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

## TL;DR

A new genotyping tool for Balantioides coli using the beta-tubulin gene reveals distinct genetic clusters and host-specific transmission patterns.

## Contribution

A novel beta-tubulin-based PCR assay was developed, offering higher specificity and detection efficiency than traditional ITS-based methods.

## Key findings

- The beta-tubulin gene classified B. coli into three genotypes, with genotype III specific to guinea pigs.
- Genotypes I and II showed cross-species transmission among pigs, cattle, and sheep.
- The assay revealed high genetic diversity and frequent recombination in the beta-tubulin locus.

## Abstract

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.

Balantioides coli, a zoonotic ciliate protozoan, poses significant challenges in molecular epidemiology due to limitations of conventional subtyping methods targeting the internal transcribed spacer (ITS) region. In this study, we developed a nested PCR assay focusing on the β-tubulin gene, validated across 56 fecal samples from pigs, cattle, sheep, and guinea pigs. The assay demonstrated 100% detection efficiency and superior specificity compared to ITS-based approaches. Phylogenetic analysis delineated three β-tubulin genotypes: genotype III exhibited strict guinea pig host specificity, while genotypes I and II displayed cross-species transmission potential among pigs, cattle, and sheep. High genetic diversity and frequent recombination events underscored evolutionary adaptability. Non-synonymous mutations in genotype III correlated with reduced trophozoite motility, suggesting host-specific adaptation. This β-tubulin-based method overcomes ITS multicopy heterogeneity, enabling precise resolution of transmission clusters and host specificity patterns. Our findings advance surveillance strategies for B. coli in veterinary and public health frameworks, highlighting the utility of functional gene markers in combating neglected zoonotic pathogens.