Online Issues

Journal Vol 49 (2)

Impact of Infections and Normal Flora in Nonhuman Primates on Drug Development

Vito G. Sasseville and Richard W. Diters

Vito G. Sasseville, DVM, PhD, DACVP, is a Veterinary Pathology Fellow in Discovery Toxicology, Research and Development at Bristol-Myers Squibb in Princeton, New Jersey; Richard W. Diters, VMD, DACVP, is Senior Principal Veterinary Pathologist in Drug Safety Evaluation at Bristol-Myers Squibb in Syracuse, New York.

Address correspondence and reprint requests to Dr. Vito G. Sasseville, Bristol-Myers Squibb Research and Development, PO Box 4000 (Mailstop 14-09), Route 206 and Provinceline Road, Princeton, NJ 08543 or email vito.sasseville@bms.com.

Abstract

Preclinical safety studies that are required for the marketing approval of a pharmaceutical include single and repeat dose studies in rodent and nonrodent species. The use of nonhuman primates (NHPs), primarily macaques, as the nonrodent species has increased in recent years, in part due to the increase in development of biopharmaceuticals and immunomodulatory agents. Depending on the source of the macaques, they may vary in genetic background, normal flora, and/or the incidence of preexisting pathogens and inflammatory conditions. As the use of alternative sources of macaques rises to meet the increased demand for these animals in biomedical research, the toxicologic pathologist should be well versed in NHP pathology to adequately assess potential drug-related effects in the context of these variations. Such knowledge is particularly important in studies involving immunomodulatory drugs as the toxicologic pathologist should anticipate which type(s) of infections are most likely to arise depending on which arm of the immune system is modulated. The purpose of this review is to discuss the immunosuppressive (e.g., simian type D retrovirus, simian immunodeficiency virus) and opportunistic viruses (e.g., cytomegalovirus, adenovirus, simian virus 40, rhesus rhadinovirus, and lymphocryptovirus), primary and opportunistic bacteria (e.g., Campylobacter spp., Shigella flexneri, Yersinia enterocolitica, Moraxella catarrhalis, Mycobacterium avium complex, enteropathogenic Escherichia coli), and parasites (e.g., Plasmodium spp., Schistosoma spp., Strongyloides fulleborni) that have had the most profound impact on the interpretation of drug safety studies and/or that may reemerge as alternative sources of NHPs are used for drug safety studies.

Key Words: hepatitis; immunomodulatory drugs; macaques; mycobacterium; opportunistic infections; plasmodium; retrovirus

Introduction

Preclinical safety studies that are required for the marketing approval of a pharmaceutical include single and repeat dose studies in two species (rodent and nonrodent) to assess reproductive toxicity, genotoxicity, local tolerance, and, for chronic use indications, carcinogenicity (ICH 1997b). The rat is the most common rodent species, and dogs are traditionally used as the nonrodent species. However, the use of nonhuman primates (NHPs¹) as the nonrodent species has increased in recent years, in part due to the increase in the development of biopharmaceuticals and immunomodulatory agents.

To ensure that studies with biopharmaceuticals are predictive of anticipated toxicities in humans, those studies need to be conducted in a relevant animal species—that is, a species in which the biopharmaceutical has a similar biologic response to that observed in humans due to the expression of a responsive orthologous drug receptor or antigen (Cavagnaro 2002; ICH 1997a). The high specificity of many biopharmaceuticals, and in particular those with monoclonal antibodies, means that most preclinical studies use nonhuman primates due to target-specific cross reactivity, relevant pharmacology, similar immune systems, and similar pharmacokinetics (Chapman et al. 2007). Moreover, the use of NHPs minimizes the potential for development of antibody responses to protein-based drug candidates (Cavagnaro 2002).

Macaques (Macaca spp.)—especially the cynomolgus macaque (Macaca fascicularis) and, to a lesser extent, the rhesus macaque (M. mulatta)—are the NHP species most widely used by pharmaceutical companies. In addition to macaques, the common marmoset (Callithrix jacchus) has been gaining in popularity for general toxicity studies, particularly in Europe, primarily due to its small size, reduced costs, comparative ease of husbandry, and lack of endemic pathogens of zoonotic concern (Mansfield 2003; Smith et al. 2001). Although other NHP species have been utilized, primarily for specialized studies, in this review we focus on cynomolgus and rhesus macaques.

In the not too distant past, many NHPs used in preclinical toxicity studies were wild caught and harbored myriad bacterial, parasitic, viral, and inflammatory diseases. These conditions, which could be asymptomatic, clinically inapparent, and immunosuppressive, could affect the interpretation of drug-related findings. However, it is important to note that this liability can be a benefit in the development of immunomodulatory drugs, as some regulators believe that the NHP's variety of preexisting pathogens/opportunistic agents, many of which have human homologues, renders it more useful than the dog in efforts to identify an immunosuppressive hazard. Nonetheless, during the last decade, improved domestic production, husbandry, and diagnostic practices have drastically reduced the incidence of these conditions.

The increased demand for NHPs for the development of immunomodulatory and biopharmaceutical drugs and for biodefense has resulted in a shortage of these animals for use in biomedical research (Mansfield 2003; Patterson and Carrion 2005). Consequently, other NHP sources, particularly in China and Southeast Asia, are being used. These new sources have introduced genetic diversity and different background rates of infection with various pathogens and inflammatory conditions (Blancher et al. 2006; Drevon-Gaillot et al. 2006; Leuchte et al. 2003). In the context of these recent developments, we describe several important historical and newly emerging infections and background flora that can affect the interpretation of drug safety studies.

Infections

Numerous viral, bacterial, and parasitic pathogens endemic in NHP populations remain clinically inapparent in immunocompetent animals and are in general mild and self-limiting; as such they do not adversely affect in-life or clinical-pathologic endpoint interpretations during routine drug safety studies. The exceptions are infections associated with the gastrointestinal tract that result in chronic enterocolitis, a condition that, albeit of low incidence, is a persistent and widespread colony problem in macaques and is most likely multifactorial in origin (Sestak et al. 2003). In addition, immunosuppression, introduced either naturally (e.g., by immunosuppressive retroviruses) or experimentally (e.g., by irradiation, immunomodulatory agents, or chemotherapeutics), can make a previously unexposed individual or group more susceptible to primary outbreaks or induce recrudescence of these pathogens. Table 1 lists the viral, bacterial, and parasitic diseases that have had the most profound impact on the interpretation of drug safety studies and/or that may reemerge with the use of alternative sources of NHPs for drug safety studies.

Viruses

Retroviruses (Simian Type D Retrovirus, Simian Immunodeficiency Virus, and Simian T Lymphotropic Virus)

Simian type D retrovirus (SRV¹) has had an impact in the pharmaceutical industry primarily because some SRV-infected animals can become viremic yet remain antibody negative, allowing infections to escape detection by routine antibody screening (Kwang et al. 1987). Facilities therefore either maintained SRV-positive animals or reinfected colonies up until the late 1990s, when virus isolation techniques, in conjunction with serologic screening and SRV polymerase chain reaction (PCR) techniques, helped to establish SRV-free colonies (Guzman et al. 1999; Lerche et al. 1994).

In the most severe form of SRV, animals can develop an immunodeficiency syndrome with a host of opportunistic infections (OIs¹), inflammatory, and proliferative/neoplastic diseases (e.g., lymphoma, retroperitoneal fibromatosis), but the more common presentation in viral-positive animals is weight loss, diarrhea, decreased lymphocyte and red blood cell counts, and an increase in the incidence, severity, and distribution of lymphoid infiltrates in various organ systems (Guzman et al. 1999; Henrickson et al. 1983). When viral-positive animals were used in drug safety studies, these characteristics of SRV infection—closely monitored and documented during the in-life, necropsy, clinical pathology, and histopathological assessments—could be confused with a drug-related effect (Guzman et al. 1999; Lerche and Osborn 2003).

Fortunately, with current diagnostic practices in the pharmaceutical industry, SRV infection is now a rare occurrence in drug safety studies. Moreover, with today's knowledge of SRV and its myriad inflammatory changes and immunosuppressive nature, the validity of any studies conducted with viral-positive animals should be questioned by regulatory agencies. Opportunistic infections secondary to retrovirus-induced immunosuppression are described in detail elsewhere in this issue (Wachtman and Mansfield 2008).

