Investigation of anaplasma species in cattle in Malatya province by reverse line blotting (RLB)

Anaplasma species are obligate intracellular rickettsial pathogens that affect the health of humans and other animals. The genus Anaplasma currently comprises 6 bacterial species mostly pathogenic to animals and/or human. Recently, two potentially novel species genetically related to A. phagocytophilum have been identified in Japan, China, and Tunisia. The aim of this study was to determine the presence and distribution of Anaplasma spp.in cattle. For this purpose, EDTA blood samples were collected in 2019 (in March and October) from 200 apparently healthy cattle farmed in 27 different willages belonging to Arguvan, Yazihan, Akçadağ district in Malatya province. DNA was screened by reverse line blot assay for the presence of the hypervariable V1 region of the 16S rRNA gene of Anaplasma/Ehrlichia species. Anaplasma spp. overall infection rates were 38.5% (77/200) by RLB. The most frequently found species was A. marginale (32.5%), followed by A. centrale (5.5%), Anaplasma/Ehrlichia catc-all (5.5%) and Anaplasma sp. Omatjanne (2.5%). A. phagocytophilum and A. bovis were not detected in any of tested animals. Eleven of 77 PCR-positive amplicons gave positive signals to the catch-all probes, but did not show any response to the species-specific probes tested. PCR-RFLP based on 16S rRNA of these amplicons provided evidence for the presence of A. phagocytophilum-like 1 and A. phagocytophilum-like 2 strains. Sequencing and phylogenetic analyses based on 16S rRNA gene validated RFLP findings, and provided evidence for circulation of two potentially novel species closely related to A. phagocytophilum in Turkish cattle. This is the first report of the presence of A. phagocytophilumlike-like strains in the country. These findings suggest that genetic diversity within the genus Anaplasma is much greater than expected, and A. phagocytophilum-like strains should be taken into account as differential diagnosis in bovine anaplasmosis. Keywords: Anaplasma phagocytophilum-like 1 and 2; Molecular epidemiology; 16S rRNA PCR-RFLP; reverse line blot; cattle