Extensive prevalence and significant genetic differentiation of Blastocystis in high- and low-altitude populations of wild rhesus macaques in China

Blastocystis is a common intestinal protist with a wide range of hosts. Thus far, 38 subtypes have been identified. In recent years, wild animals have been confronted with habitat fragmentation as well as an increasing risk of zoonotic disease transmission due to human disturbance. Only limited data are available on Blastocystis infection and subtype distribution in wild rhesus macaques in China. The aim of the present study was to investigate the prevalence and genetic diversity of Blastocystis in nine wild rhesus macaque populations in China. A total of 276 faecal samples were collected from five high-altitude populations (high-altitude group [HAG]; 2800-4100 m a.s.l.) and four low-altitude populations (low-altitude group [LAG]; 5-1000 m a.s.l) of rhesus macaques. PCR-based analysis, using a new primer pair for the amplification of a 1690-bp sequence of the small subunit ribosomal RNA (SSU rRNA) gene, was used for prevalence and genetic diversity analysis. Analysis of faecal samples revealed that Blastocystis infection was common in rhesus macaques, with an infection positivity rate of 80.1% (n = 221/276 samples). There was no significant difference (P = 0.121) in positivity rate between the LAG (84.3%) and HAG (76.8%). Overall, 33 haplotypes were obtained and classified into four subtypes (STs), of which three were potentially zoonotic subtypes (ST1, 29.7%; ST2, 16.7%; ST3, 31.9%) and one that was first identified in this study and named ST39 (12.0%). The STs were distributed differently among the rhesus macaque populations, except for ST3, which was found in all populations. Phylogenetic analyses revealed two major divergent clades of ST3 for the HAG and LAG. Genetic diversity analysis showed a high genetic diversity of ST3 (haplotype diversity: 0.846; nucleotide diversity: 0.014) in the rhesus macaques, but a high genetic differentiation (F > 0.25) and a low gene flow (Nm = 0.09) between the HAG and LAG. Our study, which is the first investigation on Blastocystis infection in multiple wild rhesus macaque populations in China, indicates a potential risk of zoonotic transmission of Blastocystis in the study areas. Blastocystis ST3 showed high genetic diversity in wild rhesus macaques and significant genetic differentiation between the HAG and LAG. Our results provide fundamental information on the genetic diversity and prevalence of Blastocystis in wild rhesus macaque populations.