Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes.

OBJECTIVES: Tick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which - European, Siberian and Far Eastern - occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve. METHODS: An ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens. RESULTS: Testing of 23 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections. CONCLUSIONS: In geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections.

Nuttalliellidae in Burmese amber: implications for tick evolution.

Ticks are composed of 3 extant families (Argasidae, Ixodidae and Nuttalliellidae) and 2 extinct families (Deinocrotonidae and Khimairidae). The Nuttalliellidae possess one extant species (Nuttalliella namaqua) limited to the Afrotropic region. A basal relationship to the hard and soft tick families and its limited distribution suggested an origin for ticks in the Afrotropics. The Deinocrotonidae has been found in Burmese amber from Myanmar and Iberian amber from Spain, suggesting a wider distribution of the lineage composed of Deinocrotonidae and Nuttalliellidae. The current study describes 8 fossils from mid-Cretaceous (ca. 100 Ma) Burmese amber: 2 Deinocroton species (Deinocroton bicornis sp. nov.; Deinocroton lacrimus sp. nov.), 5 Nuttalliella species (Nuttalliella gratae sp. nov., Nuttalliella tuberculata sp. nov., Nuttalliella placaventrala sp. nov., Nuttalliella odyssea sp. nov., Nuttalliella tropicasylvae sp. nov.) and a new genus and species (Legionaris nov. gen., Legionaris robustus sp. nov.). The argument is advanced that Deinocroton do not warrant its own family, but forms part of the Nuttalliellidae comprising 3 genera, Deinocroton, Legionaris nov. gen. and Nuttalliella). Affinities of Burmese tick fossils to the Australasian region, specifically related to rifting of the Burma terrane from northern Australia ~150 million years ago, suggest that Nuttalliella had a much wider distribution than its current limited distribution. The distribution of Nuttalliella likely stretched from Africa over Antarctica and much of Australia, suggesting that extant members of this family may still be found in Australia. Considerations for the geographic origins of ticks conclude that an Afrotropic origin can as yet not be discarded.

Ticks and tick-borne diseases from Mallorca Island, Spain.

Ixodid ticks are obligate blood-feeding arthropods and important vectors of pathogens. In Mallorca, almost no data on the tick fauna are available. Herein, we investigated ticks and tick-borne pathogens in ticks collected from dogs, a cat and humans in Mallorca as result of a citizen science project. A total of 91 ticks were received from German tourists and residents in Mallorca. Ticks were collected from March to October 2023 from dogs, cat and humans, morphologically and genetically identified and tested for pathogens by PCRs. Six tick species could be identified: Ixodes ricinus (n = 2), Ixodes ventalloi (n = 1), Hyalomma lusitanicum (n = 7), Hyalomma marginatum (n = 1), Rhipicephalus sanguineus s.l. (n = 71) and Rhipicephalus pusillus (n = 9). Rhipicephalus sanguineus s.l. adults were collected from dogs and four females from a cat and the 16S rDNA sequences identified it as Rh. sanguineus s.s. Hyalomma lusitanicum was collected from 1 human, 1 dog and 5 specimens were collected from the ground in the community of Santanyi, together with one H. marginatum male. This is the first report of Hyalomma marginatum in Mallorca. Both I. ricinus were collected from humans and I. ventalloi female was collected from a dog. All ticks tested negative for Anaplasma phagocytophilum, Coxiella spp., Francisella spp., and piroplasms. In 32/71 (45%) specimens of Rh. sanguineus s.s., Rickettsia spp. could be detected and in 18/32 (56.2%) sequenced tick DNAs R. massiliae was identified. Ixodes ventalloi female and both I. ricinus tested positive in the screening PCR, but the sequencing for the identification of the Rickettsia sp. failed.

Mitochondrial genome and nuclear ribosomal RNA analysis place Alveonasus lahorensis within the Argasinae and suggest that the genus Alveonasus is paraphyletic.

