Of Mice and Microflora: Considerations for Genetically Engineered Mice

Veterinary Pathology 49(1) 44-63 ª The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985811431446 http://vet.sagepub.com

P. M. Treuting1, C. B. Clifford2, R. S. Sellers3 and C. F. Brayton4

Abstract The phenotype of genetically engineered mice is a combination of both genetic and environmental factors that include the microflora of the mouse. The impact a particular microbe has on a mouse reflects the host–microbe interaction within the context of the mouse genotype and environment. Although often considered a confounding variable, many host–microbe interactions have resulted in the generation of novel model systems and characterization of new microbial agents. Microbes associated with overt disease in mice have been the historical focus of the laboratory animal medical and pathology community and literature. The advent of genetic engineering and the complex of mouse models have revealed previously unknown or disregarded agents that now oblige the attention of the biomedical research community. The purpose of this article is to describe and illustrate how phenotypes can be affected by microflora by focusing on the infectious diseases present in genetically engineered mouse (GEM) colonies of our collective institutions and by reviewing important agents that are rarely seen in most research facilities today. The goal is to introduce the concept of the role of microflora on phenotypes and in translational research using GEM models. Keywords animals, genetically modified, specific pathogen free, pathology, infectious disease, mice, transgenic, models, animal, review, bacterial infections and mycoses, viral diseases, parasitic diseases The impact a particular microbe has on a genetically engineered mouse (GEM) reflects the host–microbe interaction within the context of the mouse genotype and environment. The underlying supposition is that any GEM, regardless of the genetic modification, should be considered ‘‘immunovague’’ at best; vigilance on the part of veterinary pathologists and scientists is essential to identify and characterize emerging infections or outbreaks that may be responsible for or may alter phenotypes.41 Alterations in the microbial susceptibilities of GEM may be an anticipated effect (eg, in targeted disruption of key cytokines) or an unanticipated primary or secondary effect (eg, alterations in mucociliary clearance in the lung). Infectious agents that do not result in disease or persistent shedding in ‘‘normal’’ (inbred and outbred) mice can cause illness in GEM. Microflora can affect translational research through overt disease and death; however, more often the resident microflora can result in subclinical disease and alterations in physiological function (immune response), microscopic changes (increased extramedullary hematopoiesis in the spleen98), or altered study end points (xenograft tumor kinetics,81 gene expression).91 As research colonies become more stringent in defining specific pathogen-free requirements, most pathogenic agents such as Sendai virus have become significantly less prevalent. As such, new agents or those previously considered incidental or commensal are now being brought into the focus of the laboratory animal medicine and pathology community, particularly as the use of GEM increase. These microbial ‘‘emerging

diseases’’ are defined as infectious agents that have been recently discovered (eg, mouse norovirus), infectious agents that have increased incidence (eg, mouse parvovirus), or microbes with newly identified roles on a specific phenotype (eg, Helicobacter spp) (C. Clifford, unpublished data, 2011). Most of these emerging agents were discovered or recognized in parallel with the tremendously increased use of genetically modified mice. Although attention has focused on the interaction of primary or opportunistic pathogens with a mouse host (see references 2, 41, and 98 for detailed reviews), the role of normal flora or variations in normal flora should not be ignored. The mouse microbiome (defined as the microbial communities and their genetic elements in an environment such as gastrointestinal tract, see Table 1), is poorly understood but is increasingly thought to play a role in phenotype development as mammals and

1

University of Washington, Seattle, Washington Charles River Laboratories, Wilmington, Maryland 3 Albert Einstein College of Medicine, Bronx, New York 4 Johns Hopkins University, Baltimore, Maryland 2

Corresponding Author: Piper M. Treuting, Department of Comparative Medicine & Histology and Imaging Core, School of Medicine, University of Washington, T140 Health Science Center, Box 357190, Seattle, WA 98195-7190 Email: [email protected]

Treuting et al

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Table 1. Glossary of Terms Related to Genetically Engineered Mice Agent Altered Schaedler flora Barrier facility

Commensal organism Conventional facility

Conventional mice Defined flora Ectoparasite Emerging agent

Endoparasite Exclusion list

Facultative pathogen Germ-free Gnotobiotic Health report

Microbiome

Microbiota (microflora) Opportunist Parasite Pathogen Quarantine

Rederivation Sentinel

SPF

Surveillance

Synonym for a bacteria, protozoa, viruses, or parasites used when defining specific pathogen free (SPF) status of a facility (eg, list of restricted agents) Flora consisting of 8 standardized microbiota considered ‘‘normal’’ and beneficial flora.31 Aim is to protect mice from outside microbes. Exclusion lists, test practices, and protection procedures vary. The facility’s residents should be SPF for the list of excluded agents. Rigorous testing to ensure SPF status is implied. Microorganism that normally lives in symbiosis with the host without causing reduction in host fitness.114 Residents may be SPF for certain agents, but their exclusion lists generally are shorter than those for barrier facilities at the same institution. Caging is often open-top. There is less need to test for agents that are not excluded. Mice housed in conventional facilities with unknown microbial and disease status. Sometimes referred to as ‘‘dirty.’’ Animals are maintained in isolated environment and are intentionally associated with one or more known life forms, usually microorganisms such as Altered Schaedler flora. Parasite that lives on external surfaces (ie, skin, Table 2) An infectious agent in laboratory mice has recently been discovered (ie, mouse noroviruses) or is increasing in incidence (ie, mouse parvoviruses), or new information has been discovered that significantly increases awareness of the impact of the agent on research outcomes (ie, Helicobacter). Parasite that lives inside the host (eg, enteric–hepatic, Table 2) List of agents for a facility that are excluded from the rodent colonies. Incoming animals, biological products (ie, cell lines), and colony mice are routinely monitored for excluded agents. Agents on the list can vary by institution and even from one animal room to another (ie, Helicobacter-free room in a non– Helicobacter-free facility) Synonym opportunist. A microorganism capable of causing harm to the host under certain conditions. Synonym axenic. Mice with no microbiota with the possible exception of endogenous retroviruses.102 Mice with known microbiological status can be either germ free and defined flora animals.102 114 List of results from sentinel testing for excluded agents. These are used during transfer of mice between different facilities. Review of health reports from sending intuitions is usually a key step for the importation of new mice into a facility. All of the microorganisms and their genetic material within an environment. In mice, the focus is primarily the microbes within the gastrointestinal tract and their interaction with the host. Skin and lower genitourinary tracts also have unique flora. Bacteria, protozoa, and viruses within an environment that, along with their genetic material, make up the microbiome for that ecosystem (eg, colon).114 Microbiota that causes disease or colonization under certain conditions such as immunosuppression. Microorganism that causes decreased fitness to the host. In mice, usually used in context of relatively larger species such as pinworms or mites; however, on-commensal bacteria and viruses are parasites. Infectious microorganism that negatively affects the host by causing reduction in fitness. Usually used in the context of microorganisms which cause clinical disease mice. Mandatory isolation of individual mice, cages, rooms, or facilities to reduce or prevent the spread of excluded agents. Incoming quarantine is the time spent upon arrival to a new area (ie, facility or room) and is a preventative measure. Outbreak quarantine is instigated when evidence of an excluded agent is detected in the facilities mouse colonies. Used to establish SPF mice. Usually performed via embryo transfer.102 Immune-competent mice that are housed with and received soiled bedding from the colony mice the sentinels monitor. These are most often housed in pairs in a separate cage from monitored mice; however, in certain situations, sentinels may be cohoused. Exclusion from an animal facility of a unique list of microbiota. Varies by institution and may vary by rooms within an institution Specific restricted pathogens should be listed when the term is used. Unless the absent pathogens are listed and actually tested for and excluded, the term is meaningless and may be problematic when users expect SPF animals to be much ‘‘cleaner’’ than they are. SPF animals have been defined as ‘‘animals free of the pathogens specified but otherwise have undefined flora.’’102 Monitoring of colony pathogen status to ensure that excluded agents are not present. Used most often in context of sentinel mice and rodent health monitoring programs.69

SPF, specific pathogen free.

their microbiota form a symbiotic relationship.32,113,118,119 Although often considered a confounding variable, naturally occurring alterations in microflora (such as infection with an opportunistic bacteria) have resulted in the generation

of novel model systems39 and characterization of new microbial agents.57 Although the bulk of this article primarily focuses on infections with a single viral or bacterial agent, an increasing body

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Table 2. Agents by Primary Disease Phenotype When There Are Pathology Findings or Clinical Disease in Micea Viruses ENTERIC–HEPATIC Calicivirus (MNV) Coronavirus (MHV) Rotavirus (EDIM)

Bacteria

Fungi and Larger (Eukaryota)

IST BHIST BPT

ENTERIC-HEPATIC Helicobacters Citrobacter rodentium (Escherichia coli)

ENTERIC-HEPATIC Giardia muris Spironucleus muris LI Flagellates (trichomonads; chilomastix, hexamastix, etc)

IST IST CT

BHIP

Clostridium piliforme Cl difficile, perfringens Salmonella enterica Typhimurium spp

Entamoeba muris Cryptosporidium muris Cryptosporidium parvum Eimeria species Aspiculuris tetraptera Syphacia obvelata, muris Cysticercus fasciolaris Rodentolepis diminuta Rodentolepis nana

CT HIPT HIPT HIPT IO? T IO? T O? T O? T O? T

HPZ?

RESPIRATORY Pasteurella pneumotropica Mycoplasma pulmonis, etc CAR bacillus Bordetella (hinzii, avium, etc) Klebsiella pneumonia, etc Corynebacterium kutscheri Enterococcus faecalis

COT HPT HPT CO HOP HOP CIO

RESPIRATORY Pneumocystis murina

IO?

SKIN (or abscesses) Staphylococcus aureus, etc Corynebacterium bovis

COP IO I CIO CIO HPZ CIO CIO

SKIN/HAIR Trichophyton mentagrophytes Myobia musculi Myocoptes musculinus Radfordia affinis Psorergates simplex Demodex musculi Ornithonyssus bacoti Laelaps echidnina Polyplax serrata

O OPT OPT OPT HO HO OZ? O O

SEPSIS Streptococcus pneumonia, etc Pseudomonas aeruginosa Streptobacillus moniliformis Proteus mirabilis Klebsiella oxytoca, etc, spp OTHER Mycoplasma (E) coccoides Chlamydia muridarum Mycobacterium avium, etc Segmented filamentous bacteria (SFB) Defined flora

H H H ? ?

