Hematological reference values for Alouatta palliata (Primates: Atelidae) and Cebus imitator (Primates: Cebidae) from the Pacific Coast of Costa Rica / Valores de referencia hematológicos para Alouatta palliata (Primates: Atelidae) y Cebus imitator (Primates: Cebidae) de la costa Pacífica de Costa Rica

Abstract Introduction: Wildlife hematological patterns are fundamental for health monitoring, and allows elucidating variations both within and between populations. Among these, hematological parameters are particularly valuable to evaluate the health status of neotropical primate species in the wild. Objective: To define hematological reference values for two species of monkeys in Costa Rica. Methods: During 2014, we collected blood samples from free-ranging mantled howler monkeys, Alouatta palliata (17 females, 18 males) and white-faced capuchin monkeys, Cebus imitator (5 females, 7 males) in seven localities of the Costa Rican Pacific coast. Results: For both species, the hematological values were higher in males, and howler monkey populations differed significantly except for platelets. Conclusions: These hematological values, which differ by sex and locality, will help evaluate the health status of these neotropical primate populations.

Resumen Introducción: Los patrones hematológicos de la vida silvestre son fundamentales para el monitoreo de la salud y permiten dilucidar las variaciones tanto dentro como entre poblaciones. Entre estos, los parámetros hematológicos son particularmente valiosos para evaluar el estado de salud de las especies de primates neotropicales en la naturaleza. Objetivo: Definir valores de referencia hematológicos para dos especies de monos en Costa Rica. Métodos: Durante el 2014 recolectamos muestras de sangre de monos aulladores de manto, Alouatta palliata (17 hembras, 18 machos) y monos capuchinos cariblancos, Cebus imitador (5 hembras, 7 machos) en siete localidades de la costa Pacífica de Costa Rica. Resultados: Para ambas especies, los valores hematológicos fueron mayores en los machos, y las poblaciones de monos aulladores difirieron significativamente con excepción de las plaquetas. Conclusiones: Estos valores hematológicos, que difieren según el sexo y la localidad, ayudarán a evaluar el estado de salud de estas poblaciones de primates neotropicales.

Metagenomic and network analysis revealed wide distribution of antibiotic resistance genes in monkey gut microbiota.

The emergence and spread of drug-resistant microorganisms that have acquired new resistance mechanisms, leading to antibiotic resistance, continue to threaten the health of humans and animals worldwide. Non-human primates (NHPs), as close living relatives of human beings in the world, have a high degree of genetic and physiological similarity to humans. However, despite its importance, we lack a comprehensive characterization or understanding of the similarities and differences of the antibiotic resistance genes of the gut microbiome carried by non-human primates and humans. In the present study, the diversity and abundance of antibiotic resistance genes carried by the gut microbiota of cynomolgus monkeys (Macaca fascicularis) were investigated by metagenomic analysis. In total, 60 resistance types conferring resistance to 11 categories of antibiotics were identified in the gut microbiome of cynomolgus monkeys. Interestingly, the composition and abundance of ARGs carried by the gut microbiota of cynomolgus monkeys can be significantly affected by dietary changes. Moreover, we found that all ARG types carried by humans are also present in cynomolgus monkeys. The tetracycline resistance gene tet(37) is evolutionarily conserved and highly homologous. Taken together, our study provides a comprehensive overview of the diversity and richness of ARGs in the gut microbiota of cynomolgus monkeys and underlines the potentially crucial role of diet in the gut health of monkeys and humans.

Antibiotic susceptibility screening of primate-associated Clostridium ventriculi.

Clostridium ventriculi (syn. Sarcina ventriculi) is a Gram-positive opportunistic pathogen with sarcina morphology. In the case of gastrointestinal disorders, the treatment is often empirical. Due to the common occurrence in primates and the potential risk of dysbiosis; the antibiotic susceptibility screening of C. ventriculi strains isolated from guenon monkeys and crested gibbons to 58 antibiotics was performed to reduce potentially ineffective antibiotic use in case of disease. Isolates were found to be susceptible to the majority of the tested antibiotics, mainly to (fluoro)quinolones, macrolides, penicillins, and tetracyclines. The susceptibility profiles were similar despite the hosts. Tested strains showed also natural resistance to a few antibiotics on the genus level. Detected in vitro antibiotic efficiency is consistent with documented human treatment cases.

