title: Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam.
abstract: Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan; Veterinary Research Institute, Peradeniya, Sri Lanka.
authors.forename: Nilukshi
authors.initials: N
authors.lastname: Liyanagunawardena
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan; Veterinary Research Institute, Peradeniya, Sri Lanka.
authors.forename: Thillaiampalam
authors.initials: T
authors.lastname: Sivakumar
authors.affiliation: Veterinary Research Institute, Peradeniya, Sri Lanka.
authors.forename: Hemal
authors.initials: H
authors.lastname: Kothalawala
authors.affiliation: Veterinary Research Institute, Peradeniya, Sri Lanka.
authors.forename: Seekkuge Susil Priyantha
authors.initials: SS
authors.lastname: Silva
authors.affiliation: Laboratory of Molecular Genetics, Institute of Veterinary Medicine, Ulaanbaatar, Mongolia.
authors.forename: Badgar
authors.initials: B
authors.lastname: Battsetseg
authors.affiliation: Institute of Biotechnology, Hue University, Hue, Vietnam.
authors.forename: Dinh Thi Bich
authors.initials: DT
authors.lastname: Lan
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. Electronic address: yokoyama@obihiro.ac.jp.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
mesh_terms: D000818:Animals; D016793:Babesia bovis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D001404:Babesiosis / Q000469:parasitology*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D019143:Evolution, Molecular; D014644:Genetic Variation; D060005:Genotyping Techniques / Q000379:methods*; D020066:Merozoite Surface Protein 1 / Q000235:genetics*; D008986:Mongolia; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D013188:Sri Lanka; D014744:Vietnam
chemical_list: D020066:Merozoite Surface Protein 1
keywords: Asia; Babesia bovis; Genetic diversity; Type-specific PCR; msa-1