OTHER Kazachstania (Torulopsis) Klossiella muris Sarcocystis muris Nonparasitic arthropods Psocids

? HP HP ? ?

COT

Reovirus 3 (Reo3)

RESPIRATORY Paramyxovirus (Sen, MPV) CNS PicoRNAvirus (TMEV, etc)

BHST

IMMUNE OR MULTISYSTEM Adenovirus (MAV1,2) Arterivirus (LDV) Ectromelia virus Herpesvirus (MTV, MCMV) Hantaviruses LCM virus Papova (K, MPyV) Parvoviruses (MPV > MMV)

BIST BST BHIPT BIT STZ BSTZ BST BST

OTHER VIRUS , GENE Retroviruses Retroelements

B

IPT CO?

B, biological materials are a source; C, commensal; H, historical significance; I, significant disease in immunodeficient; O, opportunist; P, pathogenic (usually) in immune sufficient; S, subclinical (usually) in immune sufficient; T, tested in surveillance panels; Z, zoonotic potential; ?, significance unknown. a Agents in boldface are estimated to be more prevalent, based on references 76 and 101.

of work recognizes the multifactorial nature of disease.11,22,65 GEM are often carriers of multiple infectious agents and have a unique enteric microbiome29,113 that may variably affect the mouse depending on the genetic manipulation. Infection with 1 virus or bacterial species may alter the outcome of infection with another virus or bacterial species.22,65,74 In this review, we provide a glossary of terms related to GEM, their microbes, and microbial status specifically (Table 1). Although most of these terms will be familiar to readers, certain concepts such as the different definitions of specific pathogen free between facilities, germ free, defined flora, and conventional mice are important to understand. The tables within this article (Table 1, glossary and definitions in the context of mice; Table 2, summary of agents by primary disease type) and in supplemental materials (Supplemental Tables 1, viral agents; 2, bacterial agents; and 3, eukaryotic agents) list additional important agents, and the reader is

referred to the references for further details (for supplemental materials, please visit http://vet.sagepub.com/supplemental).

General Concepts and Definitions Mice and other macroorganisms have broad and uncharacterized flora. Although mice can be maintained germ-free or with defined flora, most facilities will house mice with undefined flora. Many institutions exclude certain agents from their colonies and house mice under specific pathogen free (SPF) conditions usually with some sort of barrier housing. SPF mice are maintained free of excluded agents by rigorous testing of sentinel mice as part of the institution health monitoring programs (surveillance, discussed below).23,21,69 Each institution, and occasionally individual mouse rooms within an institution, will have a unique list of excluded agents. Most modern institutions

Treuting et al exclude the historically important and usually pathogenic agents, for example, mouse hepatitis virus (MHV), Sendai virus, Citrobacter rodentium, Clostridium piliforme, pinworms, and mites, among others. Selection of microbes to exclude is usually based on criteria such as zoonotic potential (ie, lymphocytic choriomeningitis virus, Hantavirus, Salmonella spp), pathogens that are fatal to most mice (ie, MHV, ectromelia virus), pathogens that are not fatal but cause overt disease (ie, Pasteurella spp), and opportunists (Pneumocystis murina, Staphylococcus spp).120 Infectious agents can damage cells, tissues, or organs directly by killing cells and indirectly by immune responses that damage cells or impair normal function (eg, inflammatory cell infiltrates in lungs that impede gas exchange or infiltrates or exudates that obstruct airways). Less obvious, but equally important, is the impact of infectious agents on physiological parameters including immune responses, cancer development,104 and the generation of context-specific phenotypes (ie, disappearing phenotypes, see below). The widespread sharing of GEM between laboratories and institutions increases the potential for alterations in the mouse microbiome as well as microbial contamination. When mice become unintentionally contaminated with an agent, many parameters may be altered, often in a nonuniform fashion. Even if 100% of the mice are eventually infected, it will have been at different ages, with different infecting doses and slight individual variations in host response, even in genetically uniform mice, resulting in the stochastic effects of an uncontrolled variable. There are reports of ‘‘disappearing phenotypes’’ once mice are rederived into more stringent SPF facilities.4 For example, Smad3-deficient mice maintained free of the gram-negative enterohepatic Helicobacter spp for up to 9 months did not develop colon cancer as initially reported. Subsequent infection of Smad3-deficient mice with Helicobacter restored the colon cancer phenotype.75 The SMAD-deficient Helicobacter model is now used to explore inflammationdriven colon cancer, highlighting the utility of the interactions between microflora and GEM as a research tool.

Sources of Infection Sources of infection may be other mice, fomites (eg, bedding and experimental equipment), human caretakers, feed, and water. Biological materials can serve as a source of infection. Cell lines, antibodies, conditioned media, and serum have all been implicated as potential source of contamination (discussed in detail in the section ‘‘Biological Materials’’). Autoclaving of cages, food, and water or irradiation of diets is performed to reduce the chance of contamination through husbandry and environmental sources, and these as well as barrier facilities are not infallible. Despite stringent barrier practices and protocols, clinical disease outbreaks and changes in the microflora that alter phenotypes will happen. Contemporary mouse colony surveillance programs test for a diversity of microbial agents. Many of the tested agents are not common or likely in today’s relatively clean research mouse colonies, but they can still be found in pet rodents, wild rodents, or biological materials and may be zoonotic. Surveillance

47 programs aim to detect these before they compromise a colony or project. Detailed discussion of all of the agents tested (>30 in some comprehensive screens) is beyond the scope of this review, but references and resources for 30þ viral, bacterial, protozoan, and metazoan agents are summarized here in table format. Surveillance programs vary in their level of scrutiny, that is, how many cages or animals are surveyed by a sentinel animal, how sentinels are exposed, how many tests are done, and how often. Academic mouse-based research occurs in diverse facility and institutional environments. Replication of results depends on details of housing and husbandry conditions in a particular research setting, information that is unfortunately lacking from many reports. This oversight is being addressed with the proposed Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines,59 which require more stringent reporting of animal care conditions. In pharmaceutical settings, environmental conditions tend to be more tightly controlled in accordance with Good Laboratory Practice requirements and anticipated scrutiny from the US Food and Drug Administration. The production of novel GEM is expanding exponentially with input from both enormous international initiatives (such as the Knock-out Mouse Project [KOMP] and others) and individual laboratories. As such, there is frequent sharing of these novel mice (and their microbiomes) across the globe. Importation of rodents into facilities is usually overseen by the rodent health monitoring or attending veterinarian as part of the institutional surveillance program.69 Steps to prevent outbreaks of excluded agents usually include review of the home institution’s health reports, quarantine, and possible prophylactic treatments (eg, medicated chow) upon receiving the mice. Quarantine of incoming rodents is a keystone of surveillance programs as an attempt to reduce contamination via newly introduced mice.105 With the global nature of research and the interinstitutional sharing of mice, contamination of transferred rodents during shipment is a reality. Exposure of transport boxes to wild rodents should be expected, and thorough decontamination and housing procedures are required to prevent contamination.

Prevalent and Emerging Infectious Agents and Phenotypes in GEM Detailed descriptions of infectious diseases in mice have been discussed at length in books and review papers,2,37,41,78,98 including clinical signs and impact on research.52 The agents we cover in detail were selected as those that in our collective experiences are increasingly diagnosed in GEM models. We will focus on specific examples of viruses (eg, mouse norovirus [MNV], mouse parvovirus [MPV], MHV, bacteria (eg, Helicobacter spp; Pasteurella pneumotropica; staphylococcal, enterococcal, and streptococcal species, Corynebacterium bovis; and Sphingomonas paucimobilis), and internal and external parasites and fungi (Pneumocystis murina and gastric yeast) for

48 which the effects may be extrapolated to variety of other specific agents.

Viral Agents Viral agents can cause overt clinical diseases and even death. However, subclinical infection is the norm and affects research primarily through alterations in the immune response. By far the most prevalent viruses identified in routine serological tests are the mouse MNVs (calicivirus), with 33% of externally sourced samples testing seropositive at a large rodent diagnostic laboratory,101 although higher incidences may be encountered in some local populations.14 Antibodies to MHV and MPV are detected in fewer serum samples, approximately 1%–2% each,14,101 but these viruses are present in a high percentage of academic institutions and can have significant effects on research.14,52 Mouse norovirus. Although there are many serotypes of MNV, by far the most studied is MNV1.48 Most mouse vendors are negative for MNV; thus, the virus is surviving and perpetuating within research institutions. There is evidence that MNVs may undergo homologous recombination events, creating viruses different from the parental strain.80 Serologic crossreaction occurs for most of the strains, but MNV-1 may be distinct. Infection with one strain may not provide crossprotection against other strains.89 Although most serotypes cause persistent infection, MNV-1 and perhaps others may be cleared within several weeks. In typical mouse strains without genetic modification, MNV-associated lesions have not been identified.121 The method of transmission is thought to be fecal–oral. As a nonenveloped virus, MNV has the potential to remain active in the environment. However, MNV is readily disinfected by chlorine or ozone in drinking water and, thus, is susceptible to inactivation by oxidizing disinfectants.28,66 MNV has not been associated with clinical disease in immunecompetent mice. However, oral inoculation with a cultureadapted strain of MNV-1 (107 plaque-forming units) into 129S6/SvEv inbred mice results in very slight and transient granulocytic infiltration of the lamina propria of the duodenum at 24 hours post infection (without apoptosis of enterocytes) as well as mild increases in extramedullary hematopoiesis and lymphoid hyperplasia in the spleen.90,121 MNV1 has tropism for macrophage and dendritic cells, which are essential mediators of the innate immune system.123 In dendritic cells, the cytoplasmic helicase protein MDA5 (melanomadifferentiation-associated gene 5) has been demonstrated to be important in cell sensing of the virus.83 Resistance to infection with MNV1 has been found to rely on the transcription factor STAT-1 and interferon (IFN)-ab receptors for resistance to infection. Thus, Stat1 and Ifra1 (IFNabR) mutant mice will develop clinical disease and death after infection with MNV1, characterized by severe pneumonia, vasculitis, meningoencephalitis, hepatitis/necrosis, peritonitis, and pleuritis.57,121 Although the innate immune system is essential to protect against lethal infection by MNV, the acquired immune system

