Effects of Murine Norovirus Infection on a Mouse Model of Diet-Induced Obesity and Insulin Resistance

Murine norovirus (MNV) is prevalent in SPF mouse facilities in the United States, and we currently lack sufficient data to determine whether it should be eliminated. It is generally accepted that the virus does not cause clinical symptoms in immunocompetent mice. However, we previously reported that MNV infection alters the phenotype of a mouse model of bacteria-induced inflammatory bowel disease in part through its effects on dendritic cells. The tropism of MNV toward macrophages and dendritic cells makes MNV a potential intercurrent variable in murine models of macrophage-driven inflammatory diseases, such as obesity, insulin resistance, and atherosclerosis. Therefore, we determined whether MNV infection altered obesity and insulin resistance phenotypes in C57BL/6 mice, a widely used model of diet-induced obesity. We found that MNV did not alter weight gain, food intake, and glucose metabolism in this model, but it did induce subtle changes in lymphoid tissue. Further studies using other models of metabolic diseases are needed to provide additional information on the potential role this ‘subclinical’ virus might have on disease progression in mouse models of inflammatory diseases.Abbreviations: HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; MLN, mesenteric lymph node; MNV, murine norovirusMurine norovirus (MNV) is endemic in many SPF mouse colonies across North America,⁵ creating considerable potential for this virus to interfere with mouse models of human diseases. In addition, the presence of MNV in some mouse colonies and not in others may help explain phenotypic variability in mouse models across institutions. This virus is related to the human Norwalk virus that causes gastrointestinal inflammation in humans. Although MNV does not cause any overt illness in immunocompetent mice, significant inflammation and mortality can be induced in mice with abnormal innate immunity.⁷ Previously, we investigated the influence of MNV on the development of bacteria-induced inflammatory bowel disease in FVB.129P2-PAbcb1atm1Bor (Mdr1a^−/−) mice.⁸ We found that infection with MNV accelerated the progression of inflammatory bowel disease in this mouse model when mice were coinfected with Helicobacter bilis. In addition, infection with MNV alone altered the immune response, probably through changes in dendritic cells.⁸ These findings suggest that MNV may induce subtle changes in immune responses even in immunocompetent mice, given that MNV is known to preferentially infect macrophages and dendritic cells.²²Obesity has been defined as a disease of chronic inflammation, and in recent years, the prominent role that macrophages play in this process has been recognized.^9,10,21,24 Obesity is a risk factor for various chronic diseases that share inflammation as a critical component of the disease process, such as metabolic syndrome, diabetes, and atherosclerosis.³ Because MNV has tropism for macrophages, we wished to determine whether MNV infection influences the development of obesity and insulin resistance in a widely used animal model of diet-induced obesity. C57BL/6 mice are the most frequently used ‘wild-type’ strain and are prone to develop insulin resistance as obesity develops during high-fat feeding.¹ We hypothesized that MNV may accelerate inflammation by stimulating macrophage accumulation in adipose tissue, resulting in a more severe obesity or insulin resistance phenotype when mice are fed a high-fat diet.     

Murine Norovirus: An Intercurrent Variable in a Mouse Model of Bacteria-Induced Inflammatory Bowel Disease

Murine norovirus (MNV) has recently been recognized as a widely prevalent viral pathogen in mouse colonies and causes disease and mortality in mice with impaired innate immunity. We tested the hypothesis that MNV infection would alter disease course and immune responses in mice with inflammatory bowel disease (IBD). FVB.129P2-Abcb1a^tm1Bor N7 (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis. As compared with controls, Mdr1a−/− mice coinfected with MNV4 and H. bilis showed greater weight loss and IBD scores indicative of severe colitis, demonstrating that MNV4 can modulate the progression of IBD. Compared with controls, mice inoculated with MNV4 alone had altered levels of serum biomarkers, and flow cytometric analysis of immune cells from MNV4-infected mice showed changes in both dendritic cell (CD11c⁺) and other nonT cell (CD4⁻ CD8⁻) populations. Dendritic cells isolated from MNV4-infected mice induced higher IFNγ production by polyclonal T cells in vitro at 2 d after infection but not at later time points, indicating that MNV4 infection enhances antigen presentation by dendritic cells early after acute infection. These findings indicate that acute infection with MNV4 is immunomodulatory and alters disease progression in a mouse model of IBD.Abbreviations: DC, dendritic cell; IBD, inflammatory bowel disease; IP, IFNγ–inducible protein; MCP, macrophage chemotactic protein; MLN, mesenteric lymph node; MNV, murine norovirus; TNF, tumor necrosis factorThe genus Norovirus of the family Caliciviridae contains a large number of single-stranded, positive-sense RNA viruses that infect vertebrates, and strains have been identified in humans, cattle, swine, and (most recently) mice.^19,29,34 Murine noroviruses (MNV) are recently recognized pathogens that can cause lethal infection in immunocompromised mice that lack innate immunity.¹⁹ However, MNV did not cause clinical disease in wild-type mice or many other strains of immunodeficient mice, including those lacking the recombination-activating gene (Rag−/−) and inducible nitric oxide synthase deficient mice.^19,35,37 MNV was reported recently to be widespread in laboratory mice and may persist in immunocompetent animals, depending on the strain of MNV used.^15,16,25 Studies in Rag−/− mice and B-cell–deficient strains showed that the acquired immune system plays an important role in the clearance of MNV.^6,19,37 MNV has tropism for dendritic cells (DCs),³⁶ which are important in the presentation of antigens to T cells in draining lymph nodes and in the pathogenesis of inflammatory bowel disease (IBD). Therefore, MNV is a potential confounder for in vivo immunology studies, including murine models of IBD.Idiopathic IBD, which encompasses both ulcerative colitis and Crohn disease, is a widely studied disorder that affects approximately 1.4 million people in the United States.²⁰ Although the precise cause of human IBD has not been elucidated, studies with mouse models have demonstrated that abnormal host responses of the innate and adaptive immune systems to intestinal microbiota are important in the pathogenesis of IBD.^28,38 DCs are the sentinels of the intestinal mucosal barrier and have a pivotal role in the initiation of IBD in response to microbial ligands.³⁹ Alterations in DC responses could lead to persistence of bacterial infection, aberrant activation of the acquired immune system, and (ultimately) tissue damage.³⁸Viral stimulation of DCs leads to activation of adaptive immune responses,¹⁷ including effector T cells, and as demonstrated with murine coronavirus (mouse hepatitis virus), intercurrent viral infections in mice can alter the phenotype of mouse models of human disease.¹⁰ Additional evidence suggests that intercurrent viral infection may enhance disease in human IBD patients.^12,18 Whether infection with MNV alters DC function and, therefore, influences the progression of IBD in mouse models is unclear.Many mouse models of intestinal inflammation develop IBD that is driven by bacterial flora.^9,28 Helicobacter spp. have been shown to drive this process in several mouse models including IL10-deficient, SMAD3-deficient, severe combined immunodeficiency and T-cell–deficient mice.^4,5,13,23 FVB.129P2-Abcb1a^tm1Bor (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis.^21,22 In this report, we tested the hypothesis that infection with MNV can modulate IBD in this mouse model of bacterial-induced disease. We demonstrate that intercurrent MNV4 infection accelerates the progression of bacterial-induced IBD in the Mdr1a−/− mouse and alters the immune responses in this mouse model of IBD.     

