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 ±â€¯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 ±â€¯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 ±â€¯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.

Detection of Borrelia burgdorferi sensu stricto and Borrelia garinii DNAs in patient with Hyperkeratosis lenticularis perstans (Flegel disease).

Determination of the causative agent of erythema-like skin lesions in case of nonspecific superficial perivascular dermatitis was supported by histological examination and led to the latter diagnosis of Hyperkeratosis lenticularis perstans (Flegel disease) in patient. The presence of antibodies against Borrelia burgdorferi in patient serum was confirmed by a routine ELISA method and verified by Western blot technique. Skin biopsy and blood specimens were analyzed by PCR and multilocus sequence analysis (MLSA). Western blot method revealed IgG antibody response against two specific antigens, 17 and 83 kDa proteins. The recombinant test detected IgG antibody response against p100 and p41 antigens. The sequence analysis of amplicons from the selected genomic loci obtained from skin biopsy and serum samples revealed the presence of two species from B. burgdorferi sensu lato complex as a co-infection in this patient-B. burgdorferi sensu stricto (s.s.) and Borrelia garinii.

Lyme borreliosis--analysis of the trends in Slovakia, 1999-2008.

Lyme borreliosis (LB) presents as one of the most frequent tick-borne diseases in Europe with more than 85,000 reported cases every year. The transport of this disease on humans is by tick species of the genus Ixodes. In our work, we aimed to perform a retrospective analysis of the incidence and seasonality of Lyme borreliosis during the period 1999-2008 in Slovakia. For our analysis, we used all the relevant data about the patients with Lyme borreliosis reported in the Epidemiological Informative System of Communicable Diseases in Slovakia during the decade of 1999-2008. During the observed period, there were 7,349 reported cases of LB in Slovakia. Whereas the incidence of early localized infection did not change during the observed period, there was a significant increase in the incidence of early disseminated infection and late persistent infection of LB. Seventy per cent of all patients was infected by tick bite. LB was more frequently reported in females than in males (56.1% vs. 43.9%; p < 0.05), and the most involved age group was the productive age (15-64 years). The incidence of disseminated infection and persistent infection was rising with increasing age. Regarding the seasonality of LB, we found the highest incidence during the summer months. Comparing the situation of LB in 1999 and 2008, significant increase in the number of reported cases was in April and June and from September to November (p < 0.05). Our epidemiological analysis confirmed that Lyme borreliosis requires increased attention due to its increasing incidence.

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.

Direct detection of Borrelia burgdorferi spirochetes in patients with early disseminated Lyme borreliosis.

The detection of spirochetes in 15 patients with clinically documented early disseminated LB has been analysed when using cultivation method of the plasma or the cerebrospinal fluid, electron microscopy, commercial Western blot and detecting the DNA of the pathogen in vitro cultures by PCR-RFLP. Spirochetes were isolated in eight blood and one cerebrospinal fluid culture samples. In seven cases (47%), previous serodiagnostic laboratory tests were negative. Borrelial DNA was detected by PCR in 67% patients (9 blood samples and 1 CSF sample). Using Msel restriction fragments of PCR products of the amplified rrf-rrl region, we identified Borrelia garinii (80%), one B. afzelii isolate and one B. burgdorferi s.s.

Detection of Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi s. l. in Ticks, and wild-living animals in western and middle Slovakia.

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.