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Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques (Macaca mulatta) is a valuable nonhuman primate model of human CMV (HCMV) persistence and pathogenesis. In vivo studies predominantly use tissue culture-adapted variants of RhCMV that contain multiple genetic mutations compared to wild-type (WT) RhCMV. In many studies, animals have been inoculated by nonnatural routes (e.g., subcutaneous, intravenous) that do not recapitulate disease progression via the normative route of mucosal exposure. Accordingly, the natural history of RhCMV would be more accurately reproduced by infecting macaques with strains of RhCMV that reflect the WT genome using natural routes of mucosal transmission. Here, we tested two WT-like RhCMV strains, UCD52 and UCD59, and demonstrated that systemic infection and frequent, high-titer viral shedding in bodily fluids occurred following oral inoculation. RhCMV disseminated to a broad range of tissues, including the central nervous system and reproductive organs. Commonly infected tissues included the thymus, spleen, lymph nodes, kidneys, bladder, and salivary glands. Histological examination revealed prominent nodular hyperplasia in spleens and variable levels of lymphoid lymphofollicular hyperplasia in lymph nodes. One of six inoculated animals had limited viral dissemination and shedding, with commensurately weak antibody responses to RhCMV antigens. These data suggest that long-term RhCMV infection parameters might be restricted by local innate factors and/or de novo host immune responses in a minority of primary infections. Together, we have established an oral RhCMV infection model that mimics natural HCMV infection. The virological and immunological parameters characterized in this study will greatly inform HCMV vaccine designs for human immunization. IMPORTANCE Human cytomegalovirus (HCMV) is globally ubiquitous with high seroprevalence rates in all communities. HCMV infections can occur vertically following mother-to-fetus transmission across the placenta and horizontally following shedding of virus in bodily fluids in HCMV-infected hosts and subsequent exposure of susceptible individuals to virus-laden fluids. Intrauterine HCMV has long been recognized as an infectious threat to fetal growth and development. Since vertical HCMV infections occur following horizontal HCMV transmission to the pregnant mother, the nonhuman primate model of HCMV pathogenesis was used to characterize the virological and immunological parameters of infection following primary mucosal exposures to rhesus cytomegalovirus.

The Democratic Republic of the Congo (DRC) has a history of nonhuman primate (NHP) consumption and exposure to simian retroviruses yet little is known about the extent of zoonotic simian retroviral infections in DRC. We examined the prevalence of human T-lymphotropic viruses (HTLV), a retrovirus group of simian origin, in a large population of persons with frequent NHP exposures and a history of simian foamy virus infection. We screened plasma from 3,051 persons living in rural villages in central DRC using HTLV EIA and western blot (WB). PCR amplification of HTLV tax and LTR sequences from buffy coat DNA was used to confirm infection and to measure proviral loads (pVLs). We used phylogenetic analyses of LTR sequences to infer evolutionary histories and potential transmission clusters. Questionnaire data was analyzed in conjunction with serological and molecular data. A relatively high proportion of the study population (5.4%, n = 165) were WB seropositive: 128 HTLV-1-like, 3 HTLV-2-like, and 34 HTLV-positive but untypeable profiles. 85 persons had HTLV indeterminate WB profiles. HTLV seroreactivity was higher in females, wives, heads of households, and increased with age. HTLV-1 LTR sequences from 109 persons clustered strongly with HTLV-1 and STLV-1 subtype B from humans and simians from DRC, with most sequences more closely related to STLV-1 from Allenopithecus nigroviridis (Allen's swamp monkey). While 18 potential transmission clusters were identified, most were in different households, villages, and health zones. Three HTLV-1-infected persons were co-infected with simian foamy virus. The mean and median percentage of HTLV-1 pVLs were 5.72% and 1.53%, respectively, but were not associated with age, NHP exposure, village, or gender. We document high HTLV prevalence in DRC likely originating from STLV-1. We demonstrate regional spread of HTLV-1 in DRC with pVLs reported to be associated with HTLV disease, supporting local and national public health measures to prevent spread and morbidity.