Infection with Murine Norovirus 4 Does Not Alter Helicobacter-Induced Inflammatory Bowel Disease in Il10?/? Mice

Infection of laboratory mice with murine noroviruses (MNV) is widely prevalent. MNV alters various mouse models of disease, including the Helicobacter bilis-induced mouse model of inflammatory bowel disease (IBD) in Mdr1a^−/− mice. To further characterize the effect of MNV on IBD, we used mice deficient in the immunoregulatory cytokine IL10 (Il10^−/− mice). In vitro infection of Il10^−/− bone marrow-derived macrophages (BMDM) with MNV4 cocultured with H. bilis antigens increased the gene expression of the proinflammatory cytokines IL1β, IL6, and TNFα as compared with that of BMDM cultured with H. bilis antigens only. Therefore, to test the hypothesis that MNV4 infection increases inflammation and alters disease phenotype in H. bilis-infected Il10^−/− mice, we compared the amount and extent of inflammation in Il10^−/− mice coinfected with H. bilis and MNV4 with those of mice singly infected with H. bilis. IBD scores, incidence of IBD, or frequency of severe IBD did not differ between mice coinfected with H. bilis and MNV4 and those singly infected with H. bilis. Mice infected with MNV4 only had no appreciable IBD, comparable to uninfected mice. Our findings suggest that, unlike in Mdr1a^−/− mice, the presence of MNV4 in Il10^−/− mouse colonies is unlikely to affect the IBD phenotype in a Helicobacter-induced model. However, because MNV4 altered cytokine expression in vitro, our results highlight the importance of determining the potential influence of MNV on mouse models of inflammatory disease, given that MNV has a tropism for macrophages and dendritic cells and that infection is widely prevalent.Abbreviations: BMDM, bone marrow-derived macrophages; IBD, inflammatory bowel disease; MLN, mesenteric lymph node; MNV, murine norovirusInflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn disease, is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. In addition, patients with IBD may be at increased risk of developing colorectal cancer.^15,46 Although the exact mechanisms of disease are still not understood fully, the pathogenesis of disease is likely multifactorial, with components of the innate and adaptive immune systems, host genetics, and environmental factors (for example, the commensal gut microflora) all playing a role.^4,37,55Animal models of IBD have been used to advance our knowledge and understanding of IBD pathogenesis and treatment.^{16,20,37,38,52} One such model that has been widely used to elucidate the mechanisms of IBD is the interleukin10–deficient (Il10^−/−) mouse.^{3,5,6,20,21,29,33,57} The antiinflammatory cytokine IL10 modulates both innate and adaptive immune responses.⁴¹ Produced mainly by dendritic cells, monocytes, macrophages, and T regulatory cells, IL10 exerts its immunomodulatory effects by various mechanisms including decreasing secretion of proinflammatory cytokines (for example, interferon γ, IL1, IL2, IL6, IL12 and TNFα) and downregulating important components of innate immune responses and T-cell activation (for example, MHC class II, costimulatory molecules, and nitric oxide production) in antigen presenting cells.^14,41 As a consequence, Il10^−/− mice, which lack the suppressive effects of IL10, develop IBD in response to their commensal gut microflora or to certain microbial triggers such as Helicobacter infections.^{5,6,11,21,29,52,57}Antigen-presenting cells such as macrophages and dendritic cells play key roles in the inflammatory responses in IBD.^32,47,50 In 2003, a newly discovered murine norovirus (MNV) in laboratory mice was shown to infect macrophages and dendritic cells.^27,53 Subsequent studies indicated widespread MNV infection in laboratory mice used for biomedical research, with a serologic prevalence as high as 32%.^25,43 Members of the genus Norovirus are regarded as gastrointestinal pathogens in humans and animals, eliciting both innate and adaptive immune responses.¹⁹ Therefore, in light of the cellular (macrophages and dendritic cells) and tissue (gastrointestinal) tropisms of MNV as well as the high prevalence of MNV infection in laboratory mice, we hypothesized that MNV infection could be a potential confounder in mouse models of inflammatory diseases including IBD. In support of this idea, our laboratory recently reported that MNV infection in Mdr1a^−/− mice (FVB.129P2-Abcb1a^tm1Bor) accelerated weight loss and exacerbated IBD progression initiated by H. bilis infection.³¹ This effect potentially was mediated in part through modulating dendritic cell and cytokine responses. In addition, others have reported gastrointestinal abnormalities as a result of MNV infection in some strains of mice,^7,26,36 whereas others have described the importance of both innate and adaptive immune responses during MNV infection.^{8,9,10,28,34,36,48} Collectively, these data indicate that MNV could alter inflammatory responses in laboratory mice.Here we extended our studies of MNV beyond Mdr1a^−/− mice to Il10^−/− mice, another common animal model of IBD, to further examine the potential effect of MNV on IBD research. Disease was initiated in Il10^−/− mice with H. bilis, and we determined whether coinfection with MNV altered disease development, incidence, and severity and the production of cytokines. We demonstrated that although MNV stimulates a Th1 skewing of cytokines in Il10^−/− bone marrow-derived macrophages (BMDM) in vitro, MNV does not alter the development, incidence, or severity of disease in vivo. Therefore, although MNV may not affect disease in Il10^−/− mouse models, the virus may influence in vitro cytokine phenotypes and thus complicate interpretation of such data. To our knowledge, this report is the first to describe the evaluation of MNV infection in the Helicobacter-induced Il10^−/− mouse model of IBD.     

Effects of Murine Norovirus on Atherosclerosis in Ldlr–/– Mice Depends on the Timing of Infection

We previously reported that murine norovirus (MNV), a virus prevalent in United States research institutions, increased atherosclerotic lesion size in Ldlr^−/− mice when the mice were infected 8 wk after feeding an atherogenic diet. To determine whether the timing of MNV infection relative to atherosclerosis development altered the disease phenotype and to examine potential mechanisms by which MNV influences the disease process, we fed Ldlr^−/− mice an atherogenic diet for 16 wk. Three days after initiating the atherogenic diet, half of the mice received MNV4 and the other half vehicle only (clarified cell-culture lysate; controls). Both groups of mice developed large aortic sinus lesions (control compared with MNV4: 133 ± 8 × 10³ µm² compared with 140 ± 7 × 10³ µm²) that were not significantly different in size. Because the timing of MNV infection relative to atherosclerosis development and hypercholesterolemia differed between our previous and the current studies, we examined whether hypercholesterolemia altered MNV4-induced changes in bone-marrow–derived macrophages. MNV4 infection increased the potential of macrophages to take up and store cholesterol by increasing CD36 expression while suppressing the ABCA1 transporter. Thus, the effects of MNV4 infection on atherosclerotic lesion size appear to be dependent on the timing of the infection: MNV4 infection promotes only established lesions. This effect may be due to MNV4’s ability to increase cholesterol uptake and decrease efflux by regulating CD36 and ABCA1 protein expression.Abbreviations: ABCA1, ATP-binding cassette A1; BMDM, bone-marrow–derived macrophage; iNOS, inducible nitric oxide synthase; MNV, murine norovirusChronic viral infection, such as occurs with HIV and hepatitis C virus, has been associated with an increased risk for atherosclerosis,^2,19,46,48 although the mechanisms by which this occurs are not clearly defined. Our laboratory has been studying the effects of murine norovirus (MNV), which chronically infects immunocompetent mice, on murine models of inflammatory diseases, including atherosclerosis. MNV is a single-stranded RNA intestinal virus that belongs to the family Caliciviridae and has shown tropism toward antigen-presenting cells such as dendritic cells and macrophages.⁵⁴ Whereas human norovirus is a major cause of nonbacterial acute gastroenteritis,⁵² MNV does not cause clinical disease in immunocompetent mice.⁵⁵ However, the high prevalence of MNV in biomedical research facilities throughout the world,^42,55 combined with its tropism for antigen-presenting cells, has prompted concern regarding potential effects on disease phenotypes in murine models of human diseases. Therefore, we previously examined 2 diseases, obesity and atherosclerosis, where macrophages have critical roles.^41,42 We found that MNV infection did not influence glucose metabolism and weight gain,⁴¹ but it significantly increased the size and macrophage content of aortic sinus lesions in Ldlr^−/− mice fed an atherogenic diet.⁴² These findings suggest that MNV might be a potential tool to determine how viral infection alters the risk of atherosclerosis.Many factors influence the progression of atherosclerosis. Accordingly, we examined whether the timing of MNV infection relative to the stage of atherosclerosis progression influenced disease phenotype and evaluated potential mechanisms by which MNV could affect the disease process. To this end, we modeled the infection of macrophages by using in vitro cultures of bone-marrow–derived macrophages (BMDM).     

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.     