In contrast to SRV, macaques do not harbor simian immunodeficiency virus (SIV¹) in their native Asian habitat. African NHPs are the natural hosts for SIV and for the most part their infection is asymptomatic, whereas in Asian macaques SIV induces a lentiviral inflammatory disease and immunosuppressive AIDS-like disease (Daniel et al. 1985). SIV was first isolated in 1984 in immunodeficient captive rhesus macaques, in which the virus was most likely introduced years prior by inadvertent infection from wild-caught sooty mangabey monkeys (Sasseville et al. 1999). Thus, natural cross-species transmission of SIV from African NHPs to Asian macaques was a relatively rare event and has not significantly affected drug safety studies. Moreover, with the isolation and characterization of SIV, efficient screening practices have led to the creation of SIV-free colonies for biomedical research (Lerche et al. 1994).

Simian T lymphotropic virus 1 (STLV-1) belongs to the primate T lymphotropic virus (PTLV) group of related retroviruses that includes STLV-2, STLV type L, STLV-3, and their human T cell lymphotropic virus counterparts, which share common morphological, antigenic, biologic, and genetic features (Traina-Dorge et al. 2005). STLV is endemic in many wild and captive African and Asian monkeys and apes, with seroprevalence ranging from 0% to 80% (Andrade et al. 2003; Lerche and Osborn 2003; Sariol et al. 2006; Traina-Dorge et al. 2005).

STLV-1 is a cell-associated virus with tropism for CD4⁺ and CD8⁺ lymphocytes; transmission is via transfer of infected cells to susceptible animals (Lerche and Osborn 2003). STLV infection is usually asymptomatic, with only a small number of infected animals developing T cell lymphoma or lymphoproliferative disease (Lerche and Osborn 2003). STLV-related disease occurs only in African primate species, but altered cytokine profiles have been reported in STLV-infected macaques, which could represent a confounding variable in immunotoxicology endpoints (Lerche and Osborn 2003). As with SIV, efficient screening practices have resulted in STLV-free colonies for biomedical research (Lerche and Osborn 2003; Lerche et al. 1994).

Measles Virus

Measles virus infection typically spreads to NHPs from infected human handlers. Symptoms range from fever, rash, and conjunctivitis to mortality, secondary to transient immunosuppression (Choi et al. 1999; El Mubarak et al. 2007; Willy et al. 1999). Once established, measles virus infection spreads rapidly. In one outbreak more than 20% of the macaques in a zoo died in a 4-month period from a host of measles virus–induced OIs, including disseminated cytomegalovirus infection, adenoviral and bacterial pneumonia, and Candida albicans–associated gingivitis and esophagitis (Choi et al. 1999). PCR, virus culture, serum immunoglobulin G (IgG) and IgM levels are effective methods to assess exposure to measles virus. Animals exposed to measles have lifelong immunity, so quarantine and vaccination can be effective in the prevention of disease (Willy et al. 1999).

Opportunistic Viral Infections

Common opportunistic viral infections in macaques include cytomegalovirus, adenovirus, simian virus 40 (SV40), rhesus rhadinovirus (RRV¹), and lymphocryptovirus (Daniel et al. 1985; Desrosiers et al. 1997; Moghaddam et al. 1997). These viruses are most often associated with immunosuppressive retroviruses, experimental irradiation, or treatment with immunomodulatory or chemotherapeutic agents. The types of viral OIs that may emerge vary depending on the source of the macaque, the arm of the immune system affected, and at times the presence of an immunosuppressive coinfection. For example, viral OIs and γ-herpesvirus-induced lymphoid hyperplasia and lymphomas are more common than bacterial OIs in the context of experimental SIV infection because of SIV's depleting effects on CD4⁺ T lymphocytes and thus on cell-mediated immunity.

With selective immunomodulatory agents, one can predict, in general terms, what type(s) of OIs may occur, as exemplified by a T lymphocyte–depleting biopharmaceutical in cynomolgus macaques that was associated with γ-herpesvirus-induced B cell hyperplasia and lymphoma (Hutto et al. 2003). This finding in NHPs is not surprising as iatrogenic γ-herpesvirus-associated lymphoid hyperplasia and lymphoma in humans (usually after transplantation) have been associated with the use of various immunomodulators (Caillard et al. 2005; Norin et al. 2004).

Another factor to consider is the atypical microscopic appearance of an infection due to a diminished immune response. For instance, in SIV-infected macaques, the development of disseminated Mycobacterium avium complex (MAC¹) depends on viral strain and is correlated with the size and composition of microgranulomas (Mansfield et al. 2001). Manifestation of infection may also depend on whether it is a primary infection or reactivation of a latent infection. For instance, primary SV40 infection in immunosuppressed macaques can induce meningoencephalitis, interstitial nephritis, and pneumonitis, whereas reactivation of latent virus is associated with progressive multifocal leukoencephalopathy (PML¹) (Simon et al. 1999). Pancreatitis is a unique manifestation of adenovirus infection in immunocompromised patients and macaques (Martin et al. 1991; Niemann et al. 1993), whereas in the immunocompetent host adenovirus infection can be associated with enteritis (Sestak et al. 2003).

Other less common viral OIs that can occur in the context of immunosuppression include simian varicella virus, Cercopithecine herpesvirus 1 (B virus), and simian parvovirus (Chellman et al. 1992; Kolappaswamy et al. 2007; O'Sullivan et al. 1996). Investigators have reported the impact of parvovirus-induced anemia on the interpretation of drug safety studies (Brown 1997; O'Sullivan et al. 1996; Schroder et al. 2006), and it is likely that there are other instances that have not been publicly documented. Aside from the obvious effect of immunomodulatory compounds, other cases of simian parvovirus infection most likely occurred in association with SRV infection. The zoonotic potential of herpes B virus and simian parvovirus are probably more important than their potential impact on drug safety studies (Brown et al. 2004; Huff and Barry 2003).

With the increase in the number of immunomodulators entering clinical trials to treat chronic, nonlife-threatening diseases of the immune system, there is greater interest in adequately assessing the potential for OIs and/or lymphoproliferative diseases that might result from immune impairment (Cavagnaro 2002). For the astute toxicologic pathologist, it may be useful to identify OIs associated with chronic administration of anti-inflammatory/immunomodulatory agents as their presence may reveal the need for additional functional data from more refined drug safety–related immunotoxicology assessments (e.g., immunophenotyping, KLH and other antigen challenge, disease models). Cytomegalovirus, adenovirus, SV40, RRV, and lymphocryptovirus have a high natural seroprevalence in adult macaques. Infection with the first three is usually asymptomatic, whereas experimental infection with RRV and lymphocryptovirus in immunocompetent macaques may be mildly symptomatic—RRV can induce a mild febrile response and a lymphoproliferative disorder, and lymphocryptovirus may induce B cell hyperplasia and lymphadenopathy (Mansfield et al. 1999; Moghaddam et al. 1997).

The most important aspect of lymphocryptovirus infection has been in macaque studies with various immunomodulators, which have shown that lymphoproliferative disorders and lymphomas induced by lymphocryptovirus, the Epstein-Barr virus (EBV) γ-1-herpesvirus equivalent, model the human condition (Hutto et al. 2003; McInnes et al. 2002; Rivailler et al. 2004). Thus, for assessing the potential risk of drug-induced human immunotoxicity with immunomodulatory agents, it is essential to determine the potential for increased risks of lymphoproliferative disorders, opportunistic infections, and immune impairment in preclinical studies with NHPs (Green and Black 2000). To this end, NHPs should be screened before study initiation for lymphocryptovirus and other OIs to ensure uniform viral status among vehicle and test article treatment groups.

It is important to remember, however, that the limited number of NHPs routinely used in chronic drug safety studies may mean that such studies are not powered to identify a human hazard in the form of a relatively rare but serious OI. For example, the anti-α4 integrin monoclonal antibody natalizumab (Tysabri), which is used for the treatment of multiple sclerosis and Crohn's disease, induced PML associated with JC polyomavirus in only a small subset of patients (Berger and Koralnik 2005). Despite a high seroprevalence of the simian counterpart to JC virus, SV40, in macaques, and its association with PML in the context of SIV-induced immunosuppression, PML was not observed in preclinical studies of natalizumab in cynomolgus macaques. This absence is not surprising as the estimated incidence rate for the development of PML with natalizumab treatment is 1:1,000 patients after 18 months of therapy (Berger 2006). In addition, PML is the only OI associated with natalizumab treatment in humans, suggesting a unique interaction between JC virus and natalizumab's mechanism of action (Berger 2006).

