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.     

Transmission probabilities of mouse parvovirus 1 to sentinel mice chronically exposed to serial dilutions of contaminated bedding

Intermittent serodetection of mouse parvovirus (MPV) infections in animal facilities occurs frequently when soiled bedding sentinel mouse monitoring systems are used. We evaluated induction of seroconversion in naïve single-caged weanling ICR mice (n = 10 per group) maintained on 5-fold serially diluted contaminated bedding obtained from SCID mice persistently shedding MPV1e. Soiled bedding from the infected SCID mice was collected, diluted, and redistributed weekly to cages housing ICR mice to represent chronic exposure to MPV at varying prevalence in a research colony. Sera was collected every other week for 12 wk and evaluated for reactivity to MPV nonstructural and capsid antigens by multiplex fluorescent immunoassay. Mice were euthanized after seroconversion, and DNA extracted from lymph node and spleen was evaluated by quantitative PCR. Cumulative incidence of MPV infection for each of the 7 soiled bedding dilution groups (range, 1:5 to 1:78125 [v/v]) was 100%, 100%, 90%, 20%, 70%, 60%, and 20%, respectively. Most seropositive mice (78%) converted within the first 2 to 3 wk of soiled bedding exposure, correlating to viral exposure when mice were 4 to 7 wk of age. Viral DNA was detected in lymphoid tissues collected from all mice that were seropositive to VP2 capsid antigen, whereas viral DNA was not detected in lymphoid tissue of seronegative mice. These data indicate seroconversion occurs consistently in young mice exposed to high doses of virus equivalent to fecal MPV loads observed in acutely infected mice, whereas seroconversion is inconsistent in mice chronically exposed to lower doses of virus.Abbreviations: mfi, median fluorescent intensity; MFI, multiplex fluorescent immunoassay; MPV, mouse parvovirus; NS1, nonstructural protein 1; qPCR, quantitative PCR; SCID, severe combined immunodeficiency; VP2, viral capsid protein 2Mouse parvovirus (MPV) is among the most prevalent infectious agents detected in contemporary laboratory mouse colonies^2,7,10 and can have deleterious effects on research because of in vitro and in vivo immunomodulatory effects, tumor suppression, and contamination of cell cultures and tissues originating from mice.^11-13 The potential for MPV transmission among mice in research facilities is enhanced by its environmental stability,⁶ potential to induce persistent infection in mice,⁸ and difficult eradication from infected laboratory mouse colonies. Despite the availability of highly sensitive and specific diagnostic assays,^9,14,15 detection of MPV infections in contemporary laboratory mouse colonies remains problematic, with intermittent detection even under conditions of enzootic colony infections. The widespread use of sentinel mice exposed to soiled bedding as the primary detection system, a relatively short period of viral transmission postinfection in immunocompetent mice, and a fairly high viral dose required to induce productive infection are considered key factors that result in intermittent detection of MPV contamination in mouse colonies. As a result, MPV infections present important and costly challenges to contemporary laboratory animal research facilities.Several studies have investigated the horizontal transmission of MPV to sentinel mice. Experimentally infected SENCAR mice transmitted MPV1a to naïve sentinels both by direct contact and soiled bedding exposure, predominantly during the first 3 wk after inoculation.¹⁷ Similarly, experimentally infected Swiss Webster mice transmitted MPV1d within 2 wk to sentinels by direct contact or through various amounts of soiled bedding.¹⁸ Interestingly, transmission to sentinel mice appeared to be enhanced in mice maintained in individually ventilated caging as compared with static microisolation caging in the cited study. Naturally infected BALB/c mice when 1 mo old, but not when 2, 3, and 6 mo old, transmitted MPV to direct contact sentinels.¹⁶ Recent studies completed in our laboratory¹ indicate that C.B-17/Icr-Prkdc^scid mice inoculated with MPV1e as neonates persistently shed high levels of virus in their feces over several months. Undiluted contaminated bedding collected at any time point during this period consistently transmitted MPV1e to weanling C3H sentinel mice exposed for 2 wk. Similarly inoculated neonatal BALB/c mice shed high levels of virus, with transmission to sentinels, for only 2 wk after inoculation.¹ In all of these reports, the period of exposure of sentinel mice to soiled bedding was limited (2 wk or less), with no repeated exposure opportunities, as might be expected under field conditions with an infected colony. In the present study, we simulated a typical sentinel monitoring program and determined whether chronic exposure to various concentrations of MPV1-contaminated bedding, reflective of a broad range of disease prevalence scenarios within any given affected room, can induce seroconversion in sentinel mice.     

