Molecular detection of Apicomplexa protozoa in tissues from Alouatta guariba clamitans / Detecção molecular de protozoários Apicomplexa em tecidos de Alouatta guariba clamitans

The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.(AU)

O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.(AU)

Detection of Antimicrobial Resistance Genes in Escherichia coli Isolated from Black Howler Monkeys (Alouatta pigra) and Domestic Animals in Fragmented Rain-Forest Areas in Tabasco, Mexico.

The appearance and spread of antimicrobial resistance (AMR) in bacteria in natural environments and wildlife are related to agricultural and livestock activities and are a global health and conservation problem. We assessed the presence of AMR genes in Escherichia coli isolated from black howler monkeys (Alouatta pigra), sheep (Ovis aries), cattle (Bos taurus), and horses (Equus caballus) from a highly fragmented forest in southern Mexico. Fresh fecal samples were collected using swabs, seeded on eosin-methylene blue agar, and E. coli colonies identified by PCR; multiplex-PCR was performed on E. coli DNA for the detection of 10 AMR genes from four families (sulfonamides, tetracycline, ß-lactamase, and chloramphenicol). We detected E. coli in 94% (48/51) of fecal samples, of which 33% (16/48) tested positive for at least one AMR gene. We detected AMR genes in at least one individual from each sampled animal species, with the most prevalent genes being tet(B) 18% (9/48), sul2 14% (7/48), sul1, and blaTEM 12% (6/48). Sheep samples contained AMR genes from the four families of antibiotics detected in this study and 50% (5/10) tested positive for the presence of at least one gene. A total of 12% (2/16) of fecal samples from black howler monkeys tested positive for AMR genes. The presence of AMR genes in A. pigra and domestic animals has not been reported in the Balancán area of Tabasco, Mexico. Transmission of AMR bacteria from domestic animals to monkeys is rare; however, this is a potential health risk for wildlife and species conservation.

Molecular detection of Bifidobacterium spp. in faeces of black howler monkeys (Alouatta pigra).

BACKGROUND: Bifidobacterium genus are considered to be beneficial bacteria for their hosts; however, knowledge about the specific species that are part of the gut microbiome of howler monkeys is scarce. Polymerase chain reaction (PCR) is a useful technique for the identification of non-cultivable or difficult to grow bacterial species. With the goal of detecting species of the genus Bifidobacterium in black howler monkeys, we used PCR on DNA derived from faecal samples. METHODS: We collected and extracted DNA from 40 faecal samples. Using specific primers, we performed PCR and nested PCR to detect members of the Bifidobacterium genus and a subset of species: Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium animalis subsp. animalis. RESULTS: 97.5% (39/40) of the samples were positive for Bifidobacterium spp. We found B longum in 100% of the analysed samples. CONCLUSIONS: This is the first report of B longum in black howler monkey faeces.

Occurrence of hemotropic mycoplasmas in non-human primates (Alouatta caraya, Sapajus nigritus and Callithrix jacchus) of southern Brazil.

Hemoplasmas, the erythrocyte-associated mycoplasmas, have been detected in several primates, causing mostly subclinical infection. This study aimed to determine the prevalence of hemoplasma infection in captive and free-ranging monkeys from southern Brazil, as well as factors and hematological abnormalities associated with infection. Blood samples from 40 non-human primates (NHP) were tested for hemoplasmas and coinfections. An overall of 10/40 (25.0%) NHP tested positive for hemoplasmas using PCR-based assays, including 9/14 (64.3%) black howler monkeys (Alouatta caraya) and 1/24 (4.2%) black-horned capuchin (Sapajus nigritus). Infection was not statistically associated with anemia, but wild-born monkeys and male black howler monkeys were more likely to be positive when compared with captive-born animals and female black howler monkeys, respectively. The sequences from the black howler monkey hemoplasma were similar (94% identity) to the squirrel monkey hemoplasma ("Candidatus Mycoplasma kahanei") and were phylogenetically located in a different cluster when compared to the human hemoplasma ("Candidatus Mycoplasma haemohominis").

Patterns in Gut Microbiota Similarity Associated with Degree of Sociality among Sex Classes of a Neotropical Primate.

