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Workshop Summary: Detection, Impact, and Control of Specific Pathogens in Animal Resource Facilities
Keith G. Mansfield, Lela K. Riley, and Michael L. Kent
Abstract
Overview and Objectives
Despite advances, infectious diseases remain a threat to animal facilities, continue to affect animal health, and serve as potential confounders of experimental research. A workshop entitled Detection, Impact, and Control of Specific Pathogens in Animal Resource Facilities was sponsored by the National Center for Research Resources (NCRR) and National Institutes of Aging (NIA) and held April 23-24, 2009, at the Lister Hill Conference Center on the National Institutes of Health’s (NIH) Bethesda campus. The meeting brought together laboratory animal scientists and veterinarians with experience in fish, rodent, and nonhuman primate models to identify common issues and problems. Session speakers addressed (1) common practices and current knowledge of these species, (2) new technologies in the diagnosis of infectious diseases, (3) impact of environmental quality on infectious disease, (4) normal microbial flora in health and disease, (5) genetics and infectious disease, and (6) specific infectious agents and their impact on research. Attendees discussed current challenges and future needs, highlighting the importance of education and training, the funding of critical infrastructure and resource research, and the need for improved communication of disease risks and integration of these risks with strategic planning. NIH and NCRR have a strong record of supporting resource initiatives that have helped address many of these issues and recent efforts have focused on the building of consortium activities among such programs. This manuscript summarizes the presentations and conclusions of participants at the meeting; abstracts and a full conference report are available online (www.ncrr.nih.gov).
espite many advances in laboratory animal husbandry and welfare, infectious diseases continue to affect animal health and experimental work in important ways. Identification of the presence of infectious agents has often relied on their effect on animal and colony health. But while observations of overt morbidity and mortality in laboratory animals are still useful, the effects are frequently more subtle. When extensive host pathogen coevolution occurs, for example, microbes cause limited disease in the immunologically normal host and only become pathogenic secondary to immunodysfunction or cross-species transmission (Wachtman and Mansfield 2008). In other instances normally avirulent environmental microbes may cause disease due to genetic or experimental manipulation. In both cases, such agents may be minimally pathogenic but affect the animal’s responses to experimental or other environmental stimuli. Even more poorly understood is the role of normal microbial flora in health and disease and their influences on development, immune system ontogeny, and disease susceptibilities. For these reasons it is important for the laboratory animal scientist to understand the full spectrum of microbes that may be present in both the animal under study and its environment and to appreciate the possible impact of these microbes on myriad host responses. The workshop on Detection, Impact, and Control of Specific Pathogens in Animal Resource Facilities brought together laboratory animal scientists and veterinarians with experience in fish, rodent, and nonhuman primate (NHP1) husbandry and models to identify common issues and problems. An objective was to promote communication among research groups with the hope that lessons learned in one species might help inform and advance understanding in others. Experience with and the uses of laboratory animals vary with species and there is often a learning curve that veterinarians and laboratory scientists must overcome in efforts both to elucidate the cause of disease in animals in captivity and to define optimal husbandry conditions. Mice have been studied extensively in the laboratory and can reveal much about the identification and control of agents. The development of specific pathogen–free (SPF1) colonies, the level of environmental control, and the elimination of infectious diseases have arguably been advanced furthest in mice.
Key Words: environmental quality; fish; microbial flora; mouse; nonhuman primate (NHP); pathogen; research impact; zoonosis Keith G. Mansfield, DVM, is Associate Director for Resource and Collaborative Affairs and Associate Professor of Pathology at Harvard Medical School, and Chair of the Division of Primate Resources at the New England Regional Primate Research Center in Southborough, Massachusetts. Lela K. Riley, PhD, is Director of the Research Animal Diagnostic Laboratory and a Professor in the Department of Veterinary Pathobiology at the University of Missouri in Columbia. Michael L. Kent, PhD, is a Professor of Microbiology at Oregon State University in Corvallis. Address correspondence and reprint requests to Dr. Keith G. Mansfield, Harvard Medical School, New England Primate Research Center, PO Box 9102, Southborough, MA 01772-9012 or email keith_mansfield@hms. harvard.edu.