Irradiated Compared with Nonirradiated NSG Mice for the Development of a Human B-Cell Lymphoma Model

NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice are a superior strain for the engraftment of human tumors, as they provide an ideal model to explore the potency, toxicity, and dosage of therapeutic drugs. Although whole-body nonlethal irradiation is often performed to enhance engraftment, the need for irradiation to establish a human B-cell lymphoma model using the NSG strain has not been addressed. In the current study, a mouse model of B-cell lymphoma was established by intravenous injection of human B-cell lymphoma Z138 cells into mice with and without irradiation. Tumor development, signs of engraftment, survivability of engrafted mice, histopathology, and immunohistochemistry were evaluated. Potential sex-associated variations in the model were assessed also. Irradiation of NSG mice did not enhance tumor cell engraftment, and nonirradiated animals had increased survivability. Mice with irradiation survived for a median of 27 d before being euthanized due to signs of morbidity, whereas those without irradiation had a median survival of 35 d. Both irradiated and nonirradiated mice were normal in activity until 3 wk after the injection of cells. At that time, the mice started to show signs of lymphoma including ruffled fur, decreased activity, and hindlimb paralysis. There were no significant differences in evaluated parameters between male and female mice. Therefore, we conclude that a model of B-cell lymphoma can successfully be established by using Z138 cells in nonirradiated male and female NSG mice.Abbreviations: NHL, nonHodgkin lymphoma; NSG, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJThe National Cancer Institute defines nonHodgkin lymphoma (NHL) as cancer of lymphocytes, and it affects various organs of the immune system, including lymph nodes, spleen, and bone marrow. The several different forms of NHL include slow-progressing, fast-progressing, B-cell, and T-cell types.⁶ Mantle cell lymphoma is a rare type of aggressive B-cell lymphoma (occurring in about 6% of lymphoma patients in the United States²) and is extremely difficult to treat. Patients with mantle cell lymphoma are treated with chemotherapeutic drugs, radiotherapy and transplantation of bone marrow,⁷ but the lymphoma relapses after 3 to 4 y in nearly 50% of the patients.⁴ Therefore, it is essential to develop strategies for enhancing the therapeutic options in patients with B-cell lymphoma and specific drugs that can cure the disease or prevent its relapse. Because animal experiments enable the preclinical testing of promising therapeutics for subsequent evaluation in humans, the development of an appropriate animal model is crucial.Mice engrafted with human tumors act as a model for testing various therapeutic drugs for their potency, toxicity, and dosing.¹ Severe immunodeficient mice (SCID) mice have widely been used to disseminate tumor cells in vivo,¹³ where the cells are engrafted via intravenous injection.^10,24 These mice have been used to develop a mouse model for human Burkitt lymphoma (a type of B-cell lymphoma) by using the Daudi cell line or SU-DHL-4 cells.²³ In these experiments, hindlimb paralysis and solid tumor development were observed as characteristic signs of lymphoma in the engrafted mice.^9,23 Whereas one group observed hindlimb paralysis even without irradiation of mice, the other did not see this development in any of their nonirradiated animals.²³ However, irradiation altered the pattern of tumor growth and the animals’ responses to various chemotherapeutic drugs. It also led to variations in the animals’ immune status in general and made them more susceptible to thyomas.²³ Therefore, whether to irradiate mice prior to the injection of B-cell lymphoma cells has been a topic of debate.The development of NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice has provided a valuable tool for the development of a B-cell lymphoma model, because they lack mature B and T cells and various cytokines such as IL2, 4, 7, 9, 15, and 21, leading to impaired development of NK cells.^14,20 Some studies have shown that irradiating mice prior to the injection of various tumor cells enhances the engraftment and growth rate of the tumor,^3,21 but immunocompromised mice, especially NSG mice, are known to be sensitive to irradiation and subsequently may manifest increased morbidity and mortality.¹² In addition, the irradiation process can cause considerable distress in mice, and many institutions and IACUC require close monitoring and special care of mice after irradiation.¹⁶ In our lab, mice routinely are provided with nutritional and fluid supplements and are placed on heating pads after irradiation, to prevent dehydration and death. Given irradiation''s potential negative effect on animal health and the irradiation-associated variations reported in similar animal models, the elimination of irradiation may yield a less stressful and more reliable model for NHL.The objective of the current study was to compare irradiated and nonirradiated NSG mice as a model for a specific type of B-cell NHL, mantle cell lymphoma. We evaluated engraftment, the development of clinical signs, and survival and potential sex-associated differences in each of those parameters in both irradiated and nonirradiated NSG mice injected with Z138 (mantle cell lymphoma) cells.     

Genomic analysis and pathogenic characteristics of lymphocytic choriomeningitis virus strains isolated in Japan

Lymphocytic choriomeningitis virus (LCMV) is a zoonotic pathogen of which mice are the natural reservoir. Different strains and clones of LCMV show different pathogenicity in mice. Here we determined the complete genomic sequences of 3 LCMV strains (OQ28 and BRC which were isolated from mice in Japan and WE(ngs) which was derived from strain WE). Strains OQ28 and BRC showed high sequence homology with other LCMV strains. Although phylogenetic analyses placed these 2 Japanese strains in different subclusters, they belonged to same cluster of LCMV isolates. WE(ngs) and WE had many sequence substitutions between them but fell into same subcluster. The pathogenicity of the 3 new LCMV isolates was examined by inoculating ICR mice with 10² and 10⁴ TCID₅₀ of virus. ICR mice infected with OQ28 or WE(ngs) exhibited severe clinical signs, and some of the infected mice died. In contrast, all ICR mice infected with BRC showed no clinical signs and survived infection. Virus was detected in the blood, organs, or both of most of the surviving ICR mice inoculated with either OQ28 or WE(ngs). However, virus was below the level of detection in all ICR mice surviving infection with strain BRC. Therefore, LCMV strains OQ28 and BRC were genetically classified in the same cluster of LCMV strains but exhibited very different pathogenicity.Abbreviations: dpi, days postinfection; GP, viral glycoprotein; h, hydrophobic region; IFA, indirect fluorescent antibody assay; L, viral RNA-dependent RNA polymerase; LCMV, lymphocytic choriomeningitis virus; NP, nucleocapsid protein; UTR, untranslated region; Z, zinc-finger proteinLymphocytic choriomeningitis virus (LCMV) is a member of the genus Arenavirus in the family Arenaviridae. The genus Arenavirus is divided into 2 groups (Old World and New World arenaviruses) according to genetic and antigenic characteristics.⁴ LCMV is a member of the Old World arenavirus group, which also includes Lassa, Mopeia, Mobala, and Ippy viruses.^4,10 The LCMV genome contains 2 negative-sense single-stranded RNA segments, designated S RNA and L RNA, with approximate sizes of 3.4 kb and 7.2 kb, respectively.^30,31 Each RNA segment has an ambisense coding strategy, encoding 2 different proteins in opposite orientations. S RNA encodes the nucleocapsid protein and glycoprotein, and L RNA encodes the viral RNA-dependent RNA polymerase and a small zinc finger protein.^25,30LCMV is a zoonotic agent that is transmitted to humans via urine or saliva of infected mice (Mus musculus), which are a natural reservoir of the virus.⁴ The prevalence of LCMV in mice is 7.0% to 25.9% in Japan and 4% to 9% in Europe.^{5,17,19,20,35} Mice are naturally infected by either vertical or horizontal transmission of the virus, and infected mice usually show no clinical signs. In contrast, experimentally infected mice inoculated intraperitoneally or intracerebrally can exhibit clinical signs such as ruffled fur, half-closed eyes, hunched posture, immobility, and neurologic deficits.^4,12,19 Although human LCMV infections are generally either asymptomatic or mild, immunodeficient persons can develop spontaneous abortion, severe birth defects, aseptic meningitis, or fatal infections.^1,2,13,22,27 Therefore, LCMV is an important agent that should be monitored in facilities housing and breeding mice.LCMV strains Armstrong, Traub, and WE were isolated during the 1930s from laboratory mice and humans working in a mouse facility.⁴ Many other LCMV strains and clones used in research originated from these 3 isolates. Strains Aggressive and Docile are clones (variants) of strain UBC, which was derived from the parental strain WE, and strains E350, CA1371, 53b, and clone 13 were all derived from strain Armstrong.⁴ The lethality of strains Aggressive and Docile varies between mouse strains.³⁸ Mice inoculated with 53b develop acute infections, whereas those inoculated with clone 13 mount chronic infections, even though both of the strains were derived from strain Armstrong.²⁹ Furthermore, strain Armstrong produces more severe disease in C3H mice than do strains WE and Traub.⁴ Therefore, previous studies indicate that mice infected with different strains of LCMV exhibit differences in clinical signs and lethality.^4,7 LCMV is a noncytolytic virus and causes immune-mediated viral disease.¹² The clinical signs and lethal disease arise because virus-specific T cells attack infected cells on critical organs in infected mice.¹²Here we report the characterization of 2 LCMV strains recently isolated in Japan (strains OQ28 and BRC) and a passaged isolate of strain WE. The complete genomic sequences of these 3 strains were determined, and their phylogenetic relationship to other LCMV strains was assessed. We also evaluated the pathogenicity in ICR mice of these isolates.     

