A new protocol employing nested PCR-restriction fragment length polymorphism (RFLP) based on the flaB gene and two restriction enzymes was worked out. This protocol allows the identification of all Borrelia species transmitted by Ixodes ricinus in Europe, including Borrelia miyamotoi and 3 genetic variants of B. garinii . A dendrogram of flaB sequence similarity was in accordance with RFLP variants.

The role of red fox, Vulpes vulpes, and its associated ticks in maintaining Borrelia burgdorferi sensu lato (s.l.) was studied. A total of 1583 ticks were removed from ears of 120 infested animals and were identified as species using a nested PCR targeting the ITS2 and coxI fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga, I. ricinus, and I. hexagonus. In total, 32.4% of 943 ticks revealed Borrelia DNA and 10 species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii comprised 70% of all infections. The other eight species included B. americana, B. bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. lanei, B. spielmanii, and B. valaisiana. Analysis of tissues from 243 foxes showed that 23.5% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii, the second most prevalent species, followed by B. spielmanii. The predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with foxes. Although red foxes are exposed to a high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.

In Europe, Ixodes ricinus tick is the vector of Lyme disease spirochetes and their relatives (Borreliella genus) and Borrelia miyamotoi. However, a newly described tick I. inopinatus with similar biological features and separated from I. ricinus may act as a vector for different Borrelia species. To date, eleven Borreliella species were detected in the natural populations of I. ricinus. Recently, two North American species have been detected in ticks parasitizing bats and red foxes in Europe, i.e., B. lanei and B. californiensis pointing to the necessity for searching for them in natural tick populations. In this study, using the coxI molecular marker only I. ricinus was identified in field-collected ticks with the exception of individual specimens of Haemaphysalis concinna. Using the flaB gene and mag-trnI intergenic spacer as molecular markers 14 Borreliaceae species have been detected with various frequencies in different parts of northern Poland. Among infected ticks, the most frequent were Borreliella (Bl.) afzelii (29.4%) and Bl. garinii (20.0%), followed by Bl. spielmanii, Bl. valaisiana, Bl. lanei, Bl. californiensis, B. miyamotoi, Bl. burgdorferi, Bl. carolinensis, Bl. americana, B. turcica, Bl. lusitaniae, Bl. bissettiae and Bl. finlandensis. Three of the above-mentioned species, i.e., Bl. lanei, Bl. californiensis and B. turcica were detected in this study for the first time in the natural ixodid tick population in Europe. The existence of the newly detected spirochetes increases their total diversity in Europe and points to the necessity of careful identification and establishment of the actual distribution of all Borreliaceae species transmitted by I. ricinus.

Background: The red fox, Vulpes vulpes is the most widely distributed terrestrial carnivore species in Europe, which is parasitized by exophilic as well as nidicolous Ixodes ticks. However, the role of these canids and their tick fauna in the maintenance of Borrelia burgdorferi sensu lato (s.l.) is still insufficiently understood. Results: A total of 1,583 ticks were removed from ears of 120 infested animals and were identified to species using a nested PCR targeting the ITS2 and cox1 fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga (12%), I. ricinus (10%), and I. hexagonus (2%). In total, 32.4% of 943 ticks yielded Borrelia DNA and ten species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii composed 70% of all infections. The other eight species included B. americana, B. bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. lanei, B. spielmanii, and B. valaisiana. Analysis of 229 foxes showed that 25% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii, the second most prevalent species, followed by B. spielmanii. Conclusions: The clear predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with these wild canids. Although red foxes are exposed to high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.

The western European hedgehog (Erinaceus europaeus) and the northern white-breasted hedgehog (E. roumanicus) are natural hosts of the tick Ixodes hexagonus, the vector of tick-borne pathogens such as the Borreliella bacteria responsible for Lyme disease. The aim of this study was to identify these pathogens in ticks collected from hedgehogs in northwestern Poland and to assess their genetic diversity by molecular analysis of the detected pathogens based on the flaB gene and the mag-trnI intergenic spacer. Among 101 hedgehogs examined, 737 ticks were found on 56 (55.45%) individuals, including 501 females of I. hexagonus. Borreliella spirochete infection was confirmed in 9 females of I. hexagonus (1.8%) obtained from 4 (3.96%) hedgehogs, detecting Borreliella (Bl.) afzelii (8/89%) and Bl. spielmanii (1/11%). Phylogenetic analysis based on the flaB gene and the mag-trnI intergenic spacer showed a lack of diversity in Bl. afzelii detected in I. hexagonus ticks collected from hedgehogs as well as little diversity against reference strains detected in small mammals and ticks collected from them. The results confirm that hedgehogs play an important role in the circulation of the detected spirochete species, at least as hosts of I. hexagonus ticks infected with them, indicating their potential to spread Borreliella spirochetes.