Microbiological monitoring of laboratory mice and biocontainment in individually ventilated cages: a field study

Over recent years, the use of individually ventilated cage (IVC) rack systems in laboratory rodent facilities has increased. Since every cage in an IVC rack may be assumed to be a separate microbiological unit, comprehensive microbiological monitoring of animals kept in IVCs has become a challenging task, which may be addressed by the appropriate use of sentinel mice. Traditionally, these sentinels have been exposed to soiled bedding but more recently, the concept of exposure to exhaust air has been considered. The work reported here was aimed firstly at testing the efficiency of a sentinel-based microbiological monitoring programme under field conditions in a quarantine unit and in a multi-user unit with frequent imports of mouse colonies from various sources. Secondly, it was aimed at determining biocontainment of naturally infected mice kept in an IVC rack, which included breeding of the mice. Sentinels were exposed both to soiled bedding and to exhaust air. The mice which were used in the study carried prevalent infectious agents encountered in research animal facilities including mouse hepatitis virus (MHV), mouse parvovirus (MPV), intestinal flagellates and pinworms. Our data indicate that the sentinel-based health monitoring programme allowed rapid detection of MHV, intestinal flagellates and pinworms investigated by a combination of soiled bedding and exhaust air exposure. MHV was also detected by exposure to exhaust air only. The IVC rack used in this study provided biocontainment when infected mice were kept together with non-infected mice in separate cages in the same IVC rack.     

Role of Mouse Hepatitis Virus (MHV) Receptor Murine CEACAM1 in the Resistance of Mice to MHV Infection: Studies of Mice with Chimeric mCEACAM1a and mCEACAM1b

