Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion

There is an ongoing need to eradicate intercurrent disease from research mouse colonies. Commonly used surgical methods, however, are expensive and time-consuming. The purpose of this study was to determine the percentage of litters that could be rederived from infected mouse colonies by neonatal transfer. We immersed neonatal mice in a dilute iodine solution and transferred them to disease-free foster mothers within 48 h of birth. Donor and foster mothers were evaluated for pathogens by serology and fecal polymerase chain reaction (PCR) assay. Of 55 donor mothers, 100% were positive serologically and 59% were positive by fecal PCR for one or more tested organisms, including mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and Helicobacter hepaticus. At 4 to 6 weeks after neonatal transfer, 95% of foster mothers (which served as sentinels for the transferred pups) tested free of pathogens, the exceptions being one case of mouse parvovirus 1 and two of Helicobacter spp. We suggest that cross-fostering is a viable low-cost method for rederivation of mouse colonies contaminated with pathogens such as mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and H. hepaticus.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Embryo transfer rederivation of C.B-17/Icr-Prkdc(scid) mice experimentally infected with mouse parvovirus 1

We determined whether embryos derived from C.B-17/Icr-Prkdc(scid) (SCID) mice infected with mouse parvovirus (MPV) 1b and mated to MPV-naive B6C3F1 mice would transmit virus to naive recipient female mice and rederived progeny. Viral DNA was detected by quantitative PCR (qPCR) in lymphoid tissues, gonad, sperm, and feces of all MPV1b-inoculated SCID mice. Viral DNA was detected in 1 of 16 aliquots of embryos from infected male SCID mice and in 12 of 18 aliquots of embryos from infected female SCID mice. All recipient female mice implanted with embryos from infected SCID male mice and their progeny were negative by serology and qPCR. In contrast, 3 of 5 recipient female mice implanted with embryos from infected SCID female mice and 14 of 15 progeny mice from these recipients were seropositive by multiplex fluorescent immunoassay (MFI) for MPV capsid antigen (rVP2). All of these mice were negative by MFI for parvovirus nonstructural protein antigen (rNS1) and by qPCR, with the exception of 1 recipient female mouse that displayed weak rNS1 seroreactivity and low levels of MPV DNA in lymphoid tissues. Seroreactivity to rVP2 declined over time in all progeny mice from infected SCID female mice until all were seronegative by 20 wk of age, consistent with maternal antibody transfer. Given that the high levels of MPV contamination detected in our experimentally infected SCID mice are unlikely in naturally infected immunocompetent mice, these data indicate that embryo transfer rederivation is effective for the eradication of MPV from infected colonies.     

The effectiveness of a microisolator cage system and sentinel mice for controlling and detecting MHV and Sendai virus infections

Experiments were conducted to determine (a) whether BALB/c mice housed on soiled bedding can be used as sentinels for the detection of Sendai virus and MHV from infected mice housed in microisolators, and (b) whether the microisolator caging system protects mice against Sendai virus and MHV infections. Sentinel mice were housed in microisolator cages, exposed continuously to soiled bedding and bled at 21 and 42 days for serology. All sentinel mice were seropositive for MHV by 42 days; however, sentinel mice exposed to soiled bedding were seronegative for Sendai virus at 21 and 42 days. These results suggest that sentinels housed on soiled bedding may not detect all infectious murine viruses. This study also showed that the microisolator caging system provided an effective barrier against MHV infection at the cage level and suggests that the microisolators should protect mice against other infectious agents.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

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.     

Antibody prevents the establishment of persistent arenavirus infection in synergy with endogenous T cells.

A cardinal feature of the biology of lymphocytic choriomeningitis virus (LCMV) is its ability to establish persistent infections in mice. Persistence is usually established by infection of the mouse during the in utero or neonatal period. Susceptibility can be extended to the adult by treatment with immunosuppressive agents or by infection with immunosuppressive strains of LCMV. In this study we investigated the capacity of passively acquired anti-LCMV antibodies to prevent the establishment of persistence in both neonatal and adult mice. Suckling BALB/c mouse pups nursed by mothers immunized against LCMV before pregnancy had higher survival rates following infection than controls and withstood challenge doses of up to 400 PFU without becoming persistently infected. To establish that maternal antibody alone and not maternally derived T cells provided this protection, nonimmune mothers were infused with monoclonal anti-LCMV neutralizing antibodies within 24 h after delivering their pups. Pups nursing on these passively immunized mothers were resistant to persistent LCMV infection. The establishment of persistence in adult BALB/c mice by the immunosuppressive, macrophage-tropic LCMV variant, clone 13 was also prevented by prophylactic treatment with anti-LCMV monoclonal antibodies. However, the protection afforded by passively acquired antibody was found to be incomplete if the recipients lacked functional CD8+ T cells. While 65% of neonatal athymic (nu/nu) mice nursed by immune nu/+ dams resisted low-dose viral challenge (25 PFU), the majority of nude pups challenged with high doses of virus (100 PFU) became persistently infected. Also, protection was incomplete in beta2-microglobulin knockout mice, which lack functional CD8+ T cells, suggesting that a cooperative effect was exerted by the combination of neutralizing antibody and endogenous T cells. These results indicate that antibodies provide an effective barrier to the establishment of persistent infections in immunocompetent mice and reaffirm that vaccines which induce strong humoral responses may provide efficient protection against arenavirus infections.     

