Molecular detection of vector-borne agents in water buffaloes (Bubalus bubalis) and associated ectoparasites from Brazil.

Brazil has the largest buffalo herd outside the Asian continent, with more than 1.5 million head. As buffalo farming has become an economically important activity, studies on the occurrence of pathogens shared between cattle and buffalo are necessary. The aim of this study was to investigate the molecular occurrence of piroplasmids and bacterial agents (Anaplasmataceae and Coxiellaceae) potentially transmitted by vectors in buffaloes in southeastern Brazil using molecular methods. DNA was extracted from blood samples of 81 buffaloes, 165 Rhipicephalus microplus ticks, and 92 Haematopinus tuberculatus lice collected from buffaloes in the municipality of Passos, state of Minas Gerais, Brazil. These samples were subjected to conventional PCR (cPCR) assays based on three endogenous genes, namely gapdh from mammals, 16S rRNA from ticks, and cox-1 from lice. Positive samples from these PCR assays were then submitted to a real-time PCR (qPCR) assay targeting IS1111 of Coxiella burnetii, cPCR for Ehrlichia spp. (dsb gene), and nested-PCR assays targeting the 18S rRNA gene of Piroplasmida, sbp-2 of Babesia bovis, and rap-1α of Babesia bigemina. All buffalo and ectoparasite DNA samples tested positive in the PCR assays for the endogenous genes. All samples were negative in the qPCR for C. burnetii and nested PCR for B. bigemina (rap-1α). For Ehrlichia spp., 6/257 (2.3%) ectoparasites (five ticks and one louse) tested positive, with BLASTn analysis showing 88.04% to 100% identity with Ehrlichia minasensis. In the nPCR for piroplasmids (18S rRNA), 8/81 (9.9%) buffaloes and one tick (0.7%) tested positive, with BLASTn analysis showing > 99.9% identity with B. bovis and B. bigemina. In the nPCR for B. bovis (sbp-2), 1/81 buffalo (1.2%) was positive. This study demonstrated, for the first time, the occurrence and co-infection of tick-borne hemoparasites in buffaloes and associated ectoparasites in southeastern Brazil. The lack of positivity for E. minasensis and low molecular occurrence for B. bovis and B. bigemina among buffaloes sampled may be associated with the higher resistance of this ruminant species to R. microplus ticks and vectored pathogens.