Studies of human and domestic animal models indicate that related individuals and those that spend the most time in physical contact typically have more similar gut microbial communities. However, few studies have examined these factors in wild mammals where complex social dynamics and a variety of interacting environmental factors may impact the patterns observed in controlled systems. Here, we explore the effect of host kinship and time spent in social contact on the gut microbiota of wild, black howler monkeys (Alouatta pigra). Our results indicate that closely related individuals had less similar gut microbial communities than non-related individuals. However, the effect was small. In contrast, as previously reported in baboons and chimpanzees, individuals that spent more time in contact (0 m) and close proximity (0-1 m) had more similar gut microbial communities. This pattern was driven by adult female-adult female dyads, which generally spend more time in social contact than adult male-adult male dyads or adult male-adult female dyads. Relative abundances of individual microbial genera such as Bacteroides, Clostridium, and Streptococcus were also more similar in individuals that spent more time in contact or close proximity. Overall, our data suggest that even in arboreal primates that live in small social groups and spend a relatively low proportion of their time in physical contact, social interactions are associated with variation in gut microbiota composition. Additionally, these results demonstrate that within a given host species, subgroups of individuals may interact with the gut microbiota differently.

Phylogenetic and ecological factors impact the gut microbiota of two Neotropical primate species.

Recent studies suggest that variation in diet across time and space results in changes in the mammalian gut microbiota. This variation may ultimately impact host ecology by altering nutritional status and health. Wild animal populations provide an excellent opportunity for understanding these interactions. However, compared to clinical studies, microbial research targeting wild animals is currently limited, and many published studies focus only on a single population of a single host species. In this study we utilize fecal samples from two species of howler monkey (Alouatta pigra and A. palliata) collected at four sites to investigate factors influencing the gut microbiota at three scales: taxonomic (host species), ecosystemic (forest type), and local (habitat disturbance/season). The results demonstrate that the effect of host species on the gut microbiota is stronger than the effect of host forest type, which is stronger than the effect of habitat disturbance or seasonality. Nevertheless, within host species, gut microbiota composition differs in response to forest type, habitat disturbance, and season. Variations in the effect size of these factors are associated both with host species and environment. This information may be beneficial for understanding ecological and evolutionary questions associated with Mesoamerican howler monkeys, as well as determining conservation challenges facing each species. These mechanisms may also provide insight into the ecology of other species of howler monkeys, non-human primates, and mammals.

The gut microbiota appears to compensate for seasonal diet variation in the wild black howler monkey (Alouatta pigra).

For most mammals, including nonhuman primates, diet composition varies temporally in response to differences in food availability. Because diet influences gut microbiota composition, it is likely that the gut microbiota of wild mammals varies in response to seasonal changes in feeding patterns. Such variation may affect host digestive efficiency and, ultimately, host nutrition. In this study, we investigate the temporal variation in diet and gut microbiota composition and function in two groups (N = 13 individuals) of wild Mexican black howler monkeys (Alouatta pigra) over a 10-month period in Palenque National Park, Mexico. Temporal changes in the relative abundances of individual bacterial taxa were strongly correlated with changes in host diet. For example, the relative abundance of Ruminococcaceae was highest during periods when energy intake was lowest, and the relative abundance of Butyricicoccus was highest when young leaves and unripe fruit accounted for 68 % of the diet. Additionally, the howlers exhibited increased microbial production of energy during periods of reduced energy intake from food sources. Because we observed few changes in howler activity and ranging patterns during the course of our study, we propose that shifts in the composition and activity of the gut microbiota provided additional energy and nutrients to compensate for changes in diet. Energy and nutrient production by the gut microbiota appears to provide an effective buffer against seasonal fluctuations in energy and nutrient intake for these primates and is likely to have a similar function in other mammal species.

The role of gut microbes in satisfying the nutritional demands of adult and juvenile wild, black howler monkeys (Alouatta pigra).

In all mammals, growth, development, pregnancy, and lactation increase nutritional demands. Although primate field studies tend to focus on shifts in activity and diet as mechanisms to compensate for these demands, differences in digestive efficiency also are likely to be important. Because the gut microbiota can impact host digestive efficiency, we examined differences in activity budget, diet, and the gut microbial community among adult male (N = 4), adult female (N = 4), and juvenile (N = 5) wild black howler monkeys (Alouatta pigra) across a ten-month period in Palenque National Park, Mexico to determine how adult females and juveniles compensate for increased nutritional demands. Results indicate that adult females and juveniles consumed more protein and energy than adult males. Adult males, adult females, and juveniles also possessed distinct gut microbial communities, unrelated to diet. Juveniles exhibited a gut microbiota characterized by bacteria from the phylum Firmicutes, such as Roseburia and Ruminococcus, and demonstrated high fecal volatile fatty acid content, suggesting increased microbial contributions to host energy balances. Adult females possessed a higher Firmicutes to Bacteroidetes ratio, also suggesting increased energy production, and their gut microbiota was characterized by Lactococcus, which has been associated with folate biosynthesis. On the basis of these patterns, it appears that the gut microbiota differentially contributes to howler monkey nutrition during reproduction and growth. Determining the nutritional and energetic importance of shifts in activity, diet, and the gut microbiota in other nonhuman primate taxa, as well as humans, will transform our understanding of these life history processes and the role of host-microbe relationships in primate evolution.

Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes.

The gastrointestinal (GI) microbiome contributes significantly to host nutrition and health. However, relationships involving GI microbes, their hosts and host macrohabitats remain to be established. Here, we define clear patterns of variation in the GI microbiomes of six groups of Mexican black howler monkeys (Alouatta pigra) occupying a gradation of habitats including a continuous evergreen rainforest, an evergreen rainforest fragment, a continuous semi-deciduous forest and captivity. High throughput microbial 16S ribosomal RNA gene sequencing indicated that diversity, richness and composition of howler GI microbiomes varied with host habitat in relation to diet. Howlers occupying suboptimal habitats consumed less diverse diets and correspondingly had less diverse gut microbiomes. Quantitative real-time PCR also revealed a reduction in the number of genes related to butyrate production and hydrogen metabolism in the microbiomes of howlers occupying suboptimal habitats, which may impact host health.

Analysis of the hydrogenotrophic microbiota of wild and captive black howler monkeys (Alouatta pigra) in palenque national park, Mexico.

Intestinal methanogenesis is one of the major pathways for consumption of hydrogen produced by bacterial fermentation and is considered to affect the efficiency of host energy harvest; however, little information is available regarding the hydrogenotrophic pathways of nonhuman primates in the wild, in general, and of howler monkeys, in particular. Microbial fermentation of plant structural carbohydrates is an important feature in wild howlers owing to the high fiber and low available energy content of leaves, which make up the primary component of their diet. In contrast, captive howlers may consume greater quantities of fruits and vegetables that are higher in water, lower in fiber, and, along with commercial monkey chow commonly added to captive monkey diets, more readily digestible than the natural diet. In this study, we analyzed the composition of methanogens and sulfate-reducing bacteria (SRB) from fecal samples of black howler monkeys (Alouatta pigra) in the wild and in captivity. The hydrogenotrophic microbiota of three groups of monkeys was evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, small clone library construction, and quantitative real-time PCR. Abundance of methanogens was lower than SRB in all howler monkey groups studied. DGGE banding patterns were highly similar within each wild and captive group but distinct among groups. Desulfovibrionales-enriched DGGE showed reduced microbial diversity in the captive animals compared with their wild counterparts. Taken together, the data demonstrate that environmental or dietary changes of the host imposed by captivity likely influence the composition of intestinal hydrogenotrophs in black howler monkeys.

[Detection of yellow fever virus by reverse transcriptase polymerase chain reaction in wild monkeys: a sensitive tool for epidemiologic surveillance]. / Detección por reacción en cadena de la polimerasa de transcriptasa inversa del virus de la fiebre amarilla en monos silvestres: una herramienta sensible para la vigilancia epidemiológica.

INTRODUCTION: Yellow fever is a zoonotic infection maintained in nature by non-human primates. Appropriate surveillance with sensitive laboratory techniques is necessary to evidence viral activity in the tropical forest habitats of these primates. OBJECTIVE: Yellow fever virus was detected in hepatic tissue samples from non-human primates by reverse transcriptase polymerase chain reaction (RT-PCR) technique using specific primers for diagnosis. MATERIALS AND METHODS: Hepatic tissue samples were processed from five monkeys belonging genus Alouatta spp found dead in sylvatic areas of Cesar and Magdalena Provinces, Colombia, between December 2003 and June 2004. Samples were treated with lysis buffer prior to the isolation of viral RNA, which was then subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using yellow fever-specific primers. Simultaneously, viral proteins were identified by immunohistochemistry on parafin-embedded hepatic tissue. RESULTS: The PCR method amplified fragments of the expected size (424 bp) in four of the tested samples. In addition, these samples showed a positive reaction by immunohistochemistry, supporting the evidence that the virus was present. CONCLUSION: The detection of yellow fever virus in wild monkeys was clear evidence of enzootic activity in northern Colombia. Increased probability of yellow fever transmission among human populations is indicated due to urbanization processes as a consequence of forced migration and displacement of the human populations. Molecular tests for rapid and specific detection of yellow fever in tissue samples of non-human primates is an important tool for epidemiologic surveillance. Rapid virus identification will permit the timely activation of control systems for prevention of further cases and epidemic situations.

