Parasites of the genus Entamoeba have a wide host range including humans, and domestic and wild animals (Cui et al., 2019). In this genus, seven intestinal parasites have been reported to infect both humans and animals, i.e., Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba nuttalli, Entamoeba coli, Entamoeba hartmanni, and Entamoeba chattoni (Elsheikha et al., 2018, Ngui et al., 2020, Servián et al., 2022). Entamoeba histolytica is the only known pathogen causing amebiasis, and causes diarrhea and liver abscesses in humans and animals, resulting in 100,000 human deaths per year globally (Pillai and Kain, 2000, Stanley, 2003). Other Entamoeba spp. that may have potential pathogenicity, such as E. moshkovskii and E. nuttalli, were found to be pathogenic in mice (Shimokawa et al., 2012, Guan et al., 2018). Entamoeba dispar was able to produce liver and intestinal lesions (Oliveira et al., 2015). Entamoeba coli, E. hartmanni, and E. chattoni are not associated with any disease and are considered non-pathogenic.

As non-human primates (NHPs), macaques have a close phylogenetic relationship with humans and are commonly infected with zoonotic parasites (Jiang et al., 2023). A previous study indicated that NHPs may be a potential source of transmission of amebiasis to humans (Elsheikha et al., 2018). In China, the wild rhesus macaque (Macaca mulatta) is widely distributed and mainly occurs in Sichuan, Yunnan, Guizhou and Guangxi (Jiang et al., 1991). There have been only a few studies concerning infection with Entamoeba in wild rhesus macaque populations in China and four species, E. nuttalli, E. dispar, E. coli and E. chattoni, had been reported in the wild populations in Guiyang and Guangxi (Feng et al., 2013). In addition, infections with Entamoeba were reported in other macaque species (Guan et al., 2016, Chang et al., 2019, Zhang et al., 2019), but the pathogen E. histolytica was mainly reported in captive populations and at a low prevalence (Pu et al., 2020). The prevalence and distribution of Entamoeba spp. in wild rhesus macaques remains to be investigated.

Molecular methods are of vital importance for investigating the epidemiology of Entamoeba, especially the PCR method based on the ssrRNA gene (Cui et al., 2019). Moreover, molecular tools work best in identifying novel species of Entamoeba, and DNA sequencing can be used for further genetic analysis (Stensvold et al., 2011). The term “ribosomal lineage” (RL) was introduced to name newly discovered Entamoeba, for which the ssrRNA gene has more than 5% divergence from known species and no corresponding morphological data are available, and to date, the analysis of RLs has described 12 Entamoeba RLs from livestock and wild animals (Jacob et al., 2016, Stensvold et al., 2023b). Except for the ssrRNA gene, tRNA-linked short tandem repeats (STRs) are also well-established polymorphic markers, and have been used for genotyping of E. histolytica, E. dispar, and E. nuttalli (Weedall and Hall, 2011, Feng et al., 2014). A previous study revealed a correlation between pathogenicity and the genotypes based on tRNA-STRs of E. histolytica (Ali et al., 2008). Furthermore, recent studies on E. nuttalli in host macaques indicated that the genetic distance of E. nuttalli correlated significantly with the geographic distance of the host populations (Wei et al., 2018, Feng et al., 2019). As E. nuttalli has a close genetic relationship with E. histolytica and shows potential pathogenicity, the genotypic analysis can help in understanding the genetic differentiation of this parasite and provide basic data for future research on its pathogenicity.

This study investigated the prevalence of Entamoeba in nine wild rhesus macaque populations in China, including five populations in high altitude areas (Baiyu, Heishui, Seda, Yajiang and Daofu), and four populations in low altitude areas (Lingshui, Nanning, Fengjie and Jiangjin). Species-specific primer pairs were used for PCR amplification of the partial ssrRNA genes to identify the seven zoonotic Entamoeba spp. mentioned above. To explore the genetic differentiation of the potentially pathogenic species E. nuttalli, the genetic analysis was further performed based on the genotyping of the tRNA-STRs.