Hepatitis A Virus (Infectious Hepatitis)

Hepatitis A virus (HAV¹) is a small RNA virus in the picornavirus family that spreads by the fecal-oral route and is endemic in many developing countries; in industrialized nations it tends to occur sporadically in isolated clusters. HAV can infect a variety of Old and New World NHPs including the great apes, cynomolgus, rhesus, and stump-tailed macaques, owl monkeys (Aotus sp.), marmosets, and baboons (Balayan 1992). The reported prevalence of HAV in macaques is variable and can be high—9-35% in rhesus monkeys and 20-89% in cynomolgus monkeys (Andrade et al. 2003; Balayan 1992). Researchers have documented natural transmission of the virus between animals both in the wild and in the laboratory environment, presumably by the fecal-oral route (Andrade et al. 2003; Burke and Heisey 1984; Lankas and Jensen 1987; Le Bras et al.1984; Lemon et al. 1982). Human HAV is distinct from simian HAV and this has led to some confusion in the literature about species susceptibility.

In macaques the infection is typically mild and subclinical. Clinical pathology parameters such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) may be mildly elevated during active infection and there may be histopathologic changes consisting of increased mononuclear cell infiltration in the portal triads and minimal individual (single-cell) hepatocellular necrosis (Slighter et al. 1988), although there are reports of more severe outbreaks (Lankas and Jensen 1987; Shevtsova et al. 1987). HAV infection can alter cellular antiviral defense mechanisms (Brack et al. 2002); and immunosuppression in a cynomolgus monkey was linked to an outbreak of spontaneous HAV (Andzhaparidze et al. 1985). In both cynomolgus and rhesus macaques infections are rarely persistent (>2 years); periodic exacerbations may be characterized by ALT elevation, viral shedding, and morphologic changes in the liver (Lapin and Shevtsova 1990). Prophylactic HAV vaccination of NHPs to be used for toxicology studies is not uncommon.

Bacteria

In immunocompetent NHPs, the common bacterial infections are in general mild and self-limiting and have little impact on drug development. Exceptions are those associated with the gastrointestinal tract or bacterial OIs associated with immunosuppression. The following are examples of the more common bacterial pathogens that have an impact on drug development studies.

Campylobacter coli, Campylobacter jejuni, Shigella flexneri, and Yersinia enterocolitica

Chronic gastritis and enterocolitis, either asymptomatic or associated with chronic diarrhea, are relatively common in rhesus and cynomolgus macaques (Reindel et al. 1999; Rubio and Hubbard 2002; Sestak et al. 2003). In a study of rhesus macaques both with and without clinical symptoms of diarrhea, there was an increased incidence of bacteria (Campylobacter coli, C. jejuni, Shigella flexneri, and Yersinia enterocolitica) as well as other pathogens in fecal samples collected from the animals with clinical signs of diarrhea, suggesting that one or more of a variety of bacterial species may cause chronic diarrhea (Sestak et al. 2003). In other studies, higher incidences of Campylobacter spp. alone were associated with acute intestinal disease in NHPs (Kalashnikova et al. 2002, 2006). S. flexneri is associated with sporadic outbreaks of hemorrhagic diarrhea and death in NHPs (Lederer et al. 2005).

It is possible to control active S. flexneri infections by vigilant sampling of NHP colonies and the appropriate use of antibiotics to eliminate shedding of bacteria and spread of the disease (Black-Schultz et al. 1997; Wolfensohn 1998). However, the presence of asymptomatic carriers of S. flexneri makes it difficult to eradicate from an NHP colony. In addition, shedding of bacteria can occur or may increase under stress or immunosuppression. Y. enterocolitica infects numerous species of NHPs and can cause outbreaks of diarrhea, but its incidence, which varies considerably among NHP colonies, is generally lower than that of S. flexneri and Campylobacter spp. (Poelma et al. 1977; Sestak et al. 2003; Vore et al. 2001).

As each of these bacterial species has zoonotic potential and can cause outbreaks of diarrhea independently, macaque colonies are typically closely monitored for these pathogens and treated with antibiotics, thus overt clinical disease is rarely observed. However, sporadic outbreaks of chronic diarrhea of unknown etiology remain an issue in drug development. In addition, effects on the immune system have been documented in macaques with chronic enterocolitis and diarrhea. These effects have included elevated interleukin (IL)-1α, IL-3, and tumor necrosis factor α genes, and an increase in activated CD4⁺ and CD69⁺ T lymphocytes in gut-associated lymphoid tissues (Sestak et al. 2003).

Diarrhea in any animal awaiting assignment to a drug safety study usually eliminates it from the study and thus, depending on how many animals are affected, can limit the availability of animals for a study. Furthermore, the occurrence of diarrhea after a study has begun can adversely affect interpretation of drug-related effects. In addition, clinically inapparent gastrointestinal infections, depending on incidence and severity, can significantly affect microscopic interpretation of drug-related effects.

Helicobacter pylori

Although H. pylori is generally asymptomatic, both H. pylori and H. heilmannii–type bacteria have been associated with gastritis, peptic ulcers, gastric carcinomas, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas in humans (Nakamura et al. 2007). There are numerous reports of natural infection in macaques, with the primary microscopic findings of epithelial hyperplasia, lymphoplasmacytic infiltrates, and erosions in the antral portion of the stomach (Reindel et al. 1999). The smaller corkscrew-shaped H. pylori should be differentiated from the common spiral-shaped and larger (up to 8 μM in length) H. heilmannii–type bacteria; the latter can be quite numerous in the gastric gland lumens, but their association with inflammation remains unclear (Drevon-Gaillot et al. 2006; Reindel et al. 1999).

Tuberculosis

Tuberculosis (TB¹) caused by Mycobacterium tuberculosis in NHPs remains a concern, although TB testing and improved husbandry have greatly lowered infection rates in imported animals. In 1941 infection rates in some laboratory colonies were as high as 30% (Carlton and Hunt 1978; T-W-Fiennes 1972), whereas today TB infections in macaques used for drug development studies are rare. However, this author (R.W.D.) was involved with a contained epizootic in cynomolgus macaques that was initiated by a classically anergic animal with fulminant pulmonary tuberculosis. In this outbreak the infection was (most likely) transmitted by aerosol to involve eight animals in two different rooms and had a major impact on the outcome of these regulated toxicity studies. The monkeys that were infected by the source animal had mild contained infections limited to one or several granulomas in the lung and/or thoracic lymph nodes. Fortunately, none of the animal handlers or caretakers ever tested positive for M. tuberculosis.

This case points out that vigilance is essential in screening for TB in nonhuman primates used in drug development studies despite the reduced incidence of the disease relative to previous decades. Although the reliability of the tuberculin skin test is debatable, and other in vitro assays have been developed (Vervenne et al. 2004), the skin test, especially when repeated at 2- to 3-week intervals over a 10-week quarantine period, remains the gold standard for TB testing of NHPs (for more on TB testing, see Roberts and Andrews 2008).

Moraxella (Branhamella) catarrhalis

M. catarrhalis, part of the normal flora of the nasopharynx, has been associated with outbreaks of epistaxis ("bloody nose" syndrome) in both immunocompetent and immunocompromised macaques (Bowers et al. 2002; VandeWoude and Luzarraga 1991). Such outbreaks, which are usually mild and self-limiting, occur primarily in winter and have been attributed to lower environmental humidity levels. But in the authors' experience, certain immunomodulatory drugs can increase the incidence and severity of Moraxella-induced epistaxis, rendering the host more susceptible to secondary bacterial invasion and sepsis. Thus, in any study with an immunomodulatory drug capable of lowering the humoral immune system response, it is important to carefully monitor animals for the onset of epistaxis induced by Moraxella (or another opportunistic bacterial pathogen).

