Lyme Borreliosis with Scalp Eschar Mimicking Rickettsial Infection, Austria.

We report on a patient in Austria with scalp eschar and neck lymphadenopathy. Rickettsial etiology was excluded by culture, PCR, and serologic tests. Borrelia afzelii was identified from the eschar swab by PCR. Lyme borreliosis can mimic rickettsiosis; appropriate tests should be included in the diagnostic workup of patients with eschars.

First broad-range molecular screening of tick-borne pathogens in Ixodes (Pholeoixodes) kaiseri, with special emphasis on piroplasms.

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri.

Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/Ehrlichia (including "Candidatus Neoehrlichia"), Babesia, and Coxiella The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferisensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferisensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis, Borrelia lusitaniae, and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. "Candidatus Neoehrlichia mikurensis," Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and "Candidatus Neoehrlichia mikurensis" showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferisensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.

Molecular evidence of Ehrlichia canis and Rickettsia massiliae in ixodid ticks of carnivores from South Hungary.

To monitor the emergence of thermophilic, Mediterranean ixodid tick species and tick-borne pathogens in southern Hungary, 348 ticks were collected from shepherd dogs, red foxes and golden jackals during the summer of 2011. Golden jackals shared tick species with both the dog and the red fox in the region. Dermacentor nymphs were collected exclusively from dogs, and the sequence identification of these ticks indicated that dogs are preferred hosts of both D. reticulatus and D. marginatus nymphs, unlike previously reported. Subadults of three ixodid species were selected for reverse line blot hybridisation (RLB) analysis to screen their vector potential for 40 pathogens/groups. Results were negative for Anaplasma, Babesia and Theileria spp. Investigation of D. marginatus nymphs revealed the presence of Ehrlichia canis, Rickettsia massiliae and Borrelia afzelii for the first time in this tick species. These findings broaden the range of those tick-borne agents, which are typically transmitted by Rhipicephalus sanguineus, but may also have Dermacentor spp. as potential or alternative vectors. Ehrlichiacanis was also newly detected in Ixodes canisuga larvae from red foxes. In absence of transovarial transmission in ticks this implies that Eurasian red foxes may play a reservoir role in the epidemiology of canine ehrlichiosis.

Bacteria and protozoa with pathogenic potential in Ixodes ricinus ticks in Viennese recreational areas.

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.

Novel Protozoans in Austria Revealed through the Use of Dogs as Sentinels for Ticks and Tick-Borne Pathogens.

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.

Identification and Characterization of "Candidatus Rickettsia Thierseensis", a Novel Spotted Fever Group Rickettsia Species Detected in Austria.

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.

Transmission of Rickettsia raoultii and Rickettsia massiliae DNA by Dermacentor reticulatus and Rhipicephalus sanguineus (s.l.) ticks during artificial feeding.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The domestic pig as a potential model for Borrelia skin infection.

The skin lesion erythema migrans is a characteristic early manifestation of Lyme borreliosis in humans. However, the pathomechanisms leading to development of this erythema are not fully understood. Models that mimic human skin would enhance research in this field. Human and porcine skin structures strongly resemble each other. Therefore, we attempted to induce erythema migrans lesions in experimental Borrelia burgdorferi sensu lato infection in the skin of domestic pigs. The formation of erythema migrans-like lesions was observed after intradermal injection of these spirochetes, with the lesions forming very clearly in 2/6 animals when a strain of B. garinii was used. However, no molecular or clinical proof of systemic infection of the pigs with B. afzelii, B. burgdorferi sensu stricto, or B. garinii could be achieved.

Human granulocytic anaplasmosis acquired in Connecticut, USA, diagnosed in Vienna, Austria, 2015.

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum, an intracellular pathogen transmitted by hard ticks. We report a patient who had acquired the infection in Connecticut, USA, and was diagnosed in Vienna, Austria, using PCR methods. Imported HGA from the United States to Austria is a rare event.

Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Canine and ovine tick-borne pathogens in camels, Nigeria.

In April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively. This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection.

Tick-borne pathogens of zoonotic and veterinary importance in Nigerian cattle.

