Macaques In Thailand Research Articles

Natural selection on apical membrane antigen 1 (AMA1) of an emerging zoonotic malaria parasite Plasmodium inui

Apical membrane antigen 1 (AMA1) of malaria parasites plays an important role in host cell invasion. Antibodies to AMA1 can inhibit malaria merozoite invasion of erythrocytes while vaccine-induced specific cytotoxic T cell responses to this protein are associated with clinical protection. Polymorphisms in AMA1 of Plasmodium falciparum (PfAMA1) and P. vivax (PvAMA1) are of concern for vaccine development. To date, little is known about sequence diversity in ama1 of P. inui (Piama1), an emerging zoonotic malaria parasite. In this study, 80 complete Piama1 coding sequences were obtained from 57 macaques in Thailand that defined 60 haplotypes clustering in two phylogenetic lineages. In total, 74 nucleotide substitutions were identified and distributed unevenly across the gene. Blockwise analysis of the rates of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions did not show a significant deviation from neutrality among Thai isolates. However, significantly negative Tajima’s D values were detected in domain I and the loop region of domain II, implying purifying selection. Codon-based analysis of dN/dS has identified 12 and 14 codons under positive and negative selections, respectively. Meanwhile, 85 amino acid substitutions were identified among 80 Thai and 11 non-Thai PiAMA1 sequences. Of these, 48 substituted residues had a significant alteration in physicochemical properties, suggesting positive selection. More than half of these positively selected amino acids (32 of 48) corresponded to the predicted B-cell or T-cell epitopes, suggesting that selective pressure could be mediated by host immunity. Importantly, 14 amino acid substitutions were singletons and predicted to be deleterious that could be subject to ongoing purifying selection or elimination. Besides genetic drift and natural selection, intragenic recombination identified in domain II could generate sequence variation in Piama1. It is likely that malarial ama1 exhibits interspecies differences in evolutionary histories. Knowledge of the sequence diversity of the Piama1 locus further provides an evolutionary perspective of this important malaria vaccine candidate.

Molecular Detection and Characterization of Hemotropic Mycoplasma in Assamese Macaques (Macaca assamensis) of Northern Thailand.

Hemotropic mycoplasmas, also known as hemoplasmas, are parasitic bacteria that infect red blood cells, potentially leading to varying degrees of anemia across numerous mammalian species, including nonhuman primates. The present study aims to investigate the prevalence of hemoplasma infection and identify the species involved among free-ranging Assamese macaques (Macaca assamensis) inhabiting northern Thailand. A total of 133 blood samples were collected from Assamese macaques in Chiang Rai province, Thailand, and subjected to screening for hemoplasma infection utilizing nested PCR amplification targeting the 16S rRNA gene. Positive samples were subsequently analyzed through nucleotide sequencing and phylogenetic analysis for putative species identification. Current study results revealed that 17.3% (23/133; 95% CI 11.29-24.81) of Assamese macaques tested positive for hemoplasma infection using the nested PCR assay. Partial 16S rRNA sequences derived from hemoplasma isolates in Assamese macaques exhibited 99% homology, forming a cluster within the same phylogenetic clade as "Candidatus Mycoplasma haematomacacae," previously identified in long-tailed macaques, rhesus macaques, and Japanese macaques. These findings suggest the presence of "Ca. M. haematomacacae" not only in long-tailed macaques and rhesus macaques but also in Assamese macaques in Thailand. To our knowledge, this marks the first molecular detection of "Ca. M. haematomacacae" in Assamese macaques in Thailand. These results hold significance as they enhance our understanding of hemoplasma infection distribution among macaque populations in Thailand.

