Molecular detection of Babesia spp. in ticks sampled from asymptomatic dogs in the area of some Belgrade municipalities

Abstract

Babesiosis of domestic animals is a vector transmissible and clinically significant disease, caused by protozoa of genus Babesia and Theileria. Possible causative agents for this disease in dogs in Europe are: Babesia canis, B. gibsoni, B. vogeli and B. microti-like. Diagnostics of babesiosis of dogs was for a long time based on the visual inspection of stained blood smear under a microscope, while today there have been increasingly used molecular methods of detection in precise, species diagnostics. The objective of this work was molecular detection of the cause of babesiosis of dogs in the ticks sampled from asymptomatic dogs in the region of some Belgrade municipalities, all for better understanding of epizootiological situation. From three sites in Belgrade, there were collected 49 ticks, sampled from the dogs with no symptoms. There was carried out the determination of the ticks, and after that, DNA was isolated for molecular examination. First, theret was performed Polymerase Chain Reaction (PCR), for determining the species of the genus Babesia, and after that there was also carried out the determining of polymorphism in the length of restriction fragments (RFLP) for the purpose of the causative agent species determination. Out of the total number of the examined ticks, 18,34% were positive on Babesia spp. By RFLP method, in two cases (4,08%) B. Gibsoni was identified, while in 7 cases (14,92%) there were no restriction sites for the used enzymes, what suggests that most likely it was B. canis. The ticks positive on the cause of babesiosis were: Dermacentor reticulatus (4 cases), Rhipicephalus sanguineus (4 cases) i Ixodes ricinus (1 case). This work confirms the presence of Babesia spp. in the ticks sampled from asmptomatic dogs on the territory of Belgrade as well as the significance of PCR-RFLP method in diagnostics and identification of the causative agent of babesiosis in dogs. For the first time in Serbia, there was determined the presence of B. gibsoni in ticks (Species Rhipicephalus sanguineus).

Babezioza domaćih životinja predstavlja vektorski prenosivo i klinički veoma značajno oboljenje, uzrokovano protozoama rodova Babesia i Theileria. Uzročnici koji mogu da izazovu oboljenje kod pasa u Evropi su: Babesia canis, B. gibsoni, B. vogeli i B. microti-like. Dijagnostika babezioze pasa se dugo vremena bazirala na vizuelizaciji uzročnika pregledom obojenog krvnog razmaza pod mikroskopom, dok se danas sve više koriste molekularne metode detekcije u preciznoj, specijskoj dijagnostici. Cilj ovog rada je bila molekularna detekcija različitih uzročnika babezioze pasa u krpeljima uzorkovanim sa asimptomatskih pasa na prostoru određenih beogradskih opština, radi boljeg razumevanja epizootiološke situacije. Sa tri lokacije u Beogradu je prikupljeno 49 krpelja, uzorkovanih sa pasa bez simptoma bolesti. Izvršena je determinacja krpelja, a nakon toga je izolovana DNK za molekularna ispitivanja. Prvo je urađena reakcija lančane polimerizacije (PCR) za utvrđivanje vrsta iz roda Babesia, a nakon toga i određivanje polimorfizma u dužini restrikcionih fragmenata (RFLP) u cilju specijske identifikacije uzročnika. Od ukupnog broja ispitanih krpelja 18,34% je bilo pozitivno na Babesia spp. RFLP metodom je u dva slučaja (4,08%) identifikovana B. gibsoni, dok u 7 slučajeva (14,92%) nije bilo restrkicionih mesta za korišćene enzime, što ukazuje da se najverovatnije radilo o B. canis. Pozitivni krpelji na uzročnike babezioze su bili Dermacentor reticulatus (4 slučaja), Rhipicephalus sanguineus (4 slučaja) i Ixodes ricinus (1 slučaj). Ovaj rad potvrđuje prisustvo Babesia spp. u krpeljima uzorkovanim sa asimptomatskih pasa na teritoriji grada Beograda, kao i značaj PCR-RFLP metode u dijagnostici i identifikaciji uzročnika babezioze pasa. Prvi put u Srbiji je utvrđeno prisustvo B. gibsoni kod krpelja (vrsta Rhipicephalus sanguineus).