Overview of known non-human primate pathogens with potential to affect colonies used for toxicity testing

Abstract

The increased demand for non-human primates (NHPs) in biomedical research has resulted in alternative sources of animals being used, which has allowed for importation of animals with varying background incidences of bacterial, viral, parasitic, and fungal pathogens. This can be of minimal consequence when animals from different sources are kept isolated. However, when NHPs from different sources with varying incidences of primary and opportunistic pathogens are mixed, there can be a rapid spread of these pathogens and an increase in the seroconversion of susceptible animals. If this process occurs during the conduct of a study, interpretation of that study can be confounded. Furthermore, NHPs imported from areas enzootic for pathogens such as Plasmodium or with high incidences of human diseases such as measles and tuberculosis can introduce diseases that can be a threat to colony health, have zoonotic risk, and can severely impact study outcome. Thus, knowledge of the common primary and opportunistic NHP infections, as well as reemerging pathogens, enables the toxicologist to use information on disease status for pre-study animal selection and intelligent study design. This is particularly important when immunomodulatory compounds are being investigated. Moreover, the toxicologic pathologist well versed in the common spontaneous infections, opportunistic pathogens, and background lesions in NHPs is able to assess possible drug-related effects in drug safety studies. This review identifies the common primary and opportunistic pathogens, as well as newly emerging infections of NHPs, that can directly or indirectly affect colony health and the interpretation of drug safety studies.

Keywords::

• Non-human primate
• Macaca fascicularis
• Macaca mulatta
• opportunistic infections
• toxicology

Introduction

The use of non-human primates (NHPs) has increased in recent years, primarily due to the increase in the development of biopharmaceutical and immunomodulatory agents and of biodefense studies (Patterson and Carrion, 2005; Sasseville and Diters, 2008). To predict potential toxicities in humans, pre-clinical toxicology studies with biopharmaceuticals need to be conducted in a relevant animal species (i.e., a species in which the biopharmaceutical is pharmacologically active) (Cavagnaro, 2002; Bussiere, 2008). The high specificity of many biopharmaceuticals means that most of these pre-clinical studies use NHPs instead of the industry-standard, purpose-bred beagle dogs. This decision is driven by target-specific cross-reactivity, relevant pharmacology, similar immune systems, and similar pharmacokinetics (Chapman et al., 2007). Moreover, even when the biopharmaceutical is pharmacologically active in the rat and dog, the drug candidate may be so immunogenic in these species that longer-duration studies are impractical due to the generation of neutralizing antibodies (Cavagnaro, 2002; Bussiere, 2008). Thus, the cynomolgus macaque (Macaca fascicularis) and, to a lesser extent, the rhesus macaque (M. mulatta) are most commonly used for biopharmaceutical development. Although other NHP species have been used in drug development, such studies in the chimpanzee (Pan troglodytes) are primarily limited to rare pharmacokinetic studies or efficacy studies in which there are no other suitable animal models of human disease (e.g., hepatitis C virus [HCV]), and occasional pharmacology/toxicology studies in the common marmoset (Callithrix jacchus) (Lowenstine, 2003; Mansfield, 2003; Wong et al., 2004; Qian et al., 2007; Carroll et al., 2009). Thus, this review will focus on cynomolgus and rhesus macaques.

Up until the 1990s, macaques used in pre-clinical studies were wild-caught and were imported with numerous bacterial, viral, parasitic, fungal, and inflammatory diseases. These infections had the potential to affect adversely the interpretation of drug-related findings. In recent years, changes have been made in the husbandry of macaques maintained in research facilities, such that animals are routinely housed under controlled conditions and provided with complete nutrition, shelter from the elements, environmental enrichment, and restricted access to infected animals (Wachtman and Mansfield, 2008). However, the increased demand for macaques has resulted in a shortage of these animals for use in biomedical research (Patterson and Carrion, 2005), and other sources, such as China and Southeast Asia, are being used. These new sources have introduced genetic diversity and varying background incidences of bacterial, viral, parasitic, and fungal infections as well as inflammatory conditions (Leuchte et al., 2003; Blancher et al., 2006; Drevon-Gaillot et al., 2006).

The importation of macaques with varying background incidences of primary pathogens (any bacterial, viral, parasitic, or fungal agent capable of causing disease in the immunocompetent host) and opportunistic pathogens (any bacterial, viral, parasitic, or fungal agent usually not clinically apparent in the immunocompetent host but capable of causing disease in the immunocompromised host) can be of minimal consequence when animals from different sources are kept isolated. However, when macaques from different sources with varying incidences of pathogens are mixed, there can be a rapid spread of these pathogens to susceptible animals. If this process occurs during the conduct of a study, in-life and microscopic changes in recently infected animals can significantly confound the interpretation of that study. Furthermore, NHPs imported from areas enzootic for pathogens such as Plasmodium spp. (species of protozoal parasite that can cause malaria) or with higher incidences of human diseases such as measles and tuberculosis can introduce diseases that can be a threat to colony health, have zoonotic risk, and can severely impact study outcome. This is particularly important when immunomodulatory agents result in immunodeficient states that provide an opportunity for development of opportunistic infections.

The specific opportunistic pathogens that may emerge vary depending on the source of the macaque and which specific arm of the immune system, humoral or cell-mediated immunity, is affected. For example, in experimental simian immunodeficiency virus (SIV) infections, viral, fungal, and parasitic opportunistic pathogens and γ-herpesvirus-induced lymphoid hyperplasia and lymphomas are more common than bacterial pathogens (with the exception of higher-order bacteria such as Mycobacterium spp.). In contrast, simian type D retrovirus (SRV) and measles virus infections impair both humoral and cell-mediated immune responses, and thus, in addition to the list of opportunistic pathogens encountered with SIV-induced immunosuppression, numerous primary and opportunistic bacterial infections are also observed (Osborn et al., 1984; Maul et al., 1986; Choi et al., 1999). T-Lymphocyte-depleting biopharmaceuticals in macaques and humans have been associated with numerous viral and fungal opportunistic infections and with γ-herpesvirus-induced post-transplantation lymphoproliferative disease (van Gorder et al., 1999; Hutto et al., 2003; Norin et al., 2004; Caillard et al., 2005; Haustein et al., 2008; Maki et al., 2008). Thus, on the basis of the mechanism of action of selective immunomodulatory agents, a knowledgeable investigator should be able to predict what type(s) of primary or opportunistic agent(s) may arise at pharmacologic or suprapharmacologic doses of an immunomodulatory agent used for an efficacy or toxicology study.

