Prevalence of Four Species of Borrelia burgdorferi Sensu Lato and Coinfection with Anaplasma phagocytophila in Ixodes ricinus Ticks in Central Germany

A total of 305 Ixodes ricinus ticks collected from three areas of Thuringia in central Germany were investigated for infection with Borrelia burgdorferi sensu lato species and Anaplasma phagocytophila. Overall, 11.1% were infected with Borrelia burgdorferi and 2.3% with Anaplasma phagocytophila. Adult ticks showed a significantly higher rate of infection with both borreliae and Anaplasma phagocytophila. Borrelia garinii (55.9%) was detected most frequently, followed by Borrelia burgdorferi sensu stricto (32.4%), Borrelia afzelii (17.6%), and Borrelia valaisiana (5.9%). Four ticks had dual infection with Borrelia garinii and Borrelia burgdorferi sensu stricto. Two of the Borrelia-positive ticks were coinfected with Anaplasma phagocytophila.     

Swiss Army Survey in Switzerland to Determine the Prevalence of Francisella tularensis, Members of the Ehrlichia phagocytophila Genogroup, Borrelia burgdorferi Sensu Lato, and Tick-Borne Encephalitis Virus in Ticks

 A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.     

An<Emphasis Type="Italic"> ospA</Emphasis>-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato species and OspA types

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3–7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.     

Rate of infection ofIxodes ricinus ticks withBorrelia burgdorferi sensu stricto,Borrelia garinii,Borrelia afzelii and group VS116 in an endemic focus of Lyme disease in Italy

A study to evaluate the natural rate of infection ofIxodes ricinus withBorrelia burgdorferi sensu lato was carried out in an endemic focus of Lyme disease in the Trieste area in northern Italy. Two-hundred and twenty-seven ticks collected in ten different stations were tested individually for the presence of the spirochetes using polymerase chain reaction techniques able to identify bothBorrelia burgdorferi sensu lato and the four genospecies (Borrelia burgdorferi sensu stricto,Borrelia garinii, Borrelia afzelii and group VS116). Multiple infection of individual ticks was found. The infection rate ranged from 0–70%. Infection ofIxodes ricinus withBorrelia burgdorferi group VS116 was found for the first time in Italy in both a high and a low endemic focus of Lyme disease.     

Experimental infection of dogs with<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu stricto using<Emphasis Type="Italic"> Ixodes scapularis</Emphasis> ticks artificially infected by capillary feeding

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.     

Infestation of sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis>) resident in the Northeastern Poland by <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (L.) ticks and their infection with <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato

Sand lizards (Lacerta agilis) were trapped and examined for ticks from May to September in 2002 and 2003 in Northeastern Poland. A total of 233 Ixodes ricinus (L.) ticks (76 larvae and 157 nymphs) was found on 31 of 235 captured lizards (13.2%). The tick infestation is relatively low compared to that of mammals and passerine birds from the same area (Siński et al. 2006, Gryczyńska et al. 2002). Tick infestation depended on the month of capture, being the highest in spring. In autumn no ticks were recorded on any of the captured lizards. The oldest lizards carried the highest number of ticks but no differences related to sex of the host were found. All the collected ticks were analysed by PCR for the presence of Borrelia burgdorferi sensu lato, the etiological agents of Lyme disease. Spirochetes were detected in 11 out of 233 (4.7%) ticks tested. Genetic analysis confirmed that the spirochetes are members of the Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto genospecies. Mixed infection were not detected. The prevalence of infection was analysed in relation to months of the capture, age and sex of the lizards, but differences were not statistically significant. The obtained results suggest that lizards are probably not B. burgdorferi reservoirs, but further studies are required to confirm this.     

Infection rate ofIxodes ricinus ticks withBorrelia afzelii,Borrelia garinii,andBorrelia burgdorferi sensu stricto in Slovenia

In spring 1993,Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection withBorrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium.Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19 %): the isolation rate from adult female ticks was 35 % (23/66 ticks cultured), from adult male ticks 22 % (20/91), and from nymphal ticks 13 % (26/206). Determination of the species of 60 isolates revealed that 32 wereBorrelia afzelii (53 %), 20 wereBorrelia garinii (33 %), and 8 wereBorrelia burgdorferi sensu stricto (13 %). In the Ljubljana regionBorrelia afzelii andBorrelia garinii predominated (43 % and 40 %, respectively), whereasBorrelia burgdorferi sensu stricto constituted only 17 % of isolates. In three other regions of the countryBorrelia afzeliiwas isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species ofBorrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m².     