Molecular Epidemiology and Resistance Profile of Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Sources in Brazil.

Aims: The objectives of this study were to genotype a total of 48 Campylobacter jejuni and 39 Campylobacter coli strains isolated in Brazil from 1995 to 2016 by multilocus sequence typing (MLST) and to determine their resistance profile. The presence or points of mutation in the related resistance genes was verified. Results: By MLST, C. jejuni strains were typed into 36 STs and C. coli strains were typed into 27 STs. A total of 70.8% of C. jejuni and 35.9% of C. coli were resistant to at least one antimicrobial tested. The tet(O) gene was detected in 43.7% C. jejuni and in 12.8% C. coli. The ermB gene was not detected and one C. jejuni presented the mutation in the 23S rRNA gene. Besides, 58.3% C. jejuni presented the substitution T86I in the quinolone resistance-determining region of gyrA and 15.4% C. coli presented the substitution T38I. The cmeB gene was detected in 97.9% C. jejuni and in 97.4% C. coli. Conclusion: The presence of C. jejuni and C. coli resistant to some antimicrobial agents of clinical use is of public health concern. The presence of STs shared between Brazilian strains and isolates of different countries is of concern since it might suggest a possible spread of these shared types.

Molecular typing of Campylobacter jejuni strains: comparison among four different techniques.

This study compared the ability of pulsed-field gel electrophoresis (PFGE), flaA small variable region (SVR) sequencing, analysis of the clustered regularly interspaced short palindromic repeats locus by high resolution melting analysis (CRISPR-HRMA), and multilocus sequence typing (MLST) for typing 111 Campylobacter jejuni strains isolated from diverse sources during 20 years in Brazil. For this, we used previous results obtained by PFGE and flaA-SVR sequencing from our research group and performed CRISPR-HRMA and MLST typing for the first time. Furthermore, the discrimination index (DI) of each method was accessed. The DI for PFGE, flaA-SVR sequencing, CRISPR-HRMA, and MLST was 0.980, 0.932, 0.868, and 0.931, respectively. By PFGE and flaA-SVR sequencing, some strains from clinical and non-clinical sources and from humans and animals presented ≥ 80% similarity. Similarly, some strains from different origins presented the same ST and CRISPR-HRMA types. In conclusion, despite the different DI values, all assays provided the same epidemiological information suggesting that a potential transmission may have occurred between C. jejuni from clinical and non-clinical sources and from animals and humans in Brazil. Furthermore it was demonstrated the suitability of PFGE that should be used preferably together with MLST and/or flaA-SVR sequencing for typing C. jejuni strains.

Identification and molecular characterization of Staphylococcus aureus and multi-drug resistant MRSA from monkey faeces in China.

Staphylococcus aureus is a commensal bacterium and an important opportunistic pathogen in humans and animals. The increase in multi-drug resistant (MDR) strains of S. aureus is a growing concern due to their impact on animal health and potential for zoonotic transmission. Increasing evidence has shown that MRSA could be transmitted by faeces. The present study determined the prevalence, antibiotic resistance profile and genotypic characteristics of S. aureus isolated from monkey faecal samples in China. Thirty-eight out of 145 (26.21%) macaque faecal samples were S. aureus positive, which eight (5.5%) isolates were identified as MRSA. Antimicrobial susceptibility tests showed that most of the S. aureus isolates were resistant to tetracycline (TE, 44.74%), followed by penicillin (P, 21.05%), cefoxitin (FOX, 21.05%) and ciprofloxacin (CIP, 18.42%). The predominant spa types were t13638 (44.74%) and t189 (13.16%), which are reported to be closely associated with human infections in China. All MRSA isolates belonged to the SCCmecV type, which six of MRSA isolates were ST3268, while the other two isolates belonged to ST4981. This study for the first time describes the prevalence of S. aureus and MRSA in monkey faeces in China, indicating that faeces could be a potential factor of transmitting S. aureus between humans and monkeys.