Veterinary Pathology 49(1) is important in clearing MNV. MNV does not cause clinical disease in mice with deficits in the acquired immune system, such as Rag1- and Rag2-deficient mice; however, they fail to clear the virus, which results in prolonged infection121 (thus serving as virus reservoirs) that may affect immunology studies. In addition, coinfection of MNV with Helicobacter spp may accelerate the progression of Helicobacter-associated IBD in the Mdr1a-null mouse, despite the lack of clinical disease with MNV alone.65 MNV has been shown to increase atherosclerotic lesion size and macrophage number in a C57BL/6 model of diet-induced obesity.97 In addition, it has been demonstrated that BALB/c and C57BL/6 mice coinfected with MPV and MNV have prolonged shedding of MPV.25 In contrast to these studies in which a consequence of MNV infection was reported, no effect of MNV infection was found in immunological response to vaccinia or influenza A virus,49 in immune response to Friend retrovirus infection,1 or in body weight gain, adiposity, triglycerides, or glucose metabolism in a study of diet-induced obesity in C57BL6/J mice.96 The impacts of MNV on biomedical research are just beginning to be studied, and publications demonstrating effects on certain disease models and phenotypes will surely expand, suggesting that MNV will be added the excluded agent lists of numerous academic institutions. It should be noted, however, that these few publications on MNV already outnumber those for MPV, indicating not only that more study of MPV is needed but that the argument for exclusion of MNV from research facilities is at least as well-supported as for exclusion of MPV. Mouse hepatitis virus. MHV refers to myriad strains of an enveloped ss-RNA coronavirus with significant variation in cellular tropism, pathogenicity, and even cell receptor affinities. MHV remains one of the most common viruses of mice in contemporary research facilities.14,70,101 Common features shared among MHV strains include susceptibility to desiccation and detergent-inactivation (characteristics conferred by the lipid envelope) and a high degree of species specificity. All strains induce antibodies in immunocompetent mice that are sufficiently cross-reactive as to facilitate serological screening of mouse colonies although insufficient to prevent reinfection of mice with different MHV strains.5 Broadly speaking, MHV strains can be further classified into those strains in which infection of immunocompetent mice is limited to the intestinal tract (ie, enterotropic strains) and those strains that initially infect the respiratory tract but may then disseminate widely (ie, respiratory or polytropic strains), although strains with intermediate tropism also exist.50 Respiratory strains use CEACAM1 as the cell receptor. Mice such as the SJL strain with mutant CEACAM1 on their cell surface are highly resistant to respiratory strains of MHV, although they are fully susceptible to infection with enterotropic MHV.50 In addition to using different receptors, respiratory strains also differ from enterotropic strains in being more pathogenic and being shed in much smaller quantities.5 Perhaps because of decreased shedding, respiratory MHV appears to be much less prevalent than enterotropic MHV.50 However, cell-culture–adapted laboratory

Treuting et al strains of respiratory MHV such as JHM and A59 account for almost all of the MHV literature; relatively few studies have examined enterotropic field strains of MHV. Because respiratory MHV differs so much from enterotropic MHV, caution should be exercised in using the MHV literature to evaluate the potential for a given MHV outbreak to confound research results. As respiratory MHV is less common, readers are referred to an excellent review5 for a more complete discussion of that disease. In general, however, respiratory MHV causes foci of necrosis associated with syncytia and sometimes a mononuclear cell inflammatory response in many different tissues, including nasal epithelium, lung, liver (Figs. 1, 2), lymphoid organs, and sometimes the central nervous system.98 Enterotropic MHV infection rarely causes clinical disease in postweanling immunocompetent mice, but it is an important differential diagnosis for wasting disease in immunocompromised mice.22,48 However, even in immunocompromised mice it may not always disseminate outside of the intestinal tract.23,50 In suckling mice not protected by maternal antibodies, enterotropic MHV may cause diarrhea and mortality, thus the old acronym LIVIM (lethal intestinal virus of infant mice). The morbidity and mortality in infant mice probably result from slower cell turnover rates of the intestinal epithelium, with less ability than older mice to replace damaged cells. Grossly, infant mice may be dehydrated and the intestines filled with yellowish liquid and gas, changes similar to those described for group A rotavirus infection of infant mice.98 Microscopically, syncytia of apical epithelial cells are likely to be the only finding (Fig. 3), although syncytia may also be observed in mesenteric lymph nodes.5 Enterotropic MHV is much more contagious than respiratory MHV, being shed for longer periods of time and in greater amounts. Transgenic mice with immunological deficits may shed MHV for extended periods of up to 2 years, in the case of mice with T-cell deficiencies.108 A controlled study with enterotropic MHV-Y found that BALB/c mice shed infectious amounts of virus for longer (4 weeks) than C57BL/6 mice (2 weeks). B-cell–deficient mice shed virus for more than 3 months but had no histological lesions.23,24 In contrast, Tcell–deficient mice developed severe wasting disease after 2 weeks, requiring euthanasia at 1 month; MHV was still being shed in the feces at that time.24 Enterotropic MHV can be a copathogen with Helicobacter hepaticus.22 One study reported that gIFN-deficient mice with dual infections had less severe MHV-induced lesions during the first week (ie, acute phase) of infection but greater mortality and more severe lesions during a more chronic phase at 28 days post infection.22 Such studies of disease and transmission of infection in mice of different strains, different immune status, and different microbial health status emphasize that the typically subclinical enterotropic strains of MHV carry the potential to alter research outcomes. Parvoviruses. Parvoviruses are small-nonenveloped, ss-DNA viruses that remain among the most common exogenous viruses

49 in laboratory mice.14,70,101 Currently known parvoviruses of mice are divided into 2 groups, minute virus of mice (MVM) and MPV, the latter of which is subdivided into further serotypes, currently MPV1 through MPV5.27 Both MVM and MPV share a high degree of resistance to desiccation, heating, and solvents. In addition, natural infection of immunocompetent mice with any of the parvoviruses is clinically silent, as is natural infection of most immunodeficient mice.55 Disease was reported, however, in a line of mice with a B-lymphocyte maturational defect, NOD.h-2H4 homozygous for a targeted null mutation of immunoglobulin heavy chain, infected with a field strain of MVM, MVMm. In these mice, slow growth, deaths, and reduced fecundity were observed. Histologically, there was lymphohistiocytic inflammation in the kidneys and viral inclusions in splenic red pulp cells resembling hematopoietic progenitor cells.8,94 Experimentally, disease can be produced by intracerebral inoculation of suckling mice with the more commonly studied immunosuppressive (MVMi) or prototype (MVMp) culture-adapted strains.54 All parvoviruses of mice share several characteristics that make even subclinical, relatively nonpathogenic infections worrisome. First, like parvoviruses of other species, murine parvoviruses can only replicate in actively dividing cells. Because productive infection is cytolytic, there is concern as to interference of murine parvoviruses with oncology research or research in other areas in which active cell replication is an important feature, and another rodent parvovirus, H-1 parvovirus of rats, has been investigated as a potential anticancer agent.87 Murine parvoviruses cause further concern because they are lymphocytotropic. Concern regarding long-term influence on the immune system is amplified because both MVM and MPV are shed in the feces for long periods of time by immunodeficient or infant mice, and viral DNA can be found indefinitely in mesenteric lymph nodes and spleen. However, both MVM and MPV are very difficult to grow in culture, so most work has been done with culture-adapted strains that may behave differently than field strains. Two studies in the 1990s found altered immune function in mice experimentally infected with MPV1a, a culture-adapted strain discovered because of its effects on T-lymphocyte cultures.85,84 In these studies, BALB/ c mice were inoculated with 106 TCID intraperitoneally and 105 TCID oronasally. Infected mice had accelerated rejection of tumor allografts but decreased killing by T cells.84 Infected mice also had decreased lymphoproliferative responses in spleen and popliteal lymph node but increased proliferative responses in mesenteric lymph nodes. In a related study of BALB/c mice experimentally infected with MPV1a, infected mice had increased rejection of both allogeneic and syngeneic skin grafts, but the allogeneic-reactive T cells had decreased proliferative capacity.85 No subsequent reports have corroborated these findings with any culture adapted or field strain of MPV. At this time, no long-term immunological effects have been shown for natural infection of mice with any field strain of MVM. No alteration of tumor development or growth has been shown for any field strain of MVM or MPV. In the only study to document a consequence of infection with a field strain of

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Figure 1. Liver; mouse. Coronavirus infection. Multifocal to coalescing hepatic necrosis. Although in most modern mouse hepatitis virus infections there are few clinical signs or gross findings, necrotizing hepatitis is classically associated with infection in immunocompromised mice. In this example, there are multifocal depressed and centrally reddened foci surrounded by pale zones. Figure 2. Liver; mouse. Coronavirus infection. Histopathologically, multifocal necrosis and parenchymal collapse are evident with degeneration, necrosis, and virally induced syncytial cells (arrowhead). Hematoxylin and eosin (HE) staining. Figure 3. Small intestine; mouse. Coronavirus infection. Characteristic viral

Treuting et al MPV, the authors conducted gel electrophoresis on serum from BALB/c mice (n ¼ 3/time point) orally inoculated with 100 ID50 MPV1e or 1000 ID50 MPV5 from naturally infected mice. On day 7, the MPV1e infected mice had slightly decreased serum albumin, and on day 14 mice infected with either MPV1e or MPV5 had increased serum g-globulin. On day 28, serum a1, a2, and b globulins were increased, and albumin–globulin ratios were decreased in the MPV5 but not the MPV1e group. These changes were generally mild, and no significant changes in specific acute phase proteins were detected by ELISA.27 Because of the sparse evidence of research interference by MPV and the lack of overt disease or reproductive effects, the overall significance of MPV infection is unclear. Further complicating laboratory animal management decisions when MPV is detected in a facility is that even within MPV-positive vivaria, as few as 1% of cages may be infected if mice are housed in individually ventilated caging.73,72 Approaches to control of MVM or MPV within mouse research facilities have ranged from euthanasia of all mice with cleaning of the entire facility followed by disinfection with an oxidizing agent55 to more measured campaigns of testing and culling of infected cages or groups of cages while imposing careful biocontainment of suspect areas.73