淋巴细胞脉络丛脑膜炎病毒感染实验动物的情况概述

淋巴细胞脉络丛脑膜炎病毒（lymphocytic chorimeningtis virus,LCMV）能广泛感染啮齿类动物和人,是一种重要的人畜共患病病原。近些年LCMV感染人的检测得到加强,在实验动物中的感染率一直控制在较低水平。我国的实验动物国家标准要求豚鼠、地鼠必需检测LCMV,小鼠只在必要时检测,而国外普遍要求对大鼠等动物也作为常规检测项目。为了提高对实验动物感染LCMV的检测意识,本文对不同国家和地区LCMV感染实验动物的情况做一综述。     

Administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation

Routine testing of bedding sentinels from a barrier room revealed one mouse seropositive to ectromelia virus (EV). Results of hemagglutination-inhibition testing and western blot analysis were confirmatory for orthopoxvirus antibodies. Additional seropositive animals were not identified. Interviews indicated that replication-competent vaccinia virus (VV), Western Reserve strain (VV-WR), recently had been given to mice. Although VV-WR was not expected to spread by contact or via fomites, the case evidence suggested transmission of vaccinia via soiled bedding. In a follow-up experiment, 15 index mice were inoculated with 10(7) plaque-forming units of VV by either subcutaneous or intrarectal instillation. A dedicated contact sentinel and a bedding sentinel were provided for each index mouse. All 15 index mice were positive for antibodies when tested 22 days after inoculation. One mouse, inoculated by the subcutaneous route, appeared ill and developed lesions on the proximal portion of the tail. The contact sentinel mouse housed with this index mouse was the only sentinel to seroconvert. We conclude that VV-WR can spread to contact sentinels and potentially to bedding sentinels. The ability of other VV strains to be transmitted horizontally and the susceptibility of different mouse strains to infection merit further investigation. The use of VV in animal facilities must be managed carefully since the available serologic tests do not distinguish between VV and EV, an exotic agent of major concern to laboratory animal facilities.     

Direct non-insect-vector transmission of Leishmania parasites in mice

Animal to animal non-vector transmission of Leishmania major was investigated in Balb/c mice, a strain known for its susceptibility to this parasite. Both overt or inapparent infection (documented by positive spleen cultures) was possible after prolonged contact with infected animals. Similarly transmission of infection from infected mothers to their offspring was documented.     

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.     

Persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.     

Duration and patterns of transmission of Theiler's mouse encephalomyelitis virus infection

The duration and patterns of Theiler's murine encephalomyelitis virus (TMEV) transmission were studied in eight index mice inoculated orally. Transmission was monitored by testing for seroconversion to TMEV in sentinel mice in direct contact with index mice and in other sentinel mice in contact with bedding soiled by index mice. For the first 14 weeks after inoculation, two contact sentinels were housed with each index mouse for 1 week, then replaced with two new sentinels. For the remaining 16 weeks, contact sentinels were changed monthly. All index mice transmitted TMEV continuously (weekly) for 4 to 9 weeks. Thereafter, six index mice transmitted virus intermittently. All index mice ceased transmitting TMEV 7 to 22 weeks post-inoculation. Results obtained from sentinel mice in contact with bedding soiled by index mice were 86% concordant with those using contact sentinel mice. Seven index mice were treated with cyclophosphamide or hydrocortisone 30 weeks post-inoculation. One cyclophosphamide treated mouse reinitiated virus shedding.     

Detection of lymphocytic choriomeningitis virus antibody in colonies of laboratory animals in Japan

Indirect fluorescent antibody method was applied for a detection of lymphocytic choriomeningitis virus (LCMV) antibody in colonies of laboratory animals in Japan. The results showed that the antibody exist in SPF mice (3/152, 2.2%) and conventional mice (30/539, 5.6%) with the titers ranging from 1:10 to 1:160. The antibody was also detected in 2.2% (2/89) of Syrian hamsters, and 2.9% (2/68) of Apodemus agrarius, 21.4% (3/14) of Japanese harvest mice which have been maintained as laboratory colony for several years. However, the antibody was not demonstrated in Mongolian gerbils, Suncus murinus, guinea pigs and rats, thus far. These results indicate that LCMV infection is present in laboratory animals in Japan, and pointed out the importance of microbiological monitoring for LCMV.     