Staphylococcus aureus in Animals.

Staphylococcus aureus is a mammalian commensal and opportunistic pathogen that colonizes niches such as skin, nares and diverse mucosal membranes of about 20-30% of the human population. S. aureus can cause a wide spectrum of diseases in humans and both methicillin-sensitive and methicillin-resistant strains are common causes of nosocomial- and community-acquired infections. Despite the prevalence of literature characterising staphylococcal pathogenesis in humans, S. aureus is a major cause of infection and disease in a plethora of animal hosts leading to a significant impact on public health and agriculture. Infections in animals are deleterious to animal health, and animals can act as a reservoir for staphylococcal transmission to humans.Host-switching events between humans and animals and amongst animals are frequent and have been accentuated with the domestication and/or commercialisation of specific animal species. Host-switching is typically followed by subsequent adaptation through acquisition and/or loss of mobile genetic elements such as phages, pathogenicity islands and plasmids as well as further host-specific mutations allowing it to expand into new host populations.In this chapter, we will be giving an overview of S. aureus in animals, how this bacterial species was, and is, being transferred to new host species and the key elements thought to be involved in its adaptation to new ecological host niches. We will also highlight animal hosts as a reservoir for the development and transfer of antimicrobial resistance determinants.

Factors influencing bacterial microbiome composition in a wild non-human primate community in Taï National Park, Côte d'Ivoire.

Microbiomes impact a variety of processes including a host's ability to access nutrients and maintain health. While host species differences in microbiomes have been described across ecosystems, little is known about how microbiomes assemble, particularly in the ecological and social contexts in which they evolved. We examined gut microbiome composition in nine sympatric wild non-human primate (NHP) species. Despite sharing an environment and interspecific interactions, individuals harbored unique and persistent microbiomes influenced by host species, social group, and parentage, but surprisingly not by social relationships among members of a social group. We found a branching order of host-species networks constructed using the composition of their microbiomes as characters, which was incongruent with known NHP phylogenetic relationships, with chimpanzees (Pan troglodytes verus) sister to colobines, upon which they regularly prey. In contrast to phylogenetic clustering found in all monkey microbiomes, chimpanzee microbiomes were unique in that they exhibited patterns of phylogenetic overdispersion. This reflects unique ecological processes impacting microbiome composition in chimpanzees and future studies will elucidate the aspects of chimpanzee ecology, life history, and physiology that explain their unique microbiome community structure. Our study of contemporaneous microbiomes of all sympatric diurnal NHP in an ecosystem highlights the diverse dispersal routes shaping these complex communities.

Reversal of Chloroquine Resistance of Plasmodium vivax in Aotus Monkeys.

Chloroquine-resistant (CQR) vivax malaria has emerged as a threat to the malaria elimination agenda. The objective of this study was to assess if a combination of chloroquine (CQ) and prochlorperazine was able to reverse CQ resistance of the Plasmodium vivax AMRU-1 strain from Papua New Guinea in infected Aotus monkeys. For this purpose, in two independent experimental drug efficacy trials, a total of 18 Aotus monkeys infected with blood obtained from donor animals were randomly assigned to treatment and control groups and orally administered CQ at 10 mg/kg or prochlorperazine at 20 mg/kg, alone or in combination, for five consecutive days. Reversal of CQR was achieved in animals that received the drug combination, whereas neither drug alone produced cures. This same drug combination reverses CQR in P. falciparum and could be an alternative for treatment in humans with chloroquine-resistant P. vivax infections.

Population Genomics of Francisella tularensis subsp. holarctica and its Implication on the Eco-Epidemiology of Tularemia in Switzerland.

