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There is a "village" of people impacting research reproducibility, such as funding panels, the IACUC and its support staff, institutional leaders, investigators, veterinarians, animal facilities, and professional journals. IACUCs can contribute to research reproducibility by ensuring that reviews of animal use requests, program self-assessments and post-approval monitoring programs are sufficiently thorough, the animal model is appropriate for testing the hypothesis, animal care and use is conducted in a manner that is compliant with external and institutional requirements, and extraneous variables are minimized. The persons comprising the village also must have a shared vision that guards against reproducibility problems while simultaneously avoids being viewed as a burden to research. This review analyzes and discusses aspects of the IACUC's "must do" and "can do" activities that impact the ability of a study to be reproduced. We believe that the IACUC, with support from and when working synergistically with other entities in the village, can contribute to minimizing unintended research variables and strengthen research reproducibility.

Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.

Background. The recently identified autonomous mouse parvovirus designated mouse parvovirus-1 (MPV-1) persists in adult BALB/c mice for at least 9 weeks, infects lymphoid tissues, interferes with the ability of cloned T cells to proliferate, and exhibits immunomodulatory properties. As a consequence of these findings, the present studies were undertaken to characterize further the immunomodulatory effects of MPV-1 on T cell-mediated immune responses in vivo and in vitro. Methods. To evaluate the effect of MPV-1 infection on CD8+ T cell-mediated responses, BALB/c-H2dm2 mice were infected after transplantation of allogeneic BALB/c skin. Results. MPV-1 potentiated the rejection of allogeneic skin grafts. This potentiation was not a result of virus infecting the cellular or vascular component of the graft as determined by in situ hybridization, but was mediated by T cells. However, the proliferative capacity of alloantigen-reactive lymphocytes from graft-sensitized infected mice was diminished. MPV-1 also induced the rejection of syngeneic skin grafts, and T cells from these infected graft-sensitized mice lysed syngeneic P815 target cells. Conclusions. These results suggest that MPV-1 infection of skin-grafted mice may disrupt normal mechanisms of peripheral tolerance and provide a unique model to study virus-induced autoimmunity.

C57BL/6 (B6) mice briefly shed low levels of MPV, and transmission is inefficient. To determine whether deficits in B or T cells or in interferon γ on a B6 background increased the duration of MPV shedding or transmission, B-cell–deficient (Igh), interferon-γ–deficient (Ifnγ), B- and T-cell–deficient (Rag), and B6 mice were inoculated with MPV. At 1 and 2 wk postinoculation (wpi), 11% to 94% of mice shed MPV. From 4 to 18 wpi, 80% to 100% of Rag mice and 0% of B6 and Ifnγ mice shed MPV; Igh mice sporadically shed MPV through 20 wpi. MPV was transmitted from B6 mice and Ifnγ mice at 2 to 4 wpi. Rag and Igh mice transmitted MPV to sentinels at all or most time points, respectively, between 2 to 16 wpi. Once transmission ceased from B6, Ifnγ, and Igh mice, breeding trios were setup and showed that MPV was transmitted to offspring in only one cage of Igh mice. In another experiment, MPV shedding ceased from B6, CD8-deficient (CD8), CD4-deficient (CD4), and T-cell–receptor-deficient (TCR) mice by 2, 6, 8, and 8 wpi, respectively. MPV was transmitted to sentinels only at 1 to 4 wpi. Mesenteric lymph nodes collected from 61% to 100% of B6, Ifnγ, TCR, CD4, CD8, and Rag mice were MPV DNA-positive. In conclusion, MPV transmission did not differ between mice deficient in T cell functions or Ifnγ and B6 mice. In contrast, B-cell deficiency posed an increased risk for MPV transmission in mice.

The goal of the current studies was to determine the risk of transmission of mouse parvovirus (MPV) by caging and husbandry practices. To determine whether MPV can be transmitted during cage changes in a biologic safety cabinet without the use of disinfectants, 14 cages of Swiss Webster mice were inoculated with MPV. Cages containing infected mice were interspersed among 14 cages housing naïve Swiss Webster mice. At 1, 2, and 4 wk after inoculation of the mice, cages were changed across each row. All naïve mice housed adjacent to infected mice remained seronegative. To determine the risk of environmental contamination, nesting pads that were used to sample the room floor during husbandry procedures at 1, 2, 4, and 6 wk after inoculation of the mice were placed in cages with naïve mice. None of the mice exposed to the pads became MPV seropositive. To determine whether components from cages that had housed MPV-infected mice could transmit MPV, Swiss Webster mice were exposed to soiled bedding or used cages, drinking valves, food, cage bottoms, wire bars and filter tops, nesting material, or shelters. With the exception of drinking valves, all mice exposed to other components became MPV seropositive. Fourteen cages that had housed MPV-infected mice were washed but not autoclaved; mice housed in the washed cages did not become MPV seropositive. In conclusion, all cage components can serve as fomites, with the drinking valve being the least risky. Cage washing alone was sufficient to remove or inactivate MPV.