To identify immunodominant antigens that elicit a humoral immune response following a primary and a secondary genital infection, rhesus monkeys were inoculated cervically with Chlamydia trachomatis serovar D. Serum samples were collected and probed with a protein microarray expressing 864/894 (96.4%) of the open reading frames of the C. trachomatis serovar D genome. The antibody response to the primary infection was analyzed in 72 serum samples from 12 inoculated monkeys. The following criteria were utilized to identify immunodominant antigens: proteins found to be recognized by at least 75% (9/12) of the infected monkeys with at least 15% elevations in signal intensity from week 0 to week 8 post infection. All infected monkeys developed Chlamydia specific serum antibodies. Eight proteins satisfied the selection criteria for immunodominant antigens: CT242 (OmpH-like protein), CT541 (mip), CT681 (ompA), CT381 (artJ), CT443 (omcB), CT119 (incA), CT486 (fliY), and CT110 (groEL). Of these, three antigens, CT119, CT486 and CT381, were not previously identified as immunodominant antigens using non-human primate sera. Following the secondary infection, the antibody responses to the eight immunodominant antigens were analyzed and found to be quite different in intensity and duration to the primary infection. In conclusion, these eight immunodominant antigens can now be tested for their ability to identify individuals with a primary C. trachomatis genital infection and to design vaccine strategies to protect against a primary infection with this pathogen.

BackgroundThe microbiota plays an important role in HIV pathogenesis in humans. Microbiota can impact health through several pathways such as increasing inflammation in the gut, metabolites of bacterial origin, and microbial translocation from the gut to the periphery which contributes to systemic chronic inflammation and immune activation and the development of AIDS. Unlike HIV-infected humans, SIV-infected vervet monkeys do not experience gut dysfunction, microbial translocation, and chronic immune activation and do not progress to immunodeficiency. Here, we provide the first reported characterization of the microbial ecosystems of the gut and genital tract in a natural nonprogressing host of SIV, wild vervet monkeys from South Africa.ResultsWe characterized fecal, rectal, vaginal, and penile microbiomes in vervets from populations heavily infected with SIV from diverse locations across South Africa. Geographic site, age, and sex affected the vervet microbiome across different body sites. Fecal and vaginal microbiome showed marked stratification with three enterotypes in fecal samples and two vagitypes, which were predicted functionally distinct within each body site. External bioclimatic factors, biome type, and environmental temperature influenced microbiomes locally associated with vaginal and rectal mucosa. Several fecal microbial taxa were linked to plasma levels of immune molecules, for example, MIG was positively correlated with Lactobacillus and Escherichia/Shigella and Helicobacter, and IL-10 was negatively associated with Erysipelotrichaceae, Anaerostipes, Prevotella, and Anaerovibrio, and positively correlated with Bacteroidetes and Succinivibrio. During the chronic phase of infection, we observed a significant increase in gut microbial diversity, alterations in community composition (including a decrease in Proteobacteria/Succinivibrio in the gut) and functionality (including a decrease in genes involved in bacterial invasion of epithelial cells in the gut), and partial reversibility of acute infection-related shifts in microbial abundance observed in the fecal microbiome. As part of our study, we also developed an accurate predictor of SIV infection using fecal samples.ConclusionsThe vervets infected with SIV and humans infected with HIV differ in microbial responses to infection. These responses to SIV infection may aid in preventing microbial translocation and subsequent disease progression in vervets, and may represent host microbiome adaptations to the virus. Video Abstract.

BackgroundThe emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic prompted the need for a surveillance program to determine the viral status of the California National Primate Research Center non-human primate breeding colony, both for reasons of maintaining colony health and minimizing the risk of interference in COVID-19 and other research studies.MethodsWe collected biological samples from 10% of the rhesus macaque population for systematic testing to detect SARS-CoV-2 virus by RT-PCR and host antibody response by ELISA. Testing required the development and validation of new assays and an algorithm using in laboratory-developed and commercially available reagents and protocols.Results and conclusionsNo SARS-CoV-2 RNA or antibody was detected in this study; therefore, we have proposed a modified testing algorithm for sentinel surveillance to monitor for any future transmissions. As additional reagents and controls become available, assay development and validation will continue, leading to the enhanced sensitivity, specificity, accuracy, and efficiency of testing.

BackgroundThe emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic prompted the need for a surveillance program to determine the viral status of the California National Primate Research Center non-human primate breeding colony, both for reasons of maintaining colony health and minimizing the risk of interference in COVID-19 and other research studies.MethodsWe collected biological samples from 10% of the rhesus macaque population for systematic testing to detect SARS-CoV-2 virus by RT-PCR and host antibody response by ELISA. Testing required the development and validation of new assays and an algorithm using in laboratory-developed and commercially available reagents and protocols.Results and conclusionsNo SARS-CoV-2 RNA or antibody was detected in this study; therefore, we have proposed a modified testing algorithm for sentinel surveillance to monitor for any future transmissions. As additional reagents and controls become available, assay development and validation will continue, leading to the enhanced sensitivity, specificity, accuracy, and efficiency of testing.