Two major families exist in ticks, the Argasidae and Ixodidae. The Argasidae comprise 2 sub-families, Argasinae and Ornithodorinae. The placement into subfamilies illuminate differences in morphological and molecular systematics and is important since it provides insight into evolutionary divergence within this family. It also identifies fundamental gaps in our understanding of argasid evolution that provide directions for future research. Molecular systematics based on mitochondrial genomics and 18S/28S ribosomal RNA confirmed the placement of various genera and subgenera into the Argasinae: Argas (including Argas and Persicargas), Navis, Ogadenus, Otobius lagophilus, Proknekalia, Secretargas and the Ornithodorinae: Alectorobius, Antricola (including Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (including Microargas, Ornamentum, Ornithodoros sensu strictu, Pavlovskyella), Otobius sensu strictu, Reticulinasus and Subparmatus. The position of Alveonasus remains controversial since traditional taxonomy placed it in the Ornithodorinae, while cladistic and limited molecular analysis placed it in the Argasinae. The current study aimed to resolve the systematic position of Alveonasus using mitochondrial genomic and 18S/28S ribosomal RNA systematics by sequencing the type species Alveonasus lahorensis from Pakistan. In addition, the mitochondrial genomes for Argas reflexus and Alectorobius kelleyi are reported from Germany and the USA, respectively. The systematic data unambiguously place Alveonasus in the Argasinae and also suggest that Alveonasus may be another paraphyletic genus.

First detection and a new avian host of the tick Ixodes ventalloi Gil Collado, 1936, in Slovakia.

This study describes the first detection of Ixodes ventalloi in Slovakia. Two engorged females of I. ventalloi were collected from Dunnocks (Prunella modularis) captured in eastern Slovakia. The identification of females was based on morphological and molecular 16S rRNA gene features. Phylogenetic analysis revealed a classification of the females into distinct genogroups. Moreover, comparative morphological analysis highlighted variations between the two females, particularly in the curvature of the auriculae, the shape of coxa I, and the internal spur. These findings suggest the potential for varied phenotypes of I. ventalloi correlated with their genogroups. Nonetheless, I. ventalloi population establishment within Slovakia necessitates further investigation through flagging or drag sampling.

Exotic ticks removed from German travelers.

We report the finding of five nymphs and three adult ticks attached to German tourists while traveling the American continents. All eight specimens were morphologically identified and confirmed genetically using the 16S rRNA gene and screened for Rickettsia spp. infections. Five tick species were identified: one Amblyomma mixtum nymph from Ecuador, one Amblyomma varium nymph from Colombia, three Amblyomma coelebs nymphs from Costa Rica, one Amblyomma americanum male from the USA, one Dermacentor andersoni female and one D. andersoni male from Canada. Tick-borne microorganisms screening using the pan-Rickettsia-PCR resulted in two positive and six negative ticks. The A. mixtum nymph was positive for Rickettsia amblyommatis, while the D. andersoni female was positive for Rickettsia peacockii.

Hard ticks in Burmese amber with Australasian affinities.

Three examples of metastriate hard ticks (Ixodida: Ixodidae) with apparent affinities to modern Australasian genera are described from the mid-Cretaceous (ca. 100 Ma) Burmese amber of Myanmar. Two nymphs of Bothriocroton muelleri sp. nov. represent the oldest (and only) fossil record of this genus, living members of which are restricted to Australia and predominantly feed on monitor lizards, snakes and echidnas. A female of Archaeocroton kaufmani sp. nov. shares its basis capitulum shape with the tuatara tick Archaeocroton sphenodonti (Dumbleton, 1943), the only extant member of this genus and an endemic species for New Zealand. The presence of 2 Australasian genera in Burmese amber is consistent with a previous record of an Ixodes Latreille, 1795 tick from this deposit which resembles Australian members of this genus. They further support an emerging hypothesis that fauna of the amber forest, which may have been on an island at the time of deposition, was at least partly Gondwanan in origin. A revised evolutionary tree for Ixodida is presented compiling data from several new Burmese amber ticks described in the last few years.

Molecular characterization and phylogenetic analysis of Argas persicus (Oken, 1818) (Acari: Argasidae) from domestic birds in eastern Algeria.