Differential Susceptibility of SD and CD Rats to a Novel Rat Theilovirus

Antibodies to rat theilovirus (RTV) have been detected in rats for many years because of their serologic crossreactivity with strains of Theiler murine encephalomyelitis virus (TMEV) of mice. Little information exists regarding this pathogen, yet it is among the most common viruses detected in serologic surveys of rats used in research. In the study reported here, a novel isolate of RTV, designated RTV1, was cultured from the feces of infected rats. The RTV1 genome contained 8094 nucleotides and had approximately 95% identity with another rat theilovirus, NSG910, and 73% identity with TMEV strains. In addition, the genome size of RTV1 was similar to those of TMEV strains but larger than that reported for NSG910. Oral inoculation of Sprague–Dawley (SD) and CD male rats (n = 10 each group) with RTV1 revealed that SD rats were more susceptible than CD rats to RTV1 infection. At 14 d postinoculation, 100% of SD rats shed virus in the feces, and 70% were positive for RTV serum antibodies. By 56 d postinoculation 30% of SD rats continued to have detectable virus in the feces, and 90% had seroconverted. In contrast, in inoculated CD rats RTV was detected only in the feces at 14 d postinoculation, at which time 40% of CD rats were fecal positive. By 56 d postinoculation only 20% of CD rats had detectable RTV serum antibodies. Our data provide additional sequence information regarding a rat-specific Cardiovirus and indicate that SD rats are more susceptible than CD rats to RTV1 infection.Abbreviations: RACE, rapid amplification of cDNA ends; RTV, rat theilovirus; SD, Sprague Dawley; TMEV, Theiler murine encephalomyelitis virusFor decades it has been known that rats used in research can develop antibodies to a Cardiovirus that is antigenically similar to Theiler murine encephalomyelitis virus (TMEV) of mice.^{4,6,10,12,13,20} Recent reports on the prevalence of antibodies in rats to this Cardiovirus vary from approximately 0.6% of sera tested from research rats in North America¹⁰ to 54.4% in a survey of 18 Brazilian research facilities.^3,6,20 Multiple designations have been used to identify the Cardiovirus that infects rats, including Theiler-like virus of rats,¹³ Theiler murine encephalomyelitis virus (TMEV),²⁰ rat enterovirus,¹ rat encephalomyelitis virus,⁷ rat cardiovirus,¹⁵ and recently rat theilovirus.² We have elected to refer to the virus as rat theilovirus (RTV), consistent with 1 of the cited references,² to indicate the relation of the rat virus to TMEV of mice and to identify it as a rat-specific agent.The first report of natural infection of rats with a Cardiovirus was in 1964 with the discovery of MHG virus.¹² The finding resulted from an isolated observation in which a few rats in a large research colony displayed clinical signs indicative of central nervous system deficits, including incoordination, torticollis, circling, and tremors. The MHG virus recovered from infected rats was antigenically crossreactive with TMEV strain GDVII and had physical properties consistent with viruses in the Picornaviridae family. The virus was propagated in cell culture, and neurologic disease was reproduced when virus was inoculated into suckling mice and suckling rats.¹² Subsequent serologic studies using crossneutralization, complement fixation, and hemagglutination inhibition assays further substantiated the antigenic relatedness between MHG virus and multiple strains of TMEV.^4,11 In addition, sera from ‘normal’ rats contained antibodies to the newly identified Theiler-like virus of rats, suggesting widespread infection of the virus in research rat colonies.¹² More recently in Japan, a Theiler-like virus was isolated after intracranial inoculation of newborn Wistar rats with intestinal homogenates from TMEV GDVII-seropositive rats.¹³ Inoculated rats did not develop clinical signs of infection, but virus was cultivated in BHK21 cells from brain homogenates of the 10-d-old Wistar rats inoculated intracranially. Physiochemical properties of the virus, designated NSG910, were consistent with those of the Cardiovirus genus. Sequence analysis also showed that NSG910 was a Cardiovirus in the family Picornaviridae that was related to, but distinct from MHG virus, and strains of TMEV. This report served to further document the existence of a unique Cardiovirus of rats closely related to, but distinct from, TMEV strains.¹³ In a recent report from Brazil, neonatal mice and rats inoculated with intestinal homogenates from rats with antibodies to TMEV strain GDVII developed neurologic signs of flaccid hindlimb paralysis and tremors. In addition, brain homogenates from the affected animals were positive by RT-PCR for cardioviral RNA.²⁰Picornavirus virions are approximately 30 nm in diameter, nonenveloped, with icosahedral symmetry and a single-stranded, positive-sense RNA genome.¹⁹ Encephalomyocarditis virus and Theilovirus are 2 species of Cardiovirus in the Picornaviridae family. Encephalomyocarditis virus species includes mengovirus, Maus Elberfeld virus, and Columbia SK virus.⁷ Strains of Theilovirus species include TMEV, Vilyuisk virus, and RTV.^13,18,22 Most often studied are the TMEV strains, which are classified according to their neurovirulence after intracerebral inoculation. Included are the highly neurovirulent GD VII and FA strains²³ and the less virulent, more persistent DA, BeAn 8386, WW, and TO (Theiler original) strains.^9,17,22 Studies have shown that the virus replicates in the alimentary tract and is shed in the feces of infected mice.^15,19 Mice rarely show clinical disease when infected under natural conditions; however, neurologic manifestations have been reported.^21,24Sentinel animals typically are used to survey rodent colonies for the presence or absence of infectious agents. Outbred stocks are frequently used as sentinels because of their vigor, relatively low cost, and ability to mount a robust humoral immune response to infectious agents.^8,14 Sprague–Dawley (SD) and CD rats are 2 stocks that are commonly used as sentinels for rat colonies. The origins of the SD rat (Rattus norvegicus) date back to the 1920s as a result of mating Wistar stock with a hybrid rat stock of unknown origin. In the 1950s, an SD breeding stock was cesarean derived in an effort to improve microbiologic status. This nucleus of cesarean-derived rats formed the foundation of the CD rat stock.²⁵ Because SD and CD rat stocks have a common ancestry, they frequently are considered to be interchangeable for the purpose of sentinel animals.In the studies reported here, we isolated and propagated a novel strain of Theilovirus, referred to as RTV1, from the feces of infected SD rats. The entire genome of RTV1 was sequenced and compared with those of isolates of TMEV and NSG910, the only other isolate of RTV to be sequenced in its entirety. In addition, we evaluated the susceptibility of SD and CD outbred rats to RTV1 after oral inoculation with the virus.     

Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice

Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10^−/− mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10^−/− mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius (severe inflammation). Inflammation-associated colonic neoplasia was common in infected mice and its incidence correlated with IBD severity. Combined treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole eradicated Helicobacter in infected mice and ameliorated established IBD in both infected and noninfected mice. Infection of IL10^−/− mice with H. rodentium, H. typhlonius, or both organisms can trigger development of severe IBD that eventually leads to colonic neoplasia. The high incidence and multiplicity of neoplastic lesions in infected mice make this model well-suited for future research related to the development and chemoprevention of inflammation-associated colon cancer. The similar antiinflammatory effect of antibiotic therapy in Helicobacter-infected and -noninfected IL10^−/− mice with colitis indicates that unidentified microbiota in addition to Helicobacter drive the inflammatory process in this model. This finding suggests a complex role for both Helicobacter and other intestinal microbiota in the onset and perpetuation of IBD in these susceptible hosts.Abbreviations: IBD, Inflammatory bowel diseaseInflammatory bowel disease (IBD) is hypothesized to develop due to aberrant immune responses induced by gut microbes.⁵ IBD does not occur in germ-free IL10^−/− mice,^2,15 indicating the importance of microorganisms as environmental triggers of intestinal inflammation. However, conventionally colonized or specific pathogen-free IL10^−/− mice may develop colitis spontaneously^2,32 or in response to specific triggers such as nonsteroidal antiinflammatory drugs^3,14 or infections with certain bacteria.^6,16,18 The normal lack of ongoing immune responses against bacteria in subjects without IBD has been attributed to the immunologic tolerance that specifically downregulates immune responses against antigens derived from these bacteria. Nevertheless, despite a large number of studies, no single bacterial type has fulfilled Koch postulates and been confirmed as a cause of IBD in animals or humans.Previous studies used fluorescence in situ hybridization with probes specific for bacterial 16S rRNA combined with conventional histologic techniques to study the relationships between various species of intestinal bacteria and the mucosa in mice and humans with IBD.^33,34 Those studies showed that in normal mice, most bacterial groups are separated from the mucosal surface by either a mucus layer that excludes bacteria or, in the cecum and proximal colon, by an ‘interlaced’ layer that is composed of tightly packed bacteria. The interlaced or mucus layer thus limits the contact of the bulk of the enteric bacteria with the mucosal epithelium. In contrast, complex biofilms composed of multiple species of bacteria that were firmly adherent to the mucosal surface were identified in the majority of colon tissue samples collected from humans and mice with IBD.^33,34 The presence of a biofilm abrogates the protective effects of the normal layer of mucus and can allow luminal bacterial antigens and toxins to reach the unshielded epithelial surface, where they can trigger cascades of host inflammatory responses. Situations that cause defects in the epithelial surface or degrade the protective qualities of mucus or the interlaced layer (or both) may allow contact of bacterial antigens and adjuvants with immune cells located in the lamina propria and lead to the generation of immune responses that result in IBD.³⁴Helicobacter species are used frequently to model microbial triggers of colon inflammation, because they have previously been linked to the development of both IBD- and inflammation-associated neoplasia.^11,21,29 Most studies have been performed by using Helicobacter hepaticus or H. bilis.²⁰ However, H. typhlonius, H. rodentium, H. muridarum, H. ganmani, H. trogontum and other species^{8,12,17,29,35} can also be endemic in research animal facilities. The pathophysiologic effects of these less-common Helicobacter species are, for the most part, poorly investigated.Most rodent Helicobacter species are urease-negative and therefore preferentially colonize the intestine, but some species produce urease enzyme and can translocate to the liver or colonize the biliary system.¹³ H. typhlonius was shown to cause an enteric disease characterized by mucosal hyperplasia and associated inflammation in the cecum and colon in immunodeficient mice^11,23 and IL10^−/− mice.¹⁸ H. typhlonius is genetically related most closely to H. hepaticus, having only 2.36% difference in the 16S rRNA gene sequence, but H. typhlonius has a unique intervening sequence in this gene that makes it easily recognizable by PCR.^9,12 Molecular detection of this pathogen with PCR is rapid, sensitive and allows the detection of the early phases of infection; further enhanced sensitivity is achieved with nested primers.²² One of the most important features of PCR is that it can be performed noninvasively on fecal pellets. Data regarding the pathogenetic mechanisms of H. rodentium are scarce.^35,36 H. rodentium alone apparently does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, coinfection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical diarrheal disease in immunodeficient mice compared with mice infected with H. hepaticus alone.²³Previous reports demonstrated that H. typhlonius was capable of initiating colitis in adult IL10^−/− mice.^10,11 In those studies, colitis was relatively mild, with no development of inflammation-associated neoplasia. H. rodentium has been described to be nonpathogenic in adult wild-type mice but did enhance cytokine production in the cecum of mice also infected with H. hepaticus.²³ We recently observed rapid onset of severe IBD and a high incidence of inflammation-associated neoplasia in IL10^−/− mice that were coinfected with both H. typhlonius and H. rodentium as pups.¹⁶ The current study was undertaken to determine the relative roles of H. rodentium and H. typhlonius, individually and in combination, and age at infection in the development of colon inflammation and inflammation-associated neoplasia in IL10^−/− mice. Novel features of our model include controlled infection of the combination of H. typhlonius and H. rodentium⁹ and infection of IL10^−/− mice during the neonatal period.     

Helicobacter Infection Decreases Reproductive Performance of IL10-deficient Mice

Infections with a variety of Helicobacter species have been documented in rodent research facilities, with variable effects on rodent health. Helicobacter typhlonius has been reported to cause enteric disease in immunodeficient and IL10^−/− mice, whereas H. rodentium has only been reported to cause disease in immunodeficient mice coinfected with other Helicobacter species. The effect of Helicobacter infections on murine reproduction has not been well studied. The reproductive performance of C57BL/6 IL10^−/− female mice intentionally infected with H. typhlonius, H. rodentium, or both was compared with that of age-matched uninfected controls or similarly infected mice that received antihelicobacter therapy. The presence of Helicobacter organisms in stool and relevant tissues was detected by PCR assays. Helicobacter infection of IL10^−/− female mice markedly decreased pregnancy rates and pup survival. The number of pups surviving to weaning was greatest in noninfected mice and decreased for H. rodentium > H. typhlonius >> H. rodentium and H. typhlonius coinfected mice. Helicobacter organisms were detected by semiquantitative real-time PCR in the reproductive organs of a subset of infected mice. Treatment of infected mice with a 4-drug regimen consisting of amoxicillin, clarithromycin, metronidazole, and omeprazole increased pregnancy rates, and pup survival and dam fecundity improved. We conclude that infection with H. typhlonius, H. rodentium, or both decreased the reproductive performance of IL10^−/− mice. In addition, antihelicobacter therapy improved fecundity and enhanced pup survival.Abbreviations: qPCR, qualitative real-time PCRHelicobacter rodentium and H. typhlonius⁵ are gram-negative, urease-negative, microaerophilic flagellated bacteria.^6,20 Numerous Helicobacter species have been identified in various rodent organ systems, including portions of the gastrointestinal tract, liver, and associated biliary system.⁵ Although they often are found in the intestinal tract of immunocompetent mice without clinical disease, various Helicobacter species have been shown induce enteric disease in immunodeficient mice.^6,20 This propensity has been a useful tool in developing mouse models to study inflammatory bowel disease and colon cancer.^7,13,14Murine fecal samples submitted from a variety of institutions to the University of Missouri Research Animal Diagnostic Laboratory (Columbia, MO) between November 2001 and October 2002 showed 17% positivity for H. typhlonius and 10% positivity for H. rodentium.¹¹ H. typhlonius has been reported to cause significant enteric disease in immunodeficient and IL10^−/− mice.⁴ In contrast, H. rodentium has only been reported to cause disease in immunodeficient mice coinfected with other Helicobacter species.^16,20,21 Because these agents cause disease, they are best considered to be rodent pathogens, despite the frequency of their detection in clinically normal mice. Although most murine Helicobacter infections are subclinical, infection with H. rodentium and H. typhlonius may affect experimental studies in vivo under some circumstances. In addition, Helicobacter infections can influence murine reproduction, although this effect has not been well studied.The gastric-infecting species H. pylori influences murine pregnancy by increasing the number of fetal resorptions and producing lower fetal weights compared with those of noninfected controls.¹⁸ Induction of Th1-type responses at the endometrial level was a possible mechanism suggested for these phenomena but not further investigated. Whether intestinal-infecting Helicobacter species such as H. rodentium and H. typhlonius affect murine pregnancy in wild-type or genetically modified mice, particularly those with mutations that affect immune function, has not been determined.Mice deficient in IL10 (IL10^−/− mice) mount an exaggerated and prolonged inflammatory response resulting from their lack of circulating IL10, a cytokine that normally functions to limit inflammatory processes. Thus IL10^−/− mice may be at greater risk of adverse effects after Helicobacter infection due to their lack of IL10 to inhibit Th1 immune responses. In a breeding colony of IL10^−/− mice, those housed in a facility where H. rodentium or H. typhlonius (or both) infections were endemic appeared to have less reproductive success than those that were housed in a facility free from Helicobacter spp. These observations lead to this study to specifically determine the effect of infection with H. rodentium and H. typhlonius on the fecundity (potential reproductive capacity) of IL10^−/− mice. Because antihelicobacter drug therapy might provide a viable alternative to embryo rederivation for some strains, particularly relative to the risk and resource commitment involved with rederivation, we also investigated whether reproductive performance could be improved by the administration of commercially available antihelicobacter wafers as a method of Helicobacter eradication.     

Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2

Biology and tropism of MuAstV2Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains—a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD-Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Astroviruses are nonenveloped, positive-sense, single-stranded RNA viruses with a star-like appearance—from which the name derives—when examined by transmission electron microscopy. First identified in 1975, astroviruses are commonly associated with gastrointestinal illness in children.^1,23 They demonstrate considerable diversity, and unique strains have been identified in numerous species through advances in molecular diagnostics.^{2,4-6,11-13,18,19,21,25,27,29,30,32,35-40} This broad distribution likely resulted from cross-species transmission and subsequent adaptation to the novel host.¹¹ Clinical presentation varies among species, although most infections are asymptomatic or limited to mild gastrointestinal illness.^4,9,11 Extraintestinal disease resulting in fatal encephalitis has been described in several species (including cows, mink, and immunocompromised people).^{3,19,22,26,35}Astroviral infection of mice was first described in 1985, when an unknown astrovirus was identified by electron microscopy in the feces of nude mice.¹⁷ Since then, astroviruses have been detected in many wild and laboratory mouse populations.^12,29,30,34 Despite their prevalence, studies have been limited and their effects on host biology remains largely unknown. Murine astrovirus (MuAstV) was identified through molecular sequencing in 2012 and has since been discovered to be enzootic in numerous research and production mouse colonies.^12,29,34 Whether the strain described in 1985 was MuAstV is unknown. Immunocompetent and immunodeficient mouse strains are both susceptible to MuAstV infection, although no clinical disease and only minimal pathology are observed.^7,47 Similar to astroviruses infecting other species, MuAstV infection is frequently localized to the gastrointestinal tract.⁴⁷ A recent study demonstrated MuAstV replication in goblet cells and altered mucus production within the gastrointestinal tract, highlighting the potential effect of the virus on select research studies despite the lack of clinical disease and pathology.⁸Our group previously reported the detection of a novel murine astrovirus, murine astrovirus 2 (MuAstV2), in a laboratory mouse colony.³¹ MuAstV2 is genetically distinct from MuAstV but is closely related to a strain recently reported in wild mice.^31,43 The MuAstV2 strain identified in the laboratory mouse colony shares 89.2% nucleotide identity to a strain detected in wild mice in New York City but less than 50% nucleotide identity to MuAstV, the strain commonly isolated from laboratory mice. In addition, MuAstV2 was found to share as much as 80.8% nucleotide similarity to an astrovirus strain isolated from urban brown rats (Rattus norvegicus) in Hong Kong.^5,31 Antibodies to MuAstV2 were inadvertently detected in laboratory colony mice when a serologic immunoassay for mouse thymic virus prepared from a murine T-cell line tested positive. Further analysis showed that the mice were negative for mouse thymic virus and that the T-cell line was contaminated with a novel astrovirus strain similar to MuAstV2, resulting in the positive test. The observation that MuAstV2 did not appear to infect highly immunocompromised mice via natural exposure or experimental inoculation was highly unusual.³¹ This finding is distinct from other murine viruses, including MuAstV, given that infection of immunocompromised mice leads to persistent infection and chronic virus shedding.^12,15,16,47We sought to further understand the biology of MuAstV2 by evaluating viral shedding kinetics and tissue tropism in immunocompetent mice and to further characterize the presumptive resistance to infection observed in highly immunocompromised mice. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA after oral inoculation. Tissue and cell tropism were characterized using in-situ hybridization (ISH) and immunohistochemistry during the course of infection. We hypothesized that MuAstV2 initially infects the gastrointestinal tract, as occurs with other astroviruses, but speculated that components of the immune system were required to support infection or replication or both. Furthermore, we sought to characterize the extraintestinal spread of MuAstV2.     

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.     

Development of Standardized Insulin Treatment Protocols for Spontaneous Rodent Models of Type 1 Diabetes

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.Abbreviations: BBDP, BioBreeding diabetes-prone; BBDR, BioBreeding diabetes-resistant; NOD, nonobese diabetic; PZI, protamine zinc insulin; T1D, type 1 diabetes; VAF, viral-antibody–free; ZT, Zeitgeber timeClinical trials to prevent or reverse type 1 diabetes (T1D) are predicated on preclinical study data obtained from animal models of the disease to determine agents that exhibit efficacy and translational potential. However, according to findings published over the past several years (summarized in references 2, 17, and 31), not all preclinical T1D studies are created equal. Without a standardized screening process, the hundreds of candidate therapeutic agents in development cannot be evaluated critically for translational potential. One parameter that varies considerably from report to report in T1D reversal studies is the insulin treatment provided to diabetic NOD mice. To address the need for standardized preclinical screening of new therapeutics, the National Institute for Diabetes and Digestive and Kidney Diseases has developed the Type 1 Diabetes Preclinical Testing Program.^2,35 Under this program, a central contract testing facility (Biomedical Research Models) bridged the gap between discovery of potential therapeutics and clinical testing for efficacy in prevention or reversal of T1D. Using 2 of the best characterized models of T1D, the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse, we sought to develop standardized protocols for the treatment of diabetes with insulin to provide the best glycemic control throughout the fed and nonfed states. We began by housing these models in a viral-antibody–free (VAF) barrier facility, we created study designs approved by a scientific advisory board consisting of leaders in the field, and we performed studies by using standard operation procedures.The standard of care in patients with T1D is to attempt to maintain near-normal glucose levels, by providing exogenous insulin therapy several times daily via injection or pump after rigorous monitoring of glycemic levels and by appropriately coordinating insulin dosing with food intake. Current blood glucose control in diabetic rodent models focuses on maintaining the diabetic animal in a state of moderate hyperglycemia, with normal weight gain in the absence of severe ketonuria. This state is achieved by once-daily injections of titrated insulin doses or by implantation of continuous release insulin pellets;³⁸ however, insulin types and methods can vary widely between institutions and laboratories, yielding a wide range of glycemic control. Therefore there is marked difference between the stringent glycemic control targeted by humans with diabetes as compared with the relatively loose glycemic control afforded to rodents with diabetes. Despite the many physiologic differences between humans and rodents, glycemic control potentially can be addressed by making insulin treatment in rodents more comparable in terms of glycemic control to what is achieved currently in humans, especially given that patients with T1D will continue to administer insulin during treatment with therapeutic agents (for example, antiCD3).¹¹ The lessening of the frequency, duration, and severity of hyperglycemic events is anticipated to provide the best chance for β cells to rest (function properly) while interventions are tested.²¹ Ideally, for these studies, animals should receive sufficient insulin to maintain glycemic levels close to the normal range in control nondiabetic animals.For these studies, we focused on the 2 most widely used spontaneous rodent models of T1D: the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse.^1,12 The BBDP strain originated from a colony of outbred Wistar rats that developed spontaneous diabetes at the BioBreeding Laboratories in the 1970s. In the 1980s, the strain was acquired by the University of Massachusetts Medical School. During inbreeding, the BioBreeding diabetes- resistant (BBDR) control strain was established. Both strains are maintained at our facility and represent the most fully inbred (more than 110 generations) and characterized colonies available. BBDP rats develop T1D at 50 to 90 d of age at a frequency of approximately 85% to 90%, with equal frequency in male and female rats; the disease in BBDP rats results from autoimmune insulitis that is mediated primarily by CD4⁺ and CD8⁺ T cells and the development of autoantibodies to islet antigen. This insulitis is similar to that in human patients.¹⁸ Insulin therapy is required shortly after onset of hyperglycemia or death will occur due to ketoacidosis.¹⁹ The Gimap5 mutation in BBDP rats results in a T-cell lymphopenia and is necessary for development of T1D in BBDP rats (along with expression of a MHC class II RT1 B/D^u allele); adoptive transfer of splenocytes or regulatory T cells from BBDR rats before 35 d of age prevents the onset of diabetes in BBDP rats.^9,28,38 Alternatively, depletion of regulatory T cells from BBDR rats (which are nonlymphopenic) induces T1D in that strain.The NOD mouse strain originated from selective inbreeding of the Cataract Shionogi mouse strain and was imported from Japan to The Joslin Diabetes Center in 1984. NOD mice are now the most widely used preclinical model of T1D, in part due to the availability of genetic analysis and manipulation as well as the wide array of reagents available for mechanistic studies. The most commonly cited source for NOD mice is The Jackson Laboratory (Bar Harbor, ME), where female NOD mice develop disease at a frequency of 65% to 100% by 30 wk of age, whereas male NOD mice develop disease at a frequency of 35% to 85% (inbred for more than 83 generations). The incidence can vary from year to year³⁴ and from facility to facility depending on several factors, the most important being housing conditions.^15,26 The incidence of T1D in female NOD mice at our VAF barrier facility has been 65% to 80% over the past 3 y; this frequency can be far lower in nonVAF facilities. Diabetic NOD mice exhibit mild ketoacidosis, which allows them to survive for as long as several weeks after the onset of hyperglycemia without supportive insulin treatment. NOD mice also present with insulin resistance and a distinct stage of insulitis, referred to as peri-insulitis, that is not found in either human T1D or in diabetic BBDP rats.^5,18 Although both NOD mouse and BBDP rat models of T1D have particular advantages and disadvantages, a prudent path of drug development would include the examination of the therapeutic efficacy of novel agents in both models.^2,31To standardize and improve current testing protocols, we developed insulin treatment regimens that maintain blood glucose levels near normal levels throughout day and night activities over prolonged periods, as would be expected to occur in interventional clinical trials. We show here that whereas 2 daily injections of insulin to diabetic BBDP rats were sufficient to achieve our goal, diabetic NOD mice required continuous delivery of insulin through the implantation of osmotic pumps.     