Despite the lack of confirmed reports of an exogenous Simian betaretrovirus (SRV) isolated from baboons (Papio sp.), reports of simian endogenous gammaretrovirus (SERV) in baboons with complete genomes suggest that such viruses may be potentially infectious. In addition, serologic tests have repeatedly demonstrated antibody reactivity to SRV in baboons from multiple colonies. These findings complicate the management and use of such animals for research. To provide further insight into this situation, we performed in vitro and in vivo studies to determine if baboons are or can be infected with SRV. In our initial experiment, we were not able to isolate SRV from 6 seropositive or sero-indeterminate baboons by coculturing their peripheral blood mononuclear cells (PBMC) with macaque PBMC or permissive cell lines. In a subsequent experiment, we found that baboon PBMC infected in vitro with high dose SRV were permissive to virus replication. To test in vivo infectibil- ity, groups of naive baboons were infused intravenously with either (i) the same SRV tissue culture virus stocks used for the in vitro studies, (ii) SRV antibody positive and PCR positive macaque blood, (iii) SRV antibody positive or indeterminate, but PCR negative baboon blood, or (iv) SRV antibody and PCR negative baboon blood. Sustained SRV infection, as defined by reproducible PCR detection and/or antibody seroconversion, was confirmed in 2 of 3 baboons receiving tissue culture virus but not in any recipients of transfused blood from seropositive macaques or baboons. In conclusion, the data indicate that even though baboon cells can be infected experimentally with high doses of tissue culture grown SRV, baboons that are repeatedly SRV antibody positive and PCR negative are unlikely to be infected with exogenous SRV and thus are unlikely to transmit a virus that would threaten the SPF status of captive baboon colonies.

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94 Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface on Vif that enables adaptation while maintaining protein interactions required for potent A3 antagonism.

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non-human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon-gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.

Measles virus causes a highly infectious disease in NHP. Clinical signs range from asymptomatic to fatal, although measles virus is most well-known for its characteristic generalized maculopapular rash. Along with appropriate quarantine practices, restricted human access, and appropriate personal protective equipment, vaccines are used to combat the risk of infection. The canine distemper-measles vaccine (CDMV), administered at the manufacturers standard dose (1.0 mL IM), has been shown to be effective against clinical measles disease in rhesus macaques (Macaca mulatta). The goal of the current study was to test whether doses smaller than the manufacturers recommended dose stimulated adequate antibody production to protect against infection. We hypothesized that either 0.25 or 0.5 mL IM of CDMV would stimulate antibody production comparable to the manufacturers recommended dose. We found that the 0.25-mL dose was less effective at inducing antibodies than either the standard (1.0 mL) or 0.5-mL dose, which both yielded similar titers. The primary implication of this study informs balancing resource allocation and providing efficacious immunity. By using half the manufacturer-recommended dose, the 50% cost reduction may provide sufficient monetary incentive to implement, maintain, or modify measles vaccination programs at NHP facilities.

Diarrhea with secondary decompensation is the main cause of morbidity and mortality in captive young rhesus macaque (Macaca mulatta) colonies. Approximately 25% of diarrhea cases with secondary decompensation are considered to be idiopathic chronic diarrhea. The purpose of this study was to investigate the suspected but not systematically examined association between rotavirus infection and diarrhea with secondary decompensation among young rhesus macaques at the California National Primate Research Center (CNPRC). Blood and stool samples were collected from 89 randomly selected young animals (age range: 6 months to 1.5 years) and were tested for the presence of rotavirus antibody, and rotavirus antigen, respectively, using enzyme-linked immunosorbent assays (ELISA's). Test and clinical data were analyzed using Fisher's exact tests and multivariate logistic regression model. Our analysis indicates that rotavirus is endemic among young outdoor-housed rhesus macaques at the CNPRC. Although the relationship between detectable rotavirus antigen in stool and symptomatic diarrhea with secondary decompensation was not significant, there was a significant association between rotavirus seropositivity and a history of diarrhea with secondary decompensation within the past 6 months. While our cross-sectional and case-control study suggests an association between rotavirus infection and diarrhea with secondary decompensation among captive rhesus macaques, more extensive longitudinal studies on larger cohorts and with more intensive sample collection are needed to confirm these findings.

An adult rhesus macaque developed seizures after the induction of ischemic stroke. Initially, on the day of surgery, a focal ischemic lesion was present exclusively in the right caudate nucleus. By 48 h after stroke induction, the lesion had extended into the putamen, when a seizure was observed. Our report highlights the temporal changes in infarction of unilateral basal ganglia after acute stroke and the accompanying clinical symptoms. This unusual case may provide additional information regarding the involvement of the basal ganglia in seizures, given that prior case reports and studies usually have not described the temporal and spatial evolution of the lesion before clinical symptoms emerge.