Genetic features of DNA of <Emphasis Type="Italic">Borrelia miyamotoi</Emphasis> transmitted by <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

16S rRNA, p66 and glpQ gene fragments in Borrelia miyamotoi transmitted by Ixodes persulcatus are determined and analyzed. Specific nucleotide sequences of every locus have been shown to be identical. The highest homology level for nucleotide sequences of three loci has been shown for the sequences of strains isolated earlier in Japan. An analysis of the P66 protein amino-acid sequence has shown that the locus corresponding to the surface-exposed domain differs considerably from both Borrelia hermsii, a typical member of tick-borne relapsing fever and Borrelia lonestari, the closest related species. Three genetic variants of Borrelia miyamotoi P66 protein is characterized not only by amino-acid substitutions, but also by deletions.     

Foci of tick-borne diseases in Southwest Germany

Presently known tick-borne diseases in Germany include Lyme borreliosis, tick-borne encephalitis (TBE-virus, western subtype), Q-fever, babesiosis and presumably ehrlichiosis. Blood samples of 4,368 forestry workers in the State of Baden-Wuerttemberg (B-W), southwestern Germany, were tested for the presence of antibodies against Borrelia burgdorferi sensu lato, TBE-virus and Ehrlichia spp. (genogroup E. phagocytophila). Furthermore 12,327 ticks (Ixodes ricinus) collected in various areas of B-W were analysed by PCR and genotyping for the prevalence of pathogen RNA and DNA. The human seroprevalence rates of antibodies to B. burgdorferi sensu lato ranged from 18 % to 52 %, for TBE-virus from 0 % to 43 % and for Ehrlichia spp. from 5 % to 16 % in various counties of the State. The foci of B. burgdorferi and TBE-virus as indicated by antibody prevalence in humans are only partly overlapping with each other. The highest rates of TBE-virus antibodies are in concordance with available clinical data. However antibody prevalence up to 27 % in areas with no reports of clinical cases was found, suggesting that TBE occurs throughout the State of B-W, The prevalence of Ehrlichia spp. antibodies suggests that ehrlichiosis plays a role as a tick-borne disease in Germany, but as long as no clinical data are available, this will remain unclear.Investigations of ticks for TBE-virus (n = 9,189) by nested PCR showed prevalence rates from 0% to 2.3% and for Ehrlichia spp. (n = 1,963) from 2.6% to 3.1%. Examination of ticks (n = 3,138) for the presence of B. burgdorferi sensu lato DNA was performed by PCR and revealed prevalence rates from 13.9% up to 24%. Furthermore 1,106 samples positive for B. burgdorferi sensu lato were used for genotyping. B. afzelii DNA was found in 407 ticks (36.8%), followed by B. garinii (21.9 %), B. valaisiana (13.7%), and B. burgdorferi sensu stricto (9.9 %). Double infection was found in 6.4% and triple infection in 0.8% of the ticks. 10.5% of the positive samples could not be classified.Prevention of tick-borne diseases has to focus on behavioural intervention to reduce individual tick exposure by proper behaviour in the environment, as a large-scale control of the tick population seems impossible and thus reduction of Lyme borreliosis and TBE through tick control is unlikely. Vaccination against TBE-virus should not only be recommended for high endemic areas but also for persons with a high individual risk.     

Serological and Molecular Evidence of Human Granulocytic Ehrlichiosis Focus in the Białowieża Primeval Forest (Puszcza Białowieska), Northeastern Poland