Opportunistic Bacterial Infections

Bacterial infections commonly associated with immunosuppression include MAC, Rhodococcus equi, and enteropathogenic Escherichia coli (EPEC) (Mansfield et al. 1995, 2001). MAC is the most common disseminated bacterial disease in untreated human immunodeficiency virus (HIV)–infected patients and SIV-infected macaques (Mansfield et al. 2001). A retrospective study found that 17% of SIV-infected macaques had MAC and implicated potable water as the source of infection (Mansfield et al. 1995; Mansfield and Lackner 1997). Disseminated infection was dependent on SIV viral strain and suggests unique immune parameters in containing infection (Mansfield et al. 2001). In addition, as MAC organisms are ubiquitous in the environment, macaques are continuously exposed to them; therefore, although MAC is mostly associated with HIV- and SIV-induced immunosuppression, it should always be on the list of potential OIs in immunomodulatory drug safety studies.

Microscopically, MAC presents as focal to diffuse infiltrates of epithelioid macrophages containing abundant acid-fast bacilli in the lamina propria and submucosa of the gastrointestinal tract and in sinuses of lymph nodes, and as microgranulomas in the liver (and, rarely, other organs). The MAC lesion must be differentiated from that induced by R. equi, a soil-borne organism that can cause chronic pyogranulomatous pneumonia in foals and immunocompromised humans (Hondalus 1997). In foals and immunosuppressed macaques dissemination from the lung to the intestinal tract is common and, in the context of immunosuppressive SRV infection, R. equi infection can have a similar microscopic appearance to MAC.

EPEC infection has been observed in approximately 20% of normal healthy macaques, with diarrhea associated with SIV coinfection in both neonatal and adult macaques (Mansfield et al. 2001; Sestak et al 2003; Wachtman and Mansfield 2008).

Skin abrasions are common in NHPs and these should be closely monitored in drug safety studies with immunomodulatory drugs. Normal skin bacterial flora such as Streptococcus sp. and Staphylococcus sp. should be considered potentially pathogenic under these conditions as they are capable of invading lesions, resulting in abscesses and forming a nidus for septicemia.

Parasites

Malaria

Malaria is a widespread disease caused by protozoan organisms of the genus Plasmodium. In addition to affecting mammals, birds, and lizards in tropical and subtropical regions, malaria is a tremendous threat to human health, with hundreds of millions of individuals infected worldwide, and has emerged as a serious complication in human transplantation (Barsoum 2004). In enzootic areas, monkeys can be the sylvatic reservoir for human infection (Vythilingam et al. 2006); in the laboratory setting malaria is present in animals imported from areas where the disease is endemic, such as Asia, Africa, and South America (Le Bras et al. 1984).

The Plasmodium species of interest in natural infections of cynomolgus and rhesus macaques include P. coatneyi, P. fragile, P. knowlesi, P. inui, and P. cynomolgi. The species of monkey and Plasmodium spp. determine the individual disease outcome. For example, P. knowlesi infection is completely benign in M. iris, whereas it produces fatal infections in M. mulatta (Shadduck and Pakes 1978). The source of the primate also can influence susceptibility to Plasmodium spp.—differences have been observed between Mauritius and Philippine cynomolgus macaques (Leuchte et al. 2003; Migot-Nabias et al. 1999). Infections by multiple species of Plasmodium in a single monkey are common. Susceptibility and infection are enhanced by splenectomy and/or immunosupression and in juvenile animals. In addition, erythroid-stimulating agents, such as erythropoietin, have augmented multiplication of malarial parasites, resulting in lethal infection in murine models of malaria, but modulation of disease course has yet to be documented in NHPs (Chang et al. 2004).

The life cycle of the typical Plasmodium spp. involves a sexual development phase in the mosquito vector and asexual cycles in tissue cells and erythrocytes of the vertebrate host. The organisms produce their greatest damage to the host during schizogony in the blood, which may cause considerable destruction of red blood cells. The tissue stages are relatively harmless. In addition to red blood cell destruction, immunological perturbations may occur in lymph nodes and the spleen (immunoblast proliferation), and marked reticuloendothelial cell proliferation and increased phagocytosis are possible. Antibody-sensitized erythrocytes may cause sludging of blood in the microcirculation, leading to severe tissue hypoxia, ischemic cellular degeneration, and necrosis.

Investigators have developed and extensively studied several monkey models of human malarial conditions (Phillipp and Purcell 1995; Shadduck and Pakes 1978; Voller 1972), and have described the pathology of malaria both from natural infections and in treated monkeys (Chen et al. 2001). Both latent and active Plasmodium spp. infections may produce myriad immunologic effects and alterations in cytokines, thus it is in the best interest of scientific research to ensure that test animals are free from infection (Biswas 1999; Yang et al. 1999). In the authors' experience infections with Plasmodium spp. have had little impact in drug safety studies in immunocompetent animals. However, it is important to note that most of the macaques were from Mauritius, where malaria has been eliminated. With the increased use of NHPs from China and Southeast Asia where malaria is endemic, infection could affect drug safety studies, in particular those with immunomodulatory drugs or with erythroid-stimulating agents.

Metazoan Parasites

Although helminthic parasites—including species of Strongyloides, Oesophagostomum, Anatrichosoma, Trichostrongylus, and Gongylonema, and larval stages of nematodes (ascarids, spirurids), cestodes (hydatid, Sparganum), and pentastomids—are prevalent among wild-caught macaques, antihelminthic treatments have markedly reduced or eliminated these infestations (Karr et al. 1980; Sano et al. 1980; Wong and Conrad 1978). Strongyloides fulleborni is an exception: despite therapeutic and prophylactic use of ivermectin, researchers have reported an infection rate as high as 27% in domestic colonies of macaques and its presence is associated with cough, dermatitis, dyspnea, and diarrhea (Dufour et al. 2006; Eberhard 1981; Sestak et al. 2003). Interestingly, with a similar incidence to Strongyloides, Trichuris trichiura (whipworm) infection has not been associated with significant clinical disease, but foreign body granulomata have occasionally been observed (Drevon-Gaillot et al. 2006; Sestak et al. 2003).

Schistosomiasis is a prevalent and pathogenic disease of worldwide importance in humans and animals infected with Schistosoma spp. and has been observed in wild-caught macaques used for biomedical research. The species that are most common and have the greatest impact in humans and macaques are S. japonicum, S. mansoni, and S. hematobium, all of which have a wide geographical distribution, but the intermediate snail hosts needed to complete the life cycle are not present in the United States and so the cycle is broken. Adult schistosomes live in mesenteric and pelvic veins and cause little damage to the host, but the eggs released by females that fail to penetrate vessel walls in their attempt to reach the lumen of the intestine and bladder are responsible for most of the granulomatous inflammation associated with this disease (Cheever et al. 1974). Investigators have reported that juvenile rhesus macaques have reduced type 2 cytokine responses after primary schistosome infections, and humans infected with S. mansoni have an impaired antigen-specific Th1-type response after immunization with tetanus toxoid (Fallon et al. 2003; Sabin et al. 1996). Experimental coinfection of rhesus macaques with S. mansoni significantly increased simian-human immunodeficiency virus (SHIV) replication (Ayash-Rashkovsky et al. 2007). These studies indicate that altered immune responses associated with schistosomiasis could have a profound effect on drug safety studies and that the use of wild-caught animals is inadvisable particularly in studies with immune endpoints.

Opportunistic Parasites

Opportunistic parasitic infections secondary to retrovirus-induced immunosuppression include Cryptosporidium parvum, Enterocytozoon bieneusi, Plasmodium sp., Trichomonas sp., Acanthamoeba sp., and Toxoplasma gondii. Detailed descriptions are provided elsewhere in this issue (Gardner and Luciw 2008; Wachtman and Mansfield 2008).

Normal Flora

Numerous species of bacteria, viruses, and parasites are common, normal inhabitants of the macaque gastrointestinal tract. They vary in incidence and burden depending on the source of the macaque, its immune status, husbandry and housing practices, and diet. In general, because these organisms are observed in normal control or vehicle-treated animals, their presence in routine drug safety studies is seldom confused with a drug-related effect. However, an increased burden may be evident in a moribund animal or an animal with gastrointestinal inflammation or altered gastrointestinal transit time, and may be mistaken for a drug-related effect by an inexperienced evaluator. It is important to document increased parasite burdens, but as these organisms are rarely primary pathogens and their incidence varies considerably among animals, care must be taken not to overinterpret—the pathologist should reexamine all dose groups to determine whether any changes are due to an alteration of gut flora or may be a direct drug-related effect.