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1Abbreviations
used in this summary: NHP, nonhuman primate; SPF, specific pathogen–free
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Even so, although infectious diseases have largely been controlled in rodent facilities, novel agents such as murine norovirus remain a challenge. Stephen Barthold (Center for Comparative Medicine, University of California, Davis) reinforced these themes in his keynote address on emerging infections and future challenges in laboratory animal science. He provided a historical context illustrating the importance of infectious diseases in mice and demonstrated how infectious diseases continue to affect experimental work. Despite significant improvements in mouse husbandry, infectious diseases remain problematic and can compromise animal health or influence research in unexpected ways. This is in no small part due to the proliferation of genetically modified mice, which often have unique agent susceptibilities and disease patterns. Mouse biologists, including laboratory animal veterinarians and comparative pathologists, play a critical role in understanding the impact of infectious diseases in a variety of laboratory animal species. Dr. Barthold stressed the importance of the NCRRfunded Mutant Mouse Regional Research Centers, which provide a mechanism to address these issues by effectively serving as centers of excellence to assist the scientific community in the proper management of laboratory animals and providing venues for advanced training of veterinarians and animal scientists.
Common Practices Keith Mansfield (Harvard Medical School) presented an overview of the challenges in NHP infectious diseases and colony management. NHPs are used extensively in biomedical and translational research programs because of their close phylogenetic relationship to humans and similarities in anatomy, physiology, and immunology. While much information is available about optimal animal husbandry and veterinary care for these species, infections with viral, bacterial, fungal, and parasitic agents still present challenges in colony management. Large numbers of NHPs are imported from foreign sources each year and may harbor a variety of infections, thus serving as potential reservoirs for animal and human transmission. Dr. Mansfield summarized current efforts and strategies in (1) the establishment of domestic NHP colonies free of specific pathogens and (2) the development of microbial quality control programs based on risk assessment of the species, source, and experimental use of animals to abrogate the impact of infectious diseases (Mansfield and Kemnitz 2008). Even with these successful programs, it has been difficult to eliminate all infectious agents from NHP colonies. Facilities should therefore work to define the full spectrum of microbial agents present in a given population of animals and to understand the possible effects of these agents on pathobiology and experimental work. Lela Riley (University of Missouri) reviewed the current knowledge of pathogens and their control in rodent colonies, where a large number of bacterial, viral, and parasitic pathogens have been identified and characterized. Improved hus172
bandry standards and stringent diagnostic testing have decreased the prevalence of many of these pathogens; however, some organisms—such as mouse parvovirus, mouse hepatitis virus, Helicobacter species, and pinworms—remain prevalent in contemporary laboratory rodent colonies. In addition, scientists continue to identify new murine pathogens, such as norovirus. Reports show that the use of infected rodents alters or invalidates research findings, so it is important to eliminate pathogens from rodents used in research. But because there is a significant cost to eliminating pathogens and maintaining rodents free of all known pathogens, additional studies should focus on the impact of specific agents on various types of research in order to enable the accurate identification of infected animals and promote high-quality research. A common approach to assessing the pathogen status of laboratory rodents is to use sentinels exposed to soiled bedding; recent studies have shown, however, that many pathogens are not consistently transmitted via soiled bedding or detected via sentinels. Thus, there is a need for improved methods for detection of pathogens in laboratory animal colonies and facilities. Michael Kent (Oregon State University) presented an overview of infectious diseases in aquatic research facilities, where understanding of the etiology, modes of transmission, and virulence of aquatic pathogens lags behind that of the rodent and primate fields. The use of fish as models in biomedical research has dramatically increased in the last decade, largely led by the exploitation of the zebrafish (Danio rerio) model. Aquatic animals in confined research systems are constantly surrounded by and ingest their wastes, and thus opportunistic bacterial and protozoan infections are common in systems without optimal water quality (Kent et al. 2009). Until recently, knowledge of infectious diseases in aquatic species in research relied on data largely from aquaculture and the pet fish field, but now specific pathogens and diseases of importance to fish in the research setting are being documented as the use of aquatic models expands (http:// zebrafish.org/zirc/health/diseasemanual.php). As laboratory animal veterinarians become more involved with health surveillance in aquatic research facilities, they are learning that many biosecurity strategies used in terrestrial research systems are adaptable to aquatic systems. Indeed, researchers are now accepting that avoidance of certain pathogens in fishes is as important as in terrestrial species (Kent et al. 2009).
