Molecular identification of tick-borne pathogens (Rickettsia spp., Anaplasma phagocytophilum,Borrelia burgdorferi sensu lato,Coxiella burnetii and piroplasms) in questing and feeding hard ticks from North-Western Spain

The occurrence of tick-borne pathogens (TBPs) of human and veterinary interest was studied in questing and feeding ticks collected from wild animals in a region in North-Western Spain. A total of 529 ticks (489 questing, 40 feeding) of seven different species (386 Ixodes ricinus, 53 Haemaphysalis concinna, 27 Haemaphysalis punctata, 25 Dermacentor marginatus, 21 Haemaphysalis inermis, 15 Dermacentor reticulatus, and two Rhipicephalus bursa) were analyzed. Molecular analysis of the 16S rRNA gene in I. ricinus ticks, revealed the presence of two phylogenetic groups in the region. Most of the sequenced ticks (96%) were assigned to I. ricinus haplogroup and 4% of the ticks were phylogenetically related to I. inopinatus haplogroup. Feeding ticks were removed from 17 animals from seven wild species (seven roe deer -Capreolus capreolus-, three wolves -Canis lupus-, two Iberian red deer -Cervus elaphus hispanicus-, two European wild boar -Sus scrofa-, one Cantabrian brown bear -Ursus arctos-, one Eurasian badger -Meles meles-, and one red fox -Vulpes vulpes-). Presence of Rickettsia spp., Anaplasma phagocytophilum, piroplasms, Borrelia burgdorferi sensu lato (s.l.) and Coxiella burnetii were tested in ticks by specific PCR. A total of 92 (17.4%) of the 529 ticks analyzed were positive for at least one of the TBPs tested. Sequencing revealed the presence of the genospecies “Candidatus Rickettsia rioja”, Rickettsia raoultii, and Anaplasma phagocytophilum in both questing and feeding ticks. Rickettsia slovaca, Borrelia lusitaniae, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto and Babesia bigemina were only detected in questing ticks, while Babesia sp. badger type A, Theileria OT3 and Hepatozoon canis occurred only in engorged ticks. None of the ticks were positive for C. burnetii. The analysis of the 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants (I, X and W) circulating in the region. New host-tick-pathogen interactions have been revealed, finding for the first time the human pathogen R. raoultii in D. reticulatus removed from a Cantabrian brown bear. Co-occurrence between different TBPs were detected in 4.3% of the ticks. The association B. burgdorferi s.l./Rickettsia spp. was detected in questing ticks; and Rickettsia spp./piroplasms and A. phagocytophilum/Theileria OT3 in feeding ticks. The presence of pathogenic agents constitutes a threat to human and animal health, and should be considered in the diagnosis and treatment after a tick bite. This study increases the knowledge on TBPs diversity of medical and veterinary interest circulating between ticks and their hosts in North-Western Spain.