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD₅₀) of 10^2.5 PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10⁵ PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10⁵ PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.Differences in susceptibility to a number of viral infections have been documented among inbred mouse strains (20). These differences have been studied as models for the various degrees of susceptibility of individual humans to some viral infections. Numerous host factors have been found to be involved in such differences (2, 15). For example, allelic variations in the virus receptor and coreceptor for HIV-1 are important host factors influencing susceptibility to HIV-1 infection (36).A virus receptor is a molecule with which the virus interacts at an initial step of infection. Therefore, receptors are crucial host determinants of virus susceptibility (15, 16). A variety of receptor proteins has been identified for many different viruses, including the murine coronavirus mouse hepatitis virus (MHV) (12, 50). The principal receptor for MHV is murine carcinoembryonic antigen-related cell adhesion molecule 1 (mCEACAM1; previously called Bgp or MHVR [3]), which is in the immunoglobulin (Ig) superfamily (12, 50). Four isoforms of mCEACAM1a (1a) are expressed on the plasma membranes of a variety of murine cells and tissues (14). The two mCEACAM1 isoforms with a molecular mass of 100 to 120 kDa are composed of four Ig-like ectodomains, a transmembrane (TM) domain, and either a long or a short cytoplasmic tail (Cy) (3, 22). Two other isoforms consist of two Ig-like domains, with either long or short Cy (3, 22). The N-terminal (N) domain is responsible for virus binding (10, 24), the induction of conformational changes in the viral spike protein (S), and membrane fusion during virus entry and syncytium formation (13, 24). The replacement of the N-terminal domain of mCEACAM1a with that of the murine homolog of the poliovirus receptor (PVR) yields a functional receptor for MHV (10), and Ceacam1a-knockout mice are completely resistant to infection with the hepatotropic A59 strain of MHV (17, 25).Wild mice have two alleles of the mCeacam1 gene, called mCeacam1a and mCeacam1b. Inbred mouse strains that are homozygous for mCeacam1a, including BALB/c, C57BL/6 (B6), C3H, and A/J mice, etc., are highly susceptible to infection with strains of MHV. In contrast, the SJL line of inbred mice, which is resistant to death from MHV infection, is homozygous for the mCeacam1b allele (5, 11, 50). The most extensive differences in amino acid sequence between mCEACAM1a and mCEACAM1b are found in the N-terminal domain, where the virus-binding region is located (21, 22, 32). It was initially reported by Boyle et al. that mCEACAM1a proteins had MHV-A59 virus-binding activity in a virus overlay protein blot, while mCEACAM1b did not (5). Those authors speculated that the different viral affinities of these mCEACAM1 proteins may account for the various MHV-A59 susceptibilities of BALB/c mice compared to those of SJL mice (49). However, Yokomori and Lai (53) and Dveksler et al. (11) previously showed that when recombinant CEACAM1a and CEACAM1b proteins are expressed at high levels on cultured cells, both proteins have MHV-A59 receptor activity. Yokomori and Lai suggested that the difference in MHV susceptibility between BALB/c and SJL mice does not depend solely upon the interaction of the virus with mCEACAM1 proteins (52, 53). Dveksler et al. suggested that small differences in MHV-A59 receptor activity between mCEACAM1a and mCEACAM1b could result in very large biological differences during multiple cycles of infection in in vivo infection (11). We then quantitatively showed that recombinant mCEACAM1a expressed in BHK cells has 10- to 30-times-higher MHV-binding activity than mCEACAM1b (31). Similar results were observed in other laboratories (7, 32). Because the mCeacam1 gene is located on chromosome 7 (34) and the gene controlling MHV-A59 susceptibility and the resistance of BALB/c mice versus SJL mice is also located on chromosome 7 close to the mCeacam1 gene (40), we speculated that the mCeacam1 gene is identical to the gene that determines the susceptibility and/or resistance of mice to MHV-A59 and MHV-JHM infection.To examine the above-described hypothesis, we used progeny mice produced by crossing BALB/c and SJL mice. F₂ mice and F₁ mice backcrossed to SJL mice were examined for the mCeacam1 genotype and for MHV-JHM susceptibility (30). Mice homozygous for mCeacam1a (1a/1a) and heterozygous mice (1a/1b) were susceptible to lethal MHV-JHM infection, while mice homozygous for mCeacam1b (1b/1b) were not killed by inoculation with MHV-JHM. These data are consistent with the hypothesis that the susceptibility of mice to MHV is determined by the mCeacam1a allele (30). However, this classical genetic analysis could not prove that mCeacam1 alone determines the susceptibility or resistance of mice to MHV-JHM infection, because this methodology cannot rule out the possibility that a different unknown host gene located close to mCeacam1 on chromosome 7 could also affect MHV-JHM susceptibility. Therefore, we used gene replacement in B6 embryonic stem (ES) cells to create a mouse strain in which the exon encoding the N-terminal part of the N-terminal Ig domain of mCeacam1a was replaced with the corresponding region of mCeacam1b from SLJ mice. We bred the chimeric mCeacam1 gene on the B6 background (called B6 chimeric mCeacam1ba, or cB61ba). We compared these mice, wild-type B6 mice, and SJL mice for their susceptibilities to MHV-A59 infection. We confirmed that the expression of mCEACAM1a makes mice susceptible to lethal infection with MHV-A59. However, surprisingly, we found that cB61ba mice were profoundly resistant to MHV-A59 infection, while the virus could replicate at low levels in SJL mice in a self-limited, unapparent infection. Our results suggest that one or more as-yet-unidentified murine genes may also contribute to murine susceptibility and/or resistance to MHV-A59 infection.     

Impact of Sylvatic Plague Vaccine on Non-target Small Rodents in Grassland Ecosystems

Oral vaccination is an emerging management strategy to reduce the prevalence of high impact infectious diseases within wild animal populations. Plague is a flea-borne zoonosis of rodents that often decimates prairie dog (Cynomys spp.) colonies in the western USA. Recently, an oral sylvatic plague vaccine (SPV) was developed to protect prairie dogs from plague and aid recovery of the endangered black-footed ferret (Mustela nigripes). Although oral vaccination programs are targeted toward specific species, field distribution of vaccine-laden baits can result in vaccine uptake by non-target animals and unintended indirect effects. We assessed the impact of SPV on non-target rodents at paired vaccine and placebo-treated prairie dog colonies in four US states from 2013 to 2015. Bait consumption by non-target rodents was high (70.8%, n?=?3113), but anti-plague antibody development on vaccine plots was low (23.7%, n?=?266). In addition, no significant differences were noted in combined deer mice (Peromyscus maniculatus) and western harvest mouse (Reithrodontomys megalotis) abundance or community evenness and richness of non-target rodents between vaccine-treated and placebo plots. In our 3-year field study, we could not detect a significant positive or negative effect of SPV application on non-target rodents.     

Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus

Serologic monitoring of sentinel mice exposed to soiled bedding is a common method of detecting viral infections in mice. Because bedding transfer protocols vary, the sensitivity of this method has not been documented sufficiently. We examined the reliability of bedding transfer during various stages of infection with mouse parvovirus (MPV) and mouse hepatitis virus (MHV). Most mice exposed to bedding contaminated with MPV 0, 3, or 7 d previously seroconverted, whereas only mice exposed to bedding contaminated with MHV 4 h previously seroconverted, thus confirming the differing stabilities of these viruses. Index mice were inoculated with 30 times the infectious dose 50 (ID50) of MPV or 300 ID50 of MHV. At 3 d, 1 wk, and 2 wk postinoculation (PI), we transferred 25, 50, or 100 ml of bedding to cages of sentinel mice. Viral infection and shedding by index mice was confirmed by serology and fecal polymerase chain reaction assay. Transfer of soiled bedding between mice in static cages induced seroconversion of sentinel mice most reliably during peak viral shedding (1 wk PI for MPV and 3 d PI for MHV). Soiled bedding transfer between mice in individually ventilated cages induced a higher prevalence of sentinel seroconversion to MPV and MHV than that after transfer between mice in static cages. Our findings indicate that although soiled bedding transfer is an effective method for detecting MHV and MPV under optimal conditions, the method is less than 100% reliable under many conditions in contemporary mouse facilities.     

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.     

A Comparison of Mouse Parvovirus 1 Infection in BALB/c and C57BL/6 Mice: Susceptibility,Replication, Shedding,and Seroconversion

This study characterized the effects of challenge with a field isolate of mouse parvovirus 1 (MPV1e) in C57BL/6NCrl (B6) and BALB/cAnNCrl (C) mice. We found that C mice were more susceptible to MPV1e infection than were B6 mice; ID₅₀ were 50 to 100 times higher after gavage and 10-fold higher after intraperitoneal injection in B6 as compared with C mice. To evaluate the host strain effect on the pathogenesis of MPV1e, B6 and C mice were inoculated by gavage. Feces and tissues, including mesenteric lymph nodes (MLN), ileum, spleen and blood, were collected for analysis by quantitative PCR (qPCR) to assess infection and fecal shedding and by RT-qPCR to evaluate replication. Peak levels of MPV1e shedding, infection, and replication were on average 3.4, 4.3, and 6.2 times higher, respectively, in C than in B6 mice. Peaks occurred between 3 and 10 d after inoculation in C mice but between 5 and 14 d in B6 mice. Multiplexed fluorometric immunoassays detected seroconversion in 2 of 3 C mice at 7 d after inoculation and in all 3 B6 mice at 10 d. By 56 d after inoculation, viral replication was no longer detectable, and fecal shedding was very low; infection persisted in ileum, spleen, and MLN, with levels higher in C than B6 mice and highest in MLN. Therefore, the lower susceptibility of B6 mice, as compared with C mice, to MPV1e infection was associated with lower levels of infection, replication, and shedding and delayed seroconversion.Abbreviations: B6, C57BL/6; C, BALB/c; MFI, median fluorescence intensity; MFIA, multiplexed fluorometric immunoassay; MLN, mesenteric lymph node; MMV, mouse minute virus; MPV, mouse parvovirus; NS1, nonstructural protein 1; qPCR, quantitative PCR; r, recombinant; Rn, normalized reporter value; VP2, virus capsid protein 2Parvoviruses are small (20 to 28 nm), nonenveloped icosahedral single-stranded DNA viruses that infect a diverse range of vertebrate and arthropod species. Much of what is understood about the biology and pathogenesis of autonomous parvoviruses has been derived from studies of the original murine parvoviral isolates, particularly the prototypic and immunosuppressive strains of mouse minute virus (MMV).^9,13,32 Because autonomous parvoviruses have a requirement and predilection for proliferating cells to replicate, they are primarily teratogenic pathogens. In contrast, rodent parvovirus infections of older animals are usually asymptomatic, because the cells that divide in mature animals, such as enterocytes, lymphoreticular cells, and hematopoietic cells, are largely spared.^2,47,48 The most common parvovirus of laboratory mice, mouse parvovirus 1 (MPV1), was first isolated²⁹ from mouse T-lymphocyte cultures that had lost viability or the ability to proliferate when stimulated. In contrast to MMV,^10,27,40 MPV1 has not been shown to cause disease in newborn or immunodeficient mice^19,45 but nevertheless has been reported to modulate the immune response of infected mice.^30,31Adventitious infections of laboratory mice with MPV1 and other parvoviruses continue to occur regularly, despite biosecurity improvements that have successfully excluded once-common pathogens such as Sendai virus.^22,37,39 One reason for the continued occurrence of these infections is that nonenveloped parvovirus virions are environmentally stable and resistant to disinfection.^18,49 Furthermore, related to their tendency to persist in host tissues even after seroconversion and their predilection for dividing cells, parvoviruses have been among the most frequent viral contaminants of transplantable tumor lines and other rodent-derived biologic reagents.^34,35 Inoculation of parvovirus-contaminated biologic reagents into experimental animals can contribute to the incidence of parvoviral outbreaks. Currently, mouse populations typically are housed in microisolation cages and are monitored for MPV1 infections through the use of soiled bedding sentinels. An MPV1 infection of the principal animals may not be transmitted to sentinels when the prevalence of infection is low, as is often the case after contamination, or when the sentinels are comparatively resistant to infection because of their genetic background or age.^7,16,17 However, a recent study found that sentinel age did not affect the likelihood of MPV1 infection.¹⁷The C57BL/6 (B6) mouse strain is popular in biomedical research and is commonly used as the background strain for spontaneous and genetically engineered mutations. We and others have noted that B6 mice are less likely to be MPV1 seropositive than are mice of other strains and stocks, even in facilities where MPV1 is widespread.⁴⁴ There has been speculation that B6 mice might not seroconvert when infected with MPV1. However, data reported here and by others^7,15 indicate that B6 mice are less likely to seroconvert because they are comparatively resistant to MPV1 infection; when they become infected, they do seroconvert. The current study evaluated whether resistance of B6 mice to infection with MPV1, as compared with BALB/c (C) mice, varies with virus inoculation route and correlates with differences in the time course and levels of viral infection, replication, and shedding and of humoral immunity.Most studies of MPV1 in mice have been performed with the cultivable MPV1a strain.^{7,19,30,31,45} Cultivable murine parvoviruses are known to differ from wildtype strains genetically and in their cell tropisms, pathogenicity, and transmissibility in vivo. For example, MPV1d, a noncultivable field isolate, was more readily transmitted to sentinels than was MPV1a.¹¹ We therefore chose to perform the current experiments with MPV1e,^3,4 a representative field strain that we originally isolated from an adventitiously infected barrier colony⁴⁴ and that has been propagated only in mice.     