New Technologies Novel infectious agents and disease syndromes continue to be recognized in laboratory animal species, whether these agents cause overt disease associated with morbidity and mortality or more subtle changes that confound experimental work. The latter type of adventitious agent may be more difficult to identify and may go unrecognized in groups of animals. Gustavo Palacios (Columbia University) described new nucleic acid diagnostic methods that may assist in the rapid differential diagnosis of microbial infections and identification ILAR Journal
of previously unknown agents (Quan et al. 2008). He described a staged strategy relying on MassTag polymerase chain reaction (PCR), GreeneChip arrays, and shotgun screening that is applicable to a variety of biological samples allowing agent identification even when the microbe cannot be cultivated in vitro (Palacios et al. 2007). The GreeneChip is a particularly powerful tool that uses gene array technology to simultaneously detect a variety of viral and nonviral pathogens. The arrays, which consist of short nucleic acid sequences derived from nearly 30,000 known human and animal pathogens, have proven instrumental in the rapid recognition of human infectious disease outbreaks (Palacios et al. 2007). Matthew Myles (Research Animal Diagnostic Laboratory, University of Missouri) described another virus discovery approach using sequence-independent genome amplification. While PCR has proven a powerful tool in the detection of pathogen sequences, the approach Dr. Myles described circumvents the requirement to use primers directed at known viral sequences and relies instead on viral genome enrichment through the selective digestion of host nucleic acid before a nonspecific amplification step (Ambrose and Clewley 2006; Braham et al. 2009). With further refinement and validation the technique should have broad application in the recognition of viral agents that prove difficult to culture or identify by other means. The serologic detection of antibodies for specific pathogens remains the backbone of most laboratory animal health monitoring programs, and Joe Simmons (Charles River Laboratories) discussed advances and current challenges in the development of serological assays. In recent years the advent of xMap technology has enabled the development of multiplexed assays that allow the efficient and simultaneous detection of antibodies against multiple pathogens (Simmons 2008). Dr. Simmons stressed the importance of appropriate validation procedures and quality control in producing assays that perform consistently and reliably and the difficulty of obtaining and producing high-quality antigens. He also emphasized the necessity of interpreting laboratory tests with an understanding of the pathogenesis and epidemiology of the agent in question in order to make informed management and microbial control decisions.
Environmental Quality and Infectious Diseases Rudolf Bohm (Tulane University) discussed the impact of environmental quality on infectious diseases in NHP facilities, where environmental quality and animal housing influence exposure and transmission of infectious diseases among the animals. He described typical NHP housing configurations and their influence on the acquisition, transmission, and control of naturally occurring and experimentally induced infectious diseases. Unlike rodents and aquatic species, NHPs often have outdoor housing, which provides almost unlimited social interaction, natural breeding, and infant rearing in a complex, enriched environment, but also Volume 51, Number 2
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poses considerable problems with disease control. It is impossible to eliminate insect, vermin, and wildlife exposure and it is difficult to sanitize outdoor housing. This environment also offers less control of temperature, lighting, and humidity and makes it more difficult to monitor, diagnose, and treat individual animals. Thus housing conditions may play a fundamental role in determining the level of exposure to environmental and other pathogens. Disease outbreaks in captive fishes, including those used in research, are strongly influenced by environmental quality, as Wolfgang Vogelbein (College of William and Mary) explained. In contrast to mammals, fish are ectotherms and live in an aqueous environment that is the source of many important fish diseases and an effective medium for pathogen transmission. In addition, fish are more sensitive and adaptive than mammals to physicochemical fluctuations, and changes in their environment play a critical role in modulating disease expression. The main water quality parameters of concern in captive fishes used for research are temperature, oxygen concentration, nitrogenous wastes (primarily ammonia), carbon dioxide, pH, salinity, and, in some instances, certain chemical toxicants. Failure of disinfection in recirculating culture systems, which are widely used for small fishes (e.g., zebrafish), can lead to rapid spread of pathogens between tanks, and the failure of biological filters can cause ammonia toxicity. Flow-through systems offer many advantages as fish receive clean water at a constant flow rate, minimizing the buildup of waste products and tank-to-tank transmission of pathogens. The use of static systems is an effective way to confine pathogens to an individual tank, but is labor intensive and requires frequent water changes.