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne zoonosis. First described in the USA, it is being increasingly reported from several European countries. This study was undertaken to provide serological and molecular evidence of the occurrence of the HGE focus in the Białowieża Primeval Forest, located in northeastern Poland. To this end, the seroprevalence of HGE in this area, where Lyme borreliosis and tickborne encephalitis are highly endemic, was determined by means of an indirect immunofluorescence antibody assay. In addition, the frequency of granulocytic Ehrlichia spp. infection in Ixodes ricinus ticks from the same area was estimated using a polymerase chain reaction method with EHR 521 and EHR 747 primers, which amplified a fragment of 16S rDNA. The rate of seropositivity for HGE was 6.2% (8/130 subjects). Individuals seropositive for Lyme borreliosis were more likely to have anti-HGE antibodies than seronegative ones (P<0.05; OR=6.34, 95%CI=1.12–36.98). There was no association between self-reported frequency of tick bites or forestry employment and HGE seropositivity. Sixty of 376 (16%) Ixodes ricinus ticks tested were positive for the Ehrlichia phagocytophila genogroup by polymerase chain reaction. Ehrlichial DNA was present in 59 of 302 (19.5%) adult ticks and in 1 of 74 nymphs (1.4%). There was a significantly higher infection rate among female ticks (32.9%; 49/149) than among male ticks (6.5%; 10/153) (P<0.05). Dual infection with Ehrlichia spp. and Borrelia burgdorferi sensu lato was detected in 10 samples that were positive for ehrlichiae. The results obtained confirm the perpetuation of the HGE agent in the primeval forest ecosystem of northeastern Poland. Electronic Publication     

Molecular characterization ofBorrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates

One hundred twenty-nine Slovenian isolates ofBorrelia burgdorferi sensu lato derived from patients (69 strains) orIxodes ricinus ticks (60 strains) were characterized. All of the strains were first- or second-passage isolates obtained in 1992 and 1993 from the same endemic region. The techniques used for the molecular analysis of strains included species-specific polymerase chain reaction (PCR) typing, and pulsed-field gel electrophoretic separation of undigested andMlul-digested genomic DNA. Isolates were identified to the species level by large restriction fragment pattern (LRFP) analysis and the results compared with the species-specific PCR result. Fifty-two patient isolates (75%) were typed asBorrelia afzelii (LRFP MLa1), 6 (9%) asBorrelia garinii (LRFPs MLg1–4), and 11 (16%) asBorrelia burgdorferi sensu stricto. The latter included 9 isolates (13%) with a new LRFP that is not typical ofBorrelia burgdorferi sensu stricto and for which the designation MLx is suggested. In contrast, only 32 of 60 (53%) tick isolates were typed asBorrelia afzelii, while 20 strains (33%) were typed asBorrelia garinii and 8 strains (13%) asBorrelia burgdorferi sensu stricto. Three new LRFPs were found among theBorrelia garinii (MLg5 and 6) andBorrelia burgdorferi sensu stricto (MLb15) tick isolates. Large restriction fragment pattern analysis identified new groups ofBorrelia burgdorferi sensu lato and revealed an apparent difference in the isolation frequency of different species from patients and ticks in the same endemic region.     

<Emphasis Type="Italic">Anaplasma marginale</Emphasis> and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>: Rickettsiales pathogens of veterinary and public health significance

Anaplasma marginale and Anaplasma phagocytophilum are the most important tick-borne bacteria of veterinary and public health significance in the family Anaplasmataceae. The objective of current review is to provide knowledge on ecology and epidemiology of A. phagocytophilum and compare major similarities and differences of A. marginale and A. phagocytophilum. Bovine anaplasmosis is globally distributed tick-borne disease of livestock with great economic importance in cattle industry. A. phagocytophilum, a cosmopolitan zoonotic tick transmitted pathogen of wide mammalian hosts. The infection in domestic animals is generally referred as tick-borne fever. Concurrent infections exist in ticks, domestic and wild animals in same geographic area. All age groups are susceptible, but the prevalence increases with age. Movement of susceptible domestic animals from tick free non-endemic regions to disease endemic regions is the major risk factor of bovine anaplasmosis and tick-borne fever. Recreational activities or any other high-risk tick exposure habits as well as blood transfusion are important risk factors of human granulocytic anaplasmosis. After infection, individuals remain life-long carriers. Clinical anaplasmosis is usually diagnosed upon examination of stained blood smears. Generally, detection of serum antibodies followed by molecular diagnosis is usually recommended. There are problems of sensitivity and cross-reactivity with both the Anaplasma species during serological tests. Tetracyclines are the drugs of choice for treatment and elimination of anaplasmosis in animals and humans. Universal vaccine is not available for either A. marginale or A. phagocytophilum, effective against geographically diverse strains. Major control measures for bovine anaplasmosis and tick-borne fever include rearing of tick-resistant breeds, endemic stability, breeding Anaplasma-free herds, identification of regional vectors, domestic/wild reservoirs and control, habitat modification, biological control, chemotherapy, and vaccinations (anaplasmosis and/or tick vaccination). Minimizing the tick exposure activities, identification and control of reservoirs are important control measures for human granulocytic anaplasmosis.     

