Online Issues

Journal Vol 49 (2)

Introduction

Challenges in Microbial Quality Control for Nonhuman Primate

Keith G. Mansfield and Joseph W. Kemnitz

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. Joseph W. Kemnitz, PhD, is a professor at the Institute for Clinical and Translational Research and in the Department of Physiology at the University of Wisconsin School of Medicine and Public Health in Madison; he is also Director of the Wisconsin National Primate Research Center in Madison.

Address correspondence and reprint requests to Dr. Keith Mansfield, Harvard Medical School, New England Primate Research Center, PO Box 9102, Southborough, MA 01772-9012 or email keith_mansfield@hms.harvard.edu.

In contrast to many other laboratory animal species, infectious diseases have proven difficult to eliminate from nonhuman primate colonies and various pathogens remain a threat to animal and colony health. Moreover, despite significant advances in the diagnosis of infectious diseases, unrecognized or adventitious agents are common in nonhuman primates and have the potential to confound experimental work. This issue of the ILAR Journal is devoted to microbial quality control for nonhuman primates and addresses a number of important concerns facing veterinarians and scientists who use such animals.

The reason microbial threats have been difficult to eliminate from nonhuman primate colonies is likely multifactorial. The standard approach used in many other species to develop colonies free of specific agents has been to derive infants by cesarean section and raise them completely separated from the source colony. Such an approach has worked well with rodents and other species, but the critical role of parental input in normal primate social development has made it difficult to use this technique in a widespread fashion in nonhuman primates. Similarly, housing and breeding strategies that promote social interaction also allow the propagation of infectious agents, and significant numbers of primates are raised outside in large corrals or in a semiferal state (Figure 1). Although such environments are enriching for the animals, they complicate the control of infectious agents both between nonhuman primates and from wild animals that may be present in the environment. These factors are compounded by the animals' relatively long life span in captivity and ethical concerns over euthanizing animals solely because of the presence of an infectious agent. Finally, several important pathogens including tuberculosis, measles, and Streptococcus pneumoniae are widespread in the human population and colonies are under constant threat of reintroduction of these pathogens from human contacts.

Figure 1 Social interactions and enriching environments are critical to normal primate development but prevent the use of traditional approaches for derivation of colonies free of all potential pathogens.

Infectious diseases may adversely affect research programs in several ways. Agents may be relatively nonpathogenic in the normal host but cause zoonotic disease in human contacts. Thus B virus, simian immunodeficiency virus (SIV), simian foamy virus (SFV), and simian virus 40 (SV40) are relatively nonpathogenic in the natural host but pose a known or unknown health risk when inadvertently transmitted to humans (Cohen et al. 2002; Engels et al. 2004; Switzer et al. 2004; Wolfe et al. 2004). Agents may directly affect animal and colony health and cause overt morbidity and mortality. Simian type D retroviruses may circulate in macaque colonies and result in progressive immune dysfunction and acquisition of opportunistic infections from the environment. Microbes may cause limited disease in the immunologically normal host but become pathogenic secondary to immunodysfunction induced by concurrent infections, malnutrition, stress, or administration of immunomodulatory drugs. Finally, agents may be relatively nonpathogenic but affect an animal's responses to experimental manipulation; thus chronic parasitism may be relatively nonvirulent to the host but skew cytokine responses to exogenously administered antigens (Da'Dara et al. 2006). As animal husbandry techniques and the ability to control and eliminate some infectious agents improve, it is these last categories that have taken on increasing significance.

Clearly for some agents complete pathogen eradication and measures to prevent reintroduction are warranted to protect animals, personnel, and research programs. However, elimination of all infectious agents from nonhuman primate colonies may not be a realistic goal and in fact may not be desirable. For example, experimental work has demonstrated the fundamental role of resident gastrointestinal flora in normal mucosal morphology and immune responses (Umesaki and Setoyama 2000). Likewise, it is now recognized that common persistent viruses such as cytomegalovirus may play a role in the ontogeny and senescence of the human immune system (Koch et al. 2006; Pawelec et al. 2005). The presence in nonhuman primates of a complement of infectious agents and normal microbial flora that closely duplicate those observed in human populations may be a benefit in experimental systems, enabling more accurate modeling of a variety of disease states.

Because it has not been possible to eliminate all pathogens from NHP colonies, it is important to define the spectrum of microbial agents present in a population of animals and understand how these agents influence pathobiology and experimental work. In this paradigm it is informative to group agents into indigenous and exogenous pathogens. For indigenous pathogens or those that are present as enzootic infections in a specific species, manifestation of disease phenotype may be subtle. There has often been an extensive period of host-pathogen coevolution to allow propagation and persistence of the microbe with limited detrimental effect on the host. There are numerous examples of such host-pathogen adaptations in nonhuman primates, including the cytomegaloviruses and lymphocryptoviruses of virtually all New and Old World primates—serological evidence indicates widespread and nearly uniform infection of both captive and wild colonies. Although these viruses are readily transmitted to juveniles by 1 to 2 years of age and establish lifelong persistent infection, clinical disease is virtually unknown in the immunocompetent host. The ability of these viruses to infect and persist is dependent on complex interactions between viral determinants and the host, and often cellular signaling pathways are hijacked by the viral pathogen to accomplish this goal. In contrast, when the host's normal immune response is damaged through the introduction of a second agent (e.g., SIV, SRV, or measles virus) or through administration of immunomodulatory drugs, the host-pathogen balance is disrupted and disease states may arise that are detrimental to the animal. Thus the introduction of an immunosuppressive regime may reactivate both cytomegaloviruses and lymphocryptoviruses and cause progressive cytolytic or lymphoproliferative diseases, respectively.