Necrophilic behaviour in wild stump-tailed macaques (Macaca arctoides)

Necrophilic behavior (attempted copulation with corpses) has been scarcely reported in non-human primates, especially in the wild. Here is the first case of necrophilic behavior observed in wild stump-tailed macaques in Thailand. Six groups of total N > 460 individuals have been identified and habituated. The corpse of an adult female was found and directly observed for 2 days and by camera trap for 3 days. The cause of death could not be identified, but no prominent physical injury was detected. Within 3 days of the observation, three different males attempted copulation with the corpse. Noteworthy for this observation was that not only males in the group of the dead female but also males from different groups interacted with the corpse. Taken together, these observations suggest that some cues emanating from the corpse coupled with a nonresistant/passive orientation may have triggered these responses in the males. Given that necrophiliac responses have been scarcely reported in non-human primates, our findings provide new insight into these behaviors and to comparative thanatology in general.

Development of Quantitative Real-Time PCR and Loop-Mediated Isothermal Amplification Assays for the Surveillance and Diagnosis of Herpes B Virus Infection.

Herpes B virus (BV) is a zoonotic virus which can be transmitted from macaques to humans, which is often associated with high mortality rates. Because macaques often exhibit asymptomatic infections, individuals who come into contact with these animals face unexpected risks of BV infections. A serological test is widely performed to investigate BV infections. However, the assay's sensitivity and specificity appeared to be inadequate, and it does not necessarily indicate ongoing viral shedding. Here, we developed LAMP and qPCR assays aiming to detect BVs with a high sensitivity and specificity in various macaque species and validated them using oral swab samples collected from 97 wild cynomolgus macaques living in Thailand. Our LAMP and qPCR assays detected more than 50 and 10 copies of the target sequences per reaction, respectively. The LAMP assay could detect BV within 25 min, indicating its advantages for the rapid detection of BV. Collectively, our findings indicated that both assays developed in this study exhibit advantages and usefulness for BV surveillance and the diagnosis of BV infections in macaques. Furthermore, for the first time, we determined the partial genome sequences of BVs detected in cynomolgus macaques in Thailand. Phylogenetic analysis revealed the species-specific evolution of BV within macaques.

Socio-ecological studies of the wild stump-tailed macaques in Thailand: What we have discovered and prospects for the future

Socio-ecological studies of the wild stump-tailed macaques in Thailand: What we have discovered and prospects for the future

Zoonotic pathogens survey in free-living long-tailed macaques in Thailand

ABSTRACT Long-tailed macaques (Macaca fascicularis) are known to harbour a variety of infectious pathogens, including zoonotic species. Long-tailed macaques and humans coexist in Thailand, which creates potential for interspecies pathogen transmission. This study was conducted to assess the presence of B virus, Mycobacterium spp., simian foamy virus (SFV), hepatitis B virus (HBV), and Plasmodium spp. in 649 free-living Thai long-tailed macaques through polymerase-chain reaction. DNA of SFV (56.5%), HBV (0.3%), and Plasmodium spp. (2.2%) was detected in these macaques, whereas DNA of B virus and Mycobacterium spp. was absent. SFV infection in long-tailed macaques is broadly distributed in Thailand and is correlated with age. The HBV sequences in this study were similar to HBV sequences from orangutans. Plasmodium spp. DNA was identified as P. inui. Collectively, our results indicate that macaques can carry zoonotic pathogens, which have a public health impact. Surveillance and awareness of pathogen transmission between monkeys and humans are important.

Assessing the welfare of coconut-harvesting macaques in Thailand

Pig-tailed macaques are used by coconut farmers in Southeast Asia to harvest coconuts. We assessed the welfare of northern pig-tailed macaques Macaca leonina involved in coconut harvesting in southern Thailand. We interviewed 89 coconut farmers in three provinces focusing on quantifying basic demographics of this trade, i.e., species, where the macaques were sourced, diet, sex, and age. Independent from the interviews, we assessed the welfare of 158 working macaques through direct observations using the ‘five domains’ criteria. Based on our scoring system, the mean welfare score of 4.8 out of the maximum 12 points indicates a need for improvement. Overall, we found good agreement between the interviews data and the welfare assessments. The most important individual welfare modifications required for working macaques to obtain a good level of welfare that benefits both the farmers and macaques include: providing access to conspecifics, adding opportunities to hide from stressors, and increasing the freedom of movement. This study highlights the individual welfare concerns and necessity of legislative changes regarding working macaques and other working animals of wild origins.