This review identifies the common primary and opportunistic viral, bacterial, parasitic, and fungal infections as well as newly emerging infections of macaques that can directly or indirectly affect the interpretation of drug safety studies (Table 1).

Table 1.  Common primary and opportunistic viral, bacterial, parasitic, and fungal infections in macaque monkeys.

Primary infections	Opportunistic infections
Viral
Measles virus Hepatitis A virus Cercopithecine herpesvirus 1: Simian type D retroviruses (SRVs) Simian immunodeficiency virus (SIV) Simian T-lymphotropic viruses (STLVs)	Adenovirus Papillomavirus Simian virus 40 Cytomegalovirus Simian parvovirus Lymphocryptovirus Rhesus rhadinovirus Simian varicella virus
Bacterial
Shigella flexneri Helicobacter spp. Campylobacter coli Moraxella catarrhalis Campylobacter jejuni Yersinia enterocolitica Mycobacterium tuberculosis	Rhodococcus equi Mycobacterium avium complex Enteropathogenic Escherichia coli
Parasitic
Sarcocystis spp. Schistosoma spp. Trichuris trichiura Strongyloides fuelleborni	Trichomonas spp. Balantidium coli Plasmodium spp. Acanthamoeba spp. Toxoplasma gondii Cryptosporidium parvum Enterocytozoon bieneusi
Fungal
	Candida albicans Pneumocystis carinii Aspergillus fumigatus Histoplasma capsulatum Cryptococcus neoformans

Immunosuppressive viruses

Retroviruses

Simian type D retroviruses (genus Betaretrovirus, family Retroviridae) affect both humoral and cell-mediated immune responses, which contributes to a plethora of SRV-associated opportunistic infections (Osborn et al., 1984; Maul et al., 1986). SRV 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 PCR techniques, helped to establish SRV-free colonies (Lerche et al., 1994; Guzman et al., 1999).

Simian immunodeficiency viruses (genus Lentivirus, family Retroviridae) are among the most thoroughly studied viruses contributing to the development of opportunistic infections (Letvin and King, 1990; Hirsch and Lifson, 2000; Stump and VandeWoude, 2007). In contrast to SRV, macaques do not harbor SIV in their native Asian habitat. SIV strains naturally infect a large number of African macaque species and are usually asymptomatic in these primate species, whereas experimental infection in Asian macaques results in a lentiviral inflammatory disease and an immunodeficiency syndrome remarkably similar to human immunodeficiency virus (HIV) in humans (Daniel et al., 1985; Hirsch et al., 1995).

Simian T-lymphotropic viruses (STLVs) are members of the primate T-lymphotropic virus group of related retroviruses (STLV-2, STLV type L, STLV-3) and their human T-cell-lymphotropic virus counterparts, which share common morphological, antigenic, biological, and genetic features (Traina-Dorge et al., 2005). STLV is endemic in many wild and captive African and Asian monkeys and apes, and 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). Efficient screening practices have resulted in STLV-free colonies for biomedical research (Lerche et al., 1994; Lerche and Osborn, 2003).

Retroviral infections are described in detail elsewhere in this issue (Lerche, 2009).

Measles virus

Measles virus (genus Morbillivirus, family Paramyxoviridae) infection typically spreads to NHPs from infected human handlers, and the virus can spread rapidly within the NHP colony by the aerosol route (Willy et al., 1999). Infection in macaques is usually asymptomatic or mild, with fever, conjunctivitis, upper respiratory symptoms, and a maculopapular exanthema frequently observed (Auwaerter et al., 1999; Choi et al., 1999; Willy et al., 1999; El Mubarak et al., 2007). Acute measles infection in humans results in transient immunosuppression secondary to depletion of infected and non-infected B-lymphocytes, CD4⁺ and CD8⁺ T-lymphocytes, neutrophils, and monocytes, accompanied by a compensatory increase in numbers and activity of natural killer cells (Okada et al., 2000). Various immune dysfunctions have been linked with human measles virus–associated immunosuppression, including reduced delayed-type hypersensitivity responses, altered interleukin (IL)-12 levels, T-lymphocyte and dendritic cell functional deficits, decreased immunoglobulin production, and inhibition of interferon-γ up-regulation of the MHC-II antigen (Kerdiles et al., 2006). Similarly, in macaques, marked lymphoid depletion has been observed in lymphoid tissues, including thymus, lymph nodes, and spleen, with immunosuppression persisting for up to 6 months post-infection (McChesney et al., 1989; Wachtman and Mansfield, 2008). Opportunistic infections secondary to measles virus–induced immunosuppression include disseminated cytomegalovirus (CMV, Macacine herpesvirus 3), adenoviral and bacterial pneumonia, and candidiasis (Choi et al., 1999). An important consideration for animal husbandry is that immune dysfunction as a result of measles virus is also known to interfere with the response to Old World mammalian tuberculin (Staley et al., 1995). Although animals exposed to measles have lifelong immunity and vaccination can be effective in the prevention of disease, outbreaks from inadequate vaccination and husbandry practices continue to arise within the United States, with profound effects on toxicology study timelines and outcomes. Adequacy of vaccination programs can be assessed periodically using serologic assays.

Hepatitis A virus (infectious hepatitis)

Simian hepatitis A viruses (HAVs, genus Picornavirus, family Picornaviridae) are small RNA viruses that spread by the fecal–oral route. Simian HAV can infect a variety of Old World NHPs, including the great apes; cynomolgus, rhesus, and stump-tailed macaques; and baboons (Balayan, 1992). The prevalence of HAV in NHPs is quite variable and depends on the source of animals and husbandry practices (Balayan, 1992; Andrade et al., 2003). Antigenically, these viruses are related to human HAV yet have a significant degree of genomic heterogeneity, with four distinct simian HAVs differing not only from each other but also from all human HAV strains (Balayan, 1992). Natural cross-species transmission is primarily limited to chimpanzees and humans, although multiple species of Old World and New World monkeys can be experimentally infected (Balayan, 1992; Robertson, 2001). In macaques, natural HAV infection is usually mild or subclinical, with rare instances of severe disease and mortality (Le Bras et al., 1984; Shevtsova et al., 1987; Balayan, 1992; Lankas and Jensen, 1987). Active HAV infection can result in elevated levels of serum hepatic enzymes, alanine aminotransferase (ALT), and aspartate aminotransferase, with histopathological changes of increased periportal mononuclear cell infiltration (Slighter et al., 1988). There is one documented case, and presumably there are many other unreported cases, of active HAV infection in cynomolgus monkeys during the course of a toxicology study, in which elevations of ALT along with chronic periportal inflammation could have substantially compromised the interpretive value of the study (Slighter et al., 1988). This complication can be markedly reduced by the prophylactic HAV vaccination of NHPs used for toxicology studies.