Bacterial Agents Helicobacter spp, P. pneumotropica, and Staphylococcus aureus are the most prevalent bacterial agents isolated in laboratory mice.14,101,116 Our personal experiences reflect this, and in addition we have noted increases in isolation of Staphylococcus xylosus, Enterococcus spp, and opportunistic agents such as S. paucimobilis (usually found in soils and water51) with the explosive use of novel and usually immunodeficient (or immunovague) GEM at our collective institutions. Helicobacter spp. Disruptions in either the innate or the acquired immune system may result in Helicobacter-associated disease. Nine species of Helicobacter have been formally identified in mice, of which H. hepaticus, infecting the liver, biliary tree, colon and cecum, and H. bilis, infecting the liver, cecum, colon, and biliary tree, have gained the most attention.39 In susceptible mice (see below and references 39 and 108) the common enterohepatic Helicobacter clinical signs include weight loss, diarrhea (manifesting commonly as sticky stools), and rectal prolapse. Grossly, opaque and thickened large bowel (Fig. 4) is present in mice with proliferative typhlocolitis. Gross liver lesions are variable depending on disease chronicity and

51 host genotype. Similarly, the histological manifestations of typhlocolitis (Figs. 5, 6) and hepatitis (Fig. 7) depend on the infecting Helicobacter species and mouse genotype. Helicobacter spp have been identified worldwide in both academic and commercial mice and are not excluded from most mouse barrier facilities. Helicobacter spp have been identified as a cause for typhlocolitis (In some cases termed inflammatory bowel disease—IBD) and hepatitis as well as carcinomas in the intestine, liver, and other sites.33,38,39,92,103,111,116,122 Certain strains of immunomodified mice are most susceptible as well as spontaneously immunodeficient mice such as Rag1/Rag2 and Pkrdcscid mice. Helicobacter spp elaborate toxins, such as a cytolethal distending toxin (CDT).43 The Helicobacter CDT has been demonstrated to induce dsDNA breakage and induce apoptosis, which may be important in mediating its carcinogenic effects.43,71 The immune response to Helicobacter spp is initiated by the innate immune system, for which TLRs, NF-kb, and MyD88 signaling appears to play an important role.35,79 Th1 and Th17 responses have been identified as important in the development of colitis. Th17 cells are essential in promoting inflammation and host defenses against microbial pathogens, whereas suppression of inflammation is mediated by CD24þCD25þ regulatory T-cells.117 Th17 cells are activated by interleukin (IL)-23 and produce numerous cytokines, including IL-17, IL-17f, IL-6, IFNg, and tumor necrosis factor alpha (TNFa).9,46 Studies have suggested that IL-23, IL-17, and IFNg may play an important role in the development of Helicobacter spp colitis.61 Many genetically engineered immune modified mice are susceptible to Helicobacter-induced typhlocolitis (comprehensively reviewed in reference 39; also see tables). Critically, a background strain of mice may have a significant impact on the interaction of Helicobacter with the targeted mutation; for example, Il-10–deficient mice had more severe disease when on a 129 background compared to severity on a C57BL/6 background.6 P. pneumotropica. P. pneumotropica typically considered an opportunistic pathogen and is highly prevalent in mouse research colonies and wild mice. Transmission occurs through direct contact, likely fomites and in utero.98 Lesions in susceptible mice include pulmonary (Fig. 8) and other soft tissue abscesses, pneumonia (Fig. 9), abscessation of retrobulbar tissues, rhinitis, sinusitis, conjunctivitis, blepharitis, and otitis (Fig. 10) and should be suspected in any purulent processes in susceptible mice. Outbreaks of P. pneumotropica have been

Figure 3. (continued) syncytial cells (arrowheads) on the villi. HE staining. Figure 4. Large intestine; C57BL/6-background immunodeficient GEM. The cecum (arrowhead) and colon (C) are severely thickened and opaque. Note the lack of formed feces in the distal colon. Figure 5. Proximal colon, immunodeficient mouse infected with Helicobacter species. Histopathologically, the thickened intestine has elongated crypts with reduced goblet cells, numerous mitotic figures, dense chronic inflammatory infiltrates, and dilated crypts filled with inflammatory debris (microabscesses). There is early mild herniation of irregular crypts through the very thin submucosa (arrowhead). With more severe herniation, care must be taken to distinguish from invasion. HE. Figure 6. Anorectal junction, prolapsed rectum; mouse. Exposure of the delicate colorectal mucosa (arrowheads) to the cage environment (E) provides an entry source for bacteria into the systemic circulation. The squamous mucosa of the anus (A) is indicated. HE.

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Figure 7. Liver. Male genetically engineered mouse (GEM) infected with Helicobacter species. Chronic Helicobacter infection with marked hypertrophy and hyperplasia of intrasinusoidal cells (Kupffer, Ito, and oval cells) and hepatocyte karyo- and cytomegaly with occasional binucleated cells and intranuclear cytoplasmic invaginations (arrowhead), with variable lymphocytic and plasmacytic inflammation. Hematoxylin and eosin (HE). Figure 8. Lung; Prkdcscid. Severe cranioventral bronchopneumonia and abscessation caused by Pasteurella pneumotropica infection. Figure 9. Lung; Prkdcscid (mouse pictured in Fig. 8). A large abscess and dense inflammatory cells consolidate the lung. HE. Figure 10. Outer, middle, and inner ear; Foxn1nu (nude) mouse. There is severe neutrophilic otitis media (M) caused by P. pneumotropica. The inflammatory process often spreads to regional tissues and is a major cause of vestibular syndrome in GEM. Enterococcus, Bordetella, and Streptococcus spp have also

Treuting et al reported in numerous GEM and spontaneous immunodeficient mice (reviewed in reference 41). Staphylococcus spp. Although staphylococci are present on the skin and mucous membranes of most animals, most do not develop disease. Humans are a natural reservoir of S. aureus, and up to 20% of people may be persistently colonized.53 S. xylosus may be found as an environmental contaminant, and thus elimination of staphylococci from mouse colonies is exceedingly difficult. S. aureus and S. xylosus are gram-positive bacteria that have been associated with spontaneous disease in immune-modified mice. Superficial suppurative lesions including dermatitis (Fig. 11), blepharitis (Fig. 12), conjunctivitis, otitis, sinusitis, and pneumonia45 (unpublished observation, P.M.T.) and a necrotizing dermatitis (Fig. 11) are frequently noted.98 Abscesses caused by S. aureus are sometimes termed botryomycotic granulomas, because of aggregates of bacteria that appear similar to Actinomyces spp sulfur granules, and often have deposition of Splendore-Hoeppli material at the center of these highly suppurative abscesses (Figs. 13,14).56 Most S. aureus lesions are thought to be opportunistic and secondary to trauma. For example, penetration of hair fragments into the periodontal tissues occurs frequently in mice, especially those prone to excessive grooming that introduces Staphylococcus and other oropharyngeal flora deep in the soft tissues (Fig. 15). Superficial excoriation of the skin, for example, through cage mate aggression or trichotillomania (excessive grooming) seen in some strains (eg, C57BL/6) or disruption of the corneal epithelial barrier,60 can be a predisposing factors in colonized mice. However, in immunodeficient mice (Figs. 16,17), such as those with defects in NADPH oxidase and severe combined immune deficiency (SCID), severe pneumonia and spontaneous abscessation, particularly of the face, have been reported45 (unpublished data, 2009-2011, P.M.T., R.S.S.) NOD/LtSz-scid Il2g–null mice can develop facial abscesses as early as 7–9 weeks of age (unpublished data, 2009-2011, R.S.). Although S. aureus abscesses are apparently common in SCID mice in the research setting, reports of this disease are limited.18,56,98 Staphylococci have a number of ways to avoid immune defenses. Some of the organisms may be resistant to eradication in neutrophils through charge neutralization of their anionic cell surfaces.20 Expressions of a number of virulence factors as well as the host response to the organism play a role in the disease manifestation. In particular, the characteristic necrotizing dermatitis (Fig. 11) with superficial erosions to ulcerations and deep coagulative necrosis (burn like lesions) is due to the production of numerous proteins including exotoxins, hemolysins, proteases, collagenases, exfoliative toxins, and superantigens (enterotoxin A-C and toxic shock syndrome toxin 1) among others.98

53 C. bovis. C. bovis is a gram-positive coryneform that has been associated with skin disease in mice with T-cell (Foxn1–/–, athymic nude) and T- and B-cell (Pkrdcscid) immune deficits as well as in hairless (SKH1-Hrhr) mice with essentially normal immune function.10,19,110 Clinical disease occurs with variable morbidity and low mortality approximately 7–10 days after infection. Gross lesions are hyperkeratosis and dehydration (shriveled appearance), which persist for approximately 7 days (Figs. 18, 19). Affected Pkrdcscid mice may also appear to have a slightly puffy appearance to the facial skin. Histological changes include orthokeratotic hyperkeratosis and widespread or diffuse acanthosis that persists after hyperkeratosis is no longer grossly visible.19,110 A mild dermal mononuclear cell infiltration often accompanies the epidermal proliferation. Gram-positive coryneforms are usually readily detected in the stratum corneum and within the necks of hair follicles (Fig. 20). Infection persists indefinitely, perhaps lifelong, in the immunodeficient mice mentioned above. Immunocompetent hirsute mice, such as ICR stocks, have been reported to be transiently infected but perhaps eliminate the infection with time. Increased mortality among newborn athymic nude mice or in mice after treatment with chemotherapeutic agents has been reported.19 Control is difficult as the organism may be widespread within contaminated facilities, including being detectable on cage surfaces, within hoods, and even on doorknobs and gloves and in tumor lines.10,19,110 The mechanism by which C. bovis induces the epidermal proliferation is unknown, as is the mechanism by which the clinical signs are eventually diminished. However, whatever the mechanism, whether by defensins or other innate immune elements, mice deficient in those host defense components are likely to have increased disease. Streptococcus and Enterococcus spp. Similar to the organisms described above, these organisms may infect and cause disease in GEM. More often seen in immunodeficient mice such as the Myd88 mutant, the organisms are increasingly cultured from mice with clinical disease (eg, abscesses, meningitis, pyelonephritis, disseminated infections, and bacteremia) (unpublished data, 2008, P.M.T.).98 Historically, the pathogenic species use various proteins similar to Staphylococcus spp to impart virulence; however, in the immunodeficient mice the nonpathogenic a-hemolytic Streptococci are increasingly implicated. Typically, in the Myd88 mutant mice there is little to no host inflammatory response to a myriad of bacterial colonies that in severe cases efface tissue architecture (Supplementary Figs. S1–S3).

Parasites External parasites such as mites (fur mites: Myobia musculi, Myocoptes musculinus; follicular mite: Demodex musculi; free

Figure 10. (continued) been isolated. External ear canal (E) and inner ear (I) are indicated. HE. Figure 11. Skin; C57BL/6. There is acute coagulative necrotizing dermatitis with superficial erosions to ulcerations with adherent serocellular crust that often contain obvious bacterial colonies (Staphylococcus spp) (arrow). Deep dermal lesions include dense chronic active inflammation and fibrosis that may heal as scars. HE.

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Figure 12. C57BL/6-background–immunodeficient genetically engineered mouse (GEM). There is focally extensive severe ventrolateral facial abscessation with conjunctivitis (arrow). Regional structures such as exorbital lacrimal, parotid, sublingual, and Harderian glands as well as cervical lymph nodes, facial musculature, and cranial bones are frequently involved. Figure 13. Cross-section of head; mouse (immunodeficient). Ventral periorbital architecture is effaced by a severe suppurative inflammation with numerous centralized eosinophilic

Treuting et al living rat mite: Ornithonyssus bacoti) and lice (Polyplax serrata, the vector of Mycoplasma cocoides) are excluded from modern institutions. Outbreaks of fur mites still commonly occur, and some incoming rodent quarantines may automatically treat all mice for external and internal parasites as a result.88,107 The principle impact mites have on GEM-based research is transfer of mice between institutions, causing pruritus, self-trauma, and secondary bacterial infection (see S. aureus section above), and may be a predisposing factor for the idiopathic dermatitis of C57BL/6 substrains.2,98 Infection is via direct transfer and diagnosis is made by pelage examination, tape testing, and polymerase chain reaction (PCR). Internal parasites (Figs. 21–24) include protozoa (Spironucleus muris, Tritrichomonas muris, Trichomonas minuta and wenyoni, Octomitus pulcher, Chilomastix bettencourt, Entamoeba muris, Cryptosporidium muris and parvum, Giardia muris, Klossiella muris, and several species of Eimeria among others), microsporidia (Encephalitozoon cuniculi, extremely rare), and helminths (Oxyurids: Syphacia obvelata, Aspiculuris tetraptera; adult and larval cestodes).98 Helminths are excluded in facilities; however, as with mites, outbreaks of pinworms (oxyuriasis) are frequent enough to warrant automatic treatment of incoming rodents in many institutions because of the persistence of the eggs in the environment and the ease of transmission. Pinworms are relatively innocuous in most mice, with few reports of altered susceptibilities of GEM to parasite burden or infection with S. muris, the rat pinworm41; however, they are a top differential diagnosis for rectal prolapse. Infection with helminths has experimentally been shown to alter immunophenotype,64,47,112 and undetected natural infections could affect immunological studies.52 Transmission is fecal–oral, and eggs are often aerosolized.52 Diagnosis is via cecal or fecal examination, perineal tape tests, and PCR. The intestinal protozoa, G. muris, Eimeria, and Cryptosporidium, are extremely rare in modern facilities. However, in our experience, blooms of intestinal flagellates are apparent in histological sections of gastrointestinal tracts of immunodeficient mice (Fig. 21). The impact that these blooms may have on the host is unclear, and most often there is no observable host response or tissue damage; rarely the protozoa appear to be adherent and possibly invading ulcerated intestinal mucosa, although this has yet to be fully characterized (P.M.T., unpublished data, 2009). S. muris may have clinical manifestations in young and immunosuppressed mice.98,99 These various protozoa are definitively diagnosed by fecal wet mounts36; on tissue section they are challenging to distinguish with the

55 exception of relative size and locations within the gastrointestinal tract (Figs. 21–24), although they are readily observable.99 The relatively large trichomonads usually are found in the colon and cecum (Fig. 23), whereas the small spironucleus trophozoites are usually found the small intestinal lumen or crypts of Lieberkuhn (Fig. 22). P. murina is an opportunistic fungus transmitted by inhalation of infectious cysts that is adapted to live in the lungs of mice, even normal, immune-competent hosts.16 Overt disease, however, usually manifests with immune suppression, either by genotype or drugs, and viral infections.2 Clinical signs in immunosuppressed hosts include nonspecific sick rodent signs of weight loss and unkempt pelage and respiratory signs of dyspnea, less obvious cyanosis eventually leading to death.98 The lungs at necropsy are characteristically pale and fail to completely collapse; they have a rubbery appearance (Fig. 25). Interstitial pneumonitis with the presence of eosinophilic, foamy to honeycombed material within the alveoli is rather diagnostic (Figs. 26a) but may be challenging to identify in the face of concurrent bacterial or acidophilic macrophage pneumonias that often accompany pneumocystosis. This material contains viable and dead organisms and surfactant.17,98 Diagnosis in animal with clinical disease is possible using an impression smear at necropsy. The organisms can be visualized on a Wright’s stain, Romanowsky stain, or Grocott’s methenamine silver stain (GMS) of the impression smear. Histological staining of the 3- to 5-mm cysts with methenamine silver (Fig. 26b) or periodic acid–Schiff aids the diagnosis. Subclinical infections and confirmation of histological findings can be made with PCR. Infections with opportunistic fungi such as Paecilomyces spp and Aspergillus fumigatus, among others, are reported in mice with defects in NADPH oxidase developing pyogranulomata in multiple organs.40,44 Infections of the upper digestive (esophageal or gastric) (Figs. 27–29) have been reported98 and are infrequently diagnosed in our collective experience. However, bedding, feed, and possibly water may serve as sources of contamination82 (see ‘‘Environmental Sources and Emerging Agents’’ below).

Biological Materials In addition to the risks for disease introduction posed by incoming mice and husbandry materials, many research facilities import a wide range of cell culture lines or biologically derived

Figure 13. (continued) aggregates, Splendore-Hoeppli material (arrow) (Staphylococcal botryomycosis). Hematoxylin and eosin (HE). Figure 14. S. botryomycosis. Mouse (immunodeficient). Splendore-Hoeppli material is characterized morphologically by eosinophilic radiating-to-club-shaped hyaline material surrounding dense colonies of cocci (arrowhead). Figure 15. Lower jaw; mouse (immunodeficient ). Impaction and penetration of hair fragments (arrow) into the periodontal tissues may introduce staphylococcal and other oropharyngeal flora into the deep soft tissues. HE. Figure 16. Lung; C57BL/6-background mouse with NADPH oxidase defect (B6p47phox). There is widespread severe pneumonia (arrow, left lung most severe) and a markedly enlarged heart. The diffuse pale yellow-tan color and rubbery consistency are suggestive of acidophilic macrophage pneumonia (AMP) (confirmed histologically; see Fig. 17). AMP is a common lesion in mice with NADPH-system defects coupled with bacterial infection, particularly staphylococci. Figure 17. Lung; B6p47phox (mouse pictured in Fig. 16). A focal pulmonary abscess contains sulfur-like granules (arrow), and the adjacent lung is consolidated by severe acidophilic macrophage pneumonia characterized by accumulations of macrophages laden with acicular eosinophilic crystals, admixed with lymphohistiocytic inflammation and extracellular crystals. HE.

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Figure 18. Skin, Prkdcscid mouse. The mouse is infected with Corynebacterium bovis. Note the flakes of skin accumulating on the pinnae and rough hair coat (shriveled appearance). Figure 19. Skin; Foxn1nu (nude) mouse. The mouse is infected with C. bovis. Hyperkeratosis is noted grossly as the multifocal to coalescing yellow flakes distributed primarily on the dorsum in this individual. Figure 20. Skin; Prkdcscid mouse. Gram-positive coryneforms are readily detected in the stratum corneum. Tissue Gram staining. Figure 21. Colon; Mouse, immunodeficient. There are large numbers of fusiform to crescent-shaped trophozoites within the lumen with no histological host response. The size and location are typical of

Treuting et al reagents such as antibodies, conditioned media, and serum. These materials, loosely termed cell lines and biologicals, may be contaminated with viruses or Mycoplasma spp93,100 and should be considered as additional sources of risk whenever mice are exposed to them or to materials that have come into contact with them. Such biological materials, whether cell lines or cell-free reagents, may be contaminated with agents indigenous to the species from which they are derived, such as human viruses in human cell lines or mouse viruses in mouse cell lines or serum.67,68 They may also be contaminated by agents of other host species. For example, antibodies produced in rabbits may be contaminated with mouse viruses if the rabbit serum is purified on a column previously used for mouse serum. Similarly, human cell lines may be contaminated by mouse viruses if cultured on a feeder layer of mouse fibroblasts or if cultured with mouse serum or other biological material, for example, mouse-derived basement membrane matrix. Important considerations in assessing the level of risk posed by cell lines and biologicals may include whether they have been stored in a freezer from a time when adventitious infections were more prevalent95 and whether the material has been previously tested. One should also recognize that at this time, the American Type Culture Collection (ATCC) does not routinely test cell cultures for rodent viruses. Mycoplasma spp are the most commonly detected infectious agents in cell lines and biologicals.15,34 Fortunately, it is rare to find any Mycoplasma capable of infecting mice and, specifically, Mycoplasma pulmonis has never been reported in a cell line. In fact, cell lines contaminated with nonmouse Mycoplasma spp may be decontaminated by passaging them through mice.109 Recently, however, Mycoplasma arginini has been reported as a contaminant of cell lines and caused suppurative arthritis in Prkdcscid mice inoculated with the cells.100,115 Infection risks for humans are appropriately a major concern. Testing of human cell lines for human-origin viruses at Charles River (unpublished data, Feb 2002 to April 2010) has detected hepatitis B, not surprisingly in hepatocarcinoma lines, as well as human papillomavirus-18. Zoonotic agents detected in rodent cell lines include lymphocytic choriomeningitis virus (LCMV), which has been detected in MaTu cells (known to carry LCMV) and in baby hamster kidney (BHK) cells. Parvoviruses are among the most commonly detected endogenous rodent viruses in cell lines and biologicals, and they carry the potential to spread beyond the inoculated mice and contaminate an entire vivarium. Approximately 0.4% of cell lines and a similar percentage of other biologicals have been found to have parvoviruses, most often MPV and/or MVM. Note that any percentage prevalence cited in this article is not intended to imply a uniform risk across all cell lines or to establish an actual contamination rate for all cell lines and