Molecular Detection of Bartonella quintana among Long-Tailed Macaques (Macaca fascicularis) in Thailand.

Bartonella quintana is a zoonotic pathogen with a worldwide distribution. Humans and non-human primates are considered to be natural reservoir hosts for B. quintana. However, information on the molecular epidemiology of this organism is very limited in regard to long-tailed macaques (Macaca fascicularis) in Thailand. Therefore, this study aimed to investigate the occurrence and genetic diversity of Bartonella spp. among long-tailed macaques in Thailand. In total, 856 blood samples were collected from long-tailed macaques in Thailand. All specimens were screened for Bartonella spp. using a polymerase chain reaction (PCR) assay targeting the 16S rRNA, gltA and ftsZ genes. All positive samples were further analyzed based on nucleotide sequencing, phylogenetic analysis and multiple sequence alignment analysis. Only one macaque showed a positive result in the PCR assays based on the 16S rRNA, gltA and ftsZ genes. Nucleotide sequencing and phylogenetic analysis revealed that the obtained sequences were closely related to B. quintana previously detected in non-human primates. Single-nucleotide polymorphisms (SNPs) were detected in the gltA and ftsZ gene sequences. This study revealed that long-tailed macaques in Thailand carried B. quintana. Despite the low infection rate detected, long-tailed macaques may be a reservoir of B. quintana.

Detection and genetic characterization of "Candidatus Mycoplasma haemomacaque" infection among long-tailed macaques (Macaca fascicularis) in Thailand using broad-range nested polymerase chain reaction assay.

Background and Aim:Hemoplasmas are defined as small, epicellular parasitic bacteria that can infect the red blood cells of several mammalian species. Diseases caused by these bacteria range from asymptomatic infections to acute hemolytic anemia. However, data on hemoplasmas in non-human primates in Thailand remain to be limited. Therefore, this study aims to determine the occurrence and genetic diversity of hemoplasmas among long-tailed macaques in Thailand.Materials and Methods:Blood samples were collected from 339 long-tailed macaques in three provinces of Thailand. DNA was then extracted from the blood samples and tested for hemoplasma using broad-range nested polymerase chain reaction (PCR) based on the 16S rRNA gene. PCR-positive samples were sequenced, and phylogenetic analysis for species identification was conducted.Results:In total, 38 (11.2%) out of the 339 samples were found to be positive for hemoplasmas, based on the broad-range nested PCR assay of the 16S rRNA gene. The 16S rRNA sequences of Mycoplasma spp. were highly similar (98-99% identity) to “Candidatus Mycoplasma haemomacaque.” Furthermore, phylogenetic analysis using maximum likelihood demonstrated that the sequences were located in the same cluster of “Ca. M. haemomacaque.”Conclusion:The detection of hemoplasmas among long-tailed macaques in Thailand is reported. Genetic characterization confirmed that these hemoplasmas are closely related to “Ca. M. haemomacaque.” These results indicate that long-tailed macaques in several locations in Thailand may be infected and serve as reservoirs for this parasite.

Correction to: Dominance status and copulatory vocalizations among male stump-tailed macaques in Thailand.

In the original publication of the article the affiliations 1 and 3 were incorrectly published. The correct affiliations are given in this correction.