Opportunistic viral infections

Macaques infected with immunosuppressive retroviruses, experimentally-irradiated, or administered certain immunomodulatory or chemotherapeutic agents can develop a variety of opportunistic infections, such as adenovirus, simian virus 40 (SV40), and various simian herpesviruses, including CMV, rhesus rhadinovirus, and lymphocryptovirus (Daniel et al., 1985; Desrosiers et al., 1997; Moghaddam et al., 1997; Haustein et al., 2008; Wachtman and Mansfield, 2008). Although adenovirus, SV40, CMV, rhesus rhadinovirus, and lymphocryptovirus all have a high natural seroprevalence in adult macaques, the specific type of viral opportunistic infection encountered is primarily dependent upon the source of the macaque and the degree and type of immunomodulation. Less frequently observed opportunistic infections in young or immunosuppressed macaques include Cercopithecine herpesvirus 9 (simian varicella virus), Macacine herpesvirus 1 (B virus), papillomavirus, and simian parvovirus (SPV; Chellman et al., 1992; O’Sullivan et al., 1996; Gordon et al., 2000; Wood et al., 2004; Patterson et al., 2005; Kolappaswamy et al., 2007; Schoeb et al., 2008). Herpesvirus Herpesvirus infections are described in detail elsewhere in this issue (Simmons, 2009).

Adenovirus

Numerous serotypes of adenoviruses have been isolated from macaques. The virus is readily transmitted by aerosolization or the fecal–oral route. The prevalence of exposure in macaques varies by serotype and the individual colony under evaluation.

In most cases infection is asymptomatic, although enteritis and, rarely, fatal pneumonia have been observed in young animals (Boyce et al., 1978; Stuker et al., 1979; Sestak et al., 2003), whereas disease in immunodeficient animals may be prolonged and severe. Lesions in the liver, gastrointestinal tract, lung, and pancreas consist of epithelial cell necrosis with large basophilic intranuclear inclusions (Baskin and Soike, 1989; Ochs et al., 1991). These inclusions must be distinguished from those associated with CMV, herpes B virus, and SV40. Necrohemorrhagic tubulointerstitial nephritis, chronic active pancreatitis, and, rarely, necrotizing hepatitis have been associated with SIV infection of macaques (Martin et al., 1991; Wachtman and Mansfield, 2008; Zöller et al., 2008). Neutralizing antibodies to chimpanzee adenovirus have been detected in humans in sub-Saharan Africa, suggesting cross-species transmission (Xiang et al., 2006), but the zoonotic potential for primate-associated adenovirus remains in question.

Simian virus 40

SV40 and the related cynomolgus polyomavirus (CPV) cause common and persistent infections of many species of macaques. First isolated from primary macaque kidney cell lines that were used in polio vaccine production, SV40 has been studied extensively in the laboratory and is now a common contaminant (Butel and Lednicky, 1999). Serologic evidence indicates that both SV40 and CPV are readily transmitted in breeding colonies of macaques, with seroprevalence rates greater than 75% by 2–3 years of age (Verschoor et al., 2008). While infection is common, clinical disease has not been described in immunologically normal animals. The virus does establish latency (a state of dormancy in which the virus is not actively replicating), and either reactivation of latent virus or primary infection may result in pathology, with progressive immunodeficiency or immunosuppression. SV40 is a common opportunistic infection of rhesus macaques inoculated with SIV, in which it may cause lesions in the central nervous system (CNS), lungs, and kidneys. CNS lesions in macaques are similar to those observed in progressive multifocal leukoencephalopathy (PML) of immunodeficient humans infected with the antigenically related JC polyomavirus. In macaques, PML is characterized by locally extensive regions of demyelinization and gliosis within white matter tracts and subependymal regions (Horvath et al., 1992; Simon et al., 1999). Large basophilic intranuclear inclusions are evident primarily within oligodendrocytes and assist with the morphologic diagnosis. Infection of the kidney with SV40 results in chronic non-suppurative interstitial nephritis centered on the renal medulla and often accompanied by hyperplasia and dysplasia of collecting duct epithelium. Viral inclusions are evident within renal tubular epithelium, and localization techniques such as immunohistochemistry and in situ hybridization often reveal far more infected cells than would be found by routine stains. CPV has recently emerged as a common pathogen of cynomolgus macaques used in transplantation biology programs, in which it may produce renal, pulmonary, and hepatic lesions (van Gorder et al., 1999; Maki et al., 2008). There is currently no treatment or therapeutic prophylaxis available for polyomavirus infection. Animals can be screened serologically prior to assignment to experimental protocols if SV40 or CPV is viewed as a potential confounder.

Simian parvovirus

SPV is a recently-recognized erythrovirus within the Parvoviridae family, related antigenically to human B19 virus (O’Sullivan et al., 1994, 1996, 1997). The epizoology of SPV is poorly understood, but infection has been recognized in both cynomolgus and rhesus macaques. It appears that both viremic antibody-negative and nonviremic antibody-positive states can occur, and thus a combination of molecular detection techniques such as PCR and serologic testing must be employed in screening animals to achieve a diagnosis (Schroder et al., 2006). B19 can persist at low levels in the bone marrow of infected humans for extended periods, establishing latency, and a similar situation can be anticipated to occur in macaques with the related SPV. Both viruses target rapidly dividing cells and demonstrate a tropism for cells within the erythrocytic lineage. In immunologically normal animals, infection has not yet been associated with clinical disease, but with immunosuppression or immunodeficiency infection may cause anemia and widespread infection of erythroid cells. In bone marrow, poorly-defined eosinophilic intranuclear inclusions may be observed, in association with dyserythropoiesis; ultrastructural examination and in situ hybridization can be used to confirm the diagnosis (Brown and Young, 1997). Such infections and pathology have been observed in both SIV- and SRV-infected rhesus macaques, as well as in immunosuppressed cynomolgus macaques, in which severe clinical anemia has been diagnosed. An SPV infection can be particularly problematic in experiments in which tissues are transferred from infected to naïve animals in the context of therapeutic immunosuppression. Under these circumstances, primary infection may result in severe or fatal disease; screening animals prior to initiation of studies and selecting viral-negative animals as donors will help prevent transmission. While currently there are no specific antiviral agents, corticosteroid may help in acute hemolytic crisis.