Normal Microbial Flora in Health and Disease Susan Garges (National Human Genome Research Institute) gave an overview of the Human Microbiome Project (the microbiome consists of all the DNA, or genomes, of the microorganisms present in or on the human body). Launched in 2007 as part of the NIH’s Roadmap for Medical Research, the project is a 5-year $125 million effort that will produce a resource for researchers to explore how complex communities of microbes interact with the human body to influence health and disease (Hsiao and Fraser-Liggett 2009). The goals of the project are to catalogue the microbes that inhabit the human body, examine whether changes in the microbiome can be related to health and disease, and generate a community resource to support and enable metagenomics-based projects that investigate the role of microbial communities in human health. Researchers have sequenced more than a fifth of the planned 600 reference microbial genomes. Subsequent work will generate profiles of microbial communities in both healthy individuals and those with specific disease states, enabling investigators to determine whether changes in the microbiome at particular body sites correlate to specific diseases. 173
Animal models present unique opportunities to examine the role of microbial flora on different disease states in a highly controlled manner. Frederic Bushman (University of Pennsylvania School of Medicine) discussed recent advances in delineating the role of normal microbial flora of the macaque gut in health and disease. The macaque microbiome was analyzed to learn whether there are consistent patterns in its composition associated with lentiviral infection or other disease states. Using recently developed pyrosequencing technology investigators characterized 141,000 bacterial ribosomal sequences obtained from 100 uncultured gastrointestinal samples and examined how gastrointestinal bacterial communities are shaped in health and disease (McKenna et al. 2008). Distinctive microbial communities were associated with different anatomic sites and differed with the sex of the animal of origin. While major changes were not observed over the course of simian immunodeficient virus (SIV) infection, significant differences occurred with dietary alterations and in colitic animals. Analysis of other specific bacteria found that Campylobacter spp., a gram-negative foodborne enteric pathogen, was further enriched in colitic animals. The technique also enabled identification of unknown pathogens: bacterial profiling of a gastrointestinal disorder of unknown etiology in a culture-negative macaque found Campylobacter fetus, an emerging pathogen.
Genetics and Infectious Diseases Understanding how genetic polymorphisms affect manifestations of infectious diseases provides opportunities to elucidate host-pathogen interactions on multiple levels. Most NHP colonies represent outbred populations with considerable genetic heterogeneity. Research to determine how this heterogeneity influences disease outcome in primates is still in its infancy and has largely focused on the SIV macaque model of AIDS and the role of major histocompatibility complex (MHC) class I alleles in adaptive cellular immune responses (O’Connor et al. 2003). Roger Wiseman (Wisconsin National Primate Research Center) discussed recent advances in the use of parallel pyrosequencing as an approach to comprehensive MHC genotyping (Karl et al. 2009). Using amplicon pooling strategies with this technology it is possible to sequence MHC class I regions from 192 animals in a single run generating 100 million base pairs of data. In the future this approach can be applied to experimental cohorts, to define the role of MHC class I in immunogenicity and challenge studies, and expanded to MHC class II, killer immunoglobulin receptor, and T cell receptor transcripts. Today’s laboratory rodents are genetically and phenotypically diverse, and a detailed knowledge of the rodent’s genetic background is essential for accurate interpretation of diagnostic tests. Julie Watson (Johns Hopkins University School of Medicine) described how the genetic background influences infection phenotype in rodents. Helicobacter hepaticus and Ectromelia virus are two organisms with markedly different phenotypes in different strains. H. hepaticus 174
demonstrates different phenotypes depending on the genetic background of the mice; susceptible strains include A/JCr, C3H/HeN, BALB/cAn, DBA/2N, CBA/J, Swiss Webster, nude, SCID, Rag, IL-7, IL-10, IL-12 knockouts, and tumor suppressor gene (p53) knockouts. In contrast, C57BL/6N, B6C3F1, B6D2F, and CD2F1 mice are asymptomatic carriers. Differing manifestations include propensity for hepatocellular tumors in the A/JCr mice and sexual dimorphism in the induction of inflammatory disease (colitis in females and hepatitis in males). Investigators have shown that a comparison of resistant and sensitive strains may be useful in establishing which genes are responsible for susceptibility.