For instance, Balantidium coli, a large ciliated protozoan, is frequently observed in the lumen of the macaque cecum and colon, but despite differences in incidence (range: 13-75%) and parasitic load among cynomolgus macaques from Mauritius, Vietnam, and the Philippines, Balantidium is not associated with mucosal alteration (Drevon-Gaillot et al. 2006). In another survey from a domestic rhesus macaque colony the incidence was approximately 12% and not associated with diarrhea (Sestak et al. 2003). In the authors' experience, B. coli loads may be higher in animals with gastrointestinal disease, but this most likely represents a secondary effect to inflammation or alterations in normal gut transit times; unless there is a breach in the mucosal barrier, they are always in the gut lumen. Similarly, Trichomonas sp. may be present in the lumen or in crypts of the gastrointestinal tract of normal macaques and seldom elicits an inflammatory response, with invasive disease only rarely reported in association with SIV infection (Blanchard and Baskin 1988; Kondova et al. 2005).

Normal Flora and Immune Responses

Louis Pasteur first suggested that microorganisms may affect the development and/or function of the immune system, and since then the data have proven this theory (Hamann et al. 1998). The innate and the extremely sophisticated adaptive immune systems work synergistically together and comprise many cellular and humoral factors. Between the two arms of immunity there is great redundancy, which provides robust defenses (Cummings et al. 2004; Hamann et al. 1998).

Normal microflora are present on natural epithelial-lined body surfaces such as the skin and gastrointestinal tract and are exposed to the external environment (Tlaskalová-Hogenová et al. 2004). The large intestine is the single largest source of resident microflora (Tlaskalová-Hogenová et al. 2004). Over time the complex microbial flora of the healthy gastrointestinal (GI) tract are remarkably constant, as gastric acid, bile salts, normal motility, and mucosal IgA maintain the relative sterility of the upper portions (Marshall 1991). In the lower GI tract a complex mixture of microbes and gram-negative bacteria is a prerequisite for normal immunological development (Marshall 1991). Resident microflora contain a large number of components able to activate both innate and adaptive immunity and are therefore under strict regulatory control to avoid the risk of inflammation secondary to immune responses to commensal organisms (Tlaskalová-Hogenová et al. 2004).

The body's immune system has developed specialized regulatory, anti-inflammatory mechanisms to handle nondangerous environmental antigens and commensal microorganisms. However, the mucosal immune system also has unique innate and acquired defense mechanisms that provide a first line of protection against ingested infectious agents (Nochi and Kiyono 2006). These mechanisms ensure the integrity of the mucosal barrier and include the presence of unique types of lymphocytes and their products (including secretory IgA) and the migration and homing of cells in mucosal and glandular tissues (Tlaskalová-Hogenová et al. 2004).

The vital role of normal flora in the development of the immune system has been clearly demonstrated in germ-free animals (Tlaskalová-Hogenová et al. 2004), and studies with axenic mice have shown the same for the development of the intestinal IgA immune system (Moreau et al. 1982). A role for commensal microflora and their immunoactivating components such as lipopolysaccharides (LPS), superantigens, and bacterial DNA has been suggested in the pathogenesis of complex diseases such as inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, and colon cancer (Tlaskalová-Hogenová et al. 2004). Overgrowth of bacteria in the intestine, especially gram-negative bacteria, can result in the loss of mucosal integrity, suppression of immune responses, and altered Kupffer cell function (Marshall 1991).

Components of gram-negative bacterial cell walls such as peptidoglycan and LPS are potent activators of the immune system largely through the induction of endogenous mediators (Hamann et al. 1998). In some cases there are dramatic pathophysiological responses in the host (Hamann et al. 1998). Most dietary lectins are generally resistant to metabolism in the intestine and may be endocytosed by epithelial cells; in addition, some of them act as exogenous intestinal growth factors and can alter the bacterial flora and local hormone balance (Pusztai 1993). Lectins transported from the intestine into systemic circulation can further affect systemic hormone balance, metabolism, and health (Pusztai 1993).

Normal Flora and Drug Metabolism

The gastrointestinal tract is a complex ecosystem with a diverse and highly evolved microbial community of hundreds of different microbial species. Precise control of gastrointestinal microbiota relies on many factors, including salivary and gastric acid, biliary and pancreatic secretions, gastric and intestinal motility, local immunity, the glycocalyx and mucus layer, diet, and drugs (Batt et al. 1996). Interactions among the bacteria are also important, and may involve substrate depletion and production of bacteriocins that inhibit the growth of bacteria (Batt et al. 1996).

These normal flora represent a unique metabolic system distinct from the metabolic activity of the host's own cells (Mikov 1994). Like the body's metabolic systems, the metabolic potential of the microbial flora changes with state of health/disease, age, nutrition, and diet; differences in drug metabolism may also be linked to variations in gut flora metabolism between individuals and within the same individual over time (Mikov 1994). Local metabolic activity in the gut lumen and wall can decrease bioavailability and pharmacological activity of drugs, and factors such as the type and presence of bacteria, pH, and oxidative/conjugative enzymes all contribute to the process (Ilett et al. 1990). The cecum and colon, because of the large bacterial population, tend to have the greatest effect, while metabolism in the small intestine generally is highest in the jejunum and decreases distally (Ilett et al. 1990).

Experimental studies of the effects of diet/gut microflora on drug metabolism have mostly used rodents, but human clinical studies have shown that changes in diet can result in marked effects on drug metabolism. Although there is considerable individual variability, changing to a diet high in protein and low in carbohydrates increases the metabolism of antipyrine and theophylline, whereas shifting to an isocaloric low-protein/high-carbohydrate diet slows the metabolism of these drugs (Carr 1982). A high-protein/low-carbohydrate diet resembles that used in many animal studies to show enhanced hepatic drug metabolism (Carr 1982). Similar individual variability is seen in the response to enzyme induction by smoking (Carr 1982).

Numerous foods and food ingredients affect drug metabolism in humans and experimental animals with changes in the levels of cytochrome P450–dependent monooxygenases (Carr 1982). For example, researchers have described differences in gut microflora among vervet monkeys with different diets (Bruorton et al. 1991). The gut microflora in macaques used for drug development studies may play a significant role in the metabolism and pharmacokinetics of new drug candidates and almost certainly contributes to interanimal variability; however, at this time little is known about these effects. Thus, interpretation of drug metabolism and toxicity studies should include consideration of the role of microbial metabolism of the test article (Mikov 1994).

Selection and Screening of NHPs for Toxicology Studies

Because it is well established that macaques from China, Southeast Asia, Mauritius, and domestic sources are genetically diverse and have different background incidences of normal flora, pathogens, and inflammatory conditions, for consistency it is advisable to use macaques from one source. If this is not possible, it is preferable to use other select sources on a routine basis to gain baseline knowledge about each of them. Most importantly, it is essential to avoid mixing animals from multiple sources in a single study.

For general toxicity studies, animal selection should be based on a strict specific pathogen-free status, with screening to ensure that the animals are negative for primary pathogens including SIV, SRV, B virus, measles, HAV, and TB. There are colonies of SIV-, SRV-, and herpes B–free animals, and prophylactic vaccination of NHPs for HAV and measles has been effective. Fecal parasite evaluation and bacterial cultures along with prophylactic antibiotic and antihelminthic treatment are not mandatory, but they are useful in cases of sporadic diarrhea. For studies with immunomodulatory drugs, certain OIs such as Plasmodium spp., depending on the source of the animals, can be diagnosed via blood smear or PCR analysis and treated before study initiation. Other OIs that have a high prevalence (e.g., RRV, lymphocryptovirus, SV40, and cytomegalovirus) but are not treatable should be identified and documented with animals evenly distributed among dose treatment groups in a study.