Effects of Helicobacter Infection on Research: The Case for Eradication of Helicobacter from Rodent Research Colonies

Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.The reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome. Infections of mouse colonies are important to the scientific community because they can introduce such harmful variables. Therefore, the ultimate goal of laboratory animal facilities is to maintain disease-free animals, to eliminate those unwanted variables.Numerous pathogenic microbes can interfere with animal research (reviewed in reference 57), and colonization of mouse colonies with members of the family Helicobacteriaceae is an increasing concern for the research community. Naturally acquired Helicobacter infections have been reported in all commonly used laboratory rodent species.^{3,10,36,44,45,49,82,124} A study of mice derived from 34 commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States showed that 88% of these institutions had mouse colonies infected with 1 or more Helicobacter spp.¹⁰⁹ Approximately 59% of these mice were infected with Helicobacter hepaticus ; however monoinfections with other species also were encountered. In another study, at least 1 of 5 Helicobacter spp. was detected in 88% of the 40 mouse strains tested.⁴Surveys such as these have established that a broad range of Helicobacter spp. may be present in mouse research colonies. Several of those Helicobacter species cause disease in laboratory mice. H. hepaticus first was identified as a pathogen when it was discovered to be the cause of chronic hepatitis and hepatocellular carcinoma in mice,^26,31,116 either alone or in combination with other Helicobacter spp.⁷⁸ In addition, H. typhlonius causes intestinal inflammation in mice with immunodeficiency or defects in immune regulation;^28,37 H. muridarum has been associated with gastritis,⁸⁶ and H. bilis has been associated with hepatitis^35,38 and colitis.^60,61 Although, H. rodentium appears to be relatively nonpathogenic in wild-type and SCID mice,⁷⁸ combined infection with H. rodentium and H. typhlonius results in a high incidence of inflammation-associated neoplasia in IL10^−/− mice.^9,46 Further, it is becoming increasingly clear that the effects of Helicobacter infections are not limited to the gastrointestinal system. Helicobacter infections have been documented to directly or indirectly affect responses as diverse as reproduction, development of breast cancer, and altered immune responses to vaccines.^65,95,99 In addition to effects on rodents, Helicobacter spp. can infect other laboratory animals^{2,5,27,29,33,36,107} and can colonize different anatomic regions of the gastrointestinal system.³⁵ This review focuses on the potential effect of these organisms on in vivo experiments and biomedical research. The results summarized here emphasize the importance of knowledge of colony infection status and prevention of unintentional infections to achieve the goal of providing a consistent and reliable environment for research studies.     

The Effect of Helicobacter hepaticus Infection on Immune Responses Specific to Herpes Simplex Virus Type 1 and Characteristics of Dendritic Cells

Infection of mice with Helicobacter hepaticus is common in research colonies, yet little is known about how this persistent infection affects immunologic research. The goal of this study was to determine whether H. hepaticus infection status can modulate immune responses specific to herpes simplex virus type 1 (HSV1) and the phenotypic and functional characteristics of dendritic cells (DC) of mice. We compared virus-specific antibody and T cell-mediated responses in H. hepaticus-infected and noninfected mice that were inoculated intranasally with HSV1. The effect of H. hepaticus on the HSV1-specific antibody and T cell-mediated immune responses in superficial cervical and tracheobronchal lymph nodes (LN) did not reach statistical significance. Surface expression of the maturation-associated markers CD40, CD80, CD86, and MHC II and percentages of IL12p40- and TNFα-producing DC from spleen and colic LN in H. hepaticus-infected mice and noninfected mice were measured in separate experiments. Expression of CD40, CD86, and MHC II and percentages of IL12p40- and TNFα-producing DC from colic LN were decreased in H. hepaticus-infected mice. In contrast, H. hepaticus infection did not reduce the expression of these molecules by splenic DC. Expression of CD40, CD80, CD86, and MHC II on splenic DC from H. hepaticus-infected mice was increased after in vitro lipopolysaccharide stimulation. These results indicate that H. hepaticus infection can influence the results of immunologic assays in mice and support the use of H. hepaticus-free mice in immunologic research.Abbreviations: DC, dendritic cells; HSV1, herpes simplex virus type 1; LN, lymph nodes; MHC II, major histocompatibility complex class II; MHV, mouse hepatitis virus; OVA, ovalbumin peptide SIINFEKL; PE, phycoerythrinHelicobacter hepaticus is a gram-negative, microaerophilic, curved to spiral-shaped bacterium with bipolar, sheathed flagella. H. hepaticus was described for the first time in 1994 as the cause of chronic active hepatitis associated with a high incidence of hepatocellular neoplasms in mice on a long-term toxicology study.³⁹ Since then, H. hepaticus has been identified as a common contaminant of mouse colonies at a variety of research institutions. Although commercial breeders produce H. hepaticus-free animals, many mouse colonies at public and private research institutions still harbor H. hepaticus. A recent survey found H. hepaticus-infected mice in 59% of commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States.³⁵H. hepaticus persistently colonizes the hepatic bile canaliculi and the cecal and colonic mucosa of mice.^9,39 Infection can cause chronic active hepatitis, hepatocellular neoplasms, and typhlocolitis, which vary in severity depending on the strain, age, gender, and immune status of the mouse.^5,9,11,39 In adult immunocompetent mice, H. hepaticus infection is usually asymptomatic. However, immune-dysregulated mice can develop inflammatory bowel disease, which may present as rectal prolapse or diarrhea.¹⁶Mice initiate immune responses against H. hepaticus primarily through its interaction with Toll-like receptor 2 on antigen-presenting cells.²¹ Both systemic and local (at the site of infection) H. hepaticus-specific Th1-type immune responses are induced in immunocompetent mice.^26,40 Systemic antibody and cell-mediated immunity against the bacteria persist for at least 46 wk after experimental inoculation.⁴⁰ Gene expression profiles of cecal tissue of H. hepaticus-infected mice have shown that inflammatory responses differ depending on the mouse strain. For example, A/JCr mice had significant and prolonged expression of the Th1-type cytokines IFNγ and IL12p40 in cecal mucosa, and these expression levels persisted for at least 3 mo after H. hepaticus infection. However, C57BL/6 mice had a lesser elevation of IFNγ gene expression without an effect on IL12p40. IFN γ expression waned by 1 mo after inoculation in C57BL/6 mice.²⁵ In addition, H. hepaticus-specific secretory IgA antibodies are persistently detected in the feces of mice.⁴⁰ How these immune responses in H. hepaticus-infected mice might affect immunologic research is unknown.The goal of this study was to determine whether immune responses to herpes simplex virus type 1 (HSV1) and the phenotypic and functional characteristics of dendritic cells (DC) are altered in H. hepaticus-infected mice. The intranasal HSV1 infection model is used widely to study immune mechanisms in mice. Immunity to HSV1 consists of virus-neutralizing antibodies in the serum and virus-specific T cells in the draining LN. Superficial cervical and mediastinal LN have been described as draining LN for intranasal HSV1 infection.² The response to HSV1 infection peaks at 7 d after infection and leads to clearance of the viral load.² In this study, we compared levels of HSV1-specific antibody and T cell-mediated immune responses between H. hepaticus-infected and noninfected mice.Dendritic cells are important components of the immune system that play a role in antigen processing and presentation. On exposure to foreign antigen, DC mature and express increased levels of major histocompatibility complex class II proteins (MHC II), CD40, CD80, and CD86 on the cell surface. These maturation-associated cell surface markers interact with naive T and B cells to initiate antibody- and cell-mediated immune responses against foreign antigens.²⁷ In addition, mature DC secrete proinflammatory cytokines, including TNFα and IL12p40. These cytokines lead to increased vascular permeability, complement activity, lymphocyte activation, lymphocyte proliferation, and increased antibody production.²⁷ To determine whether infection with H. hepaticus affects characteristics of DC, we measured the expression of the maturation-associated cell surface markers CD40, CD80, CD86, and MHC II and proinflammatory cytokines IL12p40 and TNFα by DC derived from the spleen and colic LN of H. hepaticus-infected and noninfected mice. Our findings indicate that H. hepaticus infection can influence the various aspects of immune responsiveness and, therefore, must be considered as a potential variable in studies in which immune function is a measurable outcome.     