57 biologicals. However, finding a contamination in 1 out of every few hundred samples does establish that a risk of disease transmission exists and that if an institution imports large numbers of cell lines and other biologicals for use within a vivarium, the cumulative risk can be substantial.63,67 The other common virus detected in cell lines and biologicals is lactate dehydrogenase elevating virus (LDV, LDHeV).13 Of particular note with LDV is that this virus has not been routinely included in screening panels in the past. An enveloped arterivirus, LDV is primarily transmitted in the laboratory setting through infected tumors, cell lines, and other mousederived biological materials. Natural transmission usually occurs through bite wounds or sexual contact. This virus may also be transmitted transplacentally or via the milk. These latter 2 routes of infection are only clinically important if the infected animal is in the first week of infection, when viral shedding may occur via those routes. Animals remain persistently viremic after infection, but infection is unlikely to spread within most animal facilities. LDV infects macrophages and results in an increase in lactate dehydrogenase (LDH) within approximately 24 hours after infection. This increase in serum LDH persists and may be used as a screening tool for LDV infection, although LDH may also be elevated for many other reasons, including hemolysis. As viremia is persistent, PCR is often used on serum to confirm LDV infection in samples with elevated LDH. Recently, serology for LDV has been offered by rodent diagnostic laboratories. LDV infection may depress cellular immunity, increase cytokine activity, alter humoral immunity, reduce tumor growth, and alter immunity to copathogens, and cause hypergammaglobulinemia and autoantibody development.3,98 In addition, some strains develop glomerulonephritis and/or polioencephalomyelitis. Polioencephalomyelitis, which may progress to paralysis and death, occurs in susceptible strains of mice, including AKR and C58, if immunosuppressed and coinfected with endogenous n-tropic mouse leukemia virus (MuLV). However, in a recent nationwide outbreak of LDV associated with a commercial biologically derived basement membrane matrix material, paralysis associated with LDV infection was also observed in Prkdcscid mice in which the MuLV status was not determined.13 Occasional multicenter outbreaks of ectromelia, an orthopoxvirus, have been associated with use of commercial mouse serum in animal facilities.63,68 Although an enveloped virus, ectromelia may spread widely via fomites or infected animals throughout a vivarium, and it is shed in feces, in respiratory secretions, and from the skin. Resistant strains, such as C57BL/6, C57BL/10, and AKR, may show no clinical signs but serve as a source of virus for the infection of other animals. In contrast, in susceptible strains such as A, CBA, C3H, BALB/c, and DBA/2, mortality may exceed 80%.77 This mortality may

Figure 21. (continued) trichomonads, unlike the smaller Spironucleus organisms (see Fig. 29) and the primarily small intestinal Giardia spp. Hematoxylin and eosin (HE). Figure 22. Colon; mouse. Abundant Spironucleus muris trophozoites are present deep within intestinal crypts. Slide courtesy of Dr. J. M. Ward. HE. Figure 23. Colon; mouse. Entamoeba muris trophozoites (arrowhead) and cysts (arrow). Slide courtesy Dr. J. M. Ward. HE. Figure 24. Proximal colon; mouse. Numerous cross-sections of oxyurids are present with characteristic lateral alae and platymyarian musculature.

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Figure 25. Lung; C57BL/6-Rag2tm1Cgn/J. Note the failure of the lungs to collapse and the rubbery appearance with multifocal pale foci. Figure 26. Lung; mouse. (a) Pulmonary histopathological lesions of established cases of Pneumocystis murina have characteristic intraalveolar foamy to honeycombed material coupled with chronic pneumonitis. Inset; higher magnification. Hematoxylin and eosin (HE). (b) Silver stains are useful to highlight P. murina cysts, which may be present in low numbers in less advanced cases. Inset; higher magnification. GMS-sliver staining modified for P. murina. Figure 27. Nonglandular stomach; mouse. There are numerous fungal hyphae within the mucosal epithelium admixed with inflammatory cells. Figure 28. Glandular stomach; mouse. Numerous gastric yeast are evident along the superficial mucosa and stain bright blue. HE. Figure 29. Glandular stomach, mouse (higher magnification of Fig. 36). Small ovoid yeast stain darkly blue with occasional pink internal dot-like structures. These organisms stain intensely with periodic acid–Schiff. HE.

Treuting et al occur with no other clinical signs, and animals often die before they shed virus. Susceptible mice have acute hepatocellular necrosis as well as necrosis of the spleen, Peyer’s patches, thymus, and lymph nodes. Hepatocellular necrosis may be seen as white spots on the liver. Clinical signs in strains of intermediate susceptibility may include ruffled fur, hunched posture, facial edema, swelling of the limbs, conjunctivitis, cutaneous pustules, ulceration of the muzzle, limbs, ears, and tail, and the lesion that gives the virus its name, ectromelia, or partial amputation of the limbs and tail. Other viruses that have been detected in cell lines and biologicals include reovirus, polyomavirus (primarily in MethA and LLC cells), and cytomegalovirus. The presence of these uncommon viruses should serve as a reminder that although relatively few viruses appear to be circulating in contemporary populations of laboratory mice, cell lines and biologicals pose some risk for introducing other agents considered rare in modern facilities and for which testing is infrequently conducted.

Environmental Sources and Emerging Agents Historically, watering systems have been associated with the ubiquitous Pseudomonas aeruginosa or Proteus mirabilis, and acidification or chlorination of systems is used to control but not eliminate growth. With the increased use of complex and severely immunodeficient mice, biofilm-related microflora within watering systems should be considered as a likely source of emerging agents for GEM.7,26,86 With the concept that all GEM are of unknown immune status, pathologists must be alert for changes in phenotype or microflora in tissue sections that may indicate introduction of new environmental opportunistic pathogens. Recent examples from the University of Washington (UW) (P.M.T.) include a case of disseminated infection with S. paucimobilis, a gram-negative bacteria normally associated with water and soils and reported infrequently in human medical literature as a cause of nosocomial infections (abscesses, bacteremia) in immunocompromised patients.30,42,51,58 We have cultured S. paucimobilis from case of severe necrosuppurative peritonitis in a C57BL/ 6Rag2tm1Cgn/J–background GEM (Figs. S4–S6). Bulk water cultures obtained from the automatic watering unit have detected S. paucimobilis, Brevundimonas vesicularis, Pseudomonas alcaligenes, Actinomycetes, Corynebacterium genitalium, Empedobacter brevis, and other unidentified bacteria and fungus. Additionally, gastric yeast morphologically similar to Kazachstania pintolopesii62 was noted in stomach sections from numerous immunodeficient mice submitted to the UW diagnostic laboratory due to an investigator-observed increase in rectal prolapse within the colony (Figs. 28, 29). These particular mice had been housed in a Helicobacter spp–positive room with little notable clinical disease for a number of years. The gastric yeast has previously not been diagnosed in mice from the UW colonies. Although it is tempting to speculate that there are possible novel interactions between the host genotype and recently altered microflora in these anecdotal examples, the precise relationship between these potential pathogens requires

59 further study to formally implicate these agents in altered phenotypes.41 Fulfilling Koch’s postulates for newly recognized agents would be the next logical step in determining causation.

Conclusions The complexity of the interactions between the microbiota (including pathogens) is increasingly recognized for its role in the development of immune competence and disease pathogenesis.32,113,118,119 Although unrecognized infectious may result in confounding research outcomes, they may also result in new models of disease. Pathologists working with mouse models must be vigilant in unexpected changes in phenotypes that may relate to changes in pathogen status or microbiota and relate these findings to the mouse genotype as part of the process of mouse model validation.12 The combination of mouse genetics, environmental factors (including infectious agents and ‘‘normal flora’’—collectively the microbiome) must all be included in the evaluation of phenotype expression in GEM. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

References 1. Ammann CG, Messer RJ, Varvel K, et al. Effects of acute and chronic murine norovirus infections on immune responses and recovery from Friend retrovirus infection. J Virol. 2009; 83: 13037–13041. 2. Baker DG. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin Microbiol Rev. 1998; 11: 231–266. 3. Baker DG. Natural Pathogens of Laboratory Animals. Washington, DC: ASM Press; 2003. 4. Barthold SW. ‘‘Muromics’’: genomics from the perspective of the laboratory mouse. Comp Med. 2002; 52: 206–223. 5. Barthold SW, Smith A. Mouse hepatitis virus. In: Fox JG BS, Davisson MT, Newcomer CE, eds. Mouse Hepatitis Virus. Burlington: Academic Press; 2007: 141–178. 6. Berg DJ, Davidson N, Kuhn R, et al. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(þ) Th1-like responses. J Clin Invest. 1996; 98: 1010–1020. 7. Berry D, Xi C, Raskin L. Microbial ecology of drinking water distribution systems. Curr Opin Biotechnol. 2006; 17: 297–302. 8. Besselsen DG, Romero MJ, Wagner AM, et al. Identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice. J Gen Virol. 2006; 87(pt 6): 1543–1556. 9. Buonocore S, Ahern PP, Uhlig HH, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature. 2010; 464: 1371–1375.