Mouse parvovirus (MPV) remains a prevalent infection of laboratory mice. We developed 2 strategies to detect and control an active MPV infection over a 9.5-mo period. The first strategy used a test-and-cull approach in 12 rooms. After all cages corresponding to MPV-seropositive bedding sentinels were removed from the room, a naïve sentinel mouse was dedicated to every 2 to 3 rows per rack and received soiled bedding from these rows every 2 wk. All 12 rooms completed 3 consecutive negative rounds of targeted testing, which required an average of 20 wk. The second strategy used a modified quarantine approach to test unique mice that were critical for breeding. The process required removing selected cages from the seropositive rack and consolidating them to a single rack within the same room. All mice in these cages were tested by using MPV serology and fecal PCR. Cages were not moved, opened, or manipulated between sample collection and the availability of test results. The cages were relocated as a group to another room, because all mice were MPV negative. The mice were retested 3 wk after the initial testing, and all were MPV seronegative. Since the rooms were cleared 4 to 5 y ago, 7915 routine bedding sentinels and colony mice were tested from these rooms, all with negative results. These consistently negative MPV test results suggest that MPV was eliminated from these rooms, rather than driven down below the threshold of detection. These 2 strategies should be considered when confronting MPV infection.

Enzootic infection with mouse parvovirus (MPV) remains a common problem in laboratory colonies, and diagnosis of MPV infection is complicated by viral and host factors. The effect of an underlying viral infection on MPV infection has not previously been investigated. We assessed the effect of murine norovirus (MNV) infection, the most prevalent infectious agent in laboratory mice, on MPV shedding, tissue distribution and transmission. Fecal MPV shedding persisted longer in BALB/c mice infected with MNV 1 wk prior to MPV infection than in mice infected with MPV only, but transmission of MPV to soiled-bedding sentinels was not prolonged in coinfected mice. MPV DNA levels in coinfected BALB/c mice were higher in mesenteric lymph nodes and spleens at 1 and 2 wk after inoculation and in small intestines at 1 wk after inoculation compared with levels in mice infected with MPV only. In C57BL/6 mice, fecal shedding was prolonged, but no difference in soiled bedding transmission or MPV DNA levels in tissues was detected between singly and coinfected mice. MPV DNA levels in singly and coinfected SW mice were similar. MPV DNA levels were highest in SW, intermediate in BALB/c and lowest in C57BL/6 mice. MPV DNA levels in mesenteric lymph nodes of BALB/c and SW mice exceeded those in small intestines and feces, whereas the inverse occurred in C57BL/6 mice. In conclusion, MNV infection increased the duration of MPV shedding and increased MPV DNA levels in tissues of BALB/c mice.

Mouse parvovirus (MPV) infection is difficult to address because it is asymptomatic, persists for as long as 9 wk, and occurs in small subpopulations of mice. The efficacy of a PCR-based cage swabbing strategy for MPV detection was tested. On postinoculation days (PID) 3 through 63, feces were collected from MPV-infected SW mice or the wire bars and cage wall above and below the bedding were swabbed. MPV DNA was detected in all cages in all locations on PID 7 and 14 but only below the bedding on PID 21. Swabbing below the bedding detected MPV in most cages through PID 42. Sentinels exposed to soiled cages on PID 7 and 14 but not on PID 21 seroconverted. MPV was detected in feces from all cages until PID 33 and in at least 1 cage until PID 56. In BALB/c mice, MPV was detected in feces and on cage swabs on PID 5 to 14, and 80% of sentinels exposed to soiled cages on PID 7 and 14 seroconverted. In comparison, MPV infection of C57BL/6 mice was detected in feces on PID 5 to 14 and on swabs on PID 5 and 7, and 30% of sentinels exposed to soiled cages on PID 7 and 14 seroconverted. Swabbing of multiple cages in rows in which only 1 cage contained MPV-infected mice was ineffective. In conclusion, swabbing of individual cages can be used in a genotype-dependent manner as an adjunct to soiled bedding sentinels during the first 2 wk of infection.

Mouse hepatitis virus (MHV) is one of the most prevalent viruses infecting laboratory mice. Most natural infections are caused by enterotropic strains. Experiments were done to compare the pathogenesis of enterotropic strain MHV-Y in immunocompetent BALB/c and C57BL/6 mice with that in B and T cell-deficient mice. In situ hybridization was used to identify sites of virus replication, and reverse transcriptase-polymerase chain reaction analysis was used to detect viral RNA in feces and blood. MHV-Y caused acute subclinical infections restricted to the gastrointestinal tract in BALB/c and C57BL/6 mice. Viral RNA was detected in small intestine and associated lymphoid tissues of immunocompetent mice for 1 week and in cecum and colon for 2 weeks. Infected B cell-deficient mice developed chronic subclinical infection also restricted to the gastrointestinal tract. Viral RNA was detected in the small intestine, cecum, colon, and feces for 7 to 8 weeks. In contrast, infected T cell-deficient mice developed multisystemic lethal infection. During the first week, viral RNA was restricted to the gastrointestinal tract. However, by 2 weeks, mice developed peritonitis, and viral RNA was detected in mesentery and visceral peritoneum. Three to four weeks after virus inoculation, T cell-deficient mice became moribund and viral RNA was detected in multiple organ systems. These results suggest that B cells promote clearance of MHV-Y from intestinal mucosa and that T cells are required to prevent dissemination of MHV-Y from the gastrointestinal tract and associated lymphoid tissues.