Simian Betaretrovirus Infection in a Colony of Cynomolgus Monkeys (Macaca fascicularis)

Of the 419 laboratory-bred cynomolgus macaques (Macaca fascicularis) in a breeding colony at our institution, 397 (95%) exhibited antibodies or viral RNA (or both) specific for simian betaretrovirus (SRV) in plasma. Pregnant monkeys (n = 95) and their offspring were tested to evaluate maternal–infant infection with SRV. At parturition, the first group of pregnant monkeys (n = 76) was antibody-positive but RNA-negative, the second group (n = 14 monkeys) was positive for both antibody and RNA, and the last group (n = 5) was antibody-negative but RNA-positive. None of the offspring delivered from the 76 antibody-positive/RNA-negative mothers exhibited viremia at birth. Eight of the offspring (including two newborns delivered by caesarian section) from the 14 dually positive mothers exhibited SRV viremia, whereas the remaining 6 newborns from this group were not viremic. All of the offspring (including 2 newborns delivered by caesarian section) of the 5 antibody-negative/RNA-positive mothers exhibited viremia at birth. One neonatal monkey delivered by CS and two naturally delivered monkeys that were viremic at birth remained viremic at 1 to 6 mo of age and lacked SRV antibodies at weaning. Family analysis of 2 viremic mothers revealed that all 7 of their offspring exhibited SRV viremia, 6 of which were also antibody-negative. The present study demonstrates the occurrence of transplacental infection of SRV in viremic dams and infection of SRV in utero to induce immune tolerance in infant monkeys.Abbreviation: SRV, simian betaretrovirusAlthough simian betaretrovirus (SRV) causes symptoms of immunodeficiency, including anemia, tumors, and persistent refractory diarrhea, in some infected macaques,^1,7,10 most infected monkeys exhibit few or no clinical signs.² Macaques free of SRV are important in many types of experiments to avoid associated immunologic and virologic effects. Establishing an SRV-free breeding colony is paramount for a steady supply of appropriate monkeys for various experiments.⁸We previously reported that SRV-T, a novel subtype of SRV, was found in the cynomolgus colony of our institution.³ Approximately 20% of the colony monkeys tested in 2005 were viremic and shed SRV-T virus in saliva, urine, and feces.^4,5 The viruses shed by these monkeys are a potential source of horizontal SRV-T infection, as occurred in a rhesus monkey colony.^6,7 In the present study, we investigated the actual prevalence and transmission of SRV in the closed cynomolgus colony through several generations, to prevent the spread of the virus and to establish an SRV-free colony.     

Infectious Diseases in Wild Mice (Mus musculus) Collected on and around the University of Pennsylvania (Philadelphia) Campus

Laboratory mice serve as important models in biomedical research. Monitoring these animals for infections and infestations and excluding causative agents requires extensive resources. Despite advancements in detection and exclusion over the last several years, these activities remain challenging for many institutions. The infections and infestations present in laboratory mouse colonies are well documented, but their mode of introduction is not always known. One possibility is that wild rodents living near vivaria somehow transmit infections to and between the colonies. This study was undertaken to determine what infectious agents the wild mice on the University of Pennsylvania (Philadelphia) campus were carrying. Wild mice were trapped and evaluated for parasites, viruses, and selected bacteria by using histopathology, serology, and PCR-based assays. Results were compared with known infectious agents historically circulating in the vivaria housing mice on campus and were generally different. Although the ectoparasitic burdens found on the 2 populations were similar, the wild mice had a much lower incidence of endoparasites (most notably pinworms). The seroprevalence of some viral infections was also different, with a low prevalence of mouse hepatitis virus among wild mice. Wild mice had a high prevalence of murine cytomegalovirus, an agent now thought to be confined to wild mouse populations. Helicobacter DNA was amplified from more than 90% of the wild mice (59% positive for H. hepaticus). Given the results of this study, we conclude that wild mice likely are not a source of infection for many of the agents that are detected in laboratory mouse colonies at the University of Pennsylvania.Abbreviations: EDIM, epizootic diarrhea of infant mice; MAV, mouse adenovirus; MCMV, murine cytomegalovirus; MFIA, multiplex fluorescent immunoassay; MHV, mouse hepatitis virus; MNV, murine norovirus; MPV, mouse parvovirus; MVM, minute virus of mice; TMEV, Theiler mouse encephalomyelitis virusLaboratory mice constitute the most popular animal models used in biomedical research today. Like all animals, even mice housed in so-called ‘barrier’ facilities are subject to infection. The infectious agents and organisms present in laboratory mouse colonies on the University of Pennsylvania campus are known and documented by the University Laboratory Animal Resources Diagnostic Services Unit. Sentinel mice that are housed on soiled bedding from resident mouse cages are screened onsite at 3 quarterly intervals for fur mites and pinworms and for a panel of viral infections: mouse hepatitis virus (MHV); epizootic diarrhea of infant mice (EDIM) virus; minute virus of mice (MVM); mouse parvovirus (MPV); Theiler mouse encephalomyelitis virus (TMEV); and Sendai virus. Comprehensive bacteriology and parasitology assessments are performed on all sentinels once yearly during the fourth quarter. In addition, these sentinels are screened serologically for 18 viral infections, Mycoplasma pulmonis, cilia-associated respiratory bacillus, and Encephalitozoon cuniculi and by PCR for Helicobacter spp. and M. pulmonis. Mesenteric lymph nodes from sentinels monitoring barrier-maintained colonies are also screened once yearly by PCR for MPV. In addition, University Laboratory Animal Resources maintains a quarantine facility for rodents received from nonapproved sources (sources other than selected commercial breeding facilities). Mice entering the quarantine facility are housed in semirigid isolators, and contact sentinels are tested for all of the agents included in the fourth quarter comprehensive health assessment described, including PCR for MPV.Wild mice (Mus musculus) could serve as a source of infection or infestation in laboratory mouse colonies, although little is known about the prevalence of infectious diseases in wild mouse populations in Philadelphia. However, we have surveyed wild mouse populations in other geographic areas.^1,9 Significant seroprevalence of MHV, EDIM, murine cytomegalovirus (MCMV), parvovirus, and thymic virus (murid herpesvirus 3), in addition to the presence of many types of parasites and bacteria including Myocoptes spp., Myobia spp., Radfordia spp., Spironucleus spp., Giardia spp., Pasteurella pneumotropica, Pseudomonas spp., and Leptospira spp. were found in wild populations of mice from farms in southeastern Connecticut.¹ Studies of wild mouse (Mus domesticus) populations in the cereal-growing region of southeastern Australia revealed a high serologic prevalence of MHV, EDIM, and MCMV, as well as significant seroprevalence of mouse adenovirus (MAV), MPV, and reovirus type 3.⁹The goal of the current study was to expand preliminary data obtained from wild mice trapped in the University City district of Philadelphia in 2005 (which are included with the current results from a 2007 survey). These data document the prevalence of various infectious agents and parasites commonly found in populations of wild mice on the University of Pennsylvania campus in Philadelphia and are discussed in the context of infectious disease outbreaks in campus vivaria over the past 5 y.