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of Francisella tularensis subsp. holarctica, the causative agent of tularemia in Switzerland. A total of 59 Fth isolates were obtained from castor bean ticks (Ixodes ricinus), animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the Fth population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting Fth to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.

Molecular detection of fungi of public health importance in wild animals from Southern Brazil.

Some animals have an important relationship with fungal infections, and searching for pathogens in animal samples may be an opportunity for eco-epidemiological research. Since studies involving wildlife are generally restricted, using samples from road kills is an alternative. The aim of this study was to verify whether pathogenic fungi of public health importance occur in wildlife road kills from Santa Catarina State, Brazil. Organ samples (n = 1063) from 297 animals were analysed according to Polymerase Chain Reaction (PCR) using universal primers to detect fungi in general and, subsequently, using primers specific to Paracoccidioides brasiliensis, Histoplasma capsulatum and Cryptococcus spp. There were 102 samples positive for fungal species. Eight samples were positive for P. brasiliensis, three samples were positive for Cryptococcus spp. and one sample had coinfection by these two fungi. No sample was positive for Histoplasma spp. according to the molecular detection. Genetic sequencing allowed the identification of Fungal sp. in 89 samples, Cryptococcus neoformans in two samples and Aspergillus penicillioides in three samples. This study shows the importance of wild animals in the epidemiology of fungal infections and assists in the mapping of pathogen occurrence in a region that was not previously evaluated.

Pathogenic potential and genotypic diversity of Campylobacter jejuni: a neglected food-borne pathogen in Brazil.

Purpose and methodology.Campylobacter jejuni is a major zoonotic pathogen that causes food-borne gastroenteritis worldwide. However, there are only a few studies available that have molecularly characterized C. jejuni strains isolated in Brazil. The aim of this study was to genotype 111 C. jejuni strains isolated from sick humans (43), monkey faeces (19), chicken faeces (14), chicken meat (33) and sewage (2) between 1996 and 2016 in Brazil using flaA-SVR (short variable region) sequencing and PFGE. Furthermore, the presence of 16 virulence genes was analysed by PCR. RESULTS: Using PFGE and flaA-SVR sequencing, the 111 C. jejuni strains studied were grouped into three and two clusters, respectively, and some strains of different origin presented a similarity of ≥80 %. In total, 35 flaA-SVR alleles were detected. Alleles gt45, gt49 and gt57 were the most prevalent, in contrast with those frequently described in the PubMLST database. All 111 C. jejuni strains contained the genes flaA, flhA, cadF, docA, cdtA, cdtB, cdtC, iamA, ciaB, sodB, dnaJ, pldA, racR and csrA. The wlaN gene was detected in 11 strains (9.9 %), and the virB11 in just one strain (0.9 %). CONCLUSIONS: In conclusion, the pathogenic potential of the C. jejuni strains studied was highlighted by the high frequency of the majority of the virulence genes searched. The flaA-SVR sequencing and PFGE results showed that some of the strains studied presented a high genotypic similarity, suggesting potential for transmission between animal sources and humans in this country. Altogether, the results characterize further C. jejuni isolates from Brazil, an important producer and exporter of chicken meat.

Identification of bacterial infection in neotropical primates.

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.

A dynamic dose-response model to account for exposure patterns in risk assessment: a case study in inhalation anthrax.

The most commonly used dose-response models implicitly assume that accumulation of dose is a time-independent process where each pathogen has a fixed risk of initiating infection. Immune particle neutralization of pathogens, however, may create strong time dependence; i.e. temporally clustered pathogens have a better chance of overwhelming the immune particles than pathogen exposures that occur at lower levels for longer periods of time. In environmental transmission systems, we expect different routes of transmission to elicit different dose-timing patterns and thus potentially different realizations of risk. We present a dose-response model that captures time dependence in a manner that incorporates the dynamics of initial immune response. We then demonstrate the parameter estimation of our model in a dose-response survival analysis using empirical time-series data of inhalational anthrax in monkeys in which we find slight dose-timing effects. Future dose-response experiments should include varying the time pattern of exposure in addition to varying the total doses delivered. Ultimately, the dynamic dose-response paradigm presented here will improve modelling of environmental transmission systems where different systems have different time patterns of exposure.