Specific Infectious Agents and Their Impact on Research Bacterial Agents Luiz Bermudez (Oregon State University) presented an overview of mycobacteriosis and reviewed the current state of knowledge and major issues influencing the field. Mycobacterial infections are important both as overt pathogens in laboratory animals and as animal models of the human disease, which affects a large percentage of the world’s population— Mycobacterium tuberculosis is responsible for 2-3 million deaths annually. Many environmental mycobacteria are increasingly prevalent in animals and humans, as a consequence of environmental changes and human intervention. The mechanisms of pathogenesis are largely unknown, and animal models for the diverse pathogen-causing diseases vary in their ability to represent human disease and to predict therapeutic efficacy in humans. Risk factors for the development of mycobacterial infections reveal common themes in both laboratory animals and humans: genetic predisposition, environmental factors, concurrent infections, and immunomodulatory compounds. Mycobacterial diseases are a significant cause of morbidity and mortality for humans and other animals, and the development of effective therapies will increasingly depend on greater knowledge about pathogenic mechanisms. Saverio Capuano (University of Wisconsin and Wisconsin National Primate Research Center) expanded further on the health impact and diagnosis of tuberculosis in NHP colonies. The M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum, and M. canetti) consists of acid-fast, facultative, intracellular bacilli transmitted primarily through the respiratory and oral routes. There is a broad spectrum of disease manifestation from subclinical latency to rapid progression. While rhesus macaques (Macaca mulatta) are recognized as being highly susceptible, all NHPs can become infected and should be quarantined regardless of source (Roberts and Andrews 2008), especially as M. tuberculosis and M. bovis, largely controlled in domestic colonies, have repeatedly been introduced from foreign sources. New assays relying on the detection of humoral or cellular immune responses have shortcomings; enhanced diagnostic ILAR Journal
capabilities are urgently needed (Garcia et al. 2004; Lerche et al. 2008; Lyashchenko et al. 2007). In quarantine, M. tuberculosis screening should include biweekly skin tests supplemented with other diagnostic assays and radiographs. Extension of the quarantine period may be appropriate for animals obtained from high-risk sources and is necessary (for at least 90 days) in the event of a confirmed case of tuberculosis. Dr. Capuano urged investigators to demand that vendors provide quality animals and to practice rigorous preventive and quarantine methods. Christopher Whipps (State University of New York College of Environmental Science and Forestry) presented an overview of mycobacteriosis in fish, concentrating on zebrafish. Manifestation of disease ranges from sporadic deaths to epizootics with overt lesions and significant mortality. Even low-level underlying infections are important as they are confounding factors in experiments, especially with longterm and disease studies. Several different Mycobacterium species infect zebrafish and routine histological analyses are valuable for detecting the bacteria and evaluating pathological changes, but molecular tools are the most reliable for determining species identification. Virulence trials have confirmed that M. marinum and M. haemophilum cause overt disease and mortality and are of chief concern (Watral et al. 2007). Other species (e.g., M. chelonae, M. abscessus, and M. fortuitum) are associated with chronic low-level infections that may opportunistically cause disease when environmental conditions deteriorate. Control of mycobacteriosis in a fish facility is a complex problem as mycobacteria are thought to be ubiquitous in aquatic ecosystems and are present in biofilms even when fish are not infected (Whipps et al. 2008). Regular tank changes, cleaning, and maintenance of a UV sterilization system may minimize exposure to mycobacteria, especially in recirculating water systems. As with other laboratory species, an animal health monitoring program and good husbandry practices are essential to minimize the impact of background disease in studies that use zebrafish. James Fox (Massachusetts Institute of Technology Division of Comparative Medicine) presented an overview on helicobacter infections (Fox et al. 2004; Whary and Fox 2006). More than 30 helicobacters have been identified and formally named, and three dozen or more (of which 14 are known to infect rodents) await further characterization. Many of the named species colonize the lower intestinal tract of animals and humans. These helicobacters may be localized to the intestinal crypts and are often associated with diarrhea. They may cause systemic disease including colonization of the biliary tract, with induction of cholecystitis and hepatitis, and have been linked to hepatobiliary cancer (Erdman et al. 2009). Some of the species may also have zoonotic potential. A study of worldwide prevalence of helicobacter species in mice used for research found an 88% prevalence rate in academic and commercial biomedical research mouse colonies. Mounting evidence shows that prior or concurrent infections with other pathogens can modulate immunopathogenic responses and it is also increasingly evident that these enterohepatic helicobacters can influence Volume 51, Number 2
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the outcome of experimental studies in mice. For example, coinfection with H. bilis chronically downregulated proinflammatory cytokines and reduced gastric pathology due to H. pylori. Heterologous immunity to enterohepatic helicobacters played a protective role by attenuating premalignant gastric lesions in mice coinfected with H. pylori. Thus research in rodents indicates that enterohepatic helicobacters play a role in gastrointestinal and systemic disease responses; further study is necessary to better define their impact in other laboratory species and humans.
