PCR detection of Borrelia, Ehrlichia, and Rickettsia DNAs in I. ricinus ticks from Bulgaria

A total of 298 I. ricinus ticks, collected by flag from the vegetation in 2000 and 2001, were examined by the reverse line blotting technique for Borrelia, Ehrlichia, and Rickettsia DNAs [6].

Prevalence of Borrelia, Ehrlichia, and Rickettsia in 202 ticks, collected in 2000, was as follows. Overall Borrelia prevalence in adult ticks was 41% (44% in males and 39% in females) and 10% in nymphs. B. afzelii was the predominant species. Its prevalence was 23% (26 of 112) in adult ticks and 6% (5 of 90) in nymphs, repre­senting 56% (31/55) of all Borrelia-positive results. B. burgdorferi sensu stricto was detected in 15 (13%) of 112 adult ticks and in 1 (1%) of 90 nymphs. Prevalence of B. garinii was 3% in adult ticks and 7% in nymphs. B. valaisiana was detected in 3 and B. lusitaniae in other 3 of 202 examined ticks.

Overall Borrelia prevalence in adult I. ricinus ticks, collected in 2001, was 29% (21 of 72) in adult ticks (8% in males and 40% in females). No Borrelia infection was found in nymphs from 2001. B. afzelii was again the prevalent species—preva­lence rate 15% (11 of 72) in adult ticks, representing 52% (11/21) of all Borrelia- positive results. Prevalences of B. garinii and B. valaisiana were 7% and 8%, respectively.

Anaplasma phagocytophilum was detected in 36 (32%) of 112 adult ticks and in 5 (6%) of 90 nymphs, collected in 2000. Of the ticks, collected in 2001, A. phagocy­tophilum was detected in 28% of adult ticks (male 17% and female 33%) and 21% of nymphs.

Of 202 I. ricinus ticks, collected in 2000, 94 (47%) were found to carry Rickettsia DNA: 78% of males, 61% of females, and 19% of nymphs. Prevalence of R. helvetica was 28% (56/202) and prevalence of IRS4 rickettsia was 30% (60/202). R. conorii was found in only two of the ticks. Ticks, collected in 2001, showed also high Rickettsia infectivity rate, 40% (38 of 96): 83% in males, 29% in females, and 17% in nymphs.

A high proportion of Bulgarian I. ricinus ticks contains Borrelia DNA. Analysis of Borrelia prevalence revealed that ticks, collected from the same location and even in the same month (May) but in 2 adjacent years, 2000 and 2001, had differ­ent Borrelia prevalence (41% and 29%, respectively). Apart differences in overall Borrelia prevalence, there were also differences in Borrelia prevalence in males, females, and nymphs, collected in the 2 years in the same place. In ticks from 2000, Borrelia was most prevalent in adult males, less so in adult females and least so in nymphs, while in ticks from 2001, the prevalence was higher in females and lower in males and nymphs.

B. afzelii is highly prevalent among Bulgarian I. ricinus ticks, giving more than half of the Borrelia -positive results, followed by B. burgdorferi sensu stricto, B. gari- nii, B. valaisiana, and B. Iusitaniae in that order. Prevalence of coinfection is high (17-45%) in Ixodes ticks, representing double or even triple infection with Borrelia, Ehrlichia, and/or Rickettsia. Even coinfection with two different Borrelia or Ehrlichia species was often detected, showing that the tick hosts are infected with multiple tick-borne pathogens. Since these ticks were collected from vegetation, a risk for simultaneous transmission of these pathogens during the same tick bite exists.

2.