Papillomavirus

Papillomaviruses are spherical double-stranded DNA viruses with capsids composed of 72 capsomeres that have been shown to infect a variety of animals in a species-specific fashion. More than 60 strains have been identified in humans, and natural infection of a number of NHP species has been demonstrated. Sero-surveys suggest the virus is transmitted at sexual maturity, and widespread infection of colonies may occur without overt clinical signs.

When lesions are present, they are observed on mucosal surfaces or haired skin. They often have the appearance of an exophytic mass that is described as having a cauliflower-like appearance (Patterson et al., 2005). Histologically, these lesions are characterized by massive hyperplasia of the stratum spinosum and corneum. Basophilic intranuclear inclusions may be observed. Focal epithelial hyperplasia has been described in chimpanzees and is characterized by multiple sessile well-circumscribed proliferative structures within the oral mucosa. These may persist for extended periods or undergo spontaneous regression. The first macaque papillomavirus sequences were detected in a penile squamous cell carcinoma, and it has been subsequently shown that distinct strains can be transmitted sexually (Kloster et al., 1988; Wood et al., 2004). As in humans, persistent infection of female macaques may be associated with cervical dysplasia and overt cervical carcinoma (Wood et al., 2004). The impact of immune modulation on acquisition and pathogenesis of papillomavirus infection in macaques is not known. Immunohistochemistry, ultrastructural examination, and molecular assays can be used to confirm the diagnosis.

Primary bacterial infections

Due primarily to improvements in diagnosis, treatment, and prevention, the common bacterial infections in immunocompetent macaques have minimal impact on toxicology studies. Even when encountered, these infections are generally mild and self-limiting. However, infections of the gastrointestinal tract are particularly hard to eliminate entirely from macaque colonies, and thus they continue to have an impact on drug development studies. Chronic gastritis and enterocolitis are relatively common in rhesus and cynomolgus macaques (Reindel et al., 1999; Rubio and Hubbard, 2002; Sestak et al., 2003). Campylobacter coli, C. jejuni, Shigella flexneri, and Yersinia enterocolitica are commonly implicated. Since each of these bacterial species is known to cause outbreaks of diarrhea independently (Kalashnikova et al., 2002, 2006; Lederer et al., 2005) and each has zoonotic potential, colonies are closely monitored for these pathogens. When they are diagnosed, prompt quarantine procedures and appropriate antibiotic regime can usually limit the severity and spread of disease. Sestak et al. (2003) reported that rhesus macaques with clinical signs of diarrhea had an increased incidence of numerous bacterial species and other pathogens in fecal samples, suggesting that one or more of a variety of bacterial species may cause chronic diarrhea. In addition to clinical disease associated with these well-known pathogens, sporadic outbreaks of chronic diarrhea of unknown etiology remain an issue in drug development. Outbreaks of diarrhea in macaque colonies can limit the availability of animals for a study, and spontaneous outbreaks after a study starts can adversely affect interpretation of drug-related effects. Furthermore, studies with immunomodulatory drugs could be compromised because immunological effects, such as elevated IL-1, IL-3, and tumor necrosis factor- genes, and an increase in activated CD4⁺ and CD69⁺ T-lymphocytes in gut-associated lymphoid tissues have been reported in macaques with chronic enterocolitis and diarrhea (Sestak et al., 2003). Immunomodulatory drugs, even those used in transplantation that mostly target cell-mediated immunity, have been associated with numerous primary enteric and pulmonary bacterial infections (Haustein et al., 2008).

Helicobacter spp.

Helicobacter pylori and H. heilmannii bacteria have been associated with human gastritis, peptic ulcers, gastric carcinomas, and gastric mucosa–associated lymphoid tissue lymphomas (Nakamura et al., 2007). H. pylori is common in rhesus monkeys (Drazek et al., 1994; Solnick et al., 2003). Natural infection of macaques with H. pylori is for the most part asymptomatic, yet it can be associated with the microscopic findings of epithelial hyperplasia, lymphoplasmacytic infiltrates, and erosions in the antral portion of the stomach (Reindel et al., 1999). The macaque is an important animal model for investigating H. pylori–associated gastric carcinogenesis (Kodama et al., 2005; Gardner and Luciw, 2008). The smaller corkscrew-shaped H. pylori, usually located in the antral portion of the stomach, can be differentiated from H. heilmannii bacteria, which are spiral, larger, and numerous in the fundus (Reindel et al., 1999; Drevon-Gaillot et al., 2006). In one study, the presence of H. heilmannii bacteria in cynomolgus monkeys located in Mauritius, the Philippines, and Vietnam did not correlate with the severity of gastric inflammation (Reindel et al., 1999; Drevon-Gaillot et al., 2006). H. cinaedi frequently infects asymptomatic rhesus monkeys, and there has been a report of it in association with colitis and hepatitis (Fox et al., 2001; Fernandez et al, 2002)

