Comparing United States and European Union academic animal programs: Organization, operation, and services offered.

The Yale Animal Resource Cost and Benchmarking survey©, conducted in US academic Animal Research/Resource Centers (ARC), was modified to capture similar thematic information in European Union (EU; including the non-EU countries Switzerland and the UK) academic ARCs, which are members of the League of European Research Universities (LERU). Participating institutions came from Denmark, England, Finland, France, Germany, Ireland, Italy, Netherlands, Scotland, Spain, and Switzerland. Survey data analysis suggests that: (a) in LERU programs, it is common to have more than one ARC under the umbrella of a single institution with organizational "lumping" of the financial, regulatory, and/or operational tasks under one administrative unit; (b) accreditation by an outside agency (e.g., the Association for the Assessment and Accreditation of Laboratory Animal Care) is more common in US than LERU ARCs; (c) LERU ARCs are responsible for murine breeding, which contrasts with US ARCs, where ∼40% of rodent breeding is managed by researchers; (d) cryopreservation is the most frequently requested fee-for-service offering among LERU participants (75% of participants) compared to 30% of US participants; (e) like US programs, almost all LERU programs have mice and rats, but fewer LERU programs have nonhuman primates (NHPs), and none have dogs on census; (f) LERU ARCs have about an equal amount of vivarium housing and procedure space, while US facilities have twice as much housing as procedure space; (g) a higher percentage of LERU colonies are free of Helicobacter and murine norovirus compared to US colonies; and (h) more LERU ARCs used environmental microbiologic monitoring of rodent colonies compared to US programs.

Comparing United States and European Union academic animal programs: Finances.

The Yale Animal Resource Cost and Benchmarking survey, conducted in United States (US) academic animal research/resource centres (ARC), was modified to capture similar information in European Union (EU) (including the non-EU countries Switzerland and the United Kingdom) academic ARCs, who are members of the League of European Research Universities (LERU). Participating institutions came from Denmark, England, Finland, France, Germany, Ireland, Italy, Netherlands, Scotland, Spain and Switzerland. Survey data analysis suggests that (a) per diem rates have similar compositions in LERU and US programs, with >50% of the rates dedicated to recovering salary and fringe, followed by supplies (∼25%), facility costs (∼10%) and other expenses (∼15%); (b) ∼60% of US and LERU programs under-recover mouse care costs; (c) on average, LERU programs have a small positive net-operating balance, while US programs average a large deficit; (d) in LERU programs <50% of institutions fund the animal program deficit, while in US programs almost 100% of such deficits are covered by the institution; and (e) when setting per diem rates, both US and LERU programs rank cost accounting as the most influential factor. Both US and LERU programs are reluctant to raise per diem rates to the extent required to recover costs and, thus, continue to under-recover costs, resulting in the animal program being 'caught in the middle' between the competing financial challenges of investigator 'affordability' and the animal program's fiduciary responsibility to the institution.

Lack of Effect of Murine Astrovirus Infection on Dextran Sulfate-induced Colitis in NLRP3-deficient Mice.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. MuAstV is found predominantly in the gastrointestinal tract of mice. Human and turkey astroviruses have been shown to disrupt tight junctions in the intestinal epithelium. The potential of MuAstV to alter research results was tested in a well-established dextran sodium sulfate (DSS)-induced colitis model in Nod-like receptor 3 (NLRP3)-deficient mice. This model offers a direct approach to determine whether MuAstV, as a component of the mouse microbiome, contributes to the issue of poor reproducibility in murine inflammatory bowel disease research. In this model, defective inflammasome activation causes loss of epithelial integrity, resulting in leakage of intestinal bacteria and colitis. Our goal was to determine whether MuAstV, which also may affect intestinal permeability, altered the onset or severity of colitis. Male and female mice (age, 8 to 12 wk) homozygous or heterozygous for an NLRP3 mutation were inoculated orally with MuAstV or mock-inoculated with media 3 or 20 d prior to being exposed to 2% DSS in their drinking water for 9 d. MuAstV infection alone did not cause clinical signs or histopathologic changes in NLRP3-/- or NLRP3+/- mice. No significant difference was seen in weight loss, clinical disease, intestinal inflammation, edema, hyperplasia, or mucosal ulceration between MuAstV- infected and mock-infected mice that received 2% DSS for 9 d. Therefore, MuAstV does not appear to be a confounding variable in the DSS colitis model in NLRP3 mice.