In contrast to many enzootic infections, exogenous agents are those not normally found in nonhuman primate colonies and may be associated with fulminant disease upon introduction to a naïve population. A colony's exposure to the agent often results from cross-species transmission from contact animals or humans. Host-pathogen coevolution is minimal in such inadvertent or aberrant hosts and disease manifestations may be severe, as is evident in numerous examples: the transmission of Herpesvirus tamarinus to owl monkeys, M. tuberculosis to rhesus macaques, and lymphocytic choriomeningitis virus to callitrichinae (Flynn et al. 2003; Hunt and Melendez 1966; Montali et al. 1993).

SIV models in particular illustrate the differences between indigenous and exogenous agents: in the natural African primate host SIV infection is rarely associated with disease, although serological studies reveal a high rate of infection among populations and molecular data indicate that the virus is readily transmissible and replicates to high viral load. Progressive loss of CD4 T cells and subsequent immunodysfunction are not observed, likely because of as yet poorly defined host-pathogen adaptations. In contrast, cross-species transmission to Asian macaques results in destruction of CD4 T cells and a disease process that mimics human acquired immunodeficiency syndrome.

For these reasons it is essential to define the full complement of microbial agents present in a population of nonhuman primates. Disease surveillance, the underpinning of a preventive health program, is a critical component of a microbial quality control program and should include: molecular-based and immunologic assays to detect pathogens or immune responses to specific pathogens; fecal cultures and examination of samples for enteric parasites; vigorous diagnostic intervention to elucidate the cause of clinical illness in sick or diseased animals; a clinical database that integrates medical records, laboratory tests, and housing records; and routine gross and microscopic evaluation of all animals at the time of death by individuals trained in primate pathology. Serious consideration should be given to how such surveillance programs can be standardized and bridged among primate breeders, suppliers, and end users to ensure timely and efficient communication of infectious disease threats.

This issue of the ILAR Journal considers the rationale behind and principal elements of microbial quality control programs. Colonies that are specific pathogen free (SPF) of particular agents still form the foundation of successful primate husbandry and preventive health programs; Morton and colleagues (2008) consider current approaches and future directions of such SPF programs. The National Center for Research Resources and the Office of AIDS Research of the National Institutes of Health have invested heavily in the derivation and further production of macaque breeding colonies SPF for B virus as well as several important retroviruses and bacterial pathogens. Further refinement and expansion of these criteria are likely as investigators develop a better understanding of the impact of other pathogens on scientific research programs.

Roberts and Andrews (2008) provide a historical perspective on quarantine requirements as well as a detailed review of current standards and practices. With the large increase in macaques imported from Asia over the past decade these procedures have become critically important in ensuring the health and welfare of research animals.

Laboratory testing plays a vital role in clinical diagnosis and in the development of prevention and exclusion strategies. Unfortunately, there is considerable laboratoryto-laboratory variability in assay development and quality assurance programs. New assays are developed when agents are recognized as potentially important pathogens and so the clinician must be familiar with quality control measures. Simmons (2008) provides an overview of the development, application, and quality control measures for serological assays commonly used in diagnostic testing of NHP colonies.

Tuberculosis remains a constant threat to nonhuman primate colonies and has seen a resurgence as animals are imported with increasing frequency from areas of the world where disease is common in human handlers. The shortcomings of intradermal skin testing with mammalian old tuberculin have long been recognized. Lerche and colleagues (2008) describe the recent development of new assays largely made possible through systematic molecular analysis of mycobacterial agents. Although these assays are not a panacea in the diagnosis of mycobacterial diseases, used in combination they may increase the sensitivity and specificity of surveillance programs.

Sasseville and Diters (2008) consider the effect of microbial flora and adventitious agents on research programs. Nonhuman primates are used extensively in drug development and safety assessment, and their microbial flora may affect the absorption and metabolism of experimentally administered compounds. This is an area that has received little attention and yet may be a source of considerable variability. Furthermore, the presence of unrecognized infectious agents such as hepatitis A virus and simian retroviruses may adversely affect and confound interpretation of experimental data. Similarly, reactivation of latent infections may occur during progressive immunodysfunction and result in disease states not normally observed in the immunocompetent animal.

The article by Wachtman and Mansfield (2008) provides a review of opportunistic infections in immunologically compromised nonhuman primates, and Haustein and colleagues (2008) describe the impact of infections after organ transplantation. The mechanisms of immunosuppression differ, but there are parallels in the types and spectrum of opportunistic infections that occur.

Although infectious agents provide many challenges to the care and husbandry of nonhuman primates, they also offer unique opportunities for the modeling of important human diseases. Because of the close genetic relationship between humans and other members of the order Primata, the many parallels between disease manifestations in both have been useful for the development of novel models of important human conditions. Gardner and Luciw (2008) provide an extensive review of historical and current animal models of infectious disease. As genomic data and tools for their manipulation improve, further refinement in these models is likely.

Microbial quality control should be integrated with all elements of a facility's primate husbandry, veterinary care, and research programs. The elimination of all infectious diseases from nonhuman primate colonies is unlikely; instead, efforts should focus on agents that may seriously confound research programs or that pose a significant risk to animal and/or human health. Pathogen surveillance is a critical component of any microbial quality control program and serves to safeguard animal and human health as well as promote excellence in research.

Acknowledgments

This work was supported by National Institutes of Health grants RR00168, RR16020, RR000167, and RR025012. The authors do not have any commercial or other associations that might pose a conflict of interest.

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