Human pathogenic borreliae in Ixodes ricinus ticks in natural and urban ecosystem (Czech Republic)

A total of 1279 field-collected Ixodes ricinus ticks were screened for Borrelia burgdorferi sensu lato (s.l.) in a natural and an urban ecosystem of Ostrava city (Czech Republic) by using molecular methods. Minimal prevalence rate for Borrelia burgdorferi s.l. in ticks for the urban park Bělský les was found to be 13.8% (17.6% in males, 17.8% in females and 11.7% in nymphs), similarly for the natural site Proskovice was minimal prevalence 15% (12.5% in males, 20% in females and 14.9% in nymphs). Six proven human pathogenic genomic species have been recorded in the study: B. afzelii, B. garinii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, and B. spielmanii. Emerging B. spielmanii was detected for the first time in Ixodes ricinus ticks in the region. Our results highlight the need for surveillance of zoonotic tick-borne pathogens even in urban areas.     

Prevalence and incidence of antibodies to Borrelia burgdorferi and to tick-borne encephalitis virus in agricultural and forestry workers from Tuscany, Italy

The ticks Ixodes persulcatus and Ixodes ricinus are the main vectors of both Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus in Eurasia. Borrelia burgdorferi is the cause of Lyme borreliosis, and TBE is a biphasic meningoencephalitis induced by an arbovirus belonging to the flavivirus family. The principal aims of the current investigation were (i) to determine the frequency of serological evidence of Borrelia burgdorferi sensu lato and TBE infections in healthy agricultural and forestry workers, (ii) to determine the incidence of seroconversion for antibodies against Borrelia burgdorferi sensu lato and TBE virus in Tuscan workers during a 1-year survey; and (iii) to assess the occupational risk for agricultural and forestry activities in a defined area (Tuscany, Italy). A total of 412 blood samples were taken from agricultural and forestry workers, and information on age, duration of employment, and history of tick bites was collected in a questionnaire to establish the risk factors for the diseases. Three hundred sixty-five blood donors from the same region served as controls. To estimate the rate of seroconversion, 176 of the agricultural and forestry workers were tested 1 year later. IgG and IgM antibodies against Borrelia burgdorferi sensu lato and TBE virus were detected in serum by an enzyme-linked immunosorbent assay and confirmed by Western blot analysis for Borrelia burgdorferi and by a test for inhibition of hemagglutination for TBE. Antibodies against Borrelia burgdorferi were more frequent among the workers than in the control group (7.8% vs. 4.9% in the IgG-IgM enzyme-linked immunosorbent assay and 7.03% vs. 3.56% in the confirmatory test). No seropositivity was observed for TBE virus. Eighteen of 176 subjects who underwent a second blood test developed specific antibodies against Borrelia burgdorferi within 1 year.     

Prevalence of Borrelia burgdorferi Species and Identification of Borrelia valaisiana in Questing Ixodes ricinus in the Lyon Region of France as Determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

Many cases of Lyme borreliosis have been reported over the years in the region of Lyon, France. The identification and prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus were investigated by polymerase chain reaction (PCR) of the flagellin gene and restriction fragment length polymorphism (RFLP) analysis. Questing Ixodes ricinus larvae, nymphs and adults were collected by the flagging method from deciduous forests in four areas in the Lyon region of France between October 1994 and September 1995 and in June 1998. The overall prevalence of Borrelia burgdorferi sensu lato was 13.2% (91/688). No significant differences in prevalence were observed between the different stages and sex of the ixodids or between collection areas. The majority of infections were simple infections (82.4%; 75/91), most of which were due to Borrelia afzelii (41.4%), while coinfections (12.1%) were predominantly (54.5%) a combination of Borrelia valaisiana and Borrelia garinii. No tick was infected with more than two borrelial species, nor was Borrelia lusitaniae identified. The Borrelia valaisiana species was detected for the first time in France, confirming its widespread presence in Europe. This study confirms that the surroundings of Lyon are risk areas for contracting Lyme disease and that no particular clinical manifestations predominate due to the heterogeneous distribution of Borrelia genospecies. Moreover, the polymerase chain reaction restriction fragment length polymorphism analysis is a rapid and easy method for genotyping of Borrelia species. Electronic Publication     