Mycobacterium tuberculosis

Macaques are highly susceptible to M. tuberculosis, M. bovis, and M. avium (see the section on opportunistic bacterial infections), with a spectrum of disease manifestations similar to that observed in humans (Gardner and Luciw, 2008; Lin et al., 2008). M. tuberculosis can be introduced into a group from a latently infected animal or infected animal handler. Because M. tuberculosis can rapidly spread by the aerosol route and is zoonotic, it is on every list of pathogens used in the definition of specific-pathogen-free macaque colonies (Mansfield, 2005; Lerche and Simmons, 2008; Morton et al., 2008). Since the 1970s, stringent quarantine and testing practices have significantly lowered infection rates in imported animals, primarily due to the guidelines put out by the Centers for Disease Control and Prevention (CDC) and the implementation of surveillance programs in domestic primate centers (Lerche et al., 2008; Roberts and Andrews, 2008). However, natural outbreaks of tuberculosis in domestic NHPs, albeit rare, still occur. M. tuberculosis has recently reemerged in macaques imported from Asia and thus remains a constant threat to NHP colonies and animal handlers (Lerche et al., 2008; Morton et al., 2008). The reliability of the intradermal tuberculin skin test, which detects delayed-type hypersensitivity to tuberculin antigens, continues to be debated, but to date it remains the only Institute for Laboratory Animal Research/CDC-approved method for mycobacterium testing of NHPs (Lerche et al., 2008; Roberts and Andrews, 2008). As the skin test, even when repeated at 2–3-week intervals over a 10-week quarantine period, can occasionally fail to detect latent infections, efforts are ongoing to develop new, more sensitive and specific screening and diagnostic assays that can be used in conjunction with the skin test (Lerche et al., 2008; Lin et al., 2008).

Moraxella (Branhamella) catarrhalis

Moraxella catarrhalis was formerly known as Neisseria catarrhalis and is a gram-negative, oxidase-positive diplococcus. A clinical syndrome of epistaxis accompanied by upper respiratory tract symptoms has been found in association with colonization of the upper respiratory tract by M. catarrhalis in rhesus and cynomolgus macaques (VandeWoude and Luzarraga, 1991; Bowers et al., 2002). Infection is most frequently observed in the fall and winter months and may be precipitated by changes in humidity. The condition, also known as “bloody-nose syndrome,” is usually self-limiting and has been reported in several macaque species. Although it is listed here as a primary bacterial pathogen, certain immunomodulatory drugs can increase the incidence and severity of Moraxella-induced epistaxis, allowing for a more favorable environment for secondary bacterial invasion and sepsis (Sasseville and Diters, 2008). Therefore, careful monitoring of animals for the onset of Moraxella-induced epistaxis is of paramount importance in any study with immunomodulatory drugs or any other factors that could lower the humoral immune system response.

Opportunistic bacterial infections

Bacterial infections that have been observed in immunosuppressed macaques include enteropathogenic Escherichia coli, Mycobacterium avium complex (MAC), and Rhodococcus equi (Mansfield et al., 1995, 2001).

Enteropathogenic Escherichia coli

Six categories of diarrheagenic E. coli are defined, based on the underlying mechanism of disease pathogenesis, in vivo and in vitro growth characteristics, and the presence of specific genes encoding virulence factors. These include enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EaggEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffuse adherent E. coli (DAEC) (Mansfield et al., 2001a, 2001b). The prevalence of diarrheagenic E. coli in both humans and animals is probably underestimated, as most clinical laboratories do not attempt to isolate and identify lactose-fermenting organisms from fecal specimens. Diagnosis in immunodeficient humans and animals may be obscured by the presence of multiple agents, and EPEC is likely to be missed unless a specific effort is made to identify it. A systematic approach including the use of adhesion assays, biopsies, and/or molecular identification of virulence genes is required for a definitive diagnosis (Mansfield et al., 2001a).

In macaques, infection may be associated with an acute onset and persistent non-hemorrhagic diarrhea, seen primarily in infant or neonatal animals. In neotropical primates, disease is most frequently recognized as acute hemorrhagic diarrhea and may be accompanied by severe blood loss and hypovolemia. In both Old World and neotropical primates, a chronic and persistent form is also recognized (Mansfield et al., 2001a, 2001b). Transmission is by the fecal–oral route, and serologic surveys suggest most colony animals are exposed but show self-limiting clinical signs.

Morphologic findings characteristic of the attaching and effacing lesion are pathognomonic, and colonic biopsy can be a quick and efficient means of diagnosis. Histologically, the colonic surface epithelium appears irregular, with bacilli intimately associated with the apical cytoplasmic membrane and accompanied by varying degrees of colonic crypt hyperplasia. The surface epithelium often has a “cobblestone” appearance or, when accompanied by necrosis, a flattened or squamous morphology. There is a mild neutrophilic infiltrate, and individual epithelial cells with adherent bacilli often appear rounded and vacuolated. Bacilli are most frequently limited to the colonic surface epithelium, and only rarely are organisms noted in the ileum and distal jejunum. Ultrastructurally, there is effacement of normal microvillous architecture, and adherent bacilli are attached to the apical cytoplasmic membrane, with pedestal formation and rearrangement of the underlying cytoskeleton. Sequencing of the intimin (eae) gene from NHP isolates indicates a high degree of identity with human isolates, suggesting that primate EPECs represent a potential zoonotic disease.

Mycobacterium avium complex

MAC is composed of M. avium and intracellulare, and is a frequent opportunistic infection observed in immunodeficient humans and macaques. M. avium is a common environmental bacteria found in soils and moist environments. Plasmid profiles from human isolates suggest that waterborne MAC is the most common source, and macaque cases have been linked to water distribution systems using molecular techniques (von Reyn et al., 1994; Mansfield and Lackner, 1997). Animals reared outside may have a higher incidence of disease during periods of immunosuppression due to exposure to higher levels of organisms and subsequent subclinical colonization. Disease may be recognized in immunodeficient and normal NHPs. Disseminated MAC is a common clinical syndrome most frequently identified in SIV-infected macaque species (Mansfield et al., 1995). Infection generally occurs at low CD4 T-lymphocyte count (<200 cells/μL), and a gastrointestinal route of entry is suspected. Animals present with progressive diarrhea and weight loss; these may be accompanied by peripheral lymphadenopathy and hepatosplenomegaly. More than 95% of isolates are M. avium, and disseminated MAC is rare outside the context of HIV/SIV infection, suggesting that unique viral host interactions may be responsible for producing disease (Mansfield et al., 2001c).