Conclusion

In the routine conduct of drug safety studies, the toxicologic pathologist should be well versed in the types of normal flora, spontaneous infections, opportunistic pathogens, and background lesions in NHPs to appropriately identify possible drug-related effects. Such knowledge is particularly important in studies involving immunomodulatory drugs, so that the toxicologic pathologist can anticipate which type(s) of infections are most likely to arise depending on which arm of the immune system is modulated. Adequate knowledge of these conditions makes it possible to act prospectively by selecting specific pathogen-free animals before study initiation, or distributing specific pathogen-positive animals equally among all treatment groups.

In this review we have identified infections that have had the biggest impact on drug development, and we caution that many of them may resurface as alternative sources of NHPs are used to meet the increased demand for these animals in biomedical research.

Abbreviations used in this article: HAV, hepatitis A virus; MAC, Mycobacterium avium complex; NHP, nonhuman primate; OI, opportunistic infection; PML, progressive multifocal leukodystrophy; RRV, rhesus rhadinovirus; SIV, simian immunodeficiency virus; SRV, simian type D retrovirus; TB, tuberculosis

References

Andrade MR, Yee J, Barry P, Spinner A, Roberts JA, Cabello PH, Leite JP, Lerche NW. 2003. Prevalence of antibodies to selected viruses in a long-term closed breeding colony of rhesus macaques (Macaca mulatta) in Brazil. Am J Primatol 59:123-128.

Andzhaparidze AG, Poleshchuk VF, Zamiatina NA, Savinov AP, Gavrilovskaia IN. 1985. Spontaneous hepatitis in the crab-eating macaque exposed to immunodepressants. Vopr Virusol 4:468-474.

Ayash-Rashkovsky M, Chenine AL, Steele LN, Lee SJ, Song R, Ong H, Rasmussen RA, Hofmann-Lehmann R, Else JG, Augostini P, McClure HM, Secor WE, Ruprecht RM. 2007. Coinfection with Schistosoma mansoni reactivates viremia in rhesus macaques with chronic simian-human immunodeficiency virus clade C infection. Infect Immun 75:1751-1756.

Balayan MS. 1992. Natural hosts of hepatitis A virus. Vaccine 10(suppl 1):s27-s31.

Barsoum RS. 2004. Parasitic infections in human transplantation. Exp Clin Transplant 2:258-267.

Batt RM, Rutgers HC, Sancak AA. 1996. Enteric bacteria: Friend or foe? J Small Anim Pract 37:261-267.

Berger JR. 2006. Natalizumab. Drugs Today 42:639-655.

Berger JR, Koralnik IJ. 2005. Progressive multifocal leukoencephalopathy and natalizumab—unforeseen consequences. NEJM 353:414-416.

Biswas S. 1999. Patterns of parasitemia, autoantibodies, complement and circulating immune complexes in drug suppressed simian Plasmodium knowlesi malaria. Indian J Malariol 36:33-41.

Black-Schultz L, Coatney RW, Warnick CL, Swif B. 1997. Lack of reactivation of shigellosis in naturally infected enrofloxacin-treated cynomolgus monkeys after exogenous immunosuppression. Lab Anim Sci 47:602-605.

Blanchard JL, Baskin GB. 1988. Trichomonas gastritis in rhesus monkeys infected with the simian immunodeficiency virus. J Infect Dis 157:1092-1093.

Blancher A, Tisseyre P, Dutaur M, Apoil PA, Maurer C, Quesniaux V, Raulf F, Bigaud M, Abbal M. 2006. Study of cynomolgus monkey (Macaca fascicularis) MhcDRB (Mafa-DRB) polymorphism in two populations. Immunogenetics 58:269-282.

Bowers LC, Purcell JE, Plauché GB, Denoel PA, Lobet Y, Philipp MT. 2002. Assessment of the nasopharyngeal bacterial flora of rhesus macaques: Moraxella, Neisseria, haemophilus, and other genera. J Clin Microbiol 40: 4340-4342.

Brack K, Berk I, Magulski T, Lederer J, Dotzauer A, Vallbracht A. 2002. Hepatitis A virus inhibits cellular antiviral defense mechanisms induced by double-stranded RNA. J Virol 76:11920-11930.

Brown KE, Liu Z, Gallinella G, Wong S, Mills IP, O'Sullivan MG. 2004. Simian parvovirus infection: A potential zoonosis. J Infect Dis 190:1900-1907.

Brown Y. 1997. The simian parvoviruses. Rev Med Virol 7:211-218.

Bruorton MR, Davis CL, Perrin MR. 1991. Gut microflora of vervet and samango monkeys in relation to diet. Appl Environ Microbiol 57:573-578.

Burke DS, Heisey GB. 1984. Wild Malaysian cynomolgus monkeys are exposed to hepatitis A virus. Am J Trop Med Hyg 33:940-944.

Caillard S, Dharnidharka V, Agodoa L, Bohen E, Abbott K. 2005. Posttransplant lymphoproliferative disorders after renal transplantation in the United States in era of modern immunosuppression. Transplantation 80:1233-1243.

Carlton WW, Hunt RD. 1978. Bacterial diseases. In: Benirschke K, Garner FM, Jones TC, eds. Pathology of Laboratory Animals. New York: Springer-Verlag. p 1418-1427.

Carr CJ. 1982. Food and drug interactions. Ann Rev Pharmacol Toxicol 22:19-29.

Cavagnaro JA. 2002. Preclinical safety evaluation of biotechnology-derived pharmaceuticals. Nat Rev Drug Discov 1:469-475.

Chang KH, Tam M, Stevenson MM. 2004. Modulation of the course and outcome of blood-stage malaria by erythropoietin-induced reticulocytosis. J Infect Dis 189:735-743.

Chapman K, Pullen N, Graham M, Ragan I. 2007. Preclinical safety testing of monoclonal antibodies: The significance of species relevance. Nat Rev Drug Discov 6:120-126.

Cheever AW, Erickson DG, Sadun EH, von Lichtenberg F. 1974. Schistosoma japonicum infection in monkeys and baboons: Parasitological and pathological findings. Am J Trop Med Hyg 23:51-64.

Chellman GJ, Lukas VS, Eugui EM, Altera KP, Almquist SJ, Hilliard JK. 1992. Activation of B virus (Herpesvirus simiae) in chronically immunosuppressed cynomolgus monkeys. Lab Anim Sci 42:146-151.

Chen L, Li G, Lu Y, Luo Z. 2001. Histopathological changes of Macaca mulatta infected with Plasmodium knowlesi. Chin Med J 114:1073-1077.

Choi YK, Simon MA, Kim DY, Yoon BI, Kwon SW, Lee KW, Seo IB, Kim DY. 1999. Fatal measles virus infection in Japanese macaques (Macaca fuscata). Vet Pathol 36:594-600.

Cummings JH, Antoine JM, Azpiroz F, Bourdet-Sicard R, Brandtzaeg P, Calder PC, Gibson GR, Guarner F, Isolauri E, Pannemans D, Shortt C, Tuijtelaars S, Watzl B. 2004. PASSCLAIM—gut health and immunity. Eur J Nutr 43 (Suppl 2):II118-II173.

Daniel MD, Letvin NL, King NW, Kannagi M, Seghal PK, Hunt RD, Kanki P, Essex M, Desrosiers RC. 1985. Isolation of a T-cell tropic HTLV-III-like retrovirus from macaques. Science 228:1201-1204.

Desrosiers RC, Sasseville VG, Czajak SC, Zhang X, Mansfield KG, Kaur A, Johnson RP, Lackner AA, Jung JU. 1997. A herpesvirus of rhesus monkeys related to the human Kaposi's sarcoma-associated herpesvirus. J Virol 71:9764-9769.

Drevon-Gaillot E, Perron-Lepage M-F, Clement C, Burnett R. 2006. A review of background findings in cynomolgus monkeys (Macaca fascicularis) from three different geographical origins. Exp Toxicol Pathol 58:77-88.

Dufour JP, Cogswell FB, Phillippi-Falkenstein KM, Bohm RP. 2006. Comparison of efficacy of moxidectin and ivermectin in the treatment of Strongyloides fulleborni infection in rhesus macaques. J Med Primatol 35:172-176.

Eberhard ML. 1981. Intestinal parasitism in an outdoor breeding colony of Macaca mulatta. Lab Anim Sci 31:282-285.