Experimental Transmission of Karshi (Mammalian Tick-Borne Flavivirus Group) Virus by Ornithodoros Ticks >2,900 Days after Initial Virus Exposure Supports the Role of Soft Ticks as a Long-Term Maintenance Mechanism for Certain Flaviviruses

Background

Members of the mammalian tick-borne flavivirus group, including tick-borne encephalitis virus, are responsible for at least 10,000 clinical cases of tick-borne encephalitis each year. To attempt to explain the long-term maintenance of members of this group, we followed Ornithodoros parkeri, O. sonrai, and O. tartakovskyi for >2,900 days after they had been exposed to Karshi virus, a member of the mammalian tick-borne flavivirus group.

Methodology/Principal Findings

Ticks were exposed to Karshi virus either by allowing them to feed on viremic suckling mice or by intracoelomic inoculation. The ticks were then allowed to feed individually on suckling mice after various periods of extrinsic incubation to determine their ability to transmit virus by bite and to determine how long the ticks would remain infectious. The ticks remained efficient vectors of Karshi virus, even when tested >2,900 d after their initial exposure to virus, including those ticks exposed to Karshi virus either orally or by inoculation.

Conclusions/Significance

Ornithodoros spp. ticks were able to transmit Karshi virus for >2,900 days (nearly 8 years) after a single exposure to a viremic mouse. Therefore, these ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian tick-borne flavivirus group.     