Viral Agents Determination of the B virus status of individual macaques is critical for successful maintenance of specific virus–free captive breeding colonies, and Julia Hilliard (Georgia State University Viral Immunology Center) described the current laboratory practices and recent advances that enable such determinations. B virus or Macacine herpesvirus 1 is a serious zoonotic threat to individuals who work with macaque species and there have been substantial efforts in recent years to eliminate it from domestic colonies. Limitations with currently available assays, which rely on the detection of antibodies, may arise from problems with antigen type or presentation, resulting in low antigen-antibody avidity, or from the presence of abundant, nonantigenic components of the antigen lot that dilutes the B virus–specific antigen. The effectiveness of these assays may be further confounded by the nature of the antibody response in the infected host, which may include the presence of low avidity antibodies induced early after primary B virus infection, waning levels of B virus–specific antibodies due to long intervals between primary and reactivated infections, and limited antibody specificities that fail to find and bind to low concentrations of B virus–specific epitopes. Any of these conditions can result in a failure to detect infected macaques, so the use of multiple methodologies is advisable to determine the B virus status of an individual animal in a pathogen-free macaque breeding colony. Nicholas Lerche (University of California and California National Primate Research Center) presented an overview of diagnostic assays and disease associations with simian retroviruses. Exogenous retroviruses infect many species of NHPs, with a spectrum of diseases that range from subclinical infection to rapidly fatal disease, depending on a variety of host, virus, and environmental factors. Latent or subclinical infections are common and, if undetected, their effects may be subtle but seriously confound experimental work. Disease associated with retrovirus infections in NHPs is highly variable. Simian type D retrovirus infections may result in NHPs with persistent asymptomatic carriers or with overt clinical signs of disease. Simian T cell lymphotropic virus (STLV) causes disease in less than 10% of infected baboons, whereas in Asian macaques there is no documented disease. SIV infections in African NHPs are endemic but disease is rare; in contrast, in Asian species infection is sporadic and immunodeficiency is seen. Simian foamy virus is 175
nonpathogenic in NHPs. Adverse effects of undetected retrovirus infections on biomedical research include the loss of experimental subjects due to increased morbidity and mortality, virus-induced clinical abnormalities, histological lesions, alteration of physiologic parameters, and perturbations of immune responses. Effective testing options are available for the detection of both virus and antibodies. All exogenous retroviruses of NHPs are potentially infectious for humans. There is compelling evidence that HIV-1, HIV-2, and human T cell lymphotropic virus (HTLV) evolved in humans following cross-species transmission of related simian retroviruses. Simian foamy virus is emerging as the most common simian viral infection among humans with occupational exposure to NHPs (Switzer et al. 2004). Many infectious agents have documented effects on the physiology of infected rodents, thus potentially serving as confounding factors in data generated from these animals. Charlie Hsu (Merck and Co. Laboratory Animal Resources Unit) discussed the detection, impact, and control of rodent parvoviruses and noroviruses (Hsu et al. 2007; Manuel et al. 2008), the two most common viral agents that infect mice used in biomedical research and share many similar characteristics (e.g., persistent infections, fecal-oral transmission, extreme environmental stability, and high prevalence). But the viruses differ in important ways. For example, although noroviruses are shed in the feces continuously, parvoviruses are only intermittently shed, a difference that has a significant influence on transmission and detection. Furthermore, mice of different strains and age exhibit variable susceptibilities to parvovirus infection, whereas these differences have not yet been described in murine norovirus infections. Both types of virus pose management challenges for laboratory animal facilities. Rapid progress in the development of diagnostic assays and the subsequent availability of SPF rodents has facilitated the detection and control of these infectious agents.
Parasitic Agents Parasitic infections of rodents are common, but they may go unrecognized and compromise research findings (by, for example, affecting immune activation). Neil Lipman (Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College) illustrated the potential adverse impact of such infections in laboratory rodents with examples of fur mite (Mycoptes musculinus) and pinworm (Aspiculuris tetraptera and Syphacia obvelata) diagnosis and eradication from a large animal care program. While highly sensitive high-throughput technologies are available to evaluate animals for viral and bacterial agents, methods for the detection of common parasites are crude and labor intensive. Dr. Lipman emphasized the need for greater efforts to improve the diagnosis and eradication of these common parasitic infections. Keith Mansfield discussed protozoan infections of nonhuman primates. Despite significant advances in the development of SPF breeding colonies of NHPs used in biomedical research, a number of protozoan infections persist and may 176
adversely affect colony health and confound experimental work. These protozoan agents include both enteric and bloodborne pathogens and may occur as enzootic infections propagated in domestic colonies as well as exotic agents imported from nondomestic sources. They may cause overt morbidity and mortality in immunologically normal animals and may also serve as opportunistic infections during progressive immunodeficiency. Moreover, many of these protozoa are known to infect humans and pose a significant zoonotic risk to animal care staff and scientists. Improved diagnostic assays and treatment modalities are needed to eliminate these infections. Fish are susceptible to a large number and diverse spectrum of protozoan agents. John Fournie (US Environmental Protection Agency) focused on those with direct life cycles and off-host stages as these pose the greatest risk in confined research systems. He provided examples illustrating the diagnosis and treatment of ciliates (Ichthyophthirius multifiliis), dinoflagellates (Piscinoodinium pillulare), and microsporidians (Pseudoloma neurophilia), all of which may cause morbidity and mortality of species kept in captivity. As with other fish diseases, environmental quality likely plays a fundamental role in transmission and disease susceptibility.
