Branislav Pet'ko

Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic

ABSTRACT In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ∼230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

Spatial distribution of Dermacentor reticulatus tick in Slovakia in the beginning of the 21st century.

A new field survey monitoring the spatial distribution of Dermacentor (D.) reticulatus (Fabricius, 1794) tick in Slovakia was carried out in 2005-2008 in order to record changes in its distribution when compared to former studies. Last surveys on the geographical distribution were conducted in 1950s and 1970s and the presence of D. reticulatus was determined along the rivers in the south-east (Latorica) as well as in the south-west (Morava, Dunaj) Slovakia. In the present survey new areas with D. reticulatus occurrence were detected, providing evidence that this tick species has extended its range in the surroundings of its former habitats but also by at least 200 km further North and by 300 m of elevation into higher altitudes. D. reticulatus is known to transmit Babesia spp. causing babesiosis in cattle and dogs. Expansion of D. reticulatus range is therefore likely to bring a spread of babesiosis, which can be severe or fatal especially for dogs.

Climate warming and tick-borne encephalitis, Slovakia.

Increased tick-borne encephalitis (TBE) cases have been reported in central Europe. To investigate temporal trends in the altitude at which TBE cases occur in Slovakia, we analyzed the number of TBE cases during 1961–2004. Since 1980, TBE cases moved from lowlands to submountainous areas, most likely because of rising temperature.

Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia.

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Košice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. 1. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Košice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9–212 ticks per collecting hour within one flagged area (600 m2) which is 1.5–35.5 ticks per 100 m2. Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.

Detection of Borrelia burgdorferi sensu lato in lizards and their ticks from Hungary.

To investigate the involvement of lizard species in the natural cycle of Borrelia burgdorferi sensu lato (s.l.) in Hungary, a total of 186 reptiles belonging to three species--126 green lizards (Lacerta viridis), 40 Balkan wall lizards (Podarcis taurica), and 20 sand lizards (Lacerta agilis)--were captured in 2007 and 2008. All ticks removed from the lizards were Ixodes ricinus, either larvae (324/472; 68.6%) or nymphs (148/472; 31.4%). More than half (66/126; 52.4%) of L. viridis individuals were infested, and the prevalence of tick infestation on both the other two species was 35% each. All 472 I. ricinus ticks and tissue samples collected from 134 collar scales and 62 toe clips of lizards were further analyzed for the presence of B. burgdorferi s.l. with polymerase chain reaction. The amplification of B. burgdorferi s.l. DNA was successful in 8% (n = 92) of L. viridis, 9% (n = 32) of P. taurica, and 10% (n = 10) of L. agilis tissue samples. Restriction fragment length polymorphism genotyping identified the species Borrelia lusitaniae in all tested lizard samples. Prevalence of B. burgdorferi s.l. in ticks collected from L. viridis, P. taurica, and L. agilis was 8%, 2%, and 0%, respectively. Most of the infected ticks carried B. lusitaniae (74% of genotyped positives); however, Borrelia afzelii (5%) and B. burgdorferi sensu stricto (21%) were detected in ticks removed from green lizards and Balkan wall lizards, respectively. We conclude that lizards, particularly L. viridis, can be important hosts for I. ricinus larvae and nymphs; thus, they can be regarded as reservoirs of these important pathogen vectors. The role of green lizards has been confirmed, and the implication of Balkan wall lizards is suggested in the natural cycle of B. lusitaniae at our study site.

First Molecular Detection of Anaplasma phagocytophilum in European Brown Bear (Ursus arctos)

In Europe, Anaplasma phagocytophilum circulates in natural foci in a tick-host cycle. Up to now, antibodies against A. phagocytophilum as well as pathogens DNA were recorded in several domestic and wild animals. Nevertheless, the reservoir host range is still under investigation. Tissue samples from European brown bears (Ursus arctos) were tested for the presence of A. phagocytophilum DNA by a PCR amplification of 16S rRNA gene. The results of our study provides the evidence, that the range of animals involved in the circulation and maintenance of A. phagocytophilum in natural foci, is extended of another ursine carnivore, European brown bear (Ursus arctos).

New records of spotted fever group rickettsiae in Baltic region

Tick-borne rickettsiae are considered to be emerging, but there is still a lack of data on the occurrence and prevalence of the spotted fever group rickettsiae across Europe, especially in the Baltic countries. The aim of the present study was to investigate the presence of Rickettsia spp. in Dermacentor reticulatus and Ixodes ricinus ticks, as well as to determine their prevalence in various regions of Lithuania and Latvia. The prevalence of Rickettsia spp. in geographically distinct populations of D. reticulatus on transect Baltic-Adriatic regions was compared. The molecular analyses of 1859 D. reticulatus and 361 I. ricinus from 48 localities in Lithuania and Latvia revealed the presence of Rickettsia raoultii in D. reticulatus and Rickettsia helvetica in I. ricinus. The prevalence of pathogens in D. reticulatus (4.9%) and I. ricinus (17%) ranged in different locations from 0% to 36.9% and 0%-31.3%, respectively. Higher overall infection rates were detected in D. reticulatus collected in Poland (15.8%) and Slovakia (11.4%), with the prevalence range in different locations from 0% to 59.6%. Our study is the first demonstration of the presence of R. raoultii in D. reticulatus ticks in the Baltic countries and R. helvetica in questing I. ricinus ticks in Lithuania.

Immune complexes in early Lyme disease

The study investigated the presence of Borrelia-specific antibodies captured in immune complexes (ICs) in patients with early Lyme disease manifested by erythema migrans. Out of 18 patients, 15 (83.3%) tested positive for polyethylene glycol-precipitated ICs containing IgM antibodies, while only 4 (22.2%) were IgG positive. These results are in accordance with our findings obtained by standard ELISA and recombinant blot, which indicated that ICs might be used for serological diagnosis of the early disease.