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.

Diagnostic Tools for the Identification of Babesia sp. in Persistently Infected Cattle

“…Cattle, as well as buffaloes, have been screened with highly sensitive PCR assays to detect persistently infected animals in order to genetically characterize the Merozoite Surface Antigen (MSA-1) diversity in Babesia bovis populations present in cattle premises from Mexico [20], Thailand, Brazil and Ghana [21], Vietnam [22], Thailand [23], Sri Lanka, Mongolia and Vietnam [24].…”

Anaplasma marginale and A. platys Characterized from Dairy and Indigenous Cattle and Dogs in Northern Vietnam

“…In Vietnam, anaplasmosis has been investigated only recently, primarily using morphological and serological approaches with few reports of molecular identification [14–16]. A serological study conducted using the Svanovir ® A. marginale -Ab ELISA kit (SVANOVA Biotech AB, Uppsala, Sweden), revealed 28% of blood samples from lactating cows in northern Vietnam to be positive for specific antibody against A. marginale [15].…”

“…In other studies, Anaplasma spp. were suspected in animal samples from southern and central Vietnam [14,16]. There remains a need for accurate species identification for better molecular taxonomic understanding of the infections by Anaplasma spp.…”

Genetic Diversity of Babesia bovis MSA-1, MSA-2b and MSA-2c in China

“…Antigenic polymorphism has been applied as a strategy by most protozoan parasites to escape the immune response of mammalian hosts, and it is a major obstacle preventing the development of effective vaccines against these parasites [7]. In our present study, analysis of the genetic relationships of B. bovis MSA-1, MSA-2b and MSA-2c of Chinese isolates, with respect to almost all available related sequences deposited in the GenBank database (including Mongolia, Vietnam, Philippines, Thailand, Israel, Vietnam, Sri Lanka, Mexico, Argentina, Brazil, Japan and USA) was conducted [7,12,[18][19][20][21][22][23][24]26,30,31]. Phylogenetic analysis showed that the MSA-1 amino acid sequences had a wide distribution in four clades.…”