Dominance status and copulatory vocalizations among male stump-tailed macaques in Thailand

Male copulation calls sometimes play important roles in sexual strategies, attracting conspecific females or advertising their social status to conspecific males. These calls generally occur in sexually competitive societies such as harem groups and multi-male and multi-female societies. However, the call functions remain unclear because of limited availability of data sets that include a large number of male and female animals in naturalistic environments, particularly in primates. Here, we examined the possible function of male-specific copulation calls in wild stump-tailed macaques (Macaca arctoides) by analyzing the contexts and acoustic features of vocalizations. We observed 395 wild stump-tailed macaques inhabiting the Khao Krapuk Khao Taomor Non-Hunting Area in Thailand and recorded all occurrences of observed copulations. We counted 446 male-specific calls in 383 copulations recorded, and measured their acoustic characteristics. Data were categorized into three groups depending on their social status: dominant (alpha and coalition) males and non-dominant males. When comparing male status, alpha males most frequently produced copulation calls at ejaculation, coalition males produced less frequent calls than alpha males, and other non-dominant males rarely vocalized, maintaining silence even when mounting females. Acoustic analysis indicated no significant influence of status (alpha or coalition) on call number, bout duration, or further formant dispersion parameters. Our results suggest that male copulation calls of this species are social status-dependent signals. Furthermore, dominant males might actively transmit their social status and copulations to other male rivals to impede their challenging attacks, while other non-dominant males maintain silence to prevent the interference of dominants.

First molecular identification of Strongyloides fuelleborni in long-tailed macaques in Thailand and Lao People's Democratic Republic reveals considerable genetic diversity.

Strongyloides fuelleborni is a soil-transmitted nematode parasite of non-human primates. The worm is prevalent also in human populations in Africa and South-East Asia. In this study, we amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides adult males recovered from faecal samples from long-tailed macaques (Macaca fascicularis) in Thailand and Lao PDR. The prevalence in Thailand was 31.1% (55/177) and in Lao PDR it was 62.1% (41/66), with an overall prevalence of 39.5% (96/243). All 18S rRNA sequences that we obtained (n = 96) showed 100% identity with published S. fuelleborni sequences. The 96 cox1 sequences that we obtained represented 32 new haplotypes. When included with the 17 previously known haplotypes from S. fuelleborni, the cox1 sequences fell into four clusters, which had clear geographical structure. This is the first molecular confirmation of S. fuelleborni in long-tailed macaques in Thailand and Lao PDR. Clearly, awareness needs to be raised of the zoonotic potential of S. fuelleborni. A monitoring programme should be organized, taking into account the role of reservoir hosts (i.e. monkeys) in the natural background of human strongyloidiasis caused by S. fuelleborni.

Monkeys overharvest shellfish.

The use of stone tools by macaques in Thailand has reduced the size and population density of coastal shellfish; previously it was thought that tool-assisted overharvesting effects resulted uniquely from human activity.

Blastocystis subtypes detected in long-tailed macaques in Thailand—Further evidence of cryptic host specificity

Blastocystis is an enteric parasitic protist with a widespread distribution in a variety of human and non-human hosts and supposedly a source of zoonotic transmission. This study aimed to determine the frequency and distribution of Blastocystis subtypes in free-ranging Macaca fascicularis (long-tailed macaques, LTMs) in Thailand. A total of 628 faecal samples were collected from free-ranging LTMs inhabiting fourteen tourist attraction sites in Thailand. Fresh faecal samples were individually cultured in Jones’ medium, and Blastocystis-positive samples were subtyped using nuclear small subunit ribosomal DNA (SSU rDNA) sequencing. Two hundred and sixty-three (41.87%) samples were positive by culture, and 197 and 154 were successfully SSU rDNA-amplified and sequenced, respectively. Three subtypes (ST1, ST2, and ST3) comprising 19 alleles were observed. ST3 was the most common subtype detected (36.55%), followed by ST2 and ST1 (24.37% and 17.26%, respectively). Some subtype alleles not previously observed were identified. A couple of strains appeared similar to those found in humans as evidenced by SSU rDNA allelic profiles, while further evidence of cryptic host specificity emerged. This study provides the first data on Blastocystis subtypes in non-human primates in Thailand and confirms the trend observed in other Old-World countries with regard to the colonization rate and subtype distribution.