Previous research has repeatedly shown both personality and psychological stress to predict gastrointestinal disorders and chronic diarrhea in humans. The goal of the present research was to evaluate the role of personality, as well as psychological stressors (i.e., housing relocations and rearing environment), in predicting chronic diarrhea in captive Rhesus macaques, with particular attention to how personality regulated the impact of such stressors. Subjects were 1,930 R. macaques at the California National Primate Research Center reared in a variety of environments. All subjects took part in an extensive personality evaluation at approximately 90-120 days of age. Data were analyzed using generalized linear models to determine how personality, rearing condition, housing relocations, and personality by environment interactions, predicted both diarrhea risk (an animal's risk for having diarrhea at least once) and chronic diarrhea (how many repeated bouts of diarrhea an animal had after their initial bout). Much like the human literature, we found that certain personality types (i.e., nervous, gentle, vigilant, and not confident) were more likely to have chronic diarrhea, and that certain stressful environments (i.e., repeated housing relocations) increased diarrhea risk. We further found multiple interactions between personality and environment, supporting the "interactionist" perspective on personality and health. We conclude that while certain stressful environments increase risk for chronic diarrhea, the relative impact of these stressors is highly dependent on an animal's personality.

An adult rhesus macaque developed seizures after the induction of ischemic stroke. Initially, on the day of surgery, a focal ischemic lesion was present exclusively in the right caudate nucleus. By 48 h after stroke induction, the lesion had extended into the putamen, when a seizure was observed. Our report highlights the temporal changes in infarction of unilateral basal ganglia after acute stroke and the accompanying clinical symptoms. This unusual case may provide additional information regarding the involvement of the basal ganglia in seizures, given that prior case reports and studies usually have not described the temporal and spatial evolution of the lesion before clinical symptoms emerge.

Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immunocompromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.

Background:The Caribbean vervet monkey (Chlorocebus aethiops sabaeus) is a potentially valuable animal model of neurodegenerative disease. However, the trajectory of aging in vervets and its relationship to human disease is incompletely understood. Methods:To characterize biomarkers associated with neurodegeneration, we measured cerebrospinal fluid (CSF) concentrations of Aβ1-40, Aβ1-42, total tau, and p-tau181 in 329 members of a multigenerational pedigree. Linkage and genome-wide association were used to elucidate a genetic contribution to these traits. Results:Aβ1-40 concentrations were significantly correlated with age, brain total surface area, and gray matter thickness. Levels of p-tau181 were associated with cerebral volume and brain total surface area. Among the measured analytes, only CSF Aβ1-40 was heritable. No significant linkage (LOD > 3.3) was found, though suggestive linkage was highlighted on chromosomes 4 and 12. Genome-wide association identified a suggestive locus near the chromosome 4 linkage peak. Conclusions:Overall, these results support the vervet as a non-human primate model of amyloid-related neurodegeneration, such as Alzheimer's disease and cerebral amyloid angiopathy, and highlight Aβ1-40 and p-tau181 as potentially valuable biomarkers of these processes.

Background:The Caribbean vervet monkey (Chlorocebus aethiops sabaeus) is a potentially valuable animal model of neurodegenerative disease. However, the trajectory of aging in vervets and its relationship to human disease is incompletely understood. Methods:To characterize biomarkers associated with neurodegeneration, we measured cerebrospinal fluid (CSF) concentrations of Aβ1-40, Aβ1-42, total tau, and p-tau181 in 329 members of a multigenerational pedigree. Linkage and genome-wide association were used to elucidate a genetic contribution to these traits. Results:Aβ1-40 concentrations were significantly correlated with age, brain total surface area, and gray matter thickness. Levels of p-tau181 were associated with cerebral volume and brain total surface area. Among the measured analytes, only CSF Aβ1-40 was heritable. No significant linkage (LOD > 3.3) was found, though suggestive linkage was highlighted on chromosomes 4 and 12. Genome-wide association identified a suggestive locus near the chromosome 4 linkage peak. Conclusions:Overall, these results support the vervet as a non-human primate model of amyloid-related neurodegeneration, such as Alzheimer's disease and cerebral amyloid angiopathy, and highlight Aβ1-40 and p-tau181 as potentially valuable biomarkers of these processes.

A female rhesus macaque developed two episodes of generalized convulsions during transcutaneous spinal cord stimulation (TSCS) and urodynamic studies under ketamine anesthesia. The seizures took place in the absence of active TSCS and bladder pressure elevation. Ketamine anesthesia remains the primary risk factor for the convulsions during these experimental procedures.