At necropsy, there is grossly visible thickening of the small and large intestine mucosal surfaces, accompanied by enlargement of mesenteric lymph nodes. Occasionally, lymphangitis of serosal lymphatics may be evident. Hepatosplenomegaly, as well as enlargement of other lymph nodes, may be observed. In human and simian AIDS, histologic features are diagnostic and characterized by sheets of lightly pink histiocytes that infiltrate and efface normal structures in the gastrointestinal tract and lymph nodes. Scattered within the histiocytes are islands of lymphocytes and rare plasma cells. In the liver, microgranulomas (composed of histiocytes and a small number of lymphocytes) are evident. Similar granulomas may be found in multiple organs, including the lung, skin, uterus, spleen, kidney, thymus, and bone marrow. Acid-fast stains reveal large number of bacilli within histiocytes, and mycobacterial loads may approach 10⁹–10¹⁰ colony forming units per gram of tissue.

MAC infection may rarely be seen outside the context of AIDS. A recent report suggests that a high proportion of normal animals may asymptomatically harbor the organism in the mesenteric lymph nodes (Maslow et al., 2003). When disease occurs in normal animals, morphologically it resembles M. tuberculosis infection. We have observed M. avium infection in macaques with chronic obstructive pulmonary disease and in marmosets as a cause of false-positive tuberculin skin tests. In both cases, lesions were characterized by typical caseating granulomas, multinucleated giant cells, and sparse acid-fast bacilli. Definitive diagnosis could be made only through isolation of the organism and PCR sequencing of mycobacterial DNA.

Rhododococcus equi

R. equi is a gram-positive facultative anaerobe that is a common pathogen of immunocompromised animals and humans (Kedlaya et al., 2001; Kwa et al., 2001; Meijer and Prescott, 2004). The genus is related to mycobacteria and is characterized by a unique cell envelope that consists of mycolic acids linked to arabinogalactan wall polysaccharides and (glyco)lipids. The lipoarabinomannan (LAM) of R. equi is smaller than that of mycobacterial LAM. Disease is often observed in immunodeficient animals and may be seen in association with SRV-D infection. Clinical sings may be non-specific and include anorexia, weight loss, and diarrhea. Soil is an important reservoir, and lesions are observed primarily in the large intestine, lung, and draining lymph nodes and are characterized by pyogranulomatous inflammation. Gastrointestinal lesions may be ulcerative and are associated with mesenteric lymphadenopathy. With severe immunodeficiency, disease may resemble disseminated MAC, with sheets of histiocytes infiltrating the lamina propria and effacing the normal architecture.

Primary parasites

Wild-caught macaques are infected with numerous protozoal and helminth parasites. Protozoal infections are particularly important in the context of immunosuppression (see the section on opportunistic parasites). In immunocompetent macaques, tissue morphologic changes associated with protozoa are rare. Sarcocystis spp. are the exception in that, particularly in wild-caught animals, cysts are occasionally observed in the skeletal muscle, and less frequently in the cardiac and smooth muscle (Karl and Wong, 1975). There have been rare instances of active disease reported with various species of Sarcocystis, including myositis, myocarditis, fulminant sarcocystosis, and encephalitis, the latter in an immunosuppressed macaque (Hernández-Jáuregui et al., 1983; Terrell and Stookey, 1972; Lane et al., 1998; Gozalo et al., 2007; Klumpp et al., 1994).

Common helminths include species of Strongyloides, Oesophagostomum, Anatrichosoma, Trichostrongylus, and Gongylonema, and larval stages of nematodes (ascarids, spirurids), cestodes (hydatid, Sparganum), and pentastomids (Wong and Conrad, 1978; Karr et al., 1980; Sano et al., 1980). Although treatment with anti-helmintics has significantly reduced or eliminated these infestations, Strongyloides fuelleborni has been difficult to eradicate completely, with infection rates as high as 27% reported in domestic colonies of macaques (Eberhard, 1981; Sestak et al., 2003; Dufour et al., 2006). Infection of macaques with S. fuelleborni is associated with cough, dyspnea, diarrhea, and dermatitis (Eberhard, 1981; Sestak et al., 2003; Dufour et al., 2006). Trichuris trichiura is also somewhat difficult to eliminate completely from macaques, particular those animals housed outdoors, but infection does not induce significant clinical disease, although parasitic granulomas can be observed (Sestak et al., 2003; Drevon-Gaillot et al., 2006).

Schistosoma japonicum, S. mansoni, and S. hematobium

Schistosoma spp. have a wide geographical distribution and have been observed in wild-caught macaques (Cheever et al., 1974). Although schistosomiasis is a disease of worldwide importance in humans and animals, the intermediate snail hosts needed to complete the lifecycle are not present in the United States, and so the cycle is broken. Thus these species are not significant pathogens in United States colonies of macaques.

Opportunistic parasites

Opportunistic parasitic infections that can be encountered in immunosuppressed macaques include Cryptosporidium parvum, Enterocytozoon bieneusi, Plasmodium spp., Trichomonas spp., Acanthamoeba spp., and Toxoplasma gondii (Wachtman and Mansfield, 2008).

Cryptosporidium parvum

C. parvum is a common protozoal parasite of cold- and warm-blooded vertebrates. A variety of genotypes have been described that vary in their host species preference. Infection of a variety of NHPs has been recognized, and the organism is common in macaque colonies, where more than 95% of animals will be seropositive by 2–3 years of age. The organism is resilient to environmental conditions and may survive in water for prolonged periods. In most cases, clinical disease goes unrecognized in colony animals; however, in rare instances, protracted diarrhea, anorexia, and weight loss may be observed. Diagnosis can be made by examination of feces or biopsies. Fecal antigen capture tests are a useful and sensitive test combining detection of C. parvum with Giardia and Entamoeba.

The disease is more severe in immunosuppressed or immunodeficient animals in which it may cause severe diarrhea and weight loss. The organism is most commonly found in the colon but may spread to the small intestine; it is visualized as spherical 3–4 μm diameter basophilic bodies that adhere to the apical surface of cells. In severe cases, there is villous atrophy and epithelial cell hyperplasia within the gastrointestinal tract. During immunosuppression, the organism commonly invades the biliary tree, causing cholangiohepatitis, cholecystitis, and choledochitis. In the liver, there are often neutrophilic infiltrates surrounding and infiltrating involved bile ducts. These are accompanied by epithelial cell necrosis and concentric or onion-skin fibrosis in portal tracts. It can be difficult to visualize organisms in these areas, and it is best to look in regions with less inflammation. The organism may also spread to the conjunctiva, trachea, and lungs, where it can be visualized on respiratory epithelium, causing a necrotizing bronchiolitis (Baskin, 1996; Yanai et al., 2000). If doubt exists as to the identity of the organism, immunohistochemistry can be used for confirmation (Yanai et al., 2000). Disease in New World primate species is sporadic and less frequent. It may represent inadvertent introduction of the agent into colonies through contaminated food, water, or other fomites.