First molecular identification and genetic diversity of Strongyloides stercoralis and Strongyloides fuelleborni in human communities having contact with long-tailed macaques in Thailand.

The parasitic nematodes, Strongyloides stercoralis and Strongyloides fuelleborni, can infect humans and non-human primates. We amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides from humans in the study area in Thailand, where people have frequent contact with long-tailed macaques. Fresh stool samples were obtained from 213 people and were examined using the agar plate culture method. The overall prevalence of Strongyloides infection was 8.92% (19/213). From a total of 19 worms (one per infected person), 18 adult males had 18S rRNA sequences identical with that of S. stercoralis and one adult female had a sequence almost identical with that of S. fuelleborni. A median-joining network of cox1 sequences revealed nine new haplotypes from S. stercoralis, and an overall haplotype diversity (Hd) of 0.9309. The single haplotype of S. fuelleborni was also new and contributed to an overall haplotype diversity for that species of 0.9842. This is the first molecular identification of S. stercoralis and S. fuelleborni in a human community having contact with long-tailed macaques in Thailand. It is also the first report of S. fuelleborni infecting a human in Thailand.

A first report of non-invasive adenovirus detection in wild Assamese macaques in Thailand.

Several simian adenoviruses (AdVs) have been detected and isolated in various species of non-human primates with the goals of monitoring the health of wildlife and investigating their potential for zoonotic disease transmission. Here, we provide evidence of AdV infection in wild Assamese macaques (Macaca assamensis assamensis) at Phu Khieo Wildlife Sanctuary, Thailand, based on polymerase chain reaction of non-invasively collected fecal samples. Eight out of 110 fecal samples (7.3%), or five out of 87 monkeys (5.7%), showed evidence of AdV infection. All infected individuals were infants or juveniles. Phylogenetic analysis based on the sequence of hexon and polymerase genes revealed two different AdV genotypes. One genotype clustered in the human AdV-G group, while another showed 100% identity with previously reported AdVs of captive Chinese rhesus macaques (Macaca mulatta), which may be tentatively classified as a new species of AdV in non-human primates while awaiting further supporting evidence.

Preliminary study of the genetic diversity of eastern Assamese macaques (Macaca assamensis assamensis) in Thailand based on mitochondrial DNA and microsatellite markers

Human overpopulation, deforestation, invasion of agricultural areas, and livestock are the primary causes for population fragmentation of wildlife. The distribution range of species of the genus Macaca is constantly decreasing and becoming increasingly fragmented due to forest deterioration. Assamese macaques (M. assamensis) are classified as near threatened in the International Union for Conservation of Nature (IUCN) Red List of Threatened Animals (2008) and have been declared a protected wildlife animal according to Wildlife Preservation and Protection Act, B.E.2535 (1992) of Thailand. As studies of the population history and genetic diversity of Assamese macaques in Thailand are currently lacking, we aimed at a first investigation of their genetic diversity based on mitochondrial DNA [hypervariable regions 1 and 2 (HV1, HV2) and cytochrome B (CYTB) regions], as well as 15 microsatellite markers of five sampling sites distributed across Thailand. Our results indicate that Assamese macaques in Thailand are diverse, with eight maternal haplotypes and a low inbreeding coefficient in the Phu Khieo Wildlife Sanctuary (PKWS) population. Moreover, our phylogenetic and median-joining network analysis based on mitochondrial (mt)DNA suggests a population distribution in accordance with the evolutionary scenario proposed for M. sinica. Today, the population of Assamese macaques is fragmented, and conservation strategies are needed to ensure the maintenance of genetic diversity of this primate species.