Survival and transmissibility of Pasteurella pneumotropica

Survival has been determined for Pasteurella pneumotropica on various surfaces found in an animal room at 23+/-1 degrees C and 50+/-10% relative humidity. Longest survival (120 min) was found on mouse hair, shortest (< 30 min) on laboratory coat fabric. Transmission experiments were performed using sentinel animals in order to evaluate the efficiency of their use for the detection of P. pneumotropica in quarantined mice. In sentinels exposed to infected mice by close contact, P. pneumotropica was detected by culture 2 weeks post-exposure and seroconversion 3 weeks after contact. Transfer of soiled bedding from Pasteurella-infected mice did not infect sentinels within a period of 12 weeks as tested by cultivation or serum antibodies.     

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.     

Control of virus-specific CD8+ T-cell exhaustion and immune-mediated pathology by E3 ubiquitin ligase Cbl-b during chronic viral infection

A characteristic feature in the immune response to many persistent viral infections is the dysfunction or deletion of antigen-specific T cells (exhaustion). This down-regulation of virus-specific T-cell response represents a critical control mechanism that exists within T-cell activation pathways to prevent lethal disease by inappropriate responses against disseminating virus infections. However, the molecular mechanisms by which the immune system determines whether to mount a full response to such infections remain largely unexplored. Here, we have established that in the murine lymphocytic choriomeningitis virus (LCMV) model, induction of the T-cell receptor signaling inhibitor molecule E3 ligase Cbl-b is critically involved in this decision. In particular, our data revealed that Cbl-b controls the program responsible for T-cell tolerance (exhaustion) induction during a chronic viral infection. Thus, Cbl-b(-/-) mice infected with a low dose of LCMV Docile mount a strong CD8(+) T-cell response that rapidly clears the infection, and the animals remain healthy; in contrast, down-regulation of the epitope-specific CD8(+) T-cell population in persistently infected Cbl-b(-/-) mice, compared to that in chronically infected B6 mice, was significantly delayed, and this was associated with increased morbidity and eventual death in nearly 20% of the animals. Interestingly, infection of Cbl-b(-/-) mice with a moderate virus dose resulted in rapid death with 100% mortality by 7 to 8 days after infection, caused by a dysregulated antiviral T-cell response, whereas the infected B6 mice survived and remained healthy. In conclusion, our results suggest that Cbl-b is critically involved in T-cell exhaustion and prevention of lethal disease.     

Retroviruses and embryogenesis: Microinjection of Moloney leukemia virus into midgestation mouse embryos

The interaction of Moloney leukemia virus (M-MuLV) with developing post-implantation mouse embryos was studied. First, the frequency at which embryos in utero are infected by transplacental transmission with maternal virus was explored. To exclude milk transmission from the viremic mother, embryos were delivered by cesarean section prior to birth and given to normal foster mothers. None of 72 mice raised this way developed viremia. This indicates that the placenta is an efficient barrier protecting the developing embryo against infection with exogenous retroviruses. To overcome the placental barrier and to introduce virus into embryos at defined stages of differentiation, Moloney leukemia virus was microinjected directly into embryos in utero at day 8 or 9 of gestation. Between 60 and 70% of the injected embryos survived to birth and were tested for viremia at 4 weeks of age. M-MuLVspecific sequences were quantitated in organs of viremic animals derived from midgestation embryos microinjected with virus. Molecular hybridization experiments with nucleic acids extracted from different organs of these animals indicated that every cell type carried M-MuLV-specific DNA sequences and that high concentrations of M-MuLV-specific RNA sequences were present in every organ. In contrast, M-MuLV infection and expression is restricted to lymphatic tissues when animals are exposed to virus after birth or in BALB/Mo mice. These results indicate that the most important parameter determining the “target tropism” of Moloney leukemia virus infection and expression is the stage of embryogenesis and cellular differentiation at which virus infection takes place. In viremic C57BL animals derived from microinoculated embryos, the hair color changed beginning at age 6 weeks. This was not observed in animals exposed to virus after birth. All animals succumbed to MMuLV-induced leukemia at a later age. The results suggest that expression of M-MuLV may also lead to cellular dysfunctions other than leukemic transformation.     

Transforming growth factor-beta inhibits the generation of cytotoxic T cells in virus-infected mice

The immunoregulatory effects of human recombinant transforming growth factor (rTGF) beta 1 and human recombinant glioblastoma-derived T cell suppressor factor (rG-TsF)/TGF beta 2 was investigated in mice infected with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus. Starting on the day of infection, i.p. injections of 1 microgram/day or rTGF-beta 1 or rG-TsF/TGF-beta 2 suppressed the generation of virus specific CTL. The effect of TGF-beta on CTL (day 8) was less pronounced when TGF-beta treatment was delayed for 3 days after LCMV infection. rG-TsF/TGF-beta 2 also has an inhibiting effect on CTL-mediated disease in LCMV-infected mice: it prolonged the survival time of mice infected with LCMV and reduced the local swelling reaction after infection into the footpad. These results indicate that rTGF-beta 1 and rG-TsF/TGF-beta 2 influence T cell immune reactivity in vivo.     

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.