To better understand Simian betaretrovirus (SRV) seropositivity in virus-negative macaques, we transfused blood from SRV-infected or suspect donors into immunosuppressed naive recipients. Our results do not support typical SRV1-5 infection as the cause, but provide evidence for several possibilities including serological artifact, new/different SRV, or an endogenous virus.

There has been a recent surge in research on primate infectious disease ecology. Two major areas remain relatively unaddressed to date-the prevalence of enteric bacterial parasites and the role of anthropogenic environmental factors in parasite acquisition in commensally living primate populations. In this preliminary assessment, we address both these gaps by assessing the prevalence, and the role of anthropogenic factors in shaping this prevalence, of three enteric bacterial parasites-E . coli O157:H7, Salmonella sp., Shigella sp.-across populations of rhesus macaques (M. mulatta) that live commensally with humans in Dehradun, northern India. Across 10-week study period, we collected data on (1) human-macaque behavioral interactions, (2) macaque and human demographic and activity scans, and (3) macaque fecal samples from the environment at four different locations in Dehradun. Biochemical tests and morphology-based confirmations clearly established the presence of all three enteric bacterial parasites in rhesus macaques. Overall prevalence ranged from 2 to 5 %, with Shigella sp. being the most prevalent. Regression analyses linking anthropogenic factors to bacterial prevalence showed a positive association between rates of macaques eating human garbage and E. coli O157:H7 (β = 0.23, p = 0.083), but a negative association with Salmonella sp. (β = -0.17, p = 0.026). Rather, the prevalence of Salmonella sp. was positively linked to rates of macaque eating provisioned food (β = 0.0012, p = 0.058). Finally, we found no relationship between anthropogenic factors and the prevalence of Shigella sp. Our findings establish the prevalence of enteric bacterial parasites in commensal populations of primates and suggest that although anthropogenic factors are linked to bacterial prevalence, the nature of the relationships may depend on the socioecological/foraging strategies of macaques and the food sources that facilitate the environmental survival of particular types of enteric bacteria over others.

UnlabelledStaphylococcus aureus is an important pathogen of humans and animals. We genome sequenced 90 S. aureus isolates from The Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates, and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys (Chlorocebus sabaeus) in The Gambia over different time scales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by the loss of phage-carrying genes that are known to play an important role in human colonization. We also report recent anthroponotic transmission of the well-characterized human lineages sequence type 6 (ST6) and ST15 to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact, there might be an increased risk of additional interspecies exchanges of potential pathogens.ImportanceThe population structures of Staphylococcus aureus in humans and monkeys in sub-Saharan Africa have been previously described using multilocus sequence typing (MLST). However, these data lack the power to accurately infer details regarding the origin and maintenance of new adaptive lineages. Here, we describe the use of whole-genome sequencing to detect transmission of S. aureus between humans and nonhuman primates and to document the genetic changes accompanying host adaptation. We note that human-to-monkey switches tend to be more common than the reverse and that a novel monkey-associated clade is likely to have emerged from such a switch approximately 2,700 years ago. Moreover, analysis of the accessory genome provides important clues as to the genetic changes underpinning host adaptation and, in particular, shows that human-to-monkey switches tend to be associated with the loss of genes known to confer adaptation to the human host.

UnlabelledStaphylococcus aureus is an important pathogen of humans and animals. We genome sequenced 90 S. aureus isolates from The Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates, and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys (Chlorocebus sabaeus) in The Gambia over different time scales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by the loss of phage-carrying genes that are known to play an important role in human colonization. We also report recent anthroponotic transmission of the well-characterized human lineages sequence type 6 (ST6) and ST15 to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact, there might be an increased risk of additional interspecies exchanges of potential pathogens.ImportanceThe population structures of Staphylococcus aureus in humans and monkeys in sub-Saharan Africa have been previously described using multilocus sequence typing (MLST). However, these data lack the power to accurately infer details regarding the origin and maintenance of new adaptive lineages. Here, we describe the use of whole-genome sequencing to detect transmission of S. aureus between humans and nonhuman primates and to document the genetic changes accompanying host adaptation. We note that human-to-monkey switches tend to be more common than the reverse and that a novel monkey-associated clade is likely to have emerged from such a switch approximately 2,700 years ago. Moreover, analysis of the accessory genome provides important clues as to the genetic changes underpinning host adaptation and, in particular, shows that human-to-monkey switches tend to be associated with the loss of genes known to confer adaptation to the human host.

Specific pathogen free (SPF) macaques provide valuable animal models for biomedical research. In 1989, the National Center for Research Resources [now Office of Research Infrastructure Programs (ORIP)] of the National Institutes of Health initiated experimental research contracts to establish and maintain SPF colonies. The derivation and maintenance of SPF macaque colonies is a complex undertaking requiring knowledge of the biology of the agents for exclusion and normal physiology and behavior of macaques, application of the latest diagnostic technology, facilitiy management, and animal husbandry. This review provides information on the biology of the four viral agents targeted for exclusion in ORIP SPF macaque colonies, describes current state-of-the-art viral diagnostic algorithms, presents data from proficiency testing of diagnostic assays between laboratories at institutions participating in the ORIP SPF program, and outlines management strategies for maintaining the integrity of SPF colonies using results of diagnostic testing as a guide to decision making.

Concentric left ventricular hypertrophy (LVH) is a hallmark finding in hypertrophic cardiomyopathy that leads to diastolic dysfunction and variable cardiac consequences as severe as congestive heart failure and sudden cardiac death. LVH was diagnosed postmortem in a large colony of rhesus macaques (Macaca mulatta), but methods to screen and diagnose LVH in living animals are desired. We hypothesized that targeted echocardiography of macaques with a familial association of LVH would yield antemortem LVH diagnoses. We also hypothesized that cardiac biomarker levels would be higher in sudden-death LVH or occult LVH than controls and that cardiac troponin I (cTnI) levels would be higher in macaques housed outdoors than indoors. Sera were assayed for cardiac biomarkers (cTnI, C-reactive protein, creatinine kinase-MB, creatine phosphokinase, and LDH), in conjunction with echocardiography, after diagnosis by postmortem exam or from animals with different levels of exercise due to indoor compared with outdoor housing. None of the investigated biomarkers were associated with LVH. cTnI levels were significantly higher in serum collected from outdoor than indoor macaques. In addition, LVH was diagnosed in 29.4% of subjects with a familial association of LVH. These findings suggest that exercise may increase cTnI levels in rhesus macaques and that targeted echocardiography of rhesus macaques with a familial association of LVH was the most useful variable examined for disease surveillance.

Tuberculosis (TB) pathologic lesions in rhesus macaques resemble those in humans. The expression levels of several host TB candidate genes in the peripheral blood mononuclear cells (PBMCs) of six rhesus macaques experimentally infected with Mycobacterium tuberculosis were quantified pre-infection and at several dates post-infection. Quantitative measures of TB histopathology in the lungs including: granuloma count, granuloma size, volume of granulomatous and non-granulomatous lesions, and direct bacterial load, were used as the outcomes of a multi-level Bayesian regression model in which expression levels of host genes at various dates were used as predictors. The results indicate that the expression levels of TR4, CD40, CD40L, FAS (CD95) and TNF in PBMC were associated with quantitative measures of the severity of TB histopathologic lesions in the lungs of the study animals. Moreover, no reliable association between the expression levels of IFNE in PBMCs and the severity of TB lesions in the lungs of the study animals was found. In conclusion, PBMC expression profiles derived from the above-listed host genes might be appropriate biomarkers for probabilistic diagnosis and/or prognosis of TB severity in rhesus macaques.

It is currently unclear whether changes in viral communities will ever be predictable. Here we investigate whether viral communities in wildlife are inherently structured (inferring predictability) by looking at whether communities are assembled through deterministic (often predictable) or stochastic (not predictable) processes. We sample macaque faeces across nine sites in Bangladesh and use consensus PCR and sequencing to discover 184 viruses from 14 viral families. We then use network modelling and statistical null-hypothesis testing to show the presence of non-random deterministic patterns at different scales, between sites and within individuals. We show that the effects of determinism are not absolute however, as stochastic patterns are also observed. In showing that determinism is an important process in viral community assembly we conclude that it should be possible to forecast changes to some portion of a viral community, however there will always be some portion for which prediction will be unlikely.

It is currently unclear whether changes in viral communities will ever be predictable. Here we investigate whether viral communities in wildlife are inherently structured (inferring predictability) by looking at whether communities are assembled through deterministic (often predictable) or stochastic (not predictable) processes. We sample macaque faeces across nine sites in Bangladesh and use consensus PCR and sequencing to discover 184 viruses from 14 viral families. We then use network modelling and statistical null-hypothesis testing to show the presence of non-random deterministic patterns at different scales, between sites and within individuals. We show that the effects of determinism are not absolute however, as stochastic patterns are also observed. In showing that determinism is an important process in viral community assembly we conclude that it should be possible to forecast changes to some portion of a viral community, however there will always be some portion for which prediction will be unlikely.

UnlabelledAfrican green monkeys (AGMs) are naturally infected with simian immunodeficiency virus (SIV) at high prevalence levels and do not progress to AIDS. Sexual transmission is the main transmission route in AGM, while mother-to-infant transmission (MTIT) is negligible. We investigated SIV transmission in wild AGMs to assess whether or not high SIV prevalence is due to differences in mucosal permissivity to SIV (i.e., whether the genetic bottleneck of viral transmission reported in humans and macaques is also observed in AGMs in the wild). We tested 121 sabaeus AGMs (Chlorocebus sabaeus) from the Gambia and found that 53 were SIV infected (44%). By combining serology and viral load quantitation, we identified 4 acutely infected AGMs, in which we assessed the diversity of the quasispecies by single-genome amplification (SGA) and documented that a single virus variant established the infections. We thus show that natural SIV transmission in the wild is associated with a genetic bottleneck similar to that described for mucosal human immunodeficiency virus (HIV) transmission in humans. Flow cytometry assessment of the immune cell populations did not identify major differences between infected and uninfected AGM. The expression of the SIV coreceptor CCR5 on CD4+ T cells dramatically increased in adults, being higher in infected than in uninfected infant and juvenile AGMs. Thus, the limited SIV MTIT in natural hosts appears to be due to low target cell availability in newborns and infants, which supports HIV MTIT prevention strategies aimed at limiting the target cells at mucosal sites. Combined, (i) the extremely high prevalence in sexually active AGMs, (ii) the very efficient SIV transmission in the wild, and (iii) the existence of a fraction of multiparous females that remain uninfected in spite of massive exposure to SIV identify wild AGMs as an acceptable model of exposed, uninfected individuals.ImportanceWe report an extensive analysis of the natural history of SIVagm infection in its sabaeus monkey host, the African green monkey species endemic to West Africa. Virtually no study has investigated the natural history of SIV infection in the wild. The novelty of our approach is that we report for the first time that SIV infection has no discernible impact on the major immune cell populations in natural hosts, thus confirming the nonpathogenic nature of SIV infection in the wild. We also focused on the correlates of SIV transmission, and we report, also for the first time, that SIV transmission in the wild is characterized by a major genetic bottleneck, similar to that described for HIV-1 transmission in humans. Finally, we report here that the restriction of target cell availability is a major correlate of the lack of SIV transmission to the offspring in natural hosts of SIVs.

Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

Vaginal inoculation of rhesus macaques (RM) with simian immunodeficiency virus (SIV) has been used to study the biology of HIV transmission. Although the results of vaginal SIV transmission experiments could be affected by vaginal inflammation, studies to date have been conducted without regard to levels of pre-existing genital inflammation present in RM. We collected cevicovaginal secretions (CVS) from 33-36 RM during the mid menstrual cycle (day 10-20) at 2 time points approximately 8 months apart and characterized the mRNA and protein levels of inflammatory cytokines, chemokines and interferon-stimulated genes. There was extreme variability in the levels of inflammatory mediators (IFN-α, IFN-γ, IL-6, TNF, IL-1b, IP-10, MIG, IL-12 and IL-17). In most animals, the mRNA levels of the inflammatory mediators were similar in the 2 CVS samples collected 8 months apart, suggesting that genital inflammation is stable in a subset of captive female RM. At both time points the cervicovaginal microbiota had low levels of Lactobacillus and was relatively diverse with an average of 13 genera in the samples from the first time point (median 13, range 7-21) and an average of 11.5 genera in the samples from the second time point (median 11, range 5-20). Many of the macaques had similar microbiota in the samples collected 8 months apart. However, we found no correlation between specific bacterial genera and the mRNA or protein levels of the inflammatory mediators in the genital tract of RM in this study. It seems likely that results of published vaginal SIV transmission experiments in RM have been influenced by pre-existing inflammation in the animals used for the experiments.