Enterocytozoon bieneusi

E. bieneusi is a microsporidian parasite observed in a variety of vertebrate species. Microsporidia are highly evolved obligate intracellular parasites that have no metabolically active stage outside of the host. They have previously been classified as protozoan pathogens; however, recent biochemical analysis has revealed the presence of chitin, suggesting a close relationship to other fungi.

Infection of several species of macaques has been recognized; however, given the number of other vertebrates known to be infected, most NHPs are probably susceptible. Microsporidia are highly resistant to environmental influences and can survive in water or soil for years. In large colonies, asymptomatic infection is common; in a survey, nearly 20% of normal breeding animals were shedding spores at any one time (Mansfield et al., 1998). Diagnosis can be difficult but can be made based on PCR or fecal flotation with modified chromotrope stain (Carville et al., 1997).

In immunologically-normal animals, disease is infrequently recognized. The organism commonly invades the hepatobiliary system, which may represent a site of persistent infection (Mansfield et al., 1998). Chronic infection may be associated with a mild lymphoplasmacytic cholecystitis and elevations in serum alkaline phosphatase but in general is asymptomatic (Mansfield et al., 1998). In contrast, severe disease is often observed in SIV-infected immunodeficient animals, and infection may produce chronic diarrhea and wasting. In SIV-infected animals jaundice may be clinically evident, as well as hepatomegaly, in association with cholecystitis, choledochitis, and cholangiohepatitis (Mansfield et al., 1997; Chalifoux et al., 1998). Thickening of bile ducts and the gall bladder mucosa may be grossly visible, and the bile is often clear-staining; the resulting sclerosing cholangiohepatitis has a characteristic gross appearance. Histologically, the key finding is the presence of individual biliary epithelial cells that have exfoliated from the mucosal surface and lie within the lumen of the gall bladder or bile duct. Small ringlike structures or negative images representing the spores are often observed within these cells and are best visualized with a Weber’s modified trichrome. The cholangiohepatitis differs significantly from that seen with C. parvum. With E. bieneusi, there is often marked bile ductule hyperplasia, accompanied by bridging hepatic fibrosis. A lymphocytic plasmacytic infiltrate is seen, and the presence of neutrophils should suggest evaluation for concurrent C. parvum infection. Concentric fibrosis and overt necrosis are usually absent. Prevention is difficult due to the resistant nature of the spores and widespread contamination of the environment. E. bieneusi is a known human pathogen, and while infection from NHPs to humans has not been recognized, it should be considered possible.

Plasmodium spp.

Human malaria is most frequently caused by Plasmodium falciparum, P. vivax, P. malariae, and P. ovale, with hundreds of millions of individuals infected worldwide. Many species of Plasmodium (e.g., P. coatneyi, P. fragile, P. knowlesi, P. inui, and P. cynomolgi) naturally infect macaques, with a clinical presentation that varies according to the infecting species, similar to the observation in humans. In Southeast Asia, P. knowlesi has been implicated as an important plasmodium infecting humans, with macaques serving as the sylvatic reservoir (Vythilingam et al., 2006; Luchavez et al., 2008; Ng et al., 2008; White, 2008; Bronner et al., 2009). P. knowlesi of cynomolgus macaque origin has recently been implicated in the re-emergence of human infection in a country that had previously been free from disease (Ng et al., 2008). Incidence varies considerably based upon species of Plasmodium as well as the origin of the macaques. For instance, the incidence of P. inui can range from 0% in Mauritian macaques to 10–14% in Chinese- and Indonesian-origin macaques. Furthermore, differences in same-species susceptibility to Plasmodium have been observed among cynomolgus macaques from Mauritius and the Philippines (Migot-Nabias et al., 1999; Leuchte et al., 2003). Susceptibility to infection or reactivation of latent infection is enhanced by splenectomy, stress, and treatment with immunomodulatory agents. Erythropoietin and other erythroid-stimulating agents have enhanced multiplication of malarial parasites, resulting in lethal infection in murine models of malaria, but they have yet to be documented in NHPs (Chang et al., 2004). It is documented that both latent and active Plasmodium infections can cause alterations in cytokine profiles (Biswas, 1999; Yang et al., 1999). Thus, the increased use of macaques from China and southeast Asia, where malaria is endemic, could affect drug safety studies, in particular those with immunomodulatory drugs or with erythroid-stimulating agents.

Balantidium coli and Trichomonas spp.

B. coli is a large ciliated protozoan that is commonly seen in the lumen of the cecum and colon of normal macaques. The incidence and parasitic load of B. coli vary considerably according to the source of the macaque, with incidences of 13% reported in cynomolgus macaques from Mauritius and up to 75% in cynomolgus macaques from Vietnam and the Philippines (Drevon-Gaillot et al., 2006). Despite similar incidences of B. coli in cynomolgus macaques from Vietnam and the Philippines, a higher parasitic load is observed in the animals from the Philippines (Drevon-Gaillot et al., 2006). An incidence of ∼12% has been reported in one survey of a domestic rhesus macaque colony (Sestak et al., 2003). Infection is usually not associated with diarrhea or morphologic abnormalities in the intestinal mucosa (Sestak et al., 2003; Drevon-Gaillot et al., 2006). 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, within invasion of the lamina propria or submucosa rarely encountered unless there is a confounding pathology inducing mucosal ulceration. Similar to B. coli, Trichomonas spp. can frequently be observed the lumen or deep in the crypts of the gastrointestinal tract of normal macaques, rarely eliciting an inflammatory response. Invasive disease has been rarely reported in association with SIV infection (Blanchard and Baskin, 1988; Kondova et al., 2005).

