target tissues was the indicator of infection. RESULTS: Humoral immunity failed to confer protection against acute heterotypic parvovirus infection. In passive transfer studies, MPV DNA was observed occasionally in lymph nodes, intestine, or the spleen of MPV-challenged mice given homotypic antibody and kept for 6 or 28 days. Variable proportions of mice given MPV antibody and homotypic challenge had viral DNA in lymphoid tissues 56 days after virus inoculation. CONCLUSION: A mouse or colony that has sustained infection with MVM or MPV is probably fully susceptible to infection with the heterotypic virus.