Tick Dermacentor Research Articles

Deep Mitochondrial DNA Lineