The Vaginal Microbiome of Nonhuman Primates Can Be Only Transiently Altered to Become Lactobacillus Dominant without Reducing Inflammation.

The vaginal microbiome composition in humans is categorized based upon the degree to which one of four species of Lactobacillus is dominant (Lactobacillus crispatus, community state type I [CST I], Lactobacillus gasseri, CST II, Lactobacillus iners, CST III, and Lactobacillus jensenii, CST V). Women with a vaginal microbiome not dominated by one of the four Lactobacillus species tend to have a more diverse microbiome, CST IV. CSTs I, II, III, and V are common in North America and Europe and are associated with lower incidences of some pathogens, such as human immunodeficiency virus (HIV), human papillomavirus (HPV), and Gardnerella vaginalis. As a result, therapeutic interventions to change the composition of the vaginal microbiomes are under development. However, Homo sapiens is the only mammalian species which has high frequencies of Lactobacillus-dominated vaginal microbiomes. Here, we treated female nonhuman primates (NHPs) with regimens of metronidazole and high levels of L. crispatus to determine how well these animals could be colonized with L. crispatus, how this influenced the immunological milieu, and how Lactobacillus treatment influenced or was influenced by the endogenous vaginal microbiome. We find that NHPs can transiently be colonized with L. crispatus, that beta diversity and not the number of doses of L. crispatus or pretreatment with metronidazole predicts subsequent L. crispatus colonization, that L. crispatus does not alter the local immunological milieu, and that the vaginal microbiome composition was resilient, normalizing by 4 weeks after our manipulations. Overall, this study suggests these animals are not amenable to long-term L. crispatus colonization. IMPORTANCE NHPs have proven to be invaluable animal models for the study of many human infectious diseases. The use of NHPs to study the effect of the microbiome on disease transmission and susceptibility is limited due to differences between the native microbiomes of humans and NHPs. In particular, Lactobacillus dominance of the vaginal microbiome is unique to humans and remains an important risk factor in reproductive health. By assessing the extent to which NHPs can be colonized with exogenously applied L. crispatus to resemble a human vaginal microbiome and examining the effects on the vaginal microenvironment, we highlight the utility of NHPs in analysis of vaginal microbiome manipulations in the context of human disease.

Age-Related Colonic Mucosal Microbiome Community Shifts in Monkeys.

Age-related changes in gut microbiome impact host health. The interactive relationship between the microbiome and physiological systems in an aged body system remains to be clearly defined, particularly in the context of inflammation. Therefore, we aimed to evaluate systemic inflammation, microbial translocation (MT), and differences between fecal and mucosal microbiomes. Ascending colon mucosal biopsies, fecal samples, and blood samples from healthy young and old female vervet monkeys were collected for 16S rRNA gene sequencing, MT, and cytokine analyses, respectively. To demonstrate microbial co-occurrence patterns, we used Kendall's tau correlation measure of interactions between microbes. We found elevated levels of plasma LBP-1, MCP-1, and CRP in old monkeys, indicative of higher MT and systemic inflammation. Microbiome analysis revealed significant differences specific to age. At the phylum level, abundances of pathobionts such as Proteobacteria were increased in the mucosa of old monkeys. At the family level, Helicobacteriaceae was highly abundant in mucosal samples (old); in contrast, Ruminococcaceae were higher in the fecal samples of old monkeys. We found significantly lower Firmicutes:Bacteroidetes ratio and lower abundance of butyrate-producing microbes in old monkeys, consistent with less healthy profiles. Microbial community co-occurrence analysis on mucosal samples revealed 13 nodes and 41 associations in the young monkeys, but only 12 nodes and 21 associations in the old monkeys. Our findings provide novel insights into systemic inflammation and gut microbial interactions, highlight the importance of the mucosal niche, and facilitate further understanding of the decline in the stability of the microbial community with aging.

Shifts in microbial diversity, composition, and functionality in the gut and genital microbiome during a natural SIV infection in vervet monkeys.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: The 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.

Strain diversity of Treponema pallidum subsp. pertenue suggests rare interspecies transmission in African nonhuman primates.

In our most recent study, we found that in Tanzania infection with Treponema pallidum (TP) subsp. pertenue (TPE) is present in four different monkey species. In order to gain information on the diversity and epidemiological spread of the infection in Tanzanian nonhuman primates (NHP), we identified two suitable candidate genes for multi-locus sequence typing (MLST). We demonstrate the functionality of the MLST system in invasively and non-invasively collected samples. While we were not able to demonstrate frequent interspecies transmission of TPE in Tanzanian monkeys, our results show a clustering of TPE strains according to geography and not host species, which is suggestive for rare transmission events between different NHP species. In addition to the geographic stability, we describe the relative temporal stability of the strains infecting NHPs and identified multi-strain infection. Differences between TPE strains of NHP and human origin are highlighted. Our results show that antibiotic resistance does not occur in Tanzanian TPE strains of NHP origin.

Variations in the microbiome due to storage preservatives are not large enough to obscure variations due to factors such as host population, host species, body site, and captivity.

The study of the primate microbiome is critical in understanding the role of the microbial community in the host organism. To be able to isolate the main factors responsible for the differences observed in microbiomes within and between individuals, confounding factors due to technical variations need to be removed. To determine whether alterations due to preservatives outweigh differences due to factors such as host population, host species, body site, and habitat, we tested three methods (no preservative, 96% ethanol, and RNAlater) for preserving wild chimpanzee (fecal), wild lemur (fecal), wild vervet monkey (rectal, oral, nasal, otic, vaginal, and penile), and captive vervet monkey (rectal) samples. All samples were stored below - 20°C (short term) at the end of the field day and then at - 80°C until DNA extraction. Using 16S rRNA gene sequencing, we show a significant preservative effect on microbiota composition and diversity. Samples stored in ethanol and RNAlater appear to be less different compared with samples not stored in any preservative (none). Our differential analysis revealed significantly higher amounts of Enterococcaceae and Family XI in no preservative samples, Prevotellaceae and Spirochaetaceae in ethanol and RNAlater preserved samples, Oligosphaeraceae in ethanol-preserved samples, and Defluviitaleaceae in RNAlater preserved samples. While these preservative effects on the microbiome are not large enough to remove or outweigh the differences arising from biological factors (e.g., host species, body site, and habitat differences) they may promote misleading interpretations if they have large enough effect sizes compared to the biological factors (e.g., host population).

Microbial translocation into amniotic fluid of vervet monkeys is common and unrelated to adverse infant outcomes.

Amniotic fluid was collected from pregnant female African green monkeys (n = 20). Analyses indicate microbial translocation into amniotic fluid during pregnancy is typical, and microbial load reduces across gestation. Microbial translocation does not relate to infant outcome or maternal factors. Lastly, we demonstrate that sample contamination is easily introduced and detectable.

Composition and stability of the vervet monkey milk microbiome.

The human milk microbiome is vertically transmitted to offspring during the postnatal period and has emerged as a critical driver of infant immune and metabolic development. Despite this importance in humans, the milk microbiome of nonhuman primates remains largely unexplored. This dearth of comparative work precludes our ability to understand how species-specific differences in the milk microbiome may differentially drive maternal effects and limits how translational models can be used to understand the role of vertically transmitted milk microbes in human development. Here, we present the first culture-independent data on the milk microbiome of a nonhuman primate. We collected milk and matched fecal microbiome samples at early and late lactation from a cohort of captive lactating vervet monkeys (N = 15). We found that, similar to humans, the vervet monkey milk microbiome comprises a shared community of taxa that are universally present across individuals. However, unlike in humans, this shared community is dominated by the genera Lactobacillus, Bacteroides, and Prevotella. We also found that, in contrast to previous culture-dependent studies in humans, the vervet milk microbiome exhibits greater alpha-diversity than the gut microbiome across lactation. Finally, we did not find support for the translocation of microbes from the gut to the mammary gland within females (i.e., "entero-mammary pathway"). Taken together, our results show that the vervet monkey milk microbiome is taxonomically diverse, distinct from the gut microbiome, and largely stable. These findings demonstrate that the milk microbiome is a unique substrate that may selectively favor the establishment and persistence of particular microbes across lactation and highlights the need for future experimental studies on the origin of microbes in milk.

Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies.

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species.

Biofilm formation of hypermucoviscous and non-hypermucoviscous Klebsiella pneumoniae recovered from clinically affected African green monkey (Chlorocebus aethiops sabaeus).

In recent years, an emergent Klebsiella pneumoniae hypermucoviscous (HMV) phenotype has been associated with increased invasiveness and pathogenicity in primates. The HMV phenotype is characterized by different capsular serotypes, associated with several genes including the rmpA (regulator of mucoid phenotype) and magA (mucoviscosity-associated) genes. In African green monkeys (AGM) (Chlorocebus aethiops sabaeus) serotypes K1 and K5 have been implicated in fatal multisystemic abscesses. In order to better understand the epizootiology of this pathogen, the capacity of biofilm production of K. pneumoniae isolates presenting the HMV was compared to non-HMV isolates at three different temperatures (25, 30 and 37 °C). The results indicate that HMV and non-HMV isolates display similar capacity to form biofilms at the three different evaluated temperatures. Temperature appears to play a role in the formation of biofilms by K. pneumoniae presenting the HMV phenotype, where larger biofilms were formed at 37 °C than at 25 °C. Knowledge regarding local environmental sources of K. pneumoniae and the possible role of wildlife in the maintenance of this agent in the area is necessary to develop effective recommendations for the prevention and management of this disease in captive AGM populations.

Acanthamoeba and Fusarium interactions: A possible problem in keratitis.

The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoebae and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of three Acanthamoeba castellanii strains with Fusarium conidia and the possible implications on keratitis. F. conidia were internalized by A. castellanii strains and were able to germinate inside the amoebae. The co-culture with the live amoebae, as well as the amoebal culture supernatant and lysate, increased the fungal growth significantly. Moreover, live F. solani and its culture supernatant enhanced the survival of amoebae, but in a different way in each amoebal strain. The encystment of the A. castellanii strain re-isolated from rat lung was increased by the fungus. These results show that A. castellanii and F. solani interaction may have an important influence on survival of both, and specially indicate a possible effect on virulence characteristics of these microorganisms. These data suggest that the A. castellanii-F. solani interaction may cause severe impacts on keratitis.

Comparison of experimental respiratory tularemia in three nonhuman primate species.

Tularemia is a zoonotic disease caused by Francisella tularensis, which is transmitted to humans most commonly by contact with infected animals, tick bites, or inhalation of aerosolized bacteria. F. tularensis is highly infectious via the aerosol route; inhalation of as few as 10-50 organisms can cause pneumonic tularemia. Left untreated, the pneumonic form has more than >30% case-fatality rate but with early antibiotic intervention can be reduced to 3%. This study compared tularemia disease progression across three species of nonhuman primates [African green monkey (AGM), cynomolgus macaque (CM), and rhesus macaque (RM)] following aerosolized F. tularensis Schu S4 exposure. Groups of the animals exposed to various challenge doses were observed for clinical signs of infection and blood samples were analyzed to characterize the disease pathogenesis. Whereas the AGMs and CMs succumbed to disease following challenge doses of 40 and 32 colony forming units (CFU), respectively, the RM lethal dose was 276,667 CFU. Following all challenge doses that caused disease, the NHPs experienced weight loss, bacteremia, fever as early as 4 days post exposure, and tissue burden. Necrotizing-to-pyogranulomatous lesions were observed most commonly in the lung, lymph nodes, spleen, and bone marrow. Overall, the CM model consistently manifested pathological responses similar to those resulting from inhalation of F. tularensis in humans and thereby most closely emulates human tularemia disease. The RM model displayed a higher tolerance to infection and survived exposures of up to 15,593 CFU of aerosolized F. tularensis.

Selection of models for the analysis of risk-factor trees: leveraging biological knowledge to mine large sets of risk factors with application to microbiome data.

MOTIVATION: Establishment of a statistical association between microbiome features and clinical outcomes is of growing interest because of the potential for yielding insights into biological mechanisms and pathogenesis. Extracting microbiome features that are relevant for a disease is challenging and existing variable selection methods are limited due to large number of risk factor variables from microbiome sequence data and their complex biological structure. RESULTS: We propose a tree-based scanning method, Selection of Models for the Analysis of Risk factor Trees (referred to as SMART-scan), for identifying taxonomic groups that are associated with a disease or trait. SMART-scan is a model selection technique that uses a predefined taxonomy to organize the large pool of possible predictors into optimized groups, and hierarchically searches and determines variable groups for association test. We investigate the statistical properties of SMART-scan through simulations, in comparison to a regular single-variable analysis and three commonly-used variable selection methods, stepwise regression, least absolute shrinkage and selection operator (LASSO) and classification and regression tree (CART). When there are taxonomic group effects in the data, SMART-scan can significantly increase power by using bacterial taxonomic information to split large numbers of variables into groups. Through an application to microbiome data from a vervet monkey diet experiment, we demonstrate that SMART-scan can identify important phenotype-associated taxonomic features missed by single-variable analysis, stepwise regression, LASSO and CART.

Role of anaerobic bacteria in simian enteric diseases.

Screening of monkeys for anaerobic bacteria showed that the incidence of non-spore-forming anaerobes was 2.0-2.5 times higher than that of clostridia. The overwhelming majority of isolated anaerobes were saprophytes and opportunistic bacteria. The incidence of C. perfringens was higher in monkeys with enteric infections of obscure etiology and dead from these diseases than in healthy animals (48.2 vs. 36.8%).

An outbreak of Yersinia enterocolitica in a captive colony of African green monkeys (Chlorocebus aethiops sabaeus) in the Caribbean.

Yersinia enterocolitica is a zoonotic gram-negative pathogen that causes mesenteric lymphadenitis, terminal ileitis, acute gastroenteritis, and septicemia in domestic animals and primates. In 2012, 46 captive African green monkeys (Chlorocebus aethiops sabaeus) died during an outbreak of acutely fatal enteric disease over a period of 1 mo on the island of St Kitts. The affected monkeys presented with a history of mucohemorrhagic diarrhea, marked dehydration, and depression. Fifteen bacterial isolates were recovered from the spleen, liver, and lungs of affected monkeys. All isolates were identified as Y. enterocolitica by biochemical analysis and sequence comparison of the 16S rRNA gene. Phenotypic and genotypic analysis of the recovered isolates revealed homogeneity among the recovered bacteria, and all isolates gave a random amplified polymorphic DNA pattern resembling that given by genotype D under serotypes O:7,8. This outbreak represents the first isolation and characterization of Y. enterocolitica as the causative agent of fatal enteric disease in primates in the Caribbean.

Biochemical changes in cerebrospinal fluid of Chlorocebus aethiops naturally infected with zoonotic Meningonema peruzzii.

BACKGROUND: Thirty-four wild Chlorocebus aethiops monkeys were trapped for research purposes. METHODS: During routine quarantine check-up, cerebrospinal fluid (CSF) and blood were microscopically examined for parasites. Estimations of CSF protein levels were made by the biuret method and the white cell counts by the hemocytometer. RESULTS: Seven monkeys demonstrated microfilariae in blood and CSF. This was accompanied by a two- and ninefold increase in CSF total protein and white cell counts, respectively. Necropsy of one of the blood and CSF microfilariae-positive animals revealed the presence of adult worms in the brain meninges. The parasites were identified as the zoonotic filaroid nematode Meningonema peruzii. CONCLUSIONS: Wild C. aethiops monkeys developed CSF changes resulting, most probably, from infection with M. peruzii. Moreover, the monkeys could be acting as an important reservoir. The study highlights the need for epidemiological and pathogenological studies of this parasite, which is of public health significance. Moreover, C. aethiops proved to be a useful primate model for the study of this zoonotic infection.

Clinical and pathologic features of cynomolgus macaques (Macaca fascicularis) infected with aerosolized Yersinia pestis.

Since the anthrax attacks of 2001, the emphasis on developing animal models of aerosolized select agent pathogens has increased. Many scientists believe that nonhuman primate models are the most appropriate to evaluate pulmonary response to, vaccines for, and treatments for select agents such as Yersinia pestis (Y. pestis), the causative agent of plague. A recent symposium concluded that the cynomolgus macaque (Macaca fascicularis) plague model should be characterized more fully. To date, a well-characterized cynomolgus macaque model of pneumonic plague using reproducible bioaerosols of viable Y. pestis has not been published. In the current study, methods for creating reproducible bioaerosols of viable Y. pestis strain CO92 (YpCO92) and pneumonic plague models were evaluated in 22 Indonesian-origin cynomolgus macaques. Five macaques exposed to doses lower than 250 CFU remained free of any indication of plague infection. Fifteen macaques developed fever, lethargy, and anorexia indicative of clinical plague. The 2 remaining macaques died without overt clinical signs but were plague-positive on culture and demonstrated pathology consistent with plague. The lethal dose of plague in humans is reputedly less than 100 organisms; in this study, 66 CFU was the dose at which half of the macaques developed fever and clinical signs (ED(50)), The Indonesian cynomolgus macaque reproduces many aspects of human pneumonic plague and likely will provide an excellent model for studies that require a macaque model.

5' nuclease PCR assay to detect Yersinia pestis.

The 5' nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5' nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 x 10(5) copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.

Simian foamy virus isolated from an accidentally infected human individual.

Evidence for natural foamy virus (FV) infections in humans is still lacking. However, accidental infections of humans with simian FV have been demonstrated by serology and PCR, but all previous attempts to recover infectious virus in such cases have failed. Here we describe the isolation of a simian FV from peripheral blood mononuclear cells (PBMC) of a healthy animal caretaker, who acquired the virus 20 years ago from an African green monkey (AGM) bite. Properties of the human isolate such as host range in cell cultures including human PBMC and ability to induce neutralizing antibodies in the primate host proved to be similar to those of FV obtained from AGM. The genomic sequence of the isolate was found to be virtually identical to the proviral sequence present in the host lymphocytes and related to AGM isolates but distinct from those of all FV isolates handled in the laboratory. For successful virus isolation, it was essential to stimulate the host lymphocytes by phytohemagglutinin and interleukin-2 for 2 weeks prior to cocultivation with permissive cells. In contrast to the situation found in FV-infected monkeys, virus isolation from the saliva of the animal caretaker was not possible, and no evidence for FV transmission to family contacts was obtained. We conclude that, in contrast to active infection in monkeys, FV persists in a state of latency following accidental infection of humans.

Changes in normal vaginal flora of African green monkeys (Cercopithecus aethiops) during the menstrual cycle.

Changes in normal vaginal flora of African green monkeys associated with the estrous cycle were examined by the swab method. Bacteroidaceae, corynebacteria, and streptococci were predominant throughout the whole estrous cycle, although individual differences were great. It was also clear that all bacterial species tended to decrease in the menstrual phase. Lactobacilli, the most predominant bacteria in the normal vagina of humans, were detected only in very low numbers in the African green monkey, suggesting that the normal vagina of the African green monkey has a different ecosystem from that of normal human vaginal flora.

The U3 promoter region of the acutely lethal simian immunodeficiency virus clone smmPBj1.9 confers related biological activity on the apathogenic clone agm3mc.

Infection with the acutely pathogenic molecular virus clone SIVsmmPBj1.9, cloned from isolate PBj14 of simian immunodeficiency virus (SIV) from sooty mangabey monkeys (Cercocebus atys), leads to acute viral and often lethal disease within days or weeks. SIVsmmPBj1.9 has the unique property of replicating in nonstimulated peripheral blood mononuclear cells from pig-tailed macaques. In contrast, molecular virus clone SIVagm3mc of SIV from African green monkeys (Cercopithecus aethiops), which is apathogenic in its natural host and in pig-tailed macaques, is unable to grow in nonstimulated peripheral blood cells. Chimeric proviruses were constructed by exchanging defined regions of SIVagm3mc against comparable regions of SIVsmmPBj1.9. Four of five hybrid viruses generated by transfection into the CD4-positive T-cell line C8166 replicated in T-cell lines permissive for SIVagm3mc replication and in stimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Three hybrid viruses displayed the distinct biological property of SIVsmmPBj14 to replicate in nonstimulated peripheral blood cells from pig-tailed macaques and from African green monkeys. Replication in nonstimulated peripheral blood cells was dependent on the presence of the U3 promoter region of SIVsmmPBj1.9 within the viral long terminal repeat.