Vaginal inoculation of rhesus macaques (RM) with simian immunodeficiency virus (SIV) has been used to study the biology of HIV transmission. Although the results of vaginal SIV transmission experiments could be affected by vaginal inflammation, studies to date have been conducted without regard to levels of pre-existing genital inflammation present in RM. We collected cevicovaginal secretions (CVS) from 33-36 RM during the mid menstrual cycle (day 10-20) at 2 time points approximately 8 months apart and characterized the mRNA and protein levels of inflammatory cytokines, chemokines and interferon-stimulated genes. There was extreme variability in the levels of inflammatory mediators (IFN-α, IFN-γ, IL-6, TNF, IL-1b, IP-10, MIG, IL-12 and IL-17). In most animals, the mRNA levels of the inflammatory mediators were similar in the 2 CVS samples collected 8 months apart, suggesting that genital inflammation is stable in a subset of captive female RM. At both time points the cervicovaginal microbiota had low levels of Lactobacillus and was relatively diverse with an average of 13 genera in the samples from the first time point (median 13, range 7-21) and an average of 11.5 genera in the samples from the second time point (median 11, range 5-20). Many of the macaques had similar microbiota in the samples collected 8 months apart. However, we found no correlation between specific bacterial genera and the mRNA or protein levels of the inflammatory mediators in the genital tract of RM in this study. It seems likely that results of published vaginal SIV transmission experiments in RM have been influenced by pre-existing inflammation in the animals used for the experiments.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome.

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome.

The threat of zoonotic transmission of infectious agents at monkey temples highlights the necessity of investigating the prevalence of enzootic infectious agents in these primate populations. Biological samples were collected from 39 rhesus macaques at the Swoyambhu Temple and tested by enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction, or combination of these tests for evidence of infection with rhesus cytomegalovirus (RhCMV), Cercopithecine herpesvirus 1 (CHV-1), simian virus 40 (SV40), simian retrovirus (SRV), simian T-cell lymphotropic virus (STLV), simian immunodeficiency virus (SIV), and simian foamy virus (SFV). Antibody seroprevalence was 94.9% to RhCMV (37/39), 89.7% to SV40 (35/39), 64.1% to CHV-1 (25/39), and 97.4% to SFV (38/39). Humans who come into contact with macaques at Swoyambhu risk exposure to enzootic primateborne viruses. We discuss implications for public health and primate management strategies that would reduce contact between humans and primates.

The threat of zoonotic transmission of infectious agents at monkey temples highlights the necessity of investigating the prevalence of enzootic infectious agents in these primate populations. Biological samples were collected from 39 rhesus macaques at the Swoyambhu Temple and tested by enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction, or combination of these tests for evidence of infection with rhesus cytomegalovirus (RhCMV), Cercopithecine herpesvirus 1 (CHV-1), simian virus 40 (SV40), simian retrovirus (SRV), simian T-cell lymphotropic virus (STLV), simian immunodeficiency virus (SIV), and simian foamy virus (SFV). Antibody seroprevalence was 94.9% to RhCMV (37/39), 89.7% to SV40 (35/39), 64.1% to CHV-1 (25/39), and 97.4% to SFV (38/39). Humans who come into contact with macaques at Swoyambhu risk exposure to enzootic primateborne viruses. We discuss implications for public health and primate management strategies that would reduce contact between humans and primates.

Nonhuman primates are widely used in biomedical research because of their genetic, anatomic, and physiologic similarities to humans. In this setting, human contact directly with macaques or with their tissues and fluids sometimes occurs. Cercopithecine herpesvirus 1 (B virus), an alphaherpesvirus endemic in Asian macaques, is closely related to herpes simplex virus (HSV). Most macaques carry B virus without overt signs of disease. However, zoonotic infection with B virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment. Although the incidence of human infection with B virus is low, a death rate of >70% before the availability of antiviral therapy makes this virus a serious zoonotic threat. Knowledge of the clinical signs and risk factors for human B-virus disease allows early initiation of antiviral therapy and prevents severe disease or death.

Nonhuman primates are widely used in biomedical research because of their genetic, anatomic, and physiologic similarities to humans. In this setting, human contact directly with macaques or with their tissues and fluids sometimes occurs. Cercopithecine herpesvirus 1 (B virus), an alphaherpesvirus endemic in Asian macaques, is closely related to herpes simplex virus (HSV). Most macaques carry B virus without overt signs of disease. However, zoonotic infection with B virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment. Although the incidence of human infection with B virus is low, a death rate of >70% before the availability of antiviral therapy makes this virus a serious zoonotic threat. Knowledge of the clinical signs and risk factors for human B-virus disease allows early initiation of antiviral therapy and prevents severe disease or death.

A nonhuman primate model of tuberculosis that closely resembles human disease is urgently needed. We have evaluated the Philippine cynomolgus monkey, Macaca fasicularis, as a model of TB. Cynomolgus monkeys challenged intratracheally with extremely high doses of Mycobacterium tuberculosis (10(5) or 10(4) CFU) developed an acute, rapidly progressive, highly fatal multilobar pneumonia. However, monkeys challenged with moderate or low doses of M. tuberculosis (