Murine Astrovirus Infection and Transmission in Neonatal CD1 Mice.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. Our goal was to determine the duration of MuAstV infection, susceptibility of pups, and efficacy of soiled-bedding sentinels and environmental monitoring. Eight CD1 dams and their litters of 3-d-old pups and 8 CD1 dams and their litters of 13-d-old mice were inoculated orally with MuAstV. Neither dams nor offspring demonstrated any clinical signs, and MuAstV had little to no effect on weight gain in pups. MuAstV RNA was detected in feces from 15 of the 16 dams through postnatal day (PND) 21, and 9 dams were still shedding MuAstV at PND 42. MuAstV RNA was highest in intestines of mice. Low levels of MuAstV RNA were sporadically detected in the spleen, liver, and kidney. MuAstV was detected in 97% of feces from 3- to 9-wk-old mice born to infected dams. Several weanlings became pregnant, and intestines from their pups were MuAstV-negative at PND 0 through 5. Weekly swabs of cages housing MuAstV-infected mice were MuAstV-positive through PND 42. Swabs of the rear exhaust manifold of the ventilated rack were MuAstV-positive at 21 through 56 d after inoculation. In addition, 98% of sentinels that received soiled bedding from dams and their litters and 83% of sentinels that received soiled bedding from weaned mice were MuAstV-positive. Feces from most sentinels exposed to soiled bedding that had been stored for 1, 2 or 3 wk before addition of the sentinels were MuAstV-positive.

Effect of Cage-Wash Temperature on the Removal of Infectious Agents from Caging and the Detection of Infectious Agents on the Filters of Animal Bedding-Disposal Cabinets by PCR Analysis.

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.

Effect of immunodeficiency on MPV shedding and transmission.

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.

Transmission of mouse parvovirus by fomites.

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.

Transmission of mouse parvovirus to neonatal mice.

To investigate the infection of newborn mice with mouse parvovirus (MPV), a single MPV-infected mouse was added to each of 15 cages, each of which housed an uninfected breeding pair of Swiss Webster mice just before parturition. Seven litters were left with their parents, and the remaining 8 litters were fostered at postpartum day 1. All dams were shedding MPV at 1 and 2 wk after exposure. Soiled-bedding transmission did not differ between cages with and without litters. Half the foster dams but none of the fostered pups seroconverted to MPV. None of the pups left with their birth mothers had MPV DNA in their feces at 3 or 6 wk after exposure, but pups in 6 of 7 litters were MPV seropositive at 6 wk. To investigate MPV infection of older neonatal mice, 9 dams with 7-d-old litters and 9 dams with 14-d-old litters each were exposed to an MPV-infected mouse. At weaning, pups exposed to MPV at 7 or 14 d of age were shedding MPV and were seronegative but became seropositive by 6 wk of age and transmitted the infection to sentinels. In conclusion, fostering of pups had no benefit and may spread infection because the pups may act as fomites, infecting the foster dam. Infection of 7- or 14-d-old mice likely occurred because maternal antibodies had not been transferred to the progeny before they began to ingest MPV-laden feces.

Detection and control of mouse parvovirus.

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.

Effect of murine norovirus infection on mouse parvovirus 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.

A PCR-based strategy for detection of mouse parvovirus.

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.

Transmission of enterotropic mouse hepatitis virus from immunocompetent and immunodeficient mice.

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.

Pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice.

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.

Pathogenesis of mouse hepatitis virus infection in gamma interferon-deficient mice is modulated by co-infection with Helicobacter hepaticus.

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.

Assessment of static isolator cages with automatic watering when used with conventional husbandry techniques as a factor in the transmission of mouse hepatitis virus.

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.