Argas persicus (the fowl tick) is a species of soft tick commonly associated with poultry farms. It has a wide geographic distribution and colonizes different climate regions. Morphological identification of A. persicus has been reported worldwide, but genetic data regarding its molecular characterization is limited. The present study provides data for morphological identification and genetic characterization of A. persicus collected from domestic birds in traditional farms from east Algeria (Setif region). Additionally, A. persicus samples originating from Gansu province in China were included for comparative molecular study. In total, 1518 ticks collected from 30 infested farms were examined and morphologically identified as A. persicus. Furthermore, the 14 tick samples obtained from China were morphologically identified as A. persicus. Molecular analysis of 30 ticks from Algeria (one tick from each infested farm) and the 14 Chinese samples based on PCR, sequencing, and phylogenetic analysis of three mitochondrial genetic markers (16S rRNA, 12S rRNA, and cox1) confirmed morphological results where all samples belonged to the A. persicus group. However, phylogenetic analysis showed that all Algerian samples and two Chinese samples belong to A. persicus sensu stricto (s.s.), while the remaining Chinese samples represented A. persicus sensu lato (s.l.) (divergent lineage). The present study confirms the occurrence of A. persicus s.s. both in Algeria and China, as well as provides novel molecular data for a distinct Chinese lineage of A. persicus.

Revealing the Tick Microbiome: Insights into Midgut and Salivary Gland Microbiota of Female Ixodes ricinus Ticks.

The ectoparasite Ixodes ricinus is an important vector for many tick-borne diseases (TBD) in the northern hemisphere, such as Lyme borreliosis, rickettsiosis, human granulocytic anaplasmosis, or tick-borne encephalitis virus. As climate change will lead to rising temperatures in the next years, we expect an increase in tick activity, tick population, and thus in the spread of TBD. Consequently, it has never been more critical to understand relationships within the microbial communities in ticks that might contribute to the tick's fitness and the occurrence of TBD. Therefore, we analyzed the microbiota in different tick tissues such as midgut, salivary glands, and residual tick material, as well as the microbiota in complete Ixodes ricinus ticks using 16S rRNA gene amplicon sequencing. By using a newly developed DNA extraction protocol for tick tissue samples and a self-designed mock community, we were able to detect endosymbionts and pathogens that have been described in the literature previously. Further, this study displayed the usefulness of including a mock community during bioinformatic analysis to identify essential bacteria within the tick.

Atlas of ticks (Acari: Argasidae, Ixodidae) in Germany: 1st data update.

The first data update of the atlas of ticks in Germany published in 2021 is presented here. This atlas provides maps based on georeferenced tick locations of 21 species endemic in Germany as well as three tick species that are regularly imported to Germany. The data update includes the following numbers of newly georeferenced tick locations: 17 Argas reflexus, 79 Carios vespertilionis, 2 Dermacentor marginatus, 43 Dermacentor reticulatus, 4 Haemaphysalis concinna, 3 Haemaphysalis punctata, 3 Hyalomma rufipes, 3 Ixodes apronophorus, 9 Ixodes arboricola, 1 Ixodes ariadnae, 30 Ixodes canisuga, 3 Ixodes frontalis, 80 Ixodes hexagonus, 3 Ixodes lividus, 497 Ixodes ricinus/inopinatus, 1 Ixodes rugicollis, 17 Ixodes trianguliceps, 14 Ixodes vespertilionis, and 45 Rhipicephalus sanguineus sensu lato. Old and new tick findings were mapped, such as the northernmost occurrence of D. marginatus in Germany observed in 2021, but also the historical records from the first descriptions of I. apronophorus and I. arboricola, which were georeferenced here for the first time. The digital dataset of tick locations available for Germany is supplemented by 854 new tick locations. These records increase the number of tick species mapped in the federal states Bavaria, Brandenburg and Mecklenburg Western Pomerania by five each, those in Berlin and Schleswig-Holstein by four each, those in Hamburg by three, those in Baden-Wuerttemberg, Bremen, Lower Saxony, Northrhine-Westphalia, Rhineland Palatinate and Thuringia by two each, and those in Hesse, Saxony and Saxony-Anhalt by one each. Thus, the first data update of the tick atlas in Germany and the underlying digital dataset significantly improve our knowledge of the distribution of these tick species and helps to investigate the effects of climate change and habitat changes on them.