60 10. Burr HN, Lipman NS, White JR, et al. Strategies to prevent, treat, and provoke Corynebacterium-associated hyperkeratosis in athymic nude mice. J Am Assoc Lab Anim Sci. 2011; 50: 378–388. 11. Cadwell K, Patel KK, Maloney NS, et al. Virus-plussusceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell. 2010; 141: 1135–1145. 12. Cardiff RD, Rosner A, Hogarth MA, et al. Validation: the new challenge for pathology. Toxicol Pathol. 2004; 32(suppl 1): 31–39. 13. Carlson J, Garg R, Compton SR, et al. Poliomyelitis in SCID mice following injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus. JAALAS. 2008; 47: PS35. 14. Carty AJ. Opportunistic infections of mice and rats: Jacoby and Lindsey revisited. ILAR J. 2008; 49: 272–276. 15. Caviness GF, Thigpen JE, Locklear J, et al. Incidence of mycoplasma contaminants in cell cultures: detection and growth on trypticase soy agar containing 5% sheep blood. J Am Assoc Lab Anim Sci. 2008; 47: 106–106. 16. Chabe M, Aliouat-Denis CM, Delhaes L, et al. Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species. FEMS Yeast Res. 2011; 11: 2–17. 17. Chen W, Mills JW, Harmsen AG. Development and resolution of Pneumocystis carinii pneumonia in severe combined immunodeficient mice: a morphological study of host inflammatory responses. Int J Exp Pathol. 1992; 73: 709–720. 18. Clarke MC, Taylor RJ, Hall GA, et al. The occurrence in mice of facial and mandibular abscesses associated with Staphylococcus aureus. Lab Anim. 1978; 12: 121–123. 19. Clifford CB, Walton BJ, Reed TH, et al. Hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology. Lab Anim Sci. 1995; 45: 131–139. 20. Collins LV, Kristian SA, Weidenmaier C, et al. Staphylococcus aureus strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice. J Infect Dis. 2002; 186: 214–219. 21. Compton SR, Riley LK. Detection of infectious agents in laboratory rodents: traditional and molecular techniques. Comp Med. 2001; 51: 113–119. 22. Compton SR, Ball-Goodrich LJ, Zeiss CJ, et al. Pathogenesis of mouse hepatitis virus infection in gamma interferon-deficient mice is modulated by co-infection with Helicobacter hepaticus. Comp Med. 2003; 53: 197–206. 23. Compton SR, Ball-Goodrich LJ, Johnson LK, et al. Pathogenesis of enterotropic mouse hepatitis virus in immunocompetent and immunodeficient mice. Comp Med. 2004; 54: 681–689. 24. Compton SR, Ball-Goodrich LJ, Paturzo FX, et al. Transmission of enterotropic mouse hepatitis virus from immunocompetent and immunodeficient mice. Comp Med. 2004; 54: 29–35. 25. Compton SR, Paturzo FX, Macy JD. Effect of murine norovirus infection on mouse parvovirus infection. J Am Assoc Lab Anim Sci. 2010; 49: 11–21. 26. Crane LR, Tagle LC, Palutke WA. Outbreak of Pseudomonas paucimobilis in an intensive care facility. JAMA. 1981; 246: 985–987.

Veterinary Pathology 49(1) 27. Cray C, Besselsen DG, Hart JL, et al. Quantitation of acute phase proteins and protein electrophoresis in monitoring the acute inflammatory process in experimentally and naturally infected mice. Comp Med. 2010; 60: 263–271. 28. Cromeans TL, Kahler AM, Hill VR. Inactivation of adenoviruses, enteroviruses, and murine norovirus in water by free chlorine and monochloramine. Appl Environ Microbiol. 2010; 76: 1028–1033. 29. Deloris Alexander A, Orcutt R, Henry J, et al. Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment. Mamm Genome. 2006; 17: 1093–1104. 30. Dervisoglu E, Meric M, Kalender B, et al. Sphingomonas paucimobilis peritonitis: a case report and literature review. Perit Dial Int. 2008; 28: 547–550. 31. Dewhirst FE, Chien CC, Paster BJ, et al. Phylogeny of the defined murine microbiota: altered Schaedler flora. Appl Environ Microbiol. 1999; 65: 3287–3292. 32. Donohoe Dallas R, Garge N, Zhang X, et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab. 2011; 13: 517–526. 33. Donovan JC, Mayo JG, Rice JM, et al. Helicobacter-associated hepatitis of mice. Lab Anim Sci. 1993; 43: 403. 34. Drexler HG, Uphoff CC, Dirks WG, et al. Mix-ups and mycoplasma: the enemies within. Leuk Res. 2002; 26: 329–333. 35. Erdman S, Fox JG, Dangler CA, et al. Typhlocolitis in NF-kappa B-deficient mice. J Immunol. 2001; 166: 1443–1447. 36. Flynn RJ, Baker DG; American College of Laboratory Animal Medicine. Flynn’s Parasites of Laboratory Animals. 2nd ed. Ames, IO: Blackwell; 2007. 37. Fox JG. The Mouse in Biomedical Research. 2nd ed. Amsterdam; Boston: Elsevier; 2007. 38. Fox JG. Helicobacter bilis: bacterial provocateur orchestrates host immune responses to commensal flora in a model of inflammatory bowel disease. Gut. 2007; 56: 898–900. 39. Fox JG, Ge Z, Whary MT, et al. Helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer. Mucosal Immunol. 2011; 4: 22–30. 40. France MP, Muir D. An outbreak of pulmonary mycosis in respiratory burst-deficient (gp91(phox-/-))mice with concurrent acidophilic macrophage pneumonia. J Comp Pathol. 2000; 123: 190–194. 41. Franklin CL. Microbial considerations in genetically engineered mouse research. ILAR J. 2006; 47: 141–155. 42. Fredrickson JK, Balkwill DL, Romine MF, et al. Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp. J Ind Microbiol Biotechnol. 1999; 23: 273–283. 43. Ge Z, Schauer DB, Fox JG. In vivo virulence properties of bacterial cytolethal-distending toxin. Cell Microbiol. 2008; 10: 1599–1607. 44. Godfrey V. Fungal diseases in laboratory mice. In: Fox JG, ed. The Mouse in Biomedical Research. Vol. 2, 2nd ed. Amsterdam and Boston: Elsevier; 2007: 507–515. 45. Gozalo AS, Hoffmann VJ, Brinster LR, et al. Spontaneous Staphylococcus xylosus infection in mice deficient in NADPH

Treuting et al

46.

47.

48.

49.

50.

51.

52.

53.

54. 55.

56.

57.

58.

59.

60.

61.

62.

oxidase and comparison with other laboratory mouse strains. J Am Assoc Lab Anim Sci. 2010; 49: 480–486. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17producing CD4þ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005; 6: 1123–1132. Hasnain SZ, Thornton DJ, Grencis RK. Changes in the mucosal barrier during acute and chronic Trichuris muris infection. Parasite Immunol. 2011; 33: 45–55. Henderson KS. Murine norovirus, a recently discovered and highly prevalent viral agent of mice. Lab Anim (NY). 2008; 37: 314–320. Hensley SE, Pinto AK, Hickman HD, et al. Murine norovirus infection has no significant effect on adaptive immunity to vaccinia virus or influenza a virus. J Virol. 2009; 83: 7357–7360. Homberger FR, Zhang L, Barthold SW. Prevalence of enterotropic and polytropic mouse hepatitis virus in enzootically infected mouse colonies. Lab Anim Sci. 1998; 48: 50–54. Hsueh PR, Teng LJ, Yang PC, et al. Nosocomial infections caused by Sphingomonas paucimobilis: clinical features and microbiological characteristics. Clin Infect Dis. 1998; 26: 676–681. Institute of Laboratory Animal Resources (US). Committee on Infectious Diseases of Mice and Rats. Infectious Diseases of Mice and Rats. Washington, DC: National Academy Press; 1991. Iwatsuki K, Yamasaki O, Morizane S, et al. Staphylococcal cutaneous infections: invasion, evasion and aggression. J Dermatol Sci. 2006; 42: 203–214. Jacoby RO, Ball-Goodrich LJ, Besselsen DG, et al. Rodent parvovirus infections. Lab Anim Sci. 1996; 46: 370–380. Jacoby RO, Ball-Goodrich L. Parvoviruses. In: Fox JG, Barthold SW, Davisson MT, et al., eds. The Mouse in Biomedical Research. Vol. 2, 2nd ed. Burlington, MA: Academic Press; 2007: 93–103. Jacoby, RO, Fox, JG, Davisson, M. Biology and diseases of mice. In: Fox JG, Anderson LC, Loew FM, Quimby FM, eds. Laboratory Animal Medicine. 2nd ed. Amsterdam and New York: Academic Press; 2002: 35–120. Karst SM, Wobus CE, Lay M, et al. STAT1-dependent innate immunity to a Norwalk-like virus. Science. 2003; 299: 1575–1578. Kilic A, Senses Z, Kurekci AE, et al. Nosocomial outbreak of Sphingomonas paucimobilis bacteremia in a hemato/oncology unit. Jpn J Infect Dis. 2007; 60: 394–396. Kilkenny C, Browne W, Cuthill I, et al. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. J Pharmacol Pharmacother 2010; 1: 94–9. Klocke J, Barcia RN, Heimer S, et al. Spontaneous bacterial keratitis in CD36 knockout mice. Invest Ophthalmol Vis Sci. 2011; 52: 256–263. Kullberg MC, Jankovic D, Feng CG, et al. IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis. J Exp Med. 2006; 203: 2485–2494. Kurtzman CP, Robnett CJ, Ward JM, et al. Multigene phylogenetic analysis of pathogenic candida species in the Kazachstania (Arxiozyma) telluris complex and description of their ascosporic states as Kazachstania bovina sp. nov., K. heterogenica sp. nov., K.

61

63.

64.

65.

66.

67.

68.

69. 70. 71.

72.

73. 74.

75.

76.

77.

78. 79.