Mouse hepatitis virus (MHV) is the most prevalent virus that infects mice, and most MHV strains are enterotropic. Experiments were performed to elucidate the duration of enterotropic MHV-Y shedding by immunocompetent BALB/ c and C57BL/6 mice and immunocompromised B and T cell-deficient mice. Although the use of molecular diagnostics to detect MHV infection is increasing, it is unclear whether the viral RNA detected is always infectious. The ability to detect MHV-Y transmission to sentinel mice exposed directly to infected mice or to soil bedding from infected mice was compared with reverse transcriptase-polymerase chain reaction-based detection of viral RNA in the feces. The BALB/c mice developed subclinical intestinal infection, and transmitted MHV-Y for four weeks. The C57BL/6 mice also developed subclinical intestinal infection, but only transmitted virus for two weeks. The T cell-deficient mice developed severe disseminated disease by two weeks and transmitted virus for four weeks. The B cell-deficient mice developed subclinical intestinal infection and transmitted virus for longer than three months, although virus RNA was not detected in feces late in the infection. Viral RNA detected in the feces of infected mice was almost always infectious. Non-infectious RNA was detected in a few mice for several days after transmission had ceased. In addition, constant exposure of naive mice to infected mice, via the use of serial sentinels, prolonged viral transmission.

Gamma interferon-deficient (IFN-gamma KO) mice developed a wasting syndrome and were found to be co-infected with Helicobacter sp., and a new isolate of mouse hepatitis virus (MHV) designated MHV-G. The disease was characterized by pleuritis, peritonitis, hepatitis, pneumonia, and meningitis. Initial experiments used a cecal homogenate inoculum from the clinical cases that contained H. hepaticus and MHV-G to reproduce the development of peritonitis and pleuritis in IFN-gamma KO mice. In contrast, immunocompetent mice given the same inoculum developed an acute, self-limiting infection and remained clinically normal. This result confirmed the importance of IFN-gamma in preventing chronic infection and limiting viral dissemination. To understand the role of both agents in the development of peritonitis and pleuritis, IFN-gamma KO mice were infected with either agent or were co-infected with H. hepaticus and MHV-G. Infection with MHV-G induced a multisystemic infection similar to that described in the original cases, with multifocal hepatic necrosis, acute necrotizing and inflammatory lesions of the gastrointestinal tract, and acute peritonitis and pleuritis with adhesions on the serosal surfaces of the viscera. However, mice given H. hepaticus alone had minimal pathologic changes even though the organism was consistently detected in the cecum or feces. Although co-infection with H. hepaticus and MHV-G induced lesions similar to those associated with MHV-G alone, the pathogenesis of the MHV infection was modified. Helicobacter hepaticus appeared to reduce the severity of MHV-induced lesions during the acute phase of infection, and exacerbated hepatitis and meningitis at the later time point. We conclude that infection of IFN-gamma KO mice with MHV-G results in multisystemic infection with peritonitis, pleuritis, and adhesions due to the aberrant immune response in these mice. In addition, co-infection of these mice with H. hepaticus results in alterations in the pathogenesis of MHV-G infection.

This study evaluated protection against mouse hepatitis virus (MHV) afforded by static filter-top caging when automatic watering was used with conventional husbandry techniques as a labor-saving option. We fitted one side of a double-sided 72-cage rack with valves external to each cage; cages on the other side were fitted with shielded internal valves. More than 50% of the mice were breeding mice, and 30% were genetically altered. One cage of mice on each shelf on both sides of the rack was infected with MHV-A59. Each row of cages also contained one standard cage (no filter top) of uninoculated mice at various distances from the infected cage. At 2, 4, and 6 weeks after infection of the mice in the test cages, uninoculated mice in 22 cages were tested by serology, and at 8 weeks the uninoculated mice in 54 cages were tested by serology and those in 24 cages were tested by polymerase chain reaction (PCR) amplification of fecal samples to assess transmission of infection. At 8 weeks post-infection, mice in one uninoculated cage (which had a filter top and an internal valve and was adjacent to a cage of inoculated mice) was seropositive. Examination of feces by PCR revealed MHV shedding in mice in nine uninoculated cages (three lacking filter tops but with internal valve cages; two with filter tops and internal valve cages and adjacent to non-filter top cages; two non-filter-top cages with external valves; and two filter-top cages with external valves, of which one was adjacent to a non-filter-top cage). Routine husbandry using either automatic water valve system prevented (with one exception) transmission among filter-top cages for at least 6 weeks. The 10 cages where transmission occurred were non-filter-top cages (n = 5) and filter-top cages adjacent to non-filter top, infected, or sentinel cages (n = 5). These results suggest that the use of filter top-caging with automatic watering may limit MHV transmission for 6 weeks, during which immunocompetent mice would be expected to clear the virus. Our findings also suggest that long-term use of automatic watering in static filter-top cages handled using conventional husbandry techniques may not prevent transmission in the vicinity of high virus concentrations or open caging.