Molecular detection of Mycoplasma wenyonii and its closely related hemotropic Mycoplasma sp. in blood-sucking flies from a buffalo farm in Chachoengsao province, Thailand

Abstract

Bovine hemoplasmosis is a disease in buffaloes and cattle caused by hemotropic mycoplasmas or hemoplasmas. Only two bovine hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been described in several countries. Hemoplasmas induce acute hemolytic anemia or chronic infection, leading to production loss. Bovine hemoplasma DNA was also detected in blood-sucking arthropods, suggesting vector transmission in farms. To date, no studies of the molecular detection of bovine hemoplasmas in Thai buffaloes and arthropod vectors have been reported. This study aimed to study the 1-year diversity of hematophagous flies in a buffalo farm located in Chachoengsao province, Thailand, and to investigate the molecular occurrence of bovine hemoplasmas in those flies using a polymerase chain reaction (PCR) assay and sequence analyses. A total of 1,488 mosquitoes, 867 stable flies, and 312 tabanid flies were collected during this study. The most abundant mosquitoes, stable flies, and tabanid flies were Culex tritaeniorhynchus, Stomoxys calcitrans, and Tabanus megalops, respectively. A total of 249 genomic DNA samples of flies were tested using a PCR assay based on the 16S rRNA gene; 23.69% (59/249) of the insect samples were positive in this assay. Positive samples (n = 8) were subjected to bidirectional sequencing. The BLAST results showed that only three samples from Stomoxys calcitrans and two samples from Tabanus megalops showed 99.90% and 99.17% similarities to the M. wenyonii isolate B003 (MG948626/Water buffalo/Cuba) and the M. wenyonii isolate C124 (MG948625/Cattle/Cuba), respectively. This molecular occurrence of bovine hemoplasmas in blood-sucking flies suggested that those flies are the mechanical vectors for bovine hemoplasmas in Thailand. Based on the phylogenetic analysis of the 16S rRNA gene, the sequences of M. wenyonii were likely classified into two subgroups (A and B), suggesting closely related bovine hemoplasma species. Finally, the genetic analysis of the 23S rRNA gene from these two subgroups revealed that subgroup A could be M. wenynoii and subgroup B may be a subspecies of M. wenyonii or another putative novel species. However, further investigation should be conducted in buffaloes, cattle, and blood-sucking flies to gain more 16S rRNA and 23 rRNA gene sequences of bovine hemoplasmas.