Prevalence of Borrelia afzelii and Borrelia garinii Genospecies in Ixodes ricinus Ticks Collected from Three Regions in Bulgaria

The tick species <i>Ixodes ricinus</i> is the most widely distributed throughout Europe and serves as the primary vector for the spirochete <i>Borrelia burgdorferi</i> sensu lato (s.l.), which is responsible for Lyme borreliosis. The present study evaluated the prevalence of <i>B. burgdorferi</i> (s.l.) in <i>I. ricinus</i> ticks using nPCR amplification. Ticks were collected from three geographical regions in Bulgaria: the Black Sea Coast in the east, the Pleven region in the north, and the Stara Zagora region in the central south. This study focused on urban and peri-urban areas, including heavily traveled trails in parks. The results indicated statistically significant differences in infection rates, with the highest percentage of infected ticks found in Pleven (52.34%), followed by Stara Zagora (35.46%) and the lowest on the Black Sea Coast (23.08%). Furthermore, we conducted genospecies molecular identification of <i>B. afzelii</i> and <i>B. garinii</i> by PCR amplification of genospecies-specific nucleotide sequences in the 16S rRNA gene. The lowest prevalence of <i>B. afzelii</i> was recorded in Stara Zagora at 10.64%, followed by the Black Sea region at 12.5%, while the statistically highest frequency was observed in Pleven at 21.03%. The prevalence of <i>B. garinii</i> among the total number of <i>I. ricinus</i> ticks was greatest in Pleven at 15.89%, followed by the Black Sea region at 8.65%, and the lowest in the Stara Zagora region at 4.96%. Statistically significant differences were found only between Pleven and Stara Zagora (<i>p</i> = 0.002), but no significant differences in infection rates were observed between adults and nymphs in the regions examined. Overall, the prevalence of <i>B. afzelii</i> in each region exceeds that of <i>B. garinii</i>, and when considered collectively for Bulgaria, the frequency of <i>B. afzelii</i> (15.9%) is higher than that of <i>B. garinii</i> (10.89%) in infected ticks. In a controlled epidemiological context, managing the population of <i>I. ricinus</i> infected with <i>B. burgdorferi</i> s.l. can mitigate the health burden of Lyme disease, although this preventive strategy may not guarantee complete protection.