80.

pintolopesii sp. nov., and K. slooffiae sp. nov. J Clin Microbiol. 2005; 43: 101–111. Labelle P, Hahn NE, Fraser JK, et al. Mousepox detected in a research facility: case report and failure of mouse antibody production testing to identify ectromelia virus in contaminated mouse serum. Comp Med. 2009; 59: 180–186. Layland LE, Mages J, Loddenkemper C, et al. Pronounced phenotype in activated regulatory T cells during a chronic helminth infection. J Immunol. 2010; 184: 713–724. Lencioni KC, Seamons A, Treuting PM, et al. Murine norovirus: an intercurrent variable in a mouse model of bacteria-induced inflammatory bowel disease. Comp Med. 2008; 58: 522–533. Lim MY, Kim JM, Lee JE, et al. Characterization of ozone disinfection of murine norovirus. Appl Environ Microbiol. 2010; 76: 1120–1124. Lipman NS, Henderson K, Shek W. False negative results using RT-PCR for detection of lactate dehydrogenase-elevating virus in a tumor cell line. Comp Med. 2000; 50: 255–256. Lipman NS, Perkins S, Nguyen H, et al. Mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum. Comp Med. 2000; 50: 426–435. Lipman NS, Homberger FR. Rodent quality assurance testing: use of sentinel animal systems. Lab Anim (NY). 2003; 32: 36–43. Livingston RS, Riley LK. Diagnostic testing of mouse and rat colonies for infectious agents. Lab Anim (NY). 2003; 32: 44–51. Liyanage NP, Manthey KC, Dassanayake RP, et al. Helicobacter hepaticus cytolethal distending toxin causes cell death in intestinal epithelial cells via mitochondrial apoptotic pathway. Helicobacter. 2010; 15: 98–107. Macy JD, Paturzo FX, Ball-Goodrich LJ, et al. A PCR-based strategy for detection of mouse parvovirus. J Am Assoc Lab Anim Sci. 2009; 48: 263–267. Macy JD, Cameron GA, Smith PC, et al. Detection and control of mouse parvovirus. J Am Assoc Lab Anim Sci. 2011; 50: 516–522. Maggio-Price L, Shows D, Waggie K, et al. Helicobacter bilis infection accelerates and H. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr1a-/-) mice. Am J Pathol. 2002; 160: 739–751. Maggio-Price L, Treuting P, Zeng W, et al. Helicobacter infection is required for inflammation and colon cancer in SMAD3deficient mice. Cancer Res. 2006; 66: 828–838. Mahler M, Kohl W. A serological survey to evaluate contemporary prevalence of viral agents and Mycoplasma pulmonis in laboratory mice and rats in Western Europe. Lab Anim (NY). 2009; 38: 161–165. Mark R, Buller L, Fenner F. Mousepox. In: Fox JG, Barthold SW, Davisson MT, et al., eds. The Mouse in Biomedical Research. Vol. 2, 2nd ed. Burlington, MA: Academic Press; 2007: 67–92. Maronpot RR, Boorman GA, Gaul BW. Pathology of the Mouse. 1st ed. Vienna, IL: Cache River Press; 1999. Matharu KS, Mizoguchi E, Cotoner CA, et al. Toll-like receptor 4-mediated regulation of spontaneous Helicobacter-dependent colitis in IL-10-deficient mice. Gastroenterology. 2009; 137: 1380–1390; e1381–1383. Mathijs E, Denayer S, Palmeira L, et al. Novel norovirus recombinants and of GII.4 sub-lineages associated with

62

81.

82.

83. 84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

Veterinary Pathology 49(1) outbreaks between 2006 and 2010 in Belgium. Virol J. 2011; 8: 310. Matsuya Y, Kusano T, Endo S, et al. Reduced tumorigenicity by addition in vitro of Sendai virus. Eur J Cancer. 1978; 14: 837–850. Mayeux P, Dupepe L, Dunn K, et al. Massive fungal contamination in animal care facilities traced to bedding supply. Appl Environ Microbiol. 1995; 61: 2297–2301. McCartney SA, Thackray LB, Gitlin L, et al. MDA-5 Recognition of a murine norovirus. PLoS Pathog. 2008;4: e1000108. McKisic MD, Paturzo FX, Smith AL. Mouse parvovirus infection potentiates rejection of tumor allografts and modulates T cell effector functions. Transplantation. 1996; 61: 292–299. McKisic MD, Macy JD, Jr, Delano ML, et al. Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft rejection. Transplantation. 1998; 65: 1436–1446. Meier TR, Maute CJ, Cadillac JM, et al. Quantification, distribution, and possible source of bacterial biofilm in mouse automated watering systems. J Am Assoc Lab Anim Sci. 2008; 47: 63–70. Moehler MH, Zeidler M, Wilsberg V, et al. Parvovirus H-1induced tumor cell death enhances human immune response in vitro via increased phagocytosis, maturation, and crosspresentation by dendritic cells. Hum Gene Ther. 2005; 16: 996–1005. Mook DM, Benjamin KA. Use of selamectin and moxidectin in the treatment of mouse fur mites. J Am Assoc Lab Anim Sci. 2008; 47: 20–24. Muller B, Klemm U, Mas Marques A, et al. Genetic diversity and recombination of murine noroviruses in immunocompromised mice. Arch Virol. 2007; 152: 1709–1719. Mumphrey SM, Changotra H, Moore TN, et al. Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses. J Virol. 2007; 81: 3251–3263. Myles MH, Dieckgraefe BK, Criley JM, et al. Characterization of cecal gene expression in a differentially susceptible mouse model of bacterial-induced inflammatory bowel disease. Inflamm Bowel Dis. 2007; 13: 822–836. Nagamine CM, Sohn JJ, Rickman BH, et al. Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/cRag2(-/-) Apc(Min/þ) mouse. Infect Immun. 2008; 76: 2758–2766. Nakai N, Kawaguchi C, Nawa K, et al. Detection and elimination of contaminating microorganisms in transplantable tumors and cell lines. Exp Anim. 2000; 49: 309–313. Naugler, S. L., Myles, M. H., Bauer, B. A., et al. Reduced fecundity and death associated with parvovirus infection in Blymphocyte deficient mice. Contemp Top Lab Anim Sci 2001; 40: 66. Nicklas W, Staut M, Benner A. Prevalence and biochemical properties of V factor-dependent Pasteurellaceae from rodents. Zentralbl Bakteriol. 1993; 279: 114–124. Paik J, Fierce Y, Drivdahl R, et al. Effects of murine norovirus infection on a mouse model of diet-induced obesity and insulin resistance. Comp Med. 2010; 60: 189–195.

97. Paik J, Fierce Y, Mai P-O, et al. Murine norovirus increases atherosclerotic lesion size and macrophages in Ldlr-/- mice. Comp Med. 2011; 61: 330–338. 98. Percy DH, Barthold SW. Mouse. In: Percy DH, Barthold SW, eds. Pathology of Laboratory Rodents and Rabbits. 3rd ed. Ames, IA: Blackwell; 2007: 3–124. 99. Perdue KA, Copeland MK, Karjala Z, et al. Suboptimal ability of dirty-bedding sentinels to detect Spironucleus muris in a colony of mice with genetic manipulations of the adaptive immune system. J Am Assoc Lab Anim Sci. 2008; 47: 10–17. 100. Peterson NC. From bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics. ILAR J. 2008; 49: 310–315. 101. Pritchett-Corning KR, Cosentino J, et al. Contemporary prevalence of infectious agents in laboratory mice and rats. Lab Anim. 2009; 43: 165–173. 102. Rahija R. Gnotobiotics. In: Fox JG, ed. The Mouse in Biomedical Research. 2nd ed. Amsterdam and Boston: Elsevier; 2007: 217–233. 103. Rao VP, Poutahidis T, Ge Z, et al. Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice. Cancer Res. 2006; 66: 7395–7400. 104. Rao VP, Poutahidis T, Fox JG, et al. Breast cancer: should gastrointestinal bacteria be on our radar screen? Cancer Res. 2007; 67: 847–850. 105. Rehg JE, Toth LA. Rodent quarantine programs: purpose, principles, and practice. Lab Anim Sci. 1998; 48: 438–447. 106. Rehg JE, Blackman MA, Toth LA. Persistent transmission of mouse hepatitis virus by transgenic mice. Comp Med. 2001; 51: 369–374. 107. Ricart Arbona RJ, Lipman NS, Wolf FR. Treatment and eradication of murine fur mites: III. Treatment of a large mouse colony with ivermectin-compounded feed. J Am Assoc Lab Anim Sci. 2010; 49: 633–637. 108. Rogers AB, Fox JG. Inflammation and cancer, I: rodent models of infectious gastrointestinal and liver cancer. Am J Physiol Gastrointest Liver Physiol. 2004; 286: G361–G366. 109. Roseto A, Guillemin MC, Chehimi J, et al. Elimination of mycoplasma, bacteria, and fungi contaminants of hybridoma cultures by intraperitoneal passage in the mouse. Hybridoma. 1984; 3: 297–300. 110. Scanziani E, Gobbi A, Crippa L, et al. Hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice. Lab Anim. 1998; 32: 330–336. 111. Shomer NH, Dangler CA, Schrenzel MD, et al. Helicobacter bilis-induced inflammatory bowel disease in scid mice with defined flora. Infect Immun. 1997; 65: 4858–4864. 112. Smith KA, Hochweller K, Hammerling GJ, et al. Chronic helminth infection promotes immune regulation in vivo through dominance of CD11cloCD103- dendritic cells. J Immunol. 2011; 186: 7098–7109. 113. Spor A, Koren O, Ley R. Unravelling the effects of the environment and host genotype on the gut microbiome. Nat Rev Micro. 2011; 9: 279–290. 114. Stecher B, Hardt WD. The role of microbiota in infectious disease. Trends Microbiol. 2008; 16: 107–114.

Treuting et al 115. Story J, Thigpen J, Meshaw K, et al. Mycoplasma arginini associated with swollen joints in: severe combined immunodeficient mice implanted with tumor cells. J Am Assoc Lab Anim Sci. 2008; 47: 107. 116. Taylor NS, Xu S, Nambiar P, et al. Enterohepatic Helicobacter species are prevalent in mice from commercial and academic institutions in Asia, Europe, and North America. J Clin Microbiol. 2007; 45: 2166–2172. 117. Tomczak MF, Erdman SE, Poutahidis T, et al. NF-kappa B is required within the innate immune system to inhibit microflora-induced colitis and expression of IL-12 p40. J Immunol. 2003; 171: 1484–1492. 118. Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesityassociated gut microbiome with increased capacity for energy harvest. Nature. 2006; 444: 1027–1131.

63 119. Turnbaugh PJ, Ley RE, Hamady M, et al. The Human Microbiome Project. Nature. 2007; 449: 804–810. 120. Waggie KS, ed. Manual of Microbiologic Monitoring of Laboratory Animals. 2nd ed. Bethesda, MD: US Dept of Health and Human Services, Public Health Service, National Institutes of Health, National Center for Research Resources; 1994. 121. Ward JM, Wobus CE, Thackray LB, et al. Pathology of immunodeficient mice with naturally occurring murine norovirus infection. Toxicol Pathol. 2006; 34: 708–715. 122. Whary MT, Fox JG. Detection, eradication, and research implications of Helicobacter infections in laboratory rodents. Lab Anim (NY). 2006; 35: 25–27, 30–26. 123. Wobus CE, Karst SM, Thackray LB, et al. Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2004; 2: e432.