Multi-locus sequence analysis of 'Candidatus Mycoplasma haematomacacae' in free-ranging macaques from Thailand suggestive of a closer relationship to hemotropic mycoplasmas in capuchins and potential origin from bats.

Although 'Candidatus Mycoplasma haematomacacae' (formerly known as 'Candidatus Mycoplasma haemomacaque') has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize 'Ca. M. haematomacacae' among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as 'Ca. M. haematomacacae'. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that 'Ca. M. haematomacacae' infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that 'Ca. M. haematomacacae' formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of 'Ca. M. haematomacacae' from Neotropical and Old World bats. To the best of the authors' knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of 'Ca. M. haematomacacae' isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide.

Zoonotic simian malaria parasites in free-ranging Macaca fascicularis macaques and human malaria patients in Thailand, with a note on genetic characterization of recent isolates.

Despite the natural occurrences of human infections by Plasmodium knowlesi, P. cynomolgi, P. inui, and P. fieldi in Thailand, investigating the prevalence and genetic diversity of the zoonotic simian malaria parasites in macaque populations has been limited to certain areas. To address this gap, a total of 560 long-tailed macaques (Macaca fascicularis) and 20 southern pig-tailed macaques (M. nemestrina) were captured from 15 locations across 10 provinces throughout Thailand between 2018 and 2021 for investigation of malaria, as were 15 human samples residing in two simian-malaria endemic provinces, namely Songkhla and Satun, who exhibited malaria-like symptoms. Using PCR techniques targeting the mitochondrial cytb and cox1 genes coupled with DNA sequencing, 40 long-tailed macaques inhabiting five locations had mono-infections with one of the three simian malaria species. Most of the positive cases of macaque were infected with P. inui (32/40), while infections with P. cynomolgi (6/40) and P. knowlesi (2/40) were less common and confined to specific macaque populations. Interestingly, all 15 human cases were mono-infected with P. knowlesi, with one of them residing in an area with two P. knowlesi-infected macaques. Nucleotide sequence analysis showed a high level of genetic diversity in P. inui, while P. cynomolgi and P. knowlesi displayed limited genetic diversity. Phylogenetic and haplotype network analyses revealed that P. inui in this study was closely related to simian and Anopheles isolates from Peninsular Malaysia, while P. cynomolgi clustered with simian and human isolates from Asian countries. P. knowlesi, which was found in both macaques and humans in this study, was closely related to isolates from macaques, humans, and An. hackeri in Peninsular Malaysia, suggesting a sylvatic transmission cycle extending across these endemic regions. This study highlights the current hotspots for zoonotic simian malaria and sheds light on the genetic characteristics of recent isolates in both macaques and human residents in Thailand.

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

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.