Development and application of a novel beta-tubulin genotyping tool reveals host-specific transmission cluster in Balantioides coli

Balantioides coli is a zoonotic ciliated protozoan that infects humans and other mammals. Conventional and ITS-based genotyping approaches have limitations that hinder precise molecular epidemiological investigations. The objective of this study was to develop a new β-tubulin gene-based approach to enhance the detection and genotyping of B. coli. We performed single-cell isolation and whole-genome sequencing on two B. coli isolates from pigs and two from guinea pigs. We then used the β-tubulin gene sequences to design PCR primers for the new genotyping assay. We validated the assay using 56 ITS-confirmed B. coli-positive fecal DNA samples from pigs, cattle, sheep, and guinea pigs. Phylogenetic analyses were conducted using both β-tubulin and ITS sequences. The β-tubulin-based nested PCR assay exhibited 100% detection efficiency and greater specificity than ITS-based methods. Phylogenetic analysis of the β-tubulin gene sequences classified B. coli into three genotypes (I-III). Genotype III appears to be specific to guinea pigs. Genotypes I and II were found across multiple hosts, indicating potential cross-species transmission. Of the five full-length B. coli β-tubulin sequences obtained in this study, 264 polymorphic sites (19.8%) were identified, including both synonymous and non-synonymous mutations. Frequent recombination events within the β-tubulin locus were detected, indicating substantial genetic diversity. Therefore, the β-tubulin gene is a robust marker for genotyping and epidemiological studies of B. coli. The novel nested PCR assay overcomes the limitations of ITS-based methods and has produced data revealing previously unrecognized genetic diversity and host specificity patterns of B. coli.