Temporal transmission studies of mouse parvovirus 1 in BALB/c and C.B-17/Icr-Prkdc(scid) mice

Fecal shedding and transmission of mouse parvovirus 1 (MPV) to naive sentinels, breeding mates, and progeny were assessed. Neonatal SCID and BALB/c mice inoculated with MPV were evaluated over 24 wk; several mice from each strain were mated once during this period. Fecal MPV loads for each cage were determined weekly by quantitative polymerase chain reaction (PCR) analysis, and all mice were evaluated by quantitative PCR analysis of lymphoid tissues and seroconversion to MPV antigens in immunocompetent mice. Results indicated persistently high fecal shedding of MPV in SCID mice throughout the evaluation period sufficient to allow transmission to sentinels, naive breeding partners, and the progeny of infected male mice and naive partners. Lymphoid tissue viral loads in the progeny of infected female SCID mice were high at weaning but low at 6 wk of age. Infected BALB/c mice shed high levels of MPV in feces for 3 wk postinoculation, with seroconversion only in sentinels exposed during the first 2 wk postinoculation. Thereafter the feces of infected BALB/c mice and the lymphoid tissues of sentinels, naive breeding partners, and progeny intermittently contained extremely low levels of MPV DNA. Although pregnancy and lactation did not increase viral shedding in BALB/c mice, MPV exposure levels were sufficient to induce productive infection in some BALB/c progeny. These data indicate that the adaptive immune response suppresses, but does not eliminate, MPV shedding; this suppression is sufficient to inhibit infection of weanling and adult mice but allows productive infection of some progeny.     

Ten years-long survey on pathogen status of mouse and rat breeding colonies

Eleven pathogens including P. aeruginosa, Salmonella spp., E. coli O115a, c: K(B), P. pneumotropica, B. bronchiseptica, C. kutscheri, Tyzzer's organism, M. pulmonis, Sendai virus, MHV and Syphacia spp. were surveyed in 217 mouse and rat breeding colonies during 1972-1981. In conventional animals, P. pneumotropica and/or Syphacia spp. were detected in nearly 90% of 89 mouse and 64 rat colonies. Sendai virus, M. pulmonis, P. aeruginosa and MHV were positive in 51.7 to 23.6% of the colonies, and Tyzzer's organism, B. bronchiseptica and probably SDA virus were also detected in more than 10% of the rat colonies. Salmonella spp., E. coli O115a, c: K(B) and C. kutscheri were found in a few colonies. In SPF animals, P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. pneumotropica was also positive in 3 rat colonies. Infection rates of P. pneumotropica, M. pulmonis, Sendai virus and Syphacia spp. were usually higher than 40% of animals sampled from colonies contaminated with them. Accidental contaminations of SPF colonies were usually caused by P. pneumotropica and Syphacia spp.     

Kinetics of Transmission, Infectivity, and Genome Stability of Two Novel Mouse Norovirus Isolates in Breeding Mice

Murine noroviruses are a recently discovered group of viruses found within mouse research colonies in many animal facilities worldwide. In this study, we used 2 novel mouse norovirus (MNV) wildtype isolates to examine the kinetics of transmission and tissue distribution in breeding units of NOD.CB17-Prkdc^scid/J and backcrossed NOD.CB17-Prkdc^scid/J × NOD/ShiLtJ (N1) mice. Viral shedding in feces and dissemination to tissues of infected offspring mice were monitored by RT-PCR over a 6-wk period postpartum. Histologic sections of tissues from mice exposed to MNV were examined for lesions and their sera monitored for the presence of antibodies to MNV. Viruses shed in feces of parental and offspring mice were compared for sequence homology of the Orf2 gene. Studies showed that the wildtype viruses MNV5 and MNV6 behaved differently in terms of the kinetics of transmission and distribution to tissues of offspring mice. For MNV5, virus transmission from parents to offspring was not seen before 3 wk after birth, and neither isolate was transmitted between cages of infected and control mice. Susceptibility to infection was statistically different between the 2 mouse strains used in the study. Both immunodeficient NOD.CB17-Prkdc^scid/J mice and NOD.CB17-Prkdc^scid/J × NOD/ShiLtJ offspring capable of mounting an immune response shed virus in their feces throughout the 6-wk study period, but no gross or histologic lesions were present in infected tissues. Progeny viruses isolated from the feces of infected offspring showed numerous mutations in the Orf2 gene for MNV5 but not MNV6. These results confirm previous studies demonstrating that the biology of MNV in mice varies substantially with each virus isolate and mouse strain infected.Abbreviations: MNV, murine norovirus; MLN, mesenteric lymph nodes; NOD-scid, NOD.CB17-Prkdc^scid/J; VP1, viral protein 1The recent discovery of murine-specific noroviruses¹⁵ has stimulated concern in the laboratory animal health community regarding the potential for this group of viruses to cause disease in breeding colonies of mice or to negatively impact research with mice from norovirus infected colonies. Current knowledge of the biology of noroviruses in mice (MNV) is constrained by the limited number of virus isolates and mouse strains studied. One study¹⁵described the biologic and physicochemical properties of the original MNV1 isolated from mice deficient in a specific innate immune function. More recently, this innate immune deficiency has been mapped to STAT1 regulation of IFNαβ secretion.²¹Previous work¹⁵ demonstrated that inoculation of MNV1 into mouse strains deficient in the acquired immune response (129 RAG 2^−/−, B6 RAG1^−/−) resulted in the development of persistent infections with no evidence of disease, whereas inoculation of fully immunocompetent mice (129S6/SvEvTac) resulted in rapid elimination of MNV1, with viral RNA undetectable in the viscera by 3 d after inoculation. More recently, infections of outbred immunocompetent mouse strains with 3 wildtype isolates of MNV obtained from different geographic areas of the United States have been described.¹¹ Virus was detected in the feces and tissue of infected mice throughout the 8-wk study, suggesting that some isolates of MNV may persistently infect immunocompetent mice.The purpose of the present investigation was to extend the current knowledge of MNV by using 2 isolates of the virus in mouse strains that have not been previously used as infection models for MNV. We examined natural virus transmission from infected breeders to offspring, kinetics of infection within litters of infected breeding mice, and the pathogenesis of infection in breeding colonies of mice. In addition, we examined the effect of virus passage from parents to offspring on genomic stability of these 2 viral isolates. Exposure of offspring of immunodeficient mice and immunocompetent mice to the 2 different isolates of MNV resulted in different patterns of virus transmission, susceptibility to infection and kinetics of infection as shown by the progressive spread of virus within litters and in intestinal and extraintestinal tissues. MNV was shed persistently in the feces of all mice tested regardless of immune status, and viral progeny isolated from offspring mice contained genome sequence differences from the parent virus in the Orf2 gene, an area of the MNV genome known to be susceptible to mutations.     

The elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnuN/rnuN).

A nude mouse colony held in an isolation unit was found to harbor MHV despite the fact that all hygienic precautions were taken. The virus spread rapidly causing a high mortality rate predominantly in experimental animals. Moreover, we observed a high percentage of tumor regression in our tumor transplanted mice. Attempts to eliminate the MHV by repeated tumor transplantation into virus-free nude mice were unsuccessful. Since MHV has a limited host range, we transplanted, in parallel, four different lines of embryonic renal tumors (three triphasic nephroblastomas and one malignant rhabdoid tumor of the kidney) from athymic mice into athymic rats and fragments of the same tumors into "fresh" nude mice. All manipulations were performed in isolators. Detection of MHV was done twice by serological examination of six-week-old sentinels. The results showed transmission of MHV infection in the control mice under gnotobiotic conditions as previously found in the normal animal room. On the other hand, there was no evidence of infection, neither in the transplanted nude rats nor after retransplantation of tumors into nude mice. We hypothesize that the virus is harbored in the stromal cells of the murine host but not of the rat host nor in the human tumor cells. Histological comparison showed no alteration of specific tumor morphology in the different hosts.     

Contact with domestic dogs increases pathogen exposure in endangered African wild dogs (Lycaon pictus)

Background

Infectious diseases have contributed to the decline and local extinction of several wildlife species, including African wild dogs (Lycaon pictus). Mitigating such disease threats is challenging, partly because uncertainty about disease dynamics makes it difficult to identify the best management approaches. Serious impacts on susceptible populations most frequently occur when generalist pathogens are maintained within populations of abundant (often domestic) “reservoir” hosts, and spill over into less abundant host species. If this is the case, disease control directed at the reservoir host might be most appropriate. However, pathogen transmission within threatened host populations may also be important, and may not be controllable by managing another host species.

Methodology/Principal Findings

We investigated interspecific and intraspecific transmission routes, by comparing African wild dogs'' exposure to six canine pathogens with behavioural measures of their opportunities for contact with domestic dogs and with other wild dogs. Domestic dog contact was associated with exposure to canine parvovirus, Ehrlichia canis, Neospora caninum and perhaps rabies virus, but not with exposure to canine distemper virus or canine coronavirus. Contact with other wild dogs appeared not to increase the risk of exposure to any of the pathogens.

Conclusions/Significance

These findings, combined with other data, suggest that management directed at domestic dogs might help to protect wild dog populations from rabies virus, but not from canine distemper virus. However, further analyses are needed to determine the management approaches – including no intervention – which are most appropriate for each pathogen.     

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.