Divergence within the marker region of the <Emphasis Type="Italic">groESL</Emphasis> operon in <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis>

Anaplasma phagocytophilum is an obligate intracellular bacterial parasite in human and animal granulocytes. In Europe, A. phagocytophilum is transmitted by Ixodes ticks; Ixodes ricinus is the vector of the parasite in Poland. In terms of epidemiology, the identification of pathogens in ticks increasingly relies on molecular techniques. Polymerase chain reaction (PCR) with species-specific primers is a tool that allows the quick and accurate detection of pathogens in ticks, humans, or animals. DNA was extracted from the blood of Capreolus capreolus and Cervus elaphus, and amplified using the primers HS1/HS6 (external) and HS43/HSVR (internal). For sequencing, six samples from roe deer and two samples from red deer were selected, and the resulting sequences were submitted to GenBank (accession numbers DQ779568, DQ779567, EU157919, EU157920, EU157921, EU157922). These nucleotide sequences were compared with each other and five variants were distinguished in roe deer and one in red deer. A comparison of the sequences of the author’s database revealed 45 polymorphic sites of substitution character (76% transitions and 24% transversions). The homology tree revealed two groups, one with sequences only from roe deer, while the second with sequences isolated mainly from red deer, livestock animals, and humans. These strains of A. phagocytophilum are also present in Poland.     

Prevalence of serological response to <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> in farmers from eastern and central Poland

Lyme borreliosis (Lyme disease) caused by the Borrelia burgdorferi sensu lato spirochete is the most common tick-borne infection manifested by a wide spectrum of clinical symptoms. In Poland, the preventive health care does not comprise individual farmers as it is practiced in foresters. The objective of this study was to evaluate the exposure of Polish farmers to infection with B. burgdorferi, based on serological screening test and epidemiological investigation. A total of 3,597 farmers were examined for the presence of B. burgdorferi antibodies, as well as interviewed regarding exposure to ticks and prophylaxis of tick-borne diseases. The prevalence varied between 18.2 and 50.7 % suggesting a focal occurrence of borreliosis. A significant increase in the frequency of positive reactions in the oldest age ranges was observed, equaling 30.9 % in the range of 60–69 years and 53.6 % in the range of 80–91 years. The prevalence of the anti-B. burgdorferi antibodies of IgG class (14.7 %) was similar to that of IgM class (16.0 %). Seroreactivity to B. burgdorferi antigen was significantly higher in the group of farmers exposed to repeated tick bites. Significant relationships were also found between some other risk factors and occurrence of seropositive reactions to B. burgdorferi. To the best of our knowledge, this is the first study concerning seroprevalence to B. burgdorferi carried out on such a large group of farmers. Results indicate a high risk of B. burgdorferi infection among Polish farmers and associations between some risk factors and the presence of seropositive reactions.     

Spotted fever group <Emphasis Type="Italic">Rickettsia</Emphasis> in brown dog ticks <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> in southwestern Spain

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.     

Genotyping <Emphasis Type="Italic">Borrelia burgdorferi sensu lato</Emphasis> isolates from <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks in Russia and Ukraine

The four Borrelia burgdorferi sensu lato isolates obtained from I. ricinus ticks collected from the natural loci in Russia and Ukraine that had an unusual RFLP MseI pattern were studied using the sequencing of the rrfA-rrlB spacer and the rrs gene. Isolate Ir-5215 from a tick collected in southern Ukraine represented the recently described genospecies B. spielmanii, which is pathogenic for humans. The three atypical isolates Ir-3519, Ir-4721, and Ir-4812 had 100% identity with the sequence of atypical European B. burgdorferi sensu stricto strains; they constituted a subgroup of B. burgdorferi sensu stricto based on multilocus sequence analysis (MLSA). These data are indicative of the genetic heterogeneity of the current group B. burgdorferi sensu stricto.