Molecular screening of Amblyomma species (Acari: Ixodidae) imported from African countries to Egypt, with the first report of Amblyomma latum from the ball python, Python regius (Squamata: Pythonidae).

Amblyomma species are non-endemic ticks in Egypt, which have been recorded from imported animals. This study was carried out in 2022 to monitor Amblyomma spp. from dromedary camels, cattle, and snakes in Egypt. During this study, 400 camels, 200 cattle, and two snakes (Pythonidae) were inspected for tick infestation. Collected specimens were identified based on morphological characters and confirmed by phylogenetic analysis of the 12S rRNA gene. Camels were infested by adult specimens of Amblyomma variegatum and Amblyomma lepidum, but no Amblyomma spp. were collected from cattle. Amblyomma variegatum showed high genetic similarity to other A. variegatum from Guinea-Bissau and São Tomé (> 99.99%), and A. lepidum showed high genetic similarity to other A. lepidum from Israel and Sudan (99.99%). Amblyomma latum is recorded in Egypt from the ball python snake for the first time and showed high genetic similarity with South African A. latum (99.87%).

Distribution of the soft tick Carios vespertilionis in lowlands and low mountain regions of Germany.

In Germany, the knowledge about ticks infesting bats is limited, and is restricted only to a few studies, most of them dating back decades. To further improve our knowledge on ticks parasitising bats, healthy and sick bats in central Germany were examined for ticks. In total 519 larvae and one nymph of Carios vespertilionis were collected from nine bat species: Eptesicus nilssonii, Eptesicus serotinus, Myotis daubentonii, Myotis myotis, Nyctalus leisleri, Pipistrellus nathusii, Pipistrellus pygmaeus, Pipistrellus pipistrellus, and Vespertilio murinus. Either the presence of C. vespertilionis was new for some areas or it was confirmed in some federal states in central Germany. The infestation rate was mostly low (n = 1-5 larvae/bat). However, in two cases a high number of ticks was observed. The highest infestation of 97 C. vespertilionis larvae was recorded on one Parti-coloured bat (V. murinus).

Tick findings from subterranean environments in the Central German Uplands and Luxembourg reveal a predominance of male Ixodes hexagonus.

Questing ticks are usually collected by flagging or dragging. Mostly exophilic tick species are caught, such as Ixodes ricinus, the most common tick in Central Europe. In the present study, ticks collected from underground environments in the Grand Duchy of Luxembourg and in the Central German Uplands (Federal States of Hesse, Bavaria, Thuringia, Baden-Wuerttemberg, Rhineland-Palatinate, Saarland and Northrhine-Westphalia) were investigated. Six tick species were revealed among the 396 analyzed specimens: Ixodes ariadnae, Ixodes canisuga, Ixodes hexagonus, I. ricinus, Ixodes trianguliceps, and Dermacentor marginatus. Adults and immatures of I. hexagonus dominated the findings (57% of all specimens), especially in shelters acting as potential resting places of main hosts. Ixodes canisuga and I. trianguliceps were for the first time recorded in Luxembourg, and one nymph of the bat tick I. ariadnae represents only the second report for Germany. Collecting ticks in subterranean environments turned out to be a useful approach to increase knowledge about the occurrence of relatively rare tick species, including those that spend most of their lifetime on their hosts, but detach in such environmental settings.

An exotic souvenir on a German traveler returning from South Africa.

The study reports the finding of a nymph attached to a German tourist during a holiday in South Africa. The nymph specimen was morphologically identified and confirmed by sequence analysis of the 16S rRNA and 12S rRNA genes. The nymph was identified as Rhipicephalus maculatus, a rare tick reported in humans. Screening using the pan-Rickettsia-PCR for infection gave a negative result.

Haemaphysalis hoodi (Acari: Ixodidae) on a human from Yaoundé, Cameroon, and its molecular characterization.