BACKGROUND: Ticks and tick-borne diseases undermine cattle fitness and productivity in the whole of sub-Saharan Africa, including Nigeria. In this West African country, cattle are challenged by numerous tick species, especially during the wet season. Consequently, several TBDs are known to be endemic in Nigerian cattle, including anaplasmosis, babesiosis, cowdriosis and theilerioris (by Theileria mutans and Theileria velifera). To date, all investigations on cattle TBDs in Nigeria have been based on cytological examinations and/or on serological methods. This study aimed to ascertain the occurrence of tick-borne pathogens of veterinary and zoonotic importance in cattle in Nigeria using molecular approaches. METHODS: In October 2008, 704 whole blood samples were collected from indigenous cattle in the Plateau State, Nigeria. Analysis for tick-borne pathogens was conducted by means of PCR-based reverse line blotting (RLB) and sequencing targeting a panel of five genera of microorganisms (i.e. Babesia, Theileria, Anaplasma, Ehrlichia and Rickettsia spp.). RESULTS: In total, 561/704 (82.6%) animals were found infected, with 465 (69.6%) of them being infected by two or more microorganisms, with up to 77 possible combinations of pathogens detected. Theileria mutans was the most prevalent microorganism (66.3%), followed by Theileria velifera (52.4%), Theileria taurotragi (39.5%), Anaplasma marginale (39.1%), Anaplasma sp. (Omatjenne) (34.7%), Babesia bigemina (7.9%), Anaplasma centrale (6.3%), Anaplasma platys (3.9%), Rickettsia massiliae (3.5%), Babesia bovis (2.0%) and Ehrlichia ruminantium (1.1%). Calves were found significantly less infected than juvenile and adult cattle. CONCLUSIONS: This study provides updated, molecular-based information on cattle TBDs in Nigeria. The molecular approach employed allowed the diagnosis of numerous positive cases including carrier statuses, multiple infections and novel pathogen detections within the indigenous cattle population. Moreover, the RLB method here described enabled the detection of veterinary agents not only pertaining to bovine health, including also those of zoonotic importance. The high prevalence recorded for T. mutans, T. velifera, A. marginale, T. taurotragi and Anaplasma sp. (Omatjenne), suggests they may be endemically established in Nigeria, whereas the lower prevalence recorded for other microorganisms (i.e. A. centrale and B. bovis) highlights a less stable epidemiological scenario, requiring further investigations.

Novel foci of Dermacentor reticulatus ticks infected with Babesia canis and Babesia caballi in the Netherlands and in Belgium.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

No evidence of African swine fever virus replication in hard ticks.

African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe, Ixodes ricinus and Dermacentor reticulatus, have not been assessed for their vector competence for ASFV. In this study, we aimed to determine whether virus replication can occur in any of these two hard tick species (I. ricinus and/or D. reticulatus), in comparison with O. moubata (the confirmed vector), after feeding them blood containing different ASFV isolates using an improved in vitro system. DNA quantities of ASFV in these infected hard ticks were measured systematically, for 6 weeks in I. ricinus, and up to 8 weeks in D. reticulatus, and the results were compared to those obtained from O. moubata. There was evidence of virus replication in the O. moubata ticks. However, there was no evidence of virus replication in I. ricinus or D. reticulatus, even though viral DNA could be detected for up to 8 weeks after feeding in some cases. This study presents the first results on the possible vector competence of European hard (ixodid) ticks for ASFV, in a validated in vitro feeding setup. In conclusion, given the lack of evidence for virus replication under in vitro conditions, D. reticulatus and I. ricinus are unlikely to be relevant biological vectors of ASFV.

Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on dogs and on artificial membranes.

A South African strain of Ehrlichia canis was isolated and used to infect a laboratory-bred Beagle dog. Rhipicephalus sanguineus nymphs, which fed on this dog, moulted to adult ticks which carried infection rates of E. canis between 12% and 19% and were used in a series of in vivo and in vitro experiments. Five groups of 6 dogs were challenged with the infected R. sanguineus ticks, which were removed 24h, 12h, 6h or 3h after the ticks had been released onto the dogs. The animals were monitored for fever and thrombocytopenia and were considered infected if they became serologically positive for E. canis antibodies as well as PCR positive for E. canis DNA. Seven dogs became infected with E. canis in the following groups: Group 1 (24h tick challenge) 1 out of 6; Group 2 (12h) 1 of 6; Group 3 (6h) 2 of 6; Group 4 (6h) 2 of 6 and Group 5 (3h) 1 out of 6. Six of those 7 infected dogs developed fever and a significant thrombocytopenia. One dog did not show any symptoms, but seroconverted and was found PCR positive on several occasions. Five additional dogs were PCR positive on one test sample only but were not considered infected because they did not develop any specific E. canis antibodies. In vitro, R. sanguineus ticks attached and fed on bovine blood through silicone membranes with attachment rates up to 72.5% after 24h increasing to 84.2% at 72 h. The ticks transmitted E. canis as soon as 8h post application as demonstrated by E. canis DNA found in the nutritive blood medium. In conclusion, transmission of E. canis by R. sanguineus ticks starts within a few hours after attachment, which is earlier than previously thought. These findings underpin the need for acaricides to provide either a repellent, an anti-attachment and/or a rapid killing effect against ticks in order to decrease the risk of transmission of E. canis.