Detection of a large linear plasmid in Borrelia spielmanii isolate.

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.

Serological evidence of Borrelia burgdorferi sensu lato in horses and cattle from Poland and diagnostic problems of Lyme borreliosis.

In the course of epizootological research on Lyme borreliosis in animals, the serological evidence of this zoonosis in horses and cattle from different voivodships of Poland was screened. We also discussed some diagnostic problems of Lyme borreliosis resulting from, in addition to other factors, genetic and geographical heterogeneity isolates B. burgdorferi s.l. used as antigens. Using ELISA from 395 sera of horses the total mean seroprevalence for anti-Borrelia IgG antibodies 25.6% was observed. In the respective years, significant differences in the mean seroprevalence were not recorded. In the voivodships, the total mean seroprevalence and mean seroprevalence for the respective years varied from 16.6-66.6%. An analysis of seroprevalence depending on the age showed a significant difference between 0-2 year-old horses compared to older horses. The total seroprevalence in the set of 98 serum samples was lower with the strain of B. garinii (25.5%) compared to a mixture of B. burgdorferi sensu stricto with B. afzelii (36.7%) and B. afzelii (42.8%). The highest correlation of findings was reached comparing the strains of B. afzelii (South Poland) and a mixture of B. burgdorferi s. s.+B. afzelii (East Slovakia). Lower correlation was between B. garinii and mixture of B. burgdorferi s. s.+B. afzelii. On the contrary, the lowest correlation of findings was observed between the Slovak strain of B. garinii and Polish B. afzelii. In a group of 26 cow sera, the mean seroprevalence for anti-Borrelia IgG antibodies was 26.9%. In the remaining clinical signs the seroprevalence was 28.5-66.6%. In Western blot, out of 25 examined sera of horses 15 (60.0%) were positive, out of 6 cows 5 (83.3%) were positive (2 lameness, 2 phlebitis, 1 clinically healthy). The horses and cows sera recognised the proteins: 93 (MEP)-, 83-, 75-, 66-, 55-, 43-, 45-, 41 (flagellin)-, 39-, 34-, 35 (OspB) and 25-, 28 (OspC)-kDa. These results alert veterinarians to take into account the aetiology of Lyme disease in differential diagnoses.

Heterogeneity of Borrelia burgdorferi sensu lato and their reflection on immune response.

Geographically different strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto Ir 105, B. burgdorferi s.s. + B. afzelii V 123, B. garinii Ir 112 - isolates from eastern Slovakia, B. garinii K24 - isolate from western Slovakia and B. burgdorferi s.s. B 31 - American strain) were compared as antigens for serological study of Lyme borreliosis by IgG ELISA on a group of horses from eastern Slovakia. In a set of 101 horse serum samples, positivity with the use of Ir 105 strain was 53 (52.4%), with V 123 51 (51.49%), with Ir 112 48 (47.5%), with K 24 47 (46.5%) and with B 31 only 25 (24.7%). The seroprevalence between strains B 31 and Ir 105, B 31 and V 123, B 31 and Ir 112, B 31 and K 24 differed statistically significantly (test chi2, p<0.05); however, the differences between strains Ir 105, V 123, Ir 112 and K24 were insignificant. Consistency of positive and negative findings between American and Slovak strains ranged from 50.5-62.4%. Comparison of Slovak strains (Ir105, V 123, Ir 112 and K 24) consistency of positive and negative findings was higher from 79.2-95.04%. The highest consistency of findings was reached comparing strains Ir 112 and K 24, and the same high agreement of results was observed between the strains Ir 105 and V 123 and also Ir 112 and Ir 105. Higher consistency of findings of serologically examined horses with geographically close trains is in accordance with greater similarity of protein profiles of Slovak strains compared to the American strain.

Molecular evidence for Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from eastern Slovakia.

Ixodes ricinus ticks (20 males, 20 females and 20 nymphs) collected in Kosice, Slovakia were examined for the presence of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato (s.l.) by PCR. 38.3 % of the tested ticks carried single infection of B. burgdorferi s.l. and 8.3 % were infected with A. phagocytophilum. Double infection of both pathogens was detected in 5 % of tested ticks. These results indicate that both B. burgdorferi s.l. and A. phagocytophilum co-circulate in the enzootic sites of Eastern Slovakia and may cause co-infection in humans.