The genus Haemaphysalis Koch, 1844 (Acari: Ixodidae) is the second-largest genus, with more than 170 described species that primarily parasitize mammals and birds (Guglielmone et al. 2014, Guglielmone et al. 2020). Haemaphysalis species are three-host ticks, mainly distributed in southern and southeastern Asia and tropical Africa (Guglielmone et al. 2014). The present study identified a tick, Haemaphysalis hoodi Warburton & Nuttall, 1909, collected from a human in Yaoundé, Cameroon. This tick species feed on birds in sub-Saharan Africa. To the best of our knowledge, this is the second record of H. hoodi from humans. In addition, 16S ribosomal RNA and cytochrome oxidase I sequences were generated for this species for the first time. Screening pan-Rickettsia-PCR infection gave a negative result.

Morphological abnormalities in Hyalomma dromedarii and Hyalomma rufipes (Acari: Ixodidae) collected from dromedary camels (Camelus dromedarius) in Aswan, Egypt.

The present study reports anomalies in Hyalomma dromedarii and Hyalomma rufipes adults collected from dromedary camels (Camelus dromedarius) in Aswan, Egypt, between January and June 2022. A total of 52 adult ticks displayed one or several local and/or general anomalies. A wide variety of local anomalies was observed including atrophy of one or multiple legs, ectromely, absence of accessory adanal and subanal plates, fusion of adanal and accessory plates, and presence of sclerotized formation in the ventral plate, as well as abnormalities of the spiracle, anal groove, festoon, parma, and posteromedian groove. General anomalies comprised of asymmetries and gynandromorphism. Local anomalies were documented among H. dromedarii and H. rufipes, whereas general anomalies were documented only from H. dromedarii. The present work represents the first report of deuterogynander intrigue gynandromorphism in H. dromedarii, as well as the first report of morphological abnormalities in H. dromedarii and H. rufipes from Egypt.

Adaptive radiation and speciation in Rhipicephalus ticks: A medley of novel hosts, nested predator-prey food webs, off-host periods and dispersal along temperature variation gradients.

Rhipicephalus are a species-diverse genus of ticks, mainly distributed in the Afrotropics with some species in the Palearctic and Oriental regions. Current taxonomic consensus comprise nine informal species groups/lineages based on immature morphology. This work integrates biogeographic, ecological and molecular lines of evidence to better understand Rhipicephalus evolution. Phylogenetic analysis based on four genes (12S, 16S, 28S-D2 and COI) recovered five distinct clades with nine descendant clades that are generally congruent with current taxonomy, with some exceptions. Historical biogeography is inferred from molecular divergence times, ancestral distribution areas, host-use and climate niches of four phylogenetically significant bioclimatic variables (isothermality, annual, seasonal and diurnal temperature range). Novel hosts enabled host-linked dispersal events into new environments, and ticks exploited new hosts through nested predator-prey connections in food webs. Diversification was further induced by climate niche partitioning along gradients in temperature range during off-host periods. Ancestral climate niche estimates corroborated dispersal events by indicating hypothetical ancestors moved into environments with different annual and seasonal temperature ranges along latitudinal gradients. Host size for immature and adult life stages was important for dispersal and subsequent diversification rates. Clades that utilise large, mobile hosts (ungulates and carnivores) early in development have wider geographic ranges but slower diversification rates, and those utilising small, less mobile hosts (rodents, lagomorphs and afroinsectivores) early in development have smaller ranges but higher diversification rates. These findings suggest diversification is driven by a complex set of factors linked to both host-associations (host size, ranges and mobility) and climate niche partitioning along annual and seasonal temperature range gradients that vary with latitude. Moreover, competitive interactions can reinforce these processes and drive speciation. Off-host periods facilitate adaptive radiation by enabling host switches along nested predator-prey connections in food webs, but at the cost of environmental exposure that partitions niches among dispersing progenitors, disrupting geneflow and driving diversification. As such, the evolution and ecological niches of Rhipicephalus are characterised by trade-offs between on- and off-host periods, and these trade-offs interact with nested predator-prey connections in food webs, host-use at different life stages, as well as gradients in annual and seasonal temperature ranges to drive adaptive radiation and speciation.

Morphological anomalies in Rhipicephalus sanguineus s.s. (Acari: Ixodidae) collected from dogs in steppe and high plateaus regions, Algeria.

