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In two surveys conducted in April and May 2013, a total of 1148 unfed ticks belonging to two species, Ixodes ricinusand Dermacentor reticulatus,were collected by flagging lower vegetation in 15 different localities throughout the Białowieża Primeval Forest (Podlaskie voivodship; north-eastern Poland) and in its buffer zone. In order to establish their infection rate with Rickettsiaspp. individual adult ticks and pooled nymphs were tested by real-time PCR targeting the gltAgene. For the further identification of rickettsial species, positive samples were subjected for nested and semi-nested PCR targeting ompAand 16S rRNA genes, respectively, followed by sequencing analysis. Rickettsial DNA was detected in at least 279 ticks (minimum infection rate [MIR], 23.9%), including 52 nymphal and adult I. ricinus(MIR 8.6%) and 222 adult D. reticulatus(41%). Three species of SFG rickettsiae were identified: Rickettsia helveticaand ‘CandidatusR. mendelii’ in I. ricinusand R. raoultiiin D. reticulatusand I. ricinus.Moreover, unidentified Rickettsiaspp. which showed 99.4% identity, among others, with the uncultured Rickettsiasp. isolated from Cicadella viridisleafhopper, Rickettsiaendosymbiont of Lasioglossum semilucensbee and R. bellii,were detected in I. ricinus,while Rickettsiasp. 98.3-98.4% homologous to Rickettsiasecondary endosymbionts of Curculiospp. weevils was found in D. reticulatus.

Ixodes ricinusticks, collected in 1996–1998 in different Polish woodlands, were examined to assess the frequency of the occurrence of Lyme borreliosis-associated genospecies. A total of 568 samples of individual adults and 162 samples of individual (n = 48) and pooled (of 2 to 7) samples of nymphs were analysed by the polymerase chain reaction (PCR) for Borrelia burgdorferisensu lato. Spirochetes were detected in 130 adult ticks (22.9 %) and in a minimum of 32 (5.3 %) nymphs. Further identification of 153 B. burgdorferis. l.-positive samples by nested PCR using three species-specific primers revealed the occurrence of B. afzelii, B. burgdorferisensu stricto and B. garinii. Both single-species and mixed infections were noted. Single-species infections were observed in the majority of samples (n = 83/153; 54.2 %). Within this group B. afzeliiwas found in 38/153 samples (24.9%), followed by B. burgdorferisensu stricto (n =23/153; 15.0%) and B. garinii(n =22/153; 14.4%). Dual infections with B. burgdorferis.s. and B. afzeliiwere detected in 17/121 (14.0%) adults, while both B. burgdorferis.s./B. gariniiand B. afzelii/B. gariniicoinfected 11/121 (9.1%) adult ticks. Triple infection with B. burgdorferis.s., B. afzeliiand B. gariniiwas noted twice (1.6 %). In general, B. afzeliiwas found in 72/153 (47.1 %) tick samples and was the predominant species. B. burgdorferis.s. and B. gariniiwere detected in a total of 60/153 (39.2%) and 51/153 (33.3 %) samples, respectively. Although, 21 (13.7%) samples were infected by B. burgdorferis. 1. genospecies undetectable by the primers used, results of our study confirm that Lyme borreliosis pathogenic genospecies are well established in tick populations throughout Poland.