Abstract
Background: The genus Chilomastix, including C. mesnili, consists of protozoa that parasitize the gastrointestinal tracts of various host organisms, including mammals (humans and non-human primates [NHP]), birds, and amphibians. Despite its widespread presence, Chilomastix spp. are generally considered non-pathogenic, which has led to limited molecular epidemiological studies on this genus. Consequently, genetic reference data for this genus remain scarce in GenBank. In this study, we aimed to establish a molecular classification for Chilomastix spp. by investigating the genetic diversity of isolates from humans and animals in a parasite-endemic region of Indonesia. Methods: A cross-sectional molecular investigation was conducted in Wainyapu Village, Sumba Island, Indonesia. Stool samples were collected annually from 2013 to 2016 and screened using polymerase chain reaction (PCR) targeting the 18S small subunit ribosomal RNA gene (18S rRNA) of Chilomastix spp., followed by direct and subcloning sequencing. Genetic haplotypes of the partial 18S rRNA sequence (1386-1953 bp) from humans (n = 25), dogs (n = 1), pigs (n = 23), rats (n = 38), water buffaloes (n = 3), chickens (n = 10), and ducks (n = 1) were analyzed alongside reference sequences from humans, guinea pigs, leeches, frogs, and water sources using phylogenetic analyses. Results: The prevalence of Chilomastix spp. was 7.0% (25/356) in humans and 19.7% (75/380) in animals. Phylogenetic analyses revealed the following monophyletic clusters as subtypes (STs): C. mesnili ST1 (human-NHP genotype), C. mesnili ST2-1 (human genotype), and C. mesnili ST2-2 (pig genotype). In addition, C. gallinarum-like haplotypes (chicken genotype) and C. bettencourti-like haplotypes, including ST1 (rat genotype) and ST2 (rat-buffalo genotype), were also identified. Conclusions: The genetic references registered in this study, along with the revealed molecular classification of Chilomastix spp., are crucial for understanding the genetic diversity and host-specific dynamics of these parasites in endemic regions.