Vuk Vračar

Molecular detection of emerging tick-borne pathogens in Vojvodina, Serbia

Ticks play an important role in disease transmission globally due to their capability to serve as vectors for human and animal pathogens. The Republic of Serbia is an endemic area for a large number of tick-borne diseases. However, current knowledge on these diseases in Serbia is limited. The aim of this study was to investigate the presence of new emerging tick-borne pathogens in ticks collected from dogs and the vegetation from different parts of Vojvodina, Serbia. A total of 187 ticks, including 124 Rhipicephalus sanguineus, 45 Ixodes ricinus and 18 Dermacentor reticulatus were collected from dogs. In addition, 26 questing I. ricinus ticks were collected from the vegetation, using the flagging method, from 4 different localities in Vojvodina, Serbia. DNA was extracted from each tick individually and samples were tested by either conventional or real-time PCR assays for the presence of Rickettsia spp.-DNA (gltA and ompA gene fragments), Ehrlichia/Anaplasma spp.-DNA (16S rRNA gene fragment) and Hepatozoon spp./Babesia spp.-DNA (18S rRNA gene fragment). In addition, all I. ricinus DNA samples were tested for Bartonella spp.-DNA (ITS locus) by real-time PCR. In this study, the presence of novel emerging tick-borne pathogens including Rickettsia raoultii, Rickettsia massiliae, Babesia venatorum, Babesia microti, Hepatozoon canis and Candidatus Neoehrlichia mikurensis was identified for the first time in Serbia. Our findings also confirmed the presence of Rickettsia monacensis, Babesia canis and Anaplasma phagocytophilum in ticks from Serbia. The findings of the current study highlight the great diversity of tick-borne pathogens of human and animal importance in Serbia. Physicians, public health workers and veterinarians should increase alertness to the presence of these tick-borne pathogens in this country.

Molecular characterization of Borrelia strains isolated from ticks in Vojvodina

Borrelia burgdorferi sensu lato (s.l.) complex represents a group of different types of spirochete that are present globally, which causes Lyme borreliosis. The total number of types is still not final because new genetic and antigenic isolates are still being described. The clinical picture of Lyme borreliosis in people is polymorphic and is characterized with symptoms similar to flu syndromes together with erythema migrans, rheumatologic, cardiologic and neurological complications. Apart from people, dogs, horses, bovines and sheep can suffer from Lyme borreliosis. In Europe, the most important vector transmitting Lyme disease is the tick Ixodes ricinus. A description of different species of Borrelia in ticks has opened a completely new field of investigating the ecology of Lyme borreliosis. It is especially important to investigate a connection between different types of Borrelia, ticks as vectors and vertebrates as the reservoirs on various geographical localities. The aim of the work is to isolate Borrelia species from the collected ticks I. ricinus from the geographical territory of Vojvodina, and to do a molecular characterization of the isolated strains of Borrelia. A total of 12 tick pools of the type I. ricinus were cultivated in Barbour-Stoenner-Kelly-H medium with additional antibiotics and subcultivated into a modified Kelly-Pettenkofer/Preac-Mursic medium, until a clear culture is reached. For the molecular characterization of isolated strains of Borrelia, molecular methods MluI-LRFP and real-time PCR for hbb gene were used. In this research, out of 12 pools of ticks species I. ricinus 3 strains of Borrelia from the B. burgdorferi s.l. complex were isolated. All three isolates of Borrelia from ticks of the tick species I. ricinus from the territory of Vojvodina were identified as Borrelia afzelii by applying molecular methods (MluI-LRFP and real-time PCR for hbb gene). By applying MluI-LRFP all three isolated strains of B. afzelii were characterized as a subtype Mlal. In this research, like in the previous researches, we have not proved the presence of a pathogenic species Borrelia spielmanii in ticks of the species I. ricinus.

Prevalence of G class antibodies to antigens of lyme disease causes in dogs in Vojvodina, Serbia.

Lyme disease is a multisystemic disease, zoonotic in nature, caused by the Borrelia burgdorferi sensu lato complex. In the continent of Europe, these spirochetes are predominantly transmitted by ticks of the genus Ixodes. Small mammals and birds have particular significance as reservoirs of the cause of lyme disease. The objective of these epidemiological investigations was to determine the value of IgG seroprevalence to Borrelia burgdorferi and to secure the geographic distribution of seropositive dogs in Vojvodina. The investigations covered 135 dogs that were not vaccinated against lyme disease. The indirect ELISA test was used to determine IgG prevalence to Borrelia burgdorferi antigens. Reactive blood serums of dogs were tested again using the rapid immunochromatographic and immunoblot test. A seroprevalence of G class antibodies to antigens of lyme disease causes of 8.1% (11/135) was established in the examined dog population of Vojvodina. The biggest number of positive results was recorded for the South Backa District. The presented value for the seroprevalence of anti-Borrelia burgdorferi antibodies in the dog population indicates the exhistence of a significant risk of humans becoming infected with the cause of lyme disease in Vojvodina.