Opportunistic infections in non-human primates exposed to immunomodulatory biotherapeutics: considerations and case examples.

Immunomodulatory biotherapeutics are most often evaluated for safety preclinically by way of repeat dose toxicity studies in non-human primates. Since immunomodulation is expected with this class of therapeutics, and since non-human primates share many opportunistic or latent infectious agents with humans, non-human primates in these toxicity studies may present with opportunistic or recrudescent infections that would be of concern if they occurred clinically in humans. In such instances, it is suggested that non-clinical safety assessment scientists consider a series of key questions that aim to clarify the relationship of the findings to the biotherapeutic under study and the expected predictivity of the findings to the human clinical setting. In this review, relevant case examples are considered comprising (i) gammaherpesviruses-mediated B-lymphocyte proliferation associated with a T-lymphocyte depleting fusion protein; (ii) increased plasmodial hemoparasite burdens associated with a monoclonal antibody inhibitory to T-lymphocyte trafficking and macrophage function, and (iii) the expected predictivity of non-human primate models for the occurrence of encephalic polyomavirus infections.

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan.

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.

Tuberculosis as a zoonosis from a veterinary perspective.

Tuberculosis is an important disease among many zoonoses, because both Mycobacterium tuberculosis and Mycobacterium bovis, which are the major causes of tuberculosis, are highly pathogenic, infect many animal species and thus are likely to be the source of infection in humans. In particular, monkeys are highly susceptible to these bacteria and are important spreaders. Recently, two outbreaks of M. tuberculosis occurred in four different kinds of monkeys and humans were also infected with the disease in Japan. In zoos, tuberculosis was reported not only in monkeys, but also in several different kinds of animals, including elephants. Pets such as dogs and cats are believed to be generally less susceptible to M. tuberculosis, but in this article we introduce a case of infection from man to dog by close contact. Japan is one of the few countries that have been able to control M. bovis infection. In other countries, however, cases of bovine tuberculosis and human M. bovis infection have been reported, and thus further attention is still required in the future.

Quantitative, longitudinal profiling of the primate fecal microbiota reveals idiosyncratic, dynamic communities.

We used slot blot hybridization, quantitative polymerase chain reaction (qPCR), and flow cytometry microarrays to quantify specific 16S rDNAs in weekly fecal specimens from four monkeys housed in a research vivarium for periods ranging from five to 8 months. Even in these uniformly housed and fed animals the gut microbiota is idiosyncratic, very dynamic on short timescales, and shows significant positive and negative correlations among some bacteria as well as responses to heavy metal exposure. The relative quantification (fmol targets per total fmol bacterial 16S rDNA) afforded by flow cytometry microarrays agreed well with the absolute quantification (nanogram of target DNA per nanogram of fecal DNA) afforded by slot blots and qPCR. We also noted strengths and weaknesses in inter-method comparisons for DNA-based quantification of these complex bacterial communities.

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR.

In the last three decades, several monkeys reared in outdoor/indoor-outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.

Incidence of Listeria monocytogenes in wild animals in Japan.

Between 1991 and 1993, the intestinal contents and feces of wild animals in Japan were examined for the presence of Listeria. The wild animals examined included 623 mammals (11 species) and 996 birds (18 species). Listeria species were isolated from 38 (6.1%) of the 623 mammalian samples and 133 (13.4%) of 996 bird samples. The highest incidence of Listeria in the mammals was found in Japanese monkeys (20.0%) and that in birds was found in crows (43.2%). The incidence of Listeria in Japanese monkeys varied from 0 to 40.0% depending on the capture area. L. monocytogenes was isolated from II of these positive samples. Serovars 1/2a and 4b predominated in eight serotyped L. monocytogenes isolates.