Critical Challenges and Needs for Animal Model Systems Workshop speakers and participants identified a number of critical challenges and needs related to infectious disease risk and control in laboratory animal species.
Aquatic Species Identification of Important Pathogens Knowledge of which pathogens are of concern for each species is a cornerstone for the development of control strategies. At present, the most extensive knowledge base on infectious diseases of fishes is from the aquaculture field; but although there are many peer-reviewed papers, there are limits to the application of aquaculture to laboratory fishes. First, there is limited crossover between species and rearing conditions— infectious diseases in salmonids reared in netpens, or carp and catfish in ponds, pose problems quite different from those in zebrafish and medaka (Oryzias latipes) in laboratories. Moreover, underlying chronic infections that do not kill many fish may be acceptable in aquaculture but not in research as they may result in non-protocol-induced variation. Understanding modes of transmission of pathogens in laboratory fishes is also inadequate but important for the development of appropriate preventive measures. With the recent dramatic increase in the use of zebrafish, there have been about 20 or so publications on infectious diseases in zebrafish colony, but very few on other species, such as medaka. Thus research on infectious diseases of importance to a broader range of aquatic species would be quite useful, particularly studies that will lead to reductions in the impacts of such diseases. ILAR Journal
SPF Fish and Health History Documentation There is a need for the development of SPF populations of laboratory fishes, particularly for suppliers. Whereas SPF salmonids are available, the concept has only recently been adopted for zebrafish. An SPF facility for Pseudoloma neurophilia exists at Oregon State University. Soon limited lines of wild-type SPF zebrafish will be available from this laboratory through a collaboration with the Zebrafish International Resource Center (ZIRC), which is located in Eugene, Oregon, and is a main supplier of zebrafish lines to the research community. Development and Implementation of Standard Husbandry Practices Husbandry practices for aquatic research species have largely been adapted from the pet fish or aquaculture industry. Interactions of the environment with infectious diseases are particularly close with aquatics, so it is important to determine the optimum conditions for fish (e.g., rearing conditions, water quality, diet, tank size, density of fishes, temperature) that will reduce the impact of infectious diseases, particularly those caused by common opportunists. Indeed, at least one recent study has shown that crowding and stress exacerbate mycobacteriosis in zebrafish (Ramsay et al. 2009). Education and Training Two types of personnel—the institutional laboratory animal veterinarian and the husbandry technician—are key to the control of infectious diseases in aquatic research species. Yet most laboratory animal veterinarians have little training in health management for these species, and few training programs are appropriate for veterinarians.2 Indeed, the training and background of technicians working with research aquatics is often from their experience with home aquaria, as there is no formal training program for laboratory animal technicians regarding fishes and invertebrates. Increased training in both veterinary practices and husbandry for aquatic species is essential.
to support basic research on such agents is often difficult to obtain unless the pathogens can be viewed as models of human infection and disease. Because of the importance of murine models to biomedical research, there should certainly be support for work on murine infectious diseases in their own right. Methods for Improved Colony Surveillance Colony surveillance programs for infectious diseases rely heavily on the use of sentinel animals. While such sentinel programs are integral to monitoring colony health, they have significant shortcomings: (1) current housing methods and strategies may not effectively and efficiently transmit certain agents to sentinels and (2) sentinel programs are directed at detecting previously recognized pathogens for which serological and other assays are well developed. As outlined in workshop presentations (available online at www.ncrr.nih. gov), techniques have recently been developed to detect a wider range of pathogens and other methods should be explored for pathogen surveillance. Training in Mouse Pathobiology Few training opportunities prepare laboratory animal scientists and veterinarians as broad experts in rodent infectious diseases, and yet such generalists play a critical role in integrating research findings on murine pathogens with mouse biology, husbandry, and infectious disease control strategies. Speakers and participants at this workshop highlighted the importance of interpreting infectious disease risks in the context of the biology of the pathogen and host. But such efforts may be particularly difficult in mice due to the influence of genetic drift and experimental manipulation of mouse strains in producing differences in disease manifestation, detection, and control. Mouse pathobiology would greatly benefit from opportunities to enhance such integrative training.
