Autochthonous populations of Dermacentor reticulatus ticks in the Netherlands were discovered after fatal cases of babesiosis occurred in resident dogs in 2004. The presence of D. reticulatus in the Netherlands has also linked with the emergence of piroplasmosis in the resident horse population. The aim of this study was to put together results of continued surveillance of field sites and hosts for this tick in the Netherlands and also in Belgium and determine their infection status for Babesia and Theileria species. Ticks were collected from the vegetation at 11 locations between 2011 and 2013. D. reticulatus ticks were also collected from different hosts between 2007 and 2013. Ticks were screened by PCR and reverse line blot (RLB). A total of 1368 D. reticulatus ticks were collected from 4 previously known field locations and from 5 new locations in the Netherlands and from 2 sites in Belgium (one old and one new location). A total of 855 ticks collected from 8 locations in the Netherlands and 2 locations in Belgium were tested. Fourteen ticks (1,64%) collected at 4 field locations (Dintelse Gorzen, Rozenburg, Slikken van de Heen and St. Philipsland) were positive for Babesia canis, whereas two ticks were positive for Babesia caballi, one tick in the Dintelse Gorzen in the Netherlands and one tick was found positive in De Panne in Belgium. A further 1092 D. reticulatus ticks were collected between 2007 and 2013 from 40 dogs (132 ticks), two ticks from two humans, 51 ticks from 15 horses, two ticks from two cats, one tick from a roe deer, whereas most ticks (904) were collected from cattle (n = 25). Ticks were found throughout the year on dogs in nearly all provinces of the Netherlands. None of the ticks collected from these hosts were infected. D. reticulatus is continuing its spread into novel areas. The finding that some autochthonous ticks are infected with B. canis and B. caballi poses a threat to the resident dog and horse population and justifies year-round tick control measures.

Tick-borne rickettsial pathogens are emerging worldwide and pose an increased health risk to both humans and animals. A plethora of rickettsial species has been identified in ticks recovered from human and animal patients. However, the detection of rickettsial DNA in ticks does not necessarily mean that these ticks can act as vectors for these pathogens. Here, we used artificial feeding of ticks to confirm transmission of Rickettsia massiliae and Rickettsia raoultii by Rhipicephalus sanguineus (sensu lato) and Dermacentor reticulatus ticks, respectively. The speed of transmission was also determined. An artificial feeding system based on silicone membranes were used to feed adult R. sanguineus (s.l.) and D. reticulatus ticks. Blood samples from in vitro feeding units were analysed for the presence of rickettsial DNA using PCR and reverse line blot hybridisation. The attachment rate of R. sanguineus (s.l.) ticks were 40.4% at 8 h post-application, increasing to 70.2% at 72 h. Rickettsia massiliae was detected in blood samples collected 8 h after the R. sanguineus (s.l.) ticks were placed into the in vitro feeding units. D. reticulatus ticks were pre-fed on sheep and subsequently transferred to the in vitro feeding system. The attachment rate was 29.1 % at 24 h post-application, increasing to 43.6 % at 96 h. Rickettsia raoultii was detected in blood collected 24 h after D. reticulatus was placed into the feeding units. Rhipicephalus sanguineus (s.l.) and D. reticulatus ticks are vectors of R. massiliae and R. raoultii, respectively. The transmission of R. massiliae as early as 8 h after tick attachment emphasises the importance of removing ticks as soon as possible to minimise transmission. This study highlights the relevance of in vitro feeding systems to provide insight into the vectorial capacity of ticks and the dynamics of tick-borne pathogen transmission.

Continuous culture of tick cell lines has proven a valuable asset in isolating and propagating several different vector-borne pathogens, making it possible to study these microorganisms under laboratory conditions and develop serological tests to benefit public health. We describe a method for effective, cost- and labor-efficient isolation and propagation of Rickettsia raoultii using generally available laboratory equipment and Rhipicephalus microplus cells, further demonstrating the usefulness of continuous tick cell lines. R. raoultii is one of the causative agents of tick-borne lymphadenopathy (TIBOLA) and is, together with its vector Dermacentor reticulatus, emerging in novel regions of Europe, giving rise to an increased threat to general public health. Dermacentor reticulatus ticks were collected in the Donau-Auen (Lobau) national park in Vienna, Austria. The hemolymph of ten collected ticks was screened by PCR-reverse line blot for the presence of rickettsial DNA. A single tick tested positive for R. raoultii DNA and was used to infect Rhipicephalus microplus BME/CTVM2 cells. Sixty-five days after infection of the tick-cell line with an extract from a R. raoultii-infected tick, we observed intracellular bacteria in the cultured cells. On the basis of microscopy we suspected that the intracellular bacteria were a species of Rickettsia; this was confirmed by several PCRs targeting different genes. Subsequent sequencing showed 99–100 % identity with R. raoultii. Cryopreservation and resuscitation of R. raoultii was successful. After 28 days identical intracellular bacteria were microscopically observed. R. raoultii was successfully isolated and propagated from D. reticulatus ticks using R. microplus BME/CTVM2 cells. The isolated strain shows significant molecular variation compared to currently known sequences. Furthermore we show for the first time the successful cryopreservation and resuscitation of R. raoultii.

