First detection of Anaplasma phagocytophilum and enzootic focus of Theileria orientalis in cattle from Bosnia and Herzegovina

Theileria orientalis is a tick-borne intraerythrocytic protozoan parasite of cattle causing oriental or benign theileriosis. It has a worldwide distribution and is not considered a zoonotic agent, while the disease symptoms range from subclinical to severe. Theileria spp. are very common co-infecting agents with other tick borne pathogens such as Babesia spp., Anaplasma spp. including zoonotic Anaplasma phagocytophilum with rising prominence. This study is an exploratory investigation of the occurrence of Anaplasma spp. and other blood parasites in cattle from Gacko and Bileća, Herzegovina. Blood samples from 35 clinically healthy cattle were selected based on veterinary reports of tick infestation and examined microscopically, by PCR ( Babesia/Theileria ; Anaplasmataceae ) and qPCR ( A. phagocytophilum, A.platys, A.marginale ). Microscopy revealed intraerythrocytic bacterial forms in multiple samples. A. phagocytophilum DNA was detected in one and Babesia/Theileria DNA in 34 samples. Based on 16S rRNA gene sequencing, the A. phagocytophilum isolate was grouped with isolates from Europe, suggesting regional pathogen circulation. AnkA gene analysis placed the isolate in European group I, but outside the zoonotic subcluster, indicating a ruminant-associated lineage maintained primarily in wildlife– Ixodes ricinus cycles. The 18S rRNA gene sequencing showed subtle regional clustering of T.orientalis isolates. This is the first molecular confirmation of A. phagocytophilum in cattle from Eastern Herzegovina and identification of possible enzootic foci of T. orientalis infection, deeming further investigation into tick vectors and reservoir hosts in the region.