Eva Špitalská

Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health

Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis,” Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic

Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild to severe diseases in humans are associated with arthropod vectors. Dermacentor marginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rickettsia raoultii distributed across Europe. A total of 794 D. marginatus, D. reticulatus and Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep, goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR amplifying parts of gltA, ompA and sca4 genes. Next analyses of Rickettsia-positive samples by PCR–RFLP and/or sequencing showed D. reticulatus ticks were more infected with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of R. raoultii was 8.1–8.6% and 22.3–27% in D. marginatus and D. reticulatus, respectively. The prevalence of R. slovaca was 20.6–24.3% in D. marginatus and 1.7–3.4% in D. reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was evaluated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multiplication was recorded on the 7th and 8th day postinfection in Vero and L929 cells, respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. Our study describes the detection and isolation of tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and highlight their risk for humans.

Detection of Coxiella burnetii in ticks collected in Slovakia and Hungary.

A total of 235 adult ticks collected from vegetation in Slovakia and Hungary in 1998–2000 were tested for Coxiella burnetii by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). C. burnetii was identified in six ticks of Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis concinna species.

The Importance of Ixodes arboricola in Transmission of Rickettsia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi Sensu Lato in the Czech Republic, Central Europe

Wild birds are known to be a reservoir of infectious disease agents and disseminatory hosts of ticks. The purpose of this work was to obtain information about the occurrence of rickettsial, anaplasmal, and borrelial infections in some ticks that parasitize wild birds in the Czech Republic. A total of 549 subadult ticks of three species Ixodes arboricola (75.0%), Ixodes ricinus (23.1%), and Haemaphysalis concinna (1.8%) were collected from 20 species of birds (Passeriformes). Rickettsiae were detected in 44.0% larvae and 24.5% nymphs of I. arboricola collected from Parus major, Poecile palustris, and Sitta europaea. Rickettsiae-positive I. ricinus larvae (13.7%) were collected from P. major, Cyanistes caeruleus, and S. europaea, and 2.6% of nymphs from Erithacus rubecula and Prunella modularis. Comparison of sequences of a gltA gene fragment with data available in GenBank identified Rickettsia helvetica, a spotted fever rickettsia associated with human infections, and other Rickettsia spp. Anaplasma phagocytophilum was found only in two I. ricinus nymphs collected from E. rubecula and P. major. Infections with Borrelia burgdorferi sensu lato were recorded in 1.3% larvae of I. arboricola acquired from P. palustris and P. major and in 11.8% larvae and 25.0% nymphs of I. ricinus collected from P. major, P. palustris, C. caeruleus, Acrocephalus schoenobaenus, Turdus merula, Carpodacus erythrinus, Sylvia atricapilla, P. modularis, and Phylloscopus collybita. Reverse-line blot hybridization showed infections with Borrelia garinii and Borrelia valaisiana and mixed infections with these two genospecies. This is the first record of a high rate of rickettsial infection in I. arboricola subadult ticks acquired from birds in the Czech Republic and in central Europe. Our study suggests that I. arboricola, P. major, and P. palustris play important roles in circulating rickettsiae.

Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe

BackgroundCandidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it’s ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet.FindingsCandidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum.ConclusionA positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.

Emergence and genetic variability of Anaplasma species in small ruminants and ticks from Central Europe.

Anaplasmoses are common tick-borne zoonotic bacterial diseases of livestock and free-living ungulates from the genus Anaplasma that are recently emerging in Central Europe. The main aim of this study was to analyze the prevalence and genetic variability of Anaplasma phagocytophilum and Anaplasma ovis in small ruminants and questing ticks from six different sites in Slovakia and the Czech Republic using the PCR of the msp4 gene followed by the sequence analysis. At two farms from southeastern Slovakia, 66.1% small ruminants were infected with A. ovis in contrast to one positive animal from both sites in northern Slovakia. It was represented by two different genotypes. A. phagocytophilum was present in all tested flocks with the infection prevalence ranging from 0.9% to 5.7%. None of the tested questing ticks carried A. ovis. A. phagocytophilum was detected in 1.1% and 7.8% of questing Ixodes ricinus ticks collected around the farms located in southeastern and northern Slovakia, respectively. A. phagocytophilum revealed higher intraspecific diversity than A. ovis.