Failure to detect human T-lymphotropic virus antibody in wild-caught New World primates.

We conducted a study to look for a simian counterpart of human T-lymphotropic virus (HTLV) in wild-caught monkeys in the Republic of Panama. Serum specimens were obtained from 102 monkeys (Ateles fusciceps, n = 75; Alouatta villosa, n = 18; and Cebus capucinus, n = 9) captured in Panama's Darien rain forest in 1979-1980. Specimens were screened for HTLV antibody by enzyme-linked immunosorbent assay, and reactive specimens were further tested by Western blot. None of the 102 specimens were seropositive for HTLV. Our findings provide no evidence for an HTLV-like virus in New World primates from Panama, but the sample size was small, and further studies are warranted.

Sylvatic yellow fever activity in Trinidad, 1988-1989.

Of a total of 18,068 mosquitoes (361 pools) collected in south-eastern Trinidad forests from December 1988 to May 1989, 47 species belonging to 14 genera were identified. Five yellow fever virus isolates were made from Haemagogus janthinomys and one from Sabethes chloropterus. All the other pools of mosquitoes examined were negative for the virus. The mosquito isolates were made in December and January. In addition, in late February and early March, 2 infected howler monkeys (Alouatta sp.) were detected. Since March, despite continued surveillance, no yellow fever virus has been detected in mosquitoes or monkeys. There has been no reported human infection.

Sylvatic yellow fever activity in Trinidad, 1988-1989

Of a total of 18,068 mosquitoes (361 pools) collected in south-eastern Trinidad forests from December 1988 to May 1989, 47 species belonging to 14 genera were identified. Five yellow fever virus isolates were made from Haemagogus janthinomys and one from Sabethes chloropterus. All the other pools of mosquitoes examined were negative for the virus. The mosquito isolates were made in December and January. In addition, in late February and early March, 2 infected howler monkeys (Alouatta sp.) were detected. Since March, despite continued surveillance, no yellow fever virus has been detected in mosquitoes or monkeys. There has been no reported human imfection. (AU)

The known and the unknown in yellow fever ecology and epidemiology.

Earlier epidemiological concepts are discussed briefly. Attention is focussed on several of the puzzling problems in the epidemiology of the disease as seen today. The recurring outbreaks of yellow fever in regions where it has been absent, as well as the absence of yellow fever in regions where the vectors exist are discussed. Features of vaccination are discussed, including the long persistence of humoral antibodies and long duration of effective protection. The possibility of changes in virulence of yellow fever strains is mentioned. In some of the modern outbreaks, virulence for human beings appears to be lower than virulence noted some centuries ago, and a hypothesis is advanced. The possible influence of virus-protecting antibodies acquired by infection by other flaviviruses is mentioned. With respect to the vectors of yellow fever, the possibility of differing susceptibility and of different behaviour of various strains is discussed. Transovarial transmission of the virus in the vector mosquito is mentioned as a possible mechanism for long persistence of the virus in nature. Infection of ticks and transovarial transmission of virus in ticks are also mentioned. The need for a diagnostic test to differentiate immunity produced by a naturally acquired infection from immunity induced by vaccination is stressed.

The isolation of yellow fever virus from the livers of naturally infected red howler monkeys

Yellow fever virus has been isolated from the liver of seven naturally infected red howler monkeys found dead or dying in the forest. The pathological lesions in these livers were typical of yellow fever.(Summary).

Oropouche virus: a new human disease agent from Trinidad, West Indies

The isolation and laboratory studies of a new virus isolated from a human fever case in Trinidad, West Indies, are described. The virus has been named Oropouche virus after the region from which it was obtained. Oropouche virus has been to be related to Simbu virus, an agent isolated in South Africa. Neutralizing antibodies were found in the blood of a few forest workers, 8 of 26 native cebus monkeys in the Nariva Swamp and 9 of 26 howler monkeys widely distributed over the island. (Summary)

Epidemiological notes in connection with the 1954 outbreak of yellow fever in Trinidad, B.W.I

A brief history of the 1954 yellow fever outbreak in Trinidad, B.W.I., is presented. Special emphasis is laid upon difficulties in recognition of mild cases of yellow fever in the field. It is suggested that prompt recognition of the epidemic threat and early instition of control measures averted a serious epidemic. (Summary)