Development of a multilocus sequence typing tool for Balantioides coli and its genetic diversity in pigs

Balantioides coli is a globally significant zoonotic protozoan, yet effective genotyping tools such as multilocus sequence typing (MLST) remain limited for this pathogen. In this study, we screened microsatellite sequences from the whole-genome data of a pig-derived B. coli isolate (P011) and evaluated 12 candidate loci (BC-MS1-BC-MS12) using 45 porcine isolates. Five loci (BC-MS1, BC-MS5, BC-MS10, BC-MS11, and BC-MS12), which demonstrated high amplification efficiency and polymorphism, were selected to develop an MLST tool. Sequence analysis revealed 14-20 subtypes per locus, and 22 MLS subtypes were identified among the 29 isolates successfully sequenced at all five loci. The BC-MS5 locus is located in the encoding region and the transcriptional termination regulatory region of the B. coli genome, and it is the locus with the greatest variability among the five selected loci. At the BC-MS5 locus, 42 isolates were classified into five groups, with group 5 and group 1 being the dominant populations across the six provinces. No obvious geographical isolation was observed among the pig-derived B. coli isolates. This is the first development of a high-resolution MLST tool for B. coli, offering new insights into its genetic diversity and transmission dynamics. Importance: The 18S rDNA and ITS1-5.8S rDNA-ITS2 genetic markers exhibit some limitations, including low resolution in the molecular epidemiological study of Balantioides coli. Here, five microsatellite loci (BC-MS1, BC-MS5, BC-MS10, BC-MS11, and BC-MS12) with high amplification efficiency and polymorphism were selected from genomic data of B. coli to develop a multilocus sequence typing (MLST) method for the first time. Sequence analysis reveals that there are multiple MLS subtypes and groups among pig-derived isolates in China and no obvious geographical isolation. The established MLST method demonstrates higher resolution than conventional genotyping approaches relying on 18S rDNA or ITS1-5.8S rDNA-ITS2 and will serve as a useful tool for investigating the transmission dynamics and genetic diversity of this zoonotic pathogen.