Katarína Schwarzová

Detection of Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi s. l. in Ticks, and Wild‐Living Animals in Western and Middle Slovakia

Abstract:  In this study, three tick species (Ixodes ricinus, Dermacentor marginatus, and D. reticulatus), small terrestrial mammals, and game were examined by PCR for the presence of tick‐borne pathogens Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi Sensu lato.

Combined infection of Ixodes ricinus with three Borrelia burgdorferi sensu lato genotypes.

Ixodes ricinus ticks were collected by random collections from western and central Slovakia during the years 1996–98. The midgut content of 240 ticks was examined by dark-field microscopy and submitted for cultivation for the presence of borrelias. Spirochetes were found in 21 unfed and host-seeking adults and nymphs (8.8%). By the analysis of restriction fragment length polymorphism (RFLP) one sample from unfedI. ricinus male from western Slovakia was identified as a triple infection ofBorrelia burgdorferi sensu stricto,B. garinii andB. afzelii. The simultaneous presence of differentB. burgdorferi genospecies in one midgut sample (triple infection in the tick) could be observed only after the multipart amplification of denaturated DNA and subsequent pooling of the products for further analysis.

Detection of Borrelia burgdorferi sensu lato and Chlamydophila psittaci in throat and cloacal swabs from birds migrating through Slovakia.

We have screened 91 migratory birds representing 32 species during the autumn of 2003 for the presence of the zoonotic pathogensBorrelia andChlamydophila. Using polymerase chain reaction (PCR),B. burgdorferi sensu stricto was detected in cloacal swabs and, in two causes, also in throat swabs in 8 individuals (8.7 %) representing 7 birds species;B. garinii andB. afzelii were not detected.C. psittaci was detected only in cloacal swabs; 6 birds (6.6 %) from four species were found to be positive. The PCR products were sequenced and the sequences were compared phylogenetically with the gene sequences of 14Chlamydophila strains retrieved from nucleotide databases; although the sequenced DNA was only 110 bp long, all obtained sequences created a new cluster with sublines branching from a position close to the periphery of the genus. All tested samples appear distinct within the known species and were most similar toC. felis orC. abortis.

Life cycle of Rickettsia slovaca in L929 cell line studied by quantitative real‐time PCR and transmission electron microscopy

Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.

Immunochemical analysis of lipopolysaccharide-like component extracted from Borrelia burgdorferi sensu lato.

Immunoelectrophoresis and its modifications were applied to analysis of a lipopolysaccharide-like component (LPS-LC) extracted fromBorrelia garinii strains K24 and K48 isolated fromIxodes ricinus andBorrelia burgdorferi sensu stricto strain B31. A modification of the hot phenol-water method was used for isolation of LPS. Immunoelectrophoresis (IE) and crossed immunoelectrophoresis (CIE) of LPS-LC with polyclonal rabbit antisera revealed a pattern and properties partially similar to LPS from other Gramnegative bacteria.B. garinii LPS-LC formed in CIE a diffuse band extending from the start to the anode. Similarly, the shape and position of the band in IE did not show major differences from LPS of other Gramnegative bacteria. The LPS-LC extracted from the three genomic groups ofB. burgdorferi sensu lato were found to have similar immunochemical properties irrespective of their genotype origin.

Comparison of an oligo-chip based assay with PCR method to measure the prevalence of tick-borne pathogenic bacteria in central Slovakia

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.

Spirochetal non-Borrelia-microorganism isolated from Ixodes ricinus.

Spirochetal microorganisms were isolated from femaleIxodes ricinus in Slovakia. Morphological, immunochemical and molecular biological analysis showed that the microorganism shared several common antigens withBorrelia species while other genetic traits were distinct and not related toBorrelia burgdorferi sensu lato. Lyme disease patient’s serum contained antibodies reacting with antigens of this microorganism. On the one hand the cross-reacting antigens represent a risk of false positive results in laboratory diagnostics, while on the other hand they have a certain potential for vaccine development against Lyme disease.

Diversity of Coxiella-like and Francisella-like endosymbionts, and Rickettsia spp., Coxiella burnetii as pathogens in the tick populations of Slovakia, Central Europe

Ticks are important vectors of pathogens affecting humans and animals worldwide. They do not only carry pathogens but diverse commensal and symbiotic microorganisms are also present in ticks. A molecular screening for tick-borne pathogens and endosymbionts was carried out in Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis inermis questing ticks collected in Slovakia. The presence of Rickettsia spp., Coxiella burnetii, Coxiella-like and Francisella-like microorganisms was evaluated by PCR in 605 individuals and by randomly sequencing 66 samples. Four species of rickettsiae (R. raoultii, R. slovaca, R. helvetica and R. monacensis) were identified and reported with an overall prevalence range between 0.4 and 50.3% (±8.0) depending on tick species, sex and locality. Partial sequencing of the gltA gene of 5 chosen samples in H. inermis showed 99% identity with Candidatus Rickettsia hungarica. The total prevalence of C. burnetii in ticks was 2.2 ± 1.7%; bacteria were confirmed in I. ricinus and D. reticulatus ticks. The sequences from 2 D. reticulatus males and 1 I. ricinus female ticks were compared to GenBank submissions and a 99.8% match was obtained with the pathogenic C. burnetii. Coxiella-like endosymbionts were registered in all three species of ticks from all studied sites with an average prevalence of 32.7 ± 3.7%. A phylogenetic analysis of this Coxiella sp. showed that it does not group with the pathogenic C. burnetii. The prevalence of Francisella-like microorganisms in questing ticks was 47.9 ± 3.9%, however H. inermis (n = 108) were not infested. Obtained sequences were 98% identical with previously identified Francisella-like endosymbionts in D. reticulatus and I. ricinus. Coxiella-like and Francisella-like microorganisms were identified for the first time in Slovakia, they might be considered as a non-pathogenic endosymbiont of I. ricinus, D. reticulatus and H. inermis, and future investigations could aim to assess their role in these ticks. However, this work provided further data and broadened our knowledge on bacterial pathogens and endosymbionts present in ticks in Slovakia to help understanding co-infestations, combined treatments and public health issues linked to tick bites.

Erratum to: Lyme borreliosis—analysis of the trends in Slovakia, 1999–2008

The Editors apologize to the authors and readers of the paper V Svihrova et al. (issue 3, pp 270–275) where, on page 270 the addresses of authors were changed; it should read as shown below.