Seroprevalence of anti-Borrelia burgdorferi antibodies in sheep and goats from mountainous areas of Slovakia.

In the present study, domestic animals such as sheep and goats from eastern Slovakia were screened for the presence of anti-Borrelia antibodies. Seroprevalence in 181 sheep and 65 goats were carried out in 1999 and 2000. Modified ELISA method was used for detection of anti-Borrelia IgG antibodies. Seroprevalence obtained was 15.8% and 17.5% in 1999 and 2000 respectively in sheep, whereas in goats it was 17.2% and 19.4% respectively. The results suggest that these domestic species have potential to transmit the disease to other animals. Though the role of sheep and goats in Lyme disease has not yet been documented, there is great possibility of transmission of the causative agent via co-feeding to human beings.

Anti-Borrelia antibodies in rodents: important hosts in ecology of Lyme disease.

Data presented in this study focuses on the presence of anti-Borrelia antibodies in small mammals from Eastern Slovakia during 2000-2003. The total seropositivity observed was 18.78% in rodents. Amongst all species, the total seroprevalence in Apodemus flavicolis was the highest (20.87%), followed by Apodemus agrarius (19.58%) and Clethrionomys glareolus (11.11%). However, the prevalence in Apodemus flavicolis during the year 2000-2001 was higher (26.72%), which reduced to 10.60% in 2002-2003. To compare the year range of seroprevalence in other small mammals was not feasible due to the small sample number. Area-wise distribution of anti-Borrelia antibodies was even (18.75% to 20%) in this study, except in the Bot'any province (0%). This confirms the equal distribution of Borrelia spirochetes in the other 3 localities. Prevalence of anti-Borrelia antibodies during summer was significantly higher than during autumn and early spring. The overall study also reviews the importance of small mammals in Lyme disease ecology.

Wild and farm breeding cervids infections with Anaplasma phagocytophilum.

The main goal of our study was to determine the prevalence of Anaplasma phagocytophilum infections in wild cervids living in north-eastern part of Poland. Material used in the study was gathered between the years 2004- 2008. The blood samples from 106 red deer (Cervus elaphus), 32 sika deer (Cervus nippon hortulorum), 130 fallow deer (Dama dama) and 31 roe deer (Capreolus capreolus) were collected. DNA was isolated using Genomic Mini AX blood kit (A and A Biotechnology). Molecular detection of A. phagocytophilum was based on nested PCR amplification of a species-specific 16S rRNA fragment gene of A. phagocytophilum. The highest prevalence of infection was detected in Cervus elaphus, Capreolus capreolus, Cervus nippon hortulorum, there were 50.9 percent, 38.7 percent, 34.37 percent of infected animals, respectively. The lowest rate of infection was found in fallow deer (Dama dama) - ony 1.5 percent.

Polymerase chain reaction confirmation of Babesia canis canis and Anaplasma phagocytophilum in dogs suspected of babesiosis in Slovakia.

Canine babesiosis was considered an imported tick transmitted disease until the first case of autochthonous canine babesiosis in Slovakia was described in 2002. Since then, the number of cases kept increasing every year. The causative agent of babesiosis in dogs is not yet characterized; therefore, the aim of our study was to determine the agent and the rate of infection in the vector tick D. reticulatus in Slovakia. Babesia canis canis was detected in 80 out of 87 blood samples from dogs with clinical manifestations of babesiosis. Six dogs suspected of babesiosis tested positive for presence of Anaplasma phagocytophilum, and one mixed infection of B. c. canis and A. phagocytophilum was detected. B. c. canis was detected in 35.6% questing adults of D. reticulatus. The obtained sequences from blood samples showed 99.7% and from D. reticulatus, 99.4% similarity with the B. c. canis (AY072926) from dogs infected in Croatia. In our study, we characterized the agent of canine babesiosis from blood samples of naturally infected dogs and D. reticulatus, the vector tick. Further, the presence of A. phagocytophilum, bacterium responsible for the canine granulocytic anaplasmosis, was recorded in dogs for the first time in Slovakia.

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 pathogen's 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).

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.

Borrelia lusitaniae and green lizards (Lacerta viridis), Karst Region, Slovakia.

In Europe, spirochetes within the Borrelia burgdorferi sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%-94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae.

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.

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 approximately 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.