Toxoplasma gondii

Toxoplasmosis is caused by the coccidian parasite T. gondii, which can be a common pathogen of neotropical and Old World primates. Felids serve as the definitive hosts, shedding infectious oocysts from the gastrointestinal tract into the environment. These oocysts may be ingested by an intermediate host, after which the organism replicates in an extraintestinal phase, causing disease or occasionally forming cysts that can persist in tissue for extended periods. The intermediate hosts may then serve as vectors when eaten as prey. In primates, disease is most severe in callitrichids and owl monkeys, in which it may cause a fulminant, fatal disseminated infection (Seibold and Wolf, 1971; Epiphanio et al., 2000; 2003). Toxoplasmosis has also been diagnosed in an SIV-inoculated macaque, and, as in humans, immunomodulation may be associated with reactivation of tissue cysts, with the disease often targeting the CNS (Sasseville et al., 1995). Infection of NHPs may produce clinical signs that vary from asymptomatic to lethargy, anorexia, and weakness. In New World primates, a rapidly progressive disease course may be observed that includes sudden diarrhea, fever, cough, neurologic manifestations, and death (Epiphanio et al., 2000, 2003). In Old World primates, infection is often asymptomatic and may go unrecognized (Wong and Kozek, 1974). Key morphologic findings in severe disease include necrosis and a neutrophilic to mixed inflammatory infiltrate centered in the lungs, liver, and CNS. The organisms are recognized as small oval to crescent-shaped bodies, and special stains and immunohistochemistry may assist in recognizing and identifying the organisms.

Prevention of toxoplasmosis should include measures to exclude rodent vectors from animal facilities, as they serve as important intermediate hosts and may be eaten by primates. Likewise cockroaches may serve as mechanical vectors, transporting oocytes within facilities. While not a problem in most laboratory animal facilities, felids should be prevented from having direct contact with NHPs or their food.

Acanthamoebae

Amoebic infections, such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris, have been reported in immunocompromised human patients. Infections with these agents are infrequent in NHPs, but animals housed outdoors may be at increased risk as acanthamoebae are free-living in the soil and water. N. fowleri has been associated with primary amoebic meningoencephalitis in immunocompetent individuals, whereas Acanthamoeba spp. and B. mandrillaris can cause granulomatous amoebic encephalitis in immunocompromised hosts (Schuster and Visvesvara, 2004). Acute fatal meningoencephalitis has been demonstrated by experimental infection with A. culbertsoni or N. fowleri, and spontaneous acanthamoeba infection in an SIV-infected rhesus macaque was associated with necrotizing meningoencephalitis and pneumonitis (Wong et al., 1975a, 1975b; Westmoreland et al., 2004). B. mandrillaris–associated meningoencephalitis has been observed in Old World primates, but infection in macaques has not been reported (Rideout et al., 1997). Diagnosis is made by identification of trophozoites in areas of inflammation, and since it is not possible to distinguish Acanthamoeba spp. from Balamuthia spp. based on morphologic features, immuno-fluorescence and PCR can be used to identify the genus (Wachtman and Mansfield, 2008).

Opportunistic fungal infections

It is extremely rare to observe primary fungal infections in adult healthy macaques. Fungal infections that have been observed in immunosuppressed macaques include Pneumocystis carinii, Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus.

Pneumocystis carinii

P. carinii is an extracellular obligate fungal pathogen with high host specificity (Furuta et al., 1993; Gigliotti et al., 1993; Stringer et al., 2002; Norris et al., 2003). Infection of neonatal macaques is asymptomatic, with infected animals clearing the organism or becoming carriers and fulminant disease restricted to immunodeficient macaques (Demanche et al., 2005). In immunosuppressed macaques, there is a protracted asymptomatic period followed by clinical disease, including dyspnea and tachypnea (Board et al., 2003). Infection may be acquired in SIV-immunosuppressed macaques rather than by reactivation of latent infection (Vogel et al., 1993). In hematoxylin and eosin–stained sections, the organism appears as pale pink foamy material filling alveolar spaces, with minimal inflammation; at times, there can be prominent Type 2 pneumocyte hyperplasia. Infrequently, in SIV-infected macaques pneumocystis can induce a nodular pneumonia with vascular involvement and dissemination to the regional lymph nodes (Yanai et al., 1999). P. carinii stain well with Gomori methanamine silver (GMS), and immunohistochemistry can also be used to identify the organism. As juvenile carriers may play a role in propagation, prevention of pneumocystis pneumonia in macaques is difficult (Demanche et al., 2005). Due to host specificity, there is no zoonotic potential. Because separation of adult immunodeficient animals may be of some benefit, controlled air handling and reduction of animal numbers per room may limit the spread of P. carinii (Vogel et al., 1993).

Candida albicans

C. albicans is a common saprophytic agent that colonizes mucosal surfaces during or shortly after birth, and in immunocompetent animals it usually does not produce clinically apparent disease. However, a localized form (thrush) causes the formation of white plaques in the oral mucosa and esophagus in some neonates and some stressed or immunocompromised animals. It is a common opportunistic infection secondary to immunosuppressive viruses (e.g., measles virus, SIV, and HIV) and post-transplantation (Baskin et al., 1995; Choi et al., 1999; Leigh et al., 2004). These lesions have abundant pseudohyphae and blastospores, which are identified with GMS or periodic acid Schiff (PAS) stains. A disseminated form of disease termed “invasive candidiasis” is a common nosocomial infection among critically ill patients and pre-term infants (Hollenbach, 2008; Kaufman, 2008). Prevention is difficult due to the ubiquitous nature of the organism.

Histoplasma capsulatum, C. neoformans, and A. fumigatus are infrequently reported opportunistic infections in macaques (Pal et al., 1984; Baskin, 1991).

Summary

To ensure colony health and intelligent study design and to assess possible drug-related effects in drug safety studies using NHPs, it is essential that toxicologists have intimate knowledge of the common spontaneous infections, opportunistic pathogens, and background lesions of NHPs and that they are aware of some of the reemerging diseases. In addition, a thorough understanding of which types of opportunistic infections are most likely to arise, depending on which arm of the immune system is affected, is particularly helpful in studies with immunomodulatory drugs. Another important factor is that this knowledge can be used among the criteria for animal selection prior to study initiation. For many of these pathogens that are difficult to eliminate entirely from NHP populations and/or have a high incidence, it may be beneficial to identify infected and non-infected animals prior to study start and then either eliminate or treat (if possible) infected animals or distribute them equally among all treatment groups.

Acknowledgment

This work was supported by the National Center for Research Resources grants RR00168-46 (KGM).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.