Sympatric occurrence of Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks and Rickettsia and Babesia species in Slovakia

Vojka nad Dunajom in the south-west of the Slovak Republic is a locality with sympatric occurrence of 3 species of ticks. This study investigated the spatial distribution of Dermacentor reticulatus, Ixodes ricinus, and Haemaphysalis concinna ticks in this area and determined the prevalence of Babesia and Rickettsia species in questing adults of these tick species considered as potential risk for humans and animals. Ticks were collected by blanket dragging over the vegetation from September 2011 to October 2012. All ticks were subjected to DNA extraction and individually assayed with PCR-based methods targeting the gltA, sca4, 23S rRNA genes of Rickettsia spp. and the 18S rRNA gene of Babesia spp. D. reticulatus was the dominant species occurring in this area (67.7%, n=600), followed by I. ricinus (31.8%, n=282) and H. concinna (0.5%, n=4) ticks. Rickettsial infection was determined in 10.8% (n=65) and 11.7% (n=33) of D. reticulatus and I. ricinus ticks, respectively. Babesia spp. infection was confirmed in 1.8% (n=11) of D. reticulatus and 0.4% (n=1) of I. ricinus ticks. DNA of 6 different pathogenic tick-borne species, Rickettsia helvetica, Rickettsia monacensis, Rickettsia slovaca, Rickettsia raoultii, Babesia canis, and Babesia venatorum were identified in this locality with sympatric occurrence of I. ricinus, D. reticulatus, and H. concinna ticks.

Rickettsial infection in Ixodes ricinus ticks in urban and natural habitats of Slovakia.

A total of 1810 Ixodes ricinus ticks was collected from the vegetation from 2 different habitat types: urban and natural. Urban habitats were represented by cemeteries and public parks in the following towns: Bratislava, Malacky, and Martin at 150 m and 400 m above sea level. Natural habitats were selected in the mountain forest of the Martinské hole Mts. in Central Slovakia at 3 different altitudinal levels, i.e. 600 m, 800 m and 1000 ma.s.l. All ticks were tested for the presence of spotted fever group rickettsiae. The DNA of Rickettsia spp. was identified in 9% of all tested ticks. Rickettsia-infected ticks were present in both, urban and sylvatic sites at all studied altitudes. Four different species of Rickettsia were present in positive I. ricinus ticks. Rickettsia helvetica was identified in 77 out of 87 Rickettsia-positive I. ricinus ticks, followed by 8 samples that belonged to Rickettsia monacensis and 2 of the positive ticks were infected with different unidentified Rickettsia spp. Due to the association of R. helvetica and R. monacensis with human infections, it is essential to understand which species of Rickettsia circulate in the natural foci of Slovakia. Circulation of pathogenic rickettsiae in urban as well as natural habitats at different altitudinal levels in Slovakia emphasizes that infection risk is very common throughout this Central European country.

Rickettsial Agents in Slovakian Ticks (Acarina, Ixodidae) and Their Ability to Grow in Vero and L929 Cell Lines

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty‐six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA‐based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR‐positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram‐negative cell wall, and a cytoplasmic membrane.

Identification of Rickettsia africae and Wolbachia sp. in Ceratophyllus garei fleas from Passerine birds migrated from Africa.

The aim of the study was to reveal new aspects of the role of flea vector taken from migratory birds by screening of specimens with molecular biological methods. A field study was done in fishponds in Slovakia. Actually, 47 fleas were collected from reed warblers (Acrocephalus scirpaceus) and their nests. DNA was extracted and analyzed for representatives of the orders Rickettsiales. A rickettsia that shares 99.7% of identity by gltA gene with Rickettsia africae was identified in Ceratophyllus garei collected from A. scirpaceus. Moreover, two Wolbachia sp. were also detected in fleas. This is the first record of R. africae and Wolbachia sp. identified so far in Central Europe in fleas collected from migratory bird returning from Africa. This molecular study extends the geographic range and vector spectrum of arthropod-borne agents.