El Mubarak HS, Yüksel S, van Amerongen G, Mulder PG, Mukhtar MM, Osterhaus AD, de Swart RL. 2007. Infection of cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) with different wild-type measles viruses. J Gen Virol 88(Pt 7):2028-2034.

Fallon PG, Gibbons J, Vervenne RA, Richardson EJ, Fulford AJ, Kiarie S, Sturrock RF, Coulson PS, Deelder AM, Langermans JA, Thomas AW, Dunne DW. 2003. Juvenile rhesus monkeys have lower type 2 cytokine responses than adults after primary infection with Schistosoma mansoni. J Infect Dis 187:939-945.

Gardner MB, Luciw PA. 2008. Macaque models of human infectious disease. ILAR J 49:220-255.

Green JD, Black LE. 2000. Status of preclinical safety assessment for immunomodulatory biopharmaceuticals. Hum Exp Toxicol 19:208-212.

Guzman RE, Kerlin RL, Zimmerman TE. 1999. Histologic lesions in cynomolgus monkeys (Macaca fascicularis) naturally infected with simian retrovirus type D: Comparison of seropositive, virus-positive, and uninfected animals. Toxicol Pathol 27:672-677.

Hamann L, El-Samalouti V, Ulmer AJ, Flad HD, Rietschel ET. 1998. Components of gut bacteria as immunomodulators. Int J Food Microbiol 26:141-154.

Henrickson RV, Maul DH, Osborn KG, Sever JL, Madden DL, Ellingsworth LR, Anderson JH, Lowenstine LJ, Gardner MB. 1983. Epidemic of acquired immunodeficiency in rhesus monkeys. Lancet 1:388-390.

Hondalus MK. 1997. Pathogenesis and virulence of Rhodococcus equi. Vet Microbiol 56:257-268.

Huff JL, Barry PA. 2003. B-virus (Cercopithecine herpesvirus 1) infection in humans and macaques: Potential for zoonotic disease. Emerg Infect Dis 9:246-250.

Hutto D, TenHoor C, Lane J, Bernier L, Quander R, Green J. 2003. B cell hyperplasia associated with immunosuppression in cynomolgus monkeys. Vet Pathol 40:624.

ICH [International Conference on Harmonization]. 1997a. Guidance for Industry: S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. Geneva: ICH.

ICH [International Conference on Harmonization]. 1997b. Guidance for Industry: M3 Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals. Geneva: ICH.

Ilett KF, Tee LB, Reeves PT, Minchin RF. 1990. Metabolism of drugs and other xenobiotics in the gut lumen and wall. Pharmacol Ther 46:67-93.

Kalashnikova VA, Dzhikidze EK, Stasilevich ZK, Chikobava MG. 2002. Detection of Campylobacter jejuni in healthy monkeys and monkeys with enteric infections by PCR. Bull Exp Biol Med 134:299-300.

Kalashnikova VA, Dzhikidze EK, Stasilevich ZK, Krylova RI, Kebu TI. 2006. Campylobacter in the etiology of acute intestinal infections in primates. Vestn Ross Akad Med Nauk 1:6-10.

Karr SL Jr, Henrickson RV, Else JG. 1980. A survey for intestinal helminths in recently wild-caught Macaca mulatta and results of treatment with mebendazole and thiabendazole. J Med Primatol 9:200-204.

Kolappaswamy K, Mahalingam R, Traina-Dorge V, Shipley ST, Gilden DH, Kleinschmidt-Demasters BK, McLeod CG, Hungerford LL, DeTolla LJ. 2007. Disseminated simian varicella virus infection in an irradiated rhesus macaque (Macaca mulatta). J Virol 81:411-415.

Kondova I, Simon MA, Klumpp SA, MacKey J, Widmer G, Domingues HG, Persengiev SP, O'Neil SP. 2005. Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus. Vet Pathol 42:19-29.

Kwang HS, Pedersen NC, Lerche NW, Osborn KG, Marx PA, Gardner MB. 1987. Viremia, antigenemia, and serum antibodies in rhesus macaques infected with simian retrovirus type 1 and their relationship to disease course. Lab Invest 56:591-597.

Lankas GR, Jensen RD. 1987. Evidence of hepatitis A infection in immature rhesus monkeys. Vet Pathol 24:340-344.

Lapin BA, Shevtsova ZV. 1990. Persistence of spontaneous and experimental hepatitis A in rhesus macaques. Exp Pathol 39:59-60.

Le Bras JN, Larouze B, Geniteau M, Andrieu B, Dazza MC, Rodhain F. 1984. Malaria, arbovirus and hepatitis infections in Macaca fascicularis from Malaysia. Lab Anim 18:61-64.

Lederer I, Much P, Allerberger F, Voracek T, Vielgrader H. 2005. Outbreak of shigellosis in the Vienna Zoo affecting human and non-human primates. Int J Inf Dis 9:290-291.

Lemon SM, LeDuc JW, Binn LN, Escajadillo A, Ishak KG. 1982. Transmission of hepatitis A virus among recently captured Panamanian owl monkeys. J Med Virol 10:25-36.

Lerche NW, Osborn KG. 2003. Simian retrovirus infections: Potential confounding variables in primate toxicology studies. Toxicol Pathol 31(suppl):103-110.

Lerche NW, Yee YL, Jennings MB. 1994. Establishing specific retrovirus-free breeding colonies of macaques: An approach to primary screening and surveillance. Lab Anim Sci 44:217-221.

Leuchte N, Berry N, Kohler B, Almond N, LeGrand R, Thorstensson R, Titti F, Sauermann U. 2003. MhcDRB-sequences from cynomolgus macaques (Macaca fascicularis) of different origin. Tissue Antigens 63:529-537.

Mansfield K. 2003. Marmoset models commonly used in biomedical research. Comp Med 53:383-392.

Mansfield KG, Lackner AA. 1997. Simian immunodeficiency virus-inoculated macaques acquire Mycobacterium avium from potable water during AIDS. J Infect Dis 175:184-187.

Mansfield KG, Pauley D, Young HL, Lackner AA. 1995. Mycobacterium avium complex in macaques with AIDS is associated with a specific strain of simian immunodeficiency virus and prolonged survival after primary infection. J Infect Dis 172:1149-1152.

Mansfield KG, Westmoreland SV, DeBakker CD, Czajak S, Lackner AA, Desrosiers RC. 1999. Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses. J Virol 73:10320-10328.

Mansfield KG, Lin KC, Newman J, Schauer D, MacKey J, Lackner AA, Carville A. 2001. Identification of enteropathogenic Escherichia coli in simian immunodeficiency virus-infected infant and adult rhesus macaques. J Clin Microbiol 39:971-976.

Marshall JC. 1991. The ecology and immunology of the gastrointestinal tract in health and critical illness. J Hosp Infect 19 Suppl C:7-17.

Martin BJ, Dysko RC, Chrisp CE. 1991. Pancreatitis associated with simian adenovirus 23 in a rhesus monkey. Lab Anim Sci 41:382-384.

McInnes EF, Jarrett RF, Langford G, Atkinson C, Horsley J, Goddard MJ, Cozzi E, Schuurman HJ. 2002. Posttransplant lymphoproliferative disorder associated with primate gamma-herpesvirus in cynomolgus monkeys used in pig-to-primate renal xenotransplantation and primate renal allotransplantation. Transplantation 73:44-52.

Migot-Nabias F, Ollomo B, Dubreuil G, Morelli A, Domarle O, Nabias R, Georges AJ, Millet P. 1999. Plasmodium coatneyi: Differential clinical and immune responses of two populations of Macaca fascicularis from different origins. Exp Parasitol 91:30-39.

Mikov M. 1994. The metabolism of drugs by the gut flora. Eur J Drug Metab Ph 19:201-207.

Moghaddam A, Rosenzweig M, Lee-Parritz D, Annis B, Johnson RP, Wang F. 1997. An animal model for acute and persistent Epstein-Barr virus infection. Science 276:2030-2033.

Moreau MC, Raibaud P, Muller MC. 1982. Relationship between the development of the intestinal IgA immune system and the establishment of microbial flora in the digestive tract of young holoxenic mice. Ann Immunol (Paris) 133D:29-39.