The present report describes morphological anomalies in Rhipicephalus sanguineus s.s. collected from dogs between February 2017 and May 2019 in the steppic region of Djelfa and high plateau area of Bordj-Bou-Arreridj in Algeria. Out of 1043 ticks from 147 infested dogs, four adult ticks (0.38%), collected from four dogs, exhibited local morphological abnormalities. One male displayed ectromely of leg I left, and the other three specimens exhibited multiple associated anomalies in idiosomas and exoskeletons, festoons, stigmas (spiracles), as well as in adanal and accessory plates. This report provides additional exemplars of morphological abnormalities in tick populations of Africa, particularly North Africa.

Atlas of ticks (Acari: Argasidae, Ixodidae) in Germany.

An updated and increased compilation of georeferenced tick locations in Germany is presented here. This data collection extends the dataset published some years ago by another 1448 new tick locations, 900 locations of which were digitized from literature and 548 locations are published here for the first time. This means that a total of 3492 georeferenced tick locations is now available for Germany. The tick fauna of Germany includes two species of Argasidae in the genera Argas and Carios and 19 species of Ixodidae in the genera Dermacentor, Haemaphysalis, and Ixodes, altogether 21 tick species. In addition, three species of Ixodidae in the genera Hyalomma (each spring imported by migratory birds) and Rhipicephalus (occasionally imported by dogs returning from abroad with their owners) are included in the tick atlas. Of these, the georeferenced locations of 23 tick species are depicted in maps. The occurrence of the one remaining tick species, the recently described Ixodes inopinatus, is given at the level of the federal states. The most common and widespread tick species is Ixodes ricinus, with records in all 16 federal states. With the exception of Hamburg, Dermacentor reticulatus was also found in all federal states. The occurrence of the ixodid ticks Ixodes canisuga, Ixodes frontalis, Ixodes hexagonus and I. inopinatus were documented in at least 11 federal states each. The two mentioned argasid tick species were also documented in numerous federal states, the pigeon tick Argas reflexus in 11 and the bat tick Carios vespertilionis in seven federal states. The atlas of ticks in Germany and the underlying digital dataset in the supplement can be used to improve global tick maps or to study the effects of climate change and habitat alteration on the distribution of tick species.

Tick species from cattle in the Adama Region of Ethiopia and pathogens detected.

Ticks will diminish productivity among farm animals and transmit zoonotic diseases. We conducted a study to identify tick species infesting slaughter bulls from Adama City and to screen them for tick-borne pathogens. In 2016, 291 ticks were collected from 37 bulls in Adama, which were ready for slaughter. Ticks were identified morphologically. Total genomic DNA was extracted from ticks and used to test for Rickettsia spp. with real-time PCR. Species identification was done by phylogenetic analysis using sequencing that targeted the 23S-5S intergenic spacer region and ompA genes. Four tick species from two genera, Amblyomma and Rhipicephalus, were identified. Amblyomma cohaerens was the dominant species (n = 241, 82.8%), followed by Amblyomma variegatum (n = 22, 7.5%), Rhipicephalus pulchellus (n = 19, 6.5%), and Rhipicephalus decoloratus (n = 9, 3.0%). Among all ticks, 32 (11%) were positive for Rickettsia spp. and 15 (5.2%) of these were identified as R. africae comprising at least two genetic clades, occurring in A. variegatum (n = 10) and A. cohaerens (n = 5). The remainder of Rickettsia-positive samples could not be amplified due to low DNA yield. Furthermore, another 15 (5.2%) samples carried other pathogenic bacteria: Ehrlichia ruminantium (n = 9; 3.1%) in A. cohaerens, Ehrlichia sp. (n = 3; 1%) in Rh. pulchellus and A. cohaerens, Anaplasma sp. (n = 1; 0.5%) in A. cohaerens, and Neoehrlichia mikurensis (n = 2; 0.7%) in A. cohaerens. All ticks were negative for Bartonella spp., Babesia spp., Theileria spp., and Hepatozoon spp. We reported for the first time E. ruminatium, N. mikurensis, Ehrlichia sp., and Anaplasma sp. in A. cohaerens. Medically and veterinarily important pathogens were mostly detected from A. variegatum and A. cohaerens. These data are relevant for a One-health approach for monitoring and prevention of tick-borne disease transmission.