Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys-like (Rickettsiales: Anaplasmataceae) in water buffalo from eight provinces of Thailand.

BACKGROUND: Anaplasmosis, an animal disease caused by rickettsial bacteria in the genus Anaplasma, is of considerable economic importance in livestock animals in many countries worldwide. The objectives of this study were to determine the identity, prevalence, and geographic distribution of Ehrlichia and Anaplasma in naturally infected water buffalo in Thailand using PCR amplification and sequencing of the 16S ribosomal RNA and heat shock protein groEL genes. A total of 456 buffalo blood samples from Thailand were investigated. Species identification and genetic differentiation of intra-population and inter-population with the global isolates were conducted based on nucleotide sequences. Interplay between the infection and host factors was also assessed. RESULTS: Overall, 41% of water buffalo were found to be infected with rickettsial organisms in the family Anaplasmataceae, but Ehrlichia spp., Neorickettsia spp., and Wolbachia spp. were not found in any of the sequenced samples in this study. Female buffalo were more frequently infected with bacteria in the family Anaplasmataceae than males [71 out of 176 females (40.3%) versus 11 out of 47 males (23.4%)]. The Odds Ratio value indicated that the risk of infection for female buffalo was 2.2-fold higher than that for males (p < 0.05). We detected three haplotypes of A. marginale 16S rRNA gene and they were placed in a clade that was closely related to the A. marginale in buffalo in China; and cattle in Thailand, Uganda, and China. Homology searching of groEL sequences against the GenBank™ database using the BLASTn algorithm revealed that the obtained sequences had a high percentage similarity (98.36-99.62%) to A. platys sequences. The groEL sequences of three A. platys-like isolates were clustered in the same clade as the A. platys from the tick Rhipicephalus microplus in China. CONCLUSIONS: Our data showed that the apparently healthy buffalo were naturally infected by bacteria in the family Anaplasmataceae at a relatively high prevalence. We also report the finding of A. platys-like infections in water buffalo in Thailand for the first time. Water buffalo serving as the reservoir host of anaplasmosis is of concern for managing the disease control and prevention in ruminants.

Low level of genetic diversity and high occurrence of vector-borne protozoa in water buffaloes in Thailand based on 18S ribosomal RNA and mitochondrial cytochrome b genes.

Vector-borne pathogens (VBPs) pose a great risk to ruminant production through significant economic losses. Several previous studies in Thailand have mainly been focused on the health of dairy and beef cattle. Water buffaloes are one of the important ruminants in the country, but studies on their infection with VBPs remains limited. We conducted a molecular survey on blood samples from 456 buffaloes obtained from eight provinces across different geographical locations of Thailand. The PCR diagnostics indicated that 116 (25.4%) and 59 (12.9%) of these 456 samples were positive for piroplasm and Plasmodium spp., respectively, and were found in six and all eight regions, respectively, across Thailand. Co-infections of piroplasm and Plasmodium spp. were observed in 24 cases (5.26%). Babesia spp. was not detected in any of the 12 sequenced piroplasm-positive samples in the present study. Genetic comparisons and phylogenetic analyses of within and between parasite populations, based on the 18S ribosomal (r)RNA and cytochrome b (cytb) genes for T. orientalis and P. bubalis, respectively, revealed that T. orientalis shared a high similarity within its population and could be divided into four distinct haplotypes. Haplotypes 1 and 4 were placed in the same clade with the samples previously isolated from cattle in Korea, Japan, Australia, and the USA. Haplotypes 2, and 3 were novel and were placed in a separate clade not shared with the other isolates. We also confirmed our previous investigation that at least three cytb haplotypes of P. bubalis were distributed in the country with a relatively high degree of genetic polymorphisms within its population (based on cytb sequences). Type II P. bubalis was phylogenetically closely related to P. caprae in goats in Zambia and Thailand. This study improves our current understanding on the distribution, intra- and inter-population genetic diversity, and genetic relationship of piroplasms and Plasmodium spp. in water buffaloes.