Differential sequence diversity at merozoite surface protein-1 locus of Plasmodium knowlesi from humans and macaques in Thailand

To determine the genetic diversity and potential transmission routes of Plasmodium knowlesi, we analyzed the complete nucleotide sequence of the gene encoding the merozoite surface protein-1 of this simian malaria (Pkmsp-1), an asexual blood-stage vaccine candidate, from naturally infected humans and macaques in Thailand. Analysis of Pkmsp-1 sequences from humans (n=12) and monkeys (n=12) reveals five conserved and four variable domains. Most nucleotide substitutions in conserved domains were dimorphic whereas three of four variable domains contained complex repeats with extensive sequence and size variation. Besides purifying selection in conserved domains, evidence of intragenic recombination scattering across Pkmsp-1 was detected. The number of haplotypes, haplotype diversity, nucleotide diversity and recombination sites of human-derived sequences exceeded that of monkey-derived sequences. Phylogenetic networks based on concatenated conserved sequences of Pkmsp-1 displayed a character pattern that could have arisen from sampling process or the presence of two independent routes of P. knowlesi transmission, i.e. from macaques to human and from human to humans in Thailand.

Boundary zone between northern and southern pig-tailed macaques and their morphological differences

Based on previous conflicting reports that the two forms of pig-tailed macaque (northern and southern) exist as separate species, subspecies, or forms, and that their boundary zone lies in Thailand, a survey of the distribution range and morphology of pig-tailed macaques in Thailand was conducted during 2003-2010. We first conducted a questionnaire survey. Questionnaires were sent to 7,410 subdistricts throughout Thailand. We then traveled to 72 of the 123 subdistricts reporting the presence of pig-tailed macaques. However, due to a lack of reports of the presence of free-ranging pig-tailed macaques living south of the Isthmus of Kra, a survey of pet pig-tailed macaques was also conducted during 16-24 September 2011. Furthermore, 35 wild northern pig-tailed macaques inhabiting northern Thailand (13°13'N, 101°03'E) were temporarily caught and their morphological characters were measured and then compared to those of the southern form captured from Sumatra, Indonesia. Although largely allopatric, the ranges of the northern and southern pig-tailed macaques in Thailand were found to have a partially sympatric boundary at the Surat Thani-Krabi depression (8-9°30'N). Morphologically, these two forms were very distinctive, with different morphological characters such as the crown patch, the white color of the triangle above the eyes, the red streak at the external rim of the eyes, pelage color, ischial callosity, tail length and carriage, facial height, and limb length in both sexes, and patterns of sex skin swelling and reddening in females. These differences in morphological characters between the northern and southern forms should help settle the problems of their taxonomy.

Morphological and Body Color Variation in Thai Macaca fascicularis fascicularis North and South of the Isthmus of Kra

Long-tailed macaques (Macaca fascicularis fascicularis) are widely distributed in Southeast Asia and are morphologically and genetically (Tosi et al. in International Journal of Primatology 23:161–178, 2002) distinguishable on either side of the Isthmus of Kra (ca. 10.5°N). We compared the somatometry and body color of 15 local populations of long-tailed macaques in Thailand distributed over areas from 6.5°N to 16.3°N and also a Thai rhesus macaque population at 17.2°N. Limb proportions and body color variation follow the geographical trend. However, contrary to a previous report, body size does not decrease with latitude in the northern group and also in the southern (southerly distributed) rhesus macaque. Relative tail length (RTL) and color contrast in yellow between the back and thigh are the sole traits that distinctively separate the 2 groups: the southern group has a long relative tail length (RTL >125%) and small color contrast, whereas the northern group has a short RTL (<120%) and large color contrast. The southern rhesus macaques appear to have somatometric and body color traits that follow the geographical trend in long-tailed macaques, though they maintain their distinctive species-specific traits of shorter RTL (ca. 55%), shorter relative facial length, and a bipartite body color pattern. Researchers assume that the northern group of long-tailed macaques and the southern rhesus macaques had undergone partial introgression with each other. Montane refugia present during the glacial period are localities in which introgression occurred in long-tailed macaques.