Rickettsia spp. are the second most common pathogens detected in Ixodes ricinus ticks in Austria after Borrelia burgdorferi sensu lato. Species belonging to the spotted fever group (SFG) are the causative agents for tick-borne rickettsiosis across the world. So far, only four SFG Rickettsia spp. were detected in Austria, namely R. helvetica, R. raoultii, R. monacensis and R. slovaca. Here, we describe the identification of a new SFG Rickettsia species detected in an I. ricinus tick. Sequencing of various rickettsial genes revealed a nucleotide sequence similarity of 99.6%, 98.5%, 97.3% and 98.5% to the gltA, ompA, ompB, and sca4 genes, respectively, of known and validated species. Additionally, sequencing of the htrA gene and 23S-5S intergenic spacer region also only showed 99.6% and 99.2%, respectively, similarity to known species. Therefore, and in accordance with current criteria for Rickettsia species discrimination, we hereby describe a new species of the SFG with putative pathogenic potential. We propose the name “Candidatus Rickettsia thierseensis” based on the village Thiersee in the Austrian province of Tyrol, where the carrying tick was found.

Summary: Ixodes ricinus is the most relevant vector for tick-borne diseases in Austria and responsible for the transmission of Borrelia burgdorferi sensu lato (s. l.), which causes Lyme borreliosis in humans; however, also other bacteria and protozoa can be found in ticks and have the potential of infecting people and animals. In this study we collected ticks in popular recreational areas in the city of Vienna in the years 2019 and 2020 and analyzed them for the presence of such putative pathogenic microorganisms. By using reverse line blot (RLB) hybridization we detected DNA of B. burgdorferi s. l., Rickettsia spp., Babesia spp., Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum. Moreover, we also screened them for the relapsing fever spirochete Borrelia miyamotoi employing real-time PCR. The most frequently detected pathogens were B. burgdorferi s. l. in 28.6% of the ticks in 2019 and 21.3% of the ticks in 2020. The genus Rickettsia was detected in 13.8% of the ticks from 2019 and only in 4.6% from 2020. Babesia spp. were detected in 5.7% in 2019 and 4.2% in 2020. Furthermore, we detected CNM in 4.0% (2019) and 5.6% (2020), A. phagocytophilum in 0.5% (2019) and 1.3% (2020) and finally B. miyamotoi in 3.3% (2019) and 1.7% (2020). Collectively, we show that various microorganisms are prevalent in ticks collected in Vienna and identify hotspots for B. miyamotoi, which we have detected for the first time in the city.

We previously isolated and cultivated the novel Rickettsia raoultii strain Jongejan. This prompted us to ask whether this strain is unique or more widely present in Austria. To assess this issue, we retrospectively screened ticks collected from dogs in 2008. Of these collected ticks, we randomly selected 75 (47 females and 28 males) Dermacentor reticulatus, 44 (21 females, 7 males, and 16 nymphs) Haemaphysalis concinna, and 55 (52 females and 3 males) ticks of the Ixodes ricinus complex. Subsequently, these ticks were individually screened for the presence of tick-borne pathogens using the reverse line blot hybridization assay. In our current study, we detected DNA from the following microbes in D. reticulatus: Anaplasma phagocytophilum, Borrelia lusitaniae, Borrelia spielmanii, Borrelia valaisiana, and R. raoultii, all of which were R. raoultii strain Jongejan. In H. concinna, we found DNA of a Babesia sp., Rickettsia helvetica, and an organism closely related to Theileria capreoli. Lastly, I. ricinus was positive for Anaplasma phagocytophilum, Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii/Borrelia bavariensis, B. lusitaniae, B. spielmanii, B. valaisiana, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, and Theileria (Babesia) microti DNA. The detection of DNA of the Babesia sp. and an organism closely related to Theileria capreoli, both found in H. concinna ticks, is novel for Austria.