Nakamura M, Murayama SY, Serizawa H, Sekiya Y, Eguchi M, Takahashi S, Nishikawa K, Takahashi T, Matsumoto T, Yamada H, Hibi T, Tsuchimoto K, Matsui H. 2007. Candidatus Helicobacter heilmannii from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice. Infect Immun 75:1214-1222.

Niemann TH, Trigg ME, Winick N, Penick GD. 1993. Disseminated adenoviral infection presenting as acute pancreatitis. Hum Pathol 24:1145-1148.

Nochi T, Kiyono H. 2006. Innate immunity in the mucosal immune system. Curr Pharm Des 12:4203-4213.

Norin S, Kimby E, Ericzon BG, Christensson B, Sander B, Söderdahl G, Hägglund H. 2004. Posttransplant lymphoma: A single-center experience of 500 liver transplantations. Med Oncol 21:273-284.

O'Sullivan MG, Anderson DK, Lund JE, Brown WP, Green SW, Young NS, Brown KE. 1996. Clinical and epidemiological features of simian parvovirus infection in cynomolgus macaques with severe anemia. Lab Anim Sci 46:291-297.

Patterson JL, Carrion R. 2005. Demand for nonhuman primate resources in the age of biodefense. ILAR J 46:15-22.

Phillipp MT, Purcell JE. 1995. Modeling parasitic diseases in nonhuman primates: Malaria, Chagas disease, and filariasis. In: Wolfe-Coote S, ed. The Laboratory Primate. San Diego: Elsevier. p 99-104.

Poelma FG, Borst GH, Zwart P. 1977. Yersinia enterocolitica infections in non-human primates. Acta Zool Pathol Antverp 69:3-9.

Pusztai A. 1993. Dietary lectins are metabolic signals for the gut and modulate immune and hormone functions. Eur J Clin Nutr 47:691-699.

Reindel JF, Fitzgerald AL, Breider MA, Gough AW, Yan C, Mysore JV, Dubois A. 1999. An epizootic of lymphoplasmacytic gastritis attributed to Helicobacter pylori infection in cynomolgus monkeys (Macaca fascicularis). Vet Pathol 36:1-13.

Rivailler P, Carville A, Kaur A, Rao P, Quink C, Kutok JL, Westmoreland S, Klumpp S, Simon M, Aster JC, Wang F. 2004. Experimental rhesus lymphocryptovirus infection in immunosuppressed macaques: An animal model for Epstein-Barr virus pathogenesis in the immunosuppressed host. Blood 104:1482-1489.

Roberts JA, Andrews K. 2008. Nonhuman primate quarantine: Its evolution and practice. ILAR J 49:145-156.

Rubio CA, Hubbard GB. 2002. Chronic colitis in Macaca fascicularis: Similarities with chronic colitis in humans. In Vivo 16:191-195.

Sabin EA, Araujo MI, Carvalho EM, Pearce EJ. 1996. Impairment of tetanus toxoid-specific Th1-like immune responses in humans infected with Schistosoma mansoni. J Infect Dis 173:269-272

Sano M, Kino H, de Guzman TS, Ishii AI, Kino J, Tanaka T, Tsuruta M. 1980. Studies on the examination of imported laboratory monkey, Macaca fascicularis for E. histolytica and other intestinal parasites. Int J Zoonoses 7:34-39.

Sariol CA, González-Martínez J, Arana T, Gascot S, Suárez E, Maldonado E, Gerald MS, Rodríguez M, Kraiselburd EN. 2006. Differential distribution of antibodies to different viruses in young animals in the free-ranging rhesus macaques of Cayo Santiago. J Med Primatol 35:369-375.

Sasseville VG, Desrosiers RC, Morrison HG. 1999. Simian immunodeficiency viruses (retroviridae). In: Webster R, Granoff A, eds. Encyclopedia of Virology. London: Academic Press. p 1640-1647.

Schroder C, Pfeiffer S, Wu G, Azimzadeh AM, Aber A, Pierson RN, O'Sullivan MG. 2006. Simian parvovirus infection in cynomolgus monkey heart transplant recipients causes death related to severe anemia. Transplantation 81:1165-1170.

Sestak K, Merritt CK, Borda J, Saylor E, Schwamberger SR, Cogswell F, Didier ES, Didier PJ, Plauche G, Bohm RP, Aye PP, Alexa P, Ward RL, Lackner AA. 2003. Infectious agent and immune response characteristics of chronic enterocolitis in captive rhesus macaques. Infec Immun 71:4079-4086.

Shadduck JA, Pakes SP. 1978. Protozoal and metazoal diseases. In: Benirschke K, Garner FM, Jones TC, eds. Pathology of Laboratory Animals. New York: Springer-Verlag. p 1610-1612.

Shevtsova ZV, Krylova RI, Belova EG, Korzaia LI, Andzhaparidze AG. 1987. Spontaneous hepatitis A with a fatal outcome in rhesus monkeys. Vopr Virusol 32:686-690.

Simon MA, Ilyinskii PO, Baskin GB, Knight HY, Pauley DR, Lackner AA. 1999. Association of simian virus 40 with a central nervous system lesion distinct from progressive multifocal leukoencephalopathy in macaques with AIDS. Am J Pathol 154:437-446.

Slighter RG, Kimball JP, Barbolt TA, Sherer AD, Drobeck HP. 1988. Enzootic hepatitis A infection in cynomolgus monkeys (Macaca fascicularis). Am J Primatol 14:73-81.

Smith D, Trennery P, Farningham D, Klapwijk J. 2001. The selection of marmoset monkeys (Callithrix jacchus) in pharmaceutical toxicology. Lab Anim 35:117-130.

Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, Tucková L, Cukrowska B, Lodinová-Zádníková R, Kozáková H, Rossmann P, Bártová J, Sokol D, Funda DP, Borovská D, Reháková Z, Sinkora J, Hofman J, Drastich P, Kokesová A. 2004. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett 93:97-108.

Traina-Dorge VL, Lorino R, Gormus BJ, Metzger M, Telfer P, Richardson D, Robertson DL, Marx PA, Apetrei C. 2005. Molecular epidemiology of simian T-cell lymphotropic virus type 1 in wild and captive sooty mangabeys. J Virol 79:2541-2548.

T-W-Fiennes RN. 1972. Tuberculosis. In: T-W-Fiennes RN, ed. Pathology of Simian Primates, Part II. Basel: S Karger. p 314-334.

VandeWoude SJ, Luzarraga MB. 1991. The role of Branhamella catarrhalis in the 'bloody-nose syndrome' of cynomolgus macaques. Lab Anim Sci 41:401-406.

Vervenne RA, Jones SL, van Soolingen D, van der Laan T, Andersen P, Heidt PJ, Thomas AW, Langermans JA. 2004. TB diagnosis in non-human primates: Comparison of two interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection. Vet Immunol Immunop 100:61-71.

Voller A. 1972. Plasmodium and hepatocystis. In: T-W-Fiennes RN, ed. Pathology of Simian Primates, Part II. Basel: S Karger. p 57-73.

Vore SJ, Peele PD, Barrow PA, Bradfield JF, Pryor WH Jr. 2001. A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric Yersinia. J Med Primatol 30:20-25.

Vythilingam I, Tan CH, Asmad M, Chan ST, Lee KS, Singh B. 2006. Natural transmission of Plasmodium knowlesi to humans by Anopheles latens in Sarawak, Malaysia. Trans R Soc Trop Med Hyg 100:1087-1088.

Wachtman LM, Mansfield KG. 2008. Opportunistic infections in immunologically compromised nonhuman primates. ILAR J 49:191-208.

Willy ME, Woodward RA, Thornton VB, Wolff AV, Flynn BM, Heath JL, Villamarzo YS, Smith S, Bellini WJ, Rota PA. 1999. Management of a measles outbreak among Old World nonhuman primates. Lab Anim Sci 49:42-48.

Wolfensohn S. 1998. Shigella infection in macaque colonies: Case report of an eradication and control program. Lab Anim Sci 48:330-333.

Wong MM, Conrad HD. 1978. Prevalence of metazoan parasite infections in five species of Asian macaques. Lab Anim Sci 28:412-416.

Yang C, Xiao L, Tongren JE, Sullivan J, Lal AA, Collins WE. 1999. Cytokine production in rhesus monkeys infected with Plasmodium coatneyi. Am J Trop Med Hyg 61:226-229.