Nonhuman Primates Strategic Plan for NHP Resources
Rodents Infectious Agent Knowledge Base Arguably more is known about the spectrum of infectious agents and their control in mice than in any other laboratory species. Yet despite this experience and expertise, considerable gaps remain. Novel infectious agents continue to emerge in mouse colonies and yet there is little understanding of their impact on colony health or experimental work. Funding 2Two
exceptions are AquaVet (www.aquavet.info/) and the Mount Desert Island Marine Lab course on Health and Colony Management of Laboratory Fish (www.mdibl.org/courses/fishhealth09.shtml).
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The continued heavy reliance of US researchers on imported nonhuman primates poses a substantial infectious disease risk to both domestic colonies and human contacts. A longterm strategic plan should be developed for US NHP resources that considers the threat and impact of infectious diseases on biomedical research programs and reduces dependency on the importation of animals. NCRR has supported the establishment of SPF breeding colonies of NHPs and such efforts should continue in order to ensure an adequate supply of well-characterized animals to meet national biomedical research needs. Bridging of infectious disease monitoring and prevention programs between domestic and foreign sources to enhance communication of infectious 177
disease risks would assist in the recognition of emerging or reemerging infectious diseases and help to protect these resources. Improved Diagnostics for Common NHP Pathogens Despite current efforts, several infectious diseases remain problematic. Improved diagnostic tests and serological screening tools for common infections would support the management of these threats. In particular, current in vivo and in vitro tests for the M. tuberculosis complex lack both the sensitivity to detect latent infections and the specificity to distinguish atypical infections. Likewise, current assays for detection of serological responses to B virus may miss a subset of acutely and chronically infected animals. Both agents may affect animal health and pose serious zoonotic risk to animal handlers. In addition to these well-recognized infections, a number of emerging agents have been identified in NHPs and reliable diagnostic assays are not readily available. Improved diagnostics are urgently needed. Advanced Training in the Diagnosis and Management of NHP Infectious Diseases Enhanced opportunities for advanced training in the clinical management and diagnosis of primate infectious diseases are required to ensure that individuals with appropriate expertise are available to manage infectious disease threats in NHP colonies. Diagnosis of primate infectious diseases requires (1) knowledge of background lesions present in a population of animals and (2) the ability to integrate clinical, laboratory, and pathology findings. Furthermore, it is often necessary to develop appropriate diagnostic assays as they are not available from commercial sources. The career development of diagnosticians with training in molecular biology and infectious diseases and a firm understanding of primate husbandry and veterinary care should be fostered. Resource-Related Research Novel infectious agents continue to be recognized in NHP colonies and their impact on experimental work and animal health is often unknown. The development of rational control methods requires an understanding of transmission, epizoology, and pathogenesis of primate disease. Opportunities and funding to enhance resource-related research on emergent NHP infectious diseases will be critical in determining the potential impact of infectious agents on colony health and research programs.
Conclusions and Common Themes Infectious diseases remain a threat to many laboratory animal species. The workshop presentations illustrated common themes among diverse species and research programs 178
and highlighted the need for advanced training opportunities, mechanisms to promote resource-related research, improved communication of infectious disease risks, and strategic planning for future requirements. There was universal agreement on the importance of continuing efforts to advance the education and training of veterinarians, technicians, and scientists on the risks associated with infectious diseases; efforts should be multifaceted and include formal training in traditional laboratory animal medicine and pathology residencies as well as ongoing educational opportunities for scientific staff—in particular, investigators should be educated about the potential confounding and disruptive impacts of infections on research using laboratory animals. Training in the clinical aspects of infectious disease diagnosis and control represents a critical resource to the national biomedical research community. Mechanisms to promote and support resource-related research should be sought in order to improve diagnostic assays, elucidate the epidemiology and pathogenesis of diseases, and develop SPF colonies when appropriate. Research on important laboratory animal infectious diseases should be a priority as these infections may adversely affect a variety of research programs. Laboratory animal resource research has often been difficult to support through traditional funding mechanisms; it may be beneficial to explore partnerships that bring together academic and private constituents affected by these issues. NIH and NCRR have had a strong record in supporting Center and resource programs that have helped address many of the issues discussed at this workshop. Recent efforts have focused on consortium activities among Center programs that bridge expertise, resources, and assets. Such consortium activities should be fostered and will likely play a key role in laboratory animal resources and the management of infectious disease risk.
Acknowledgments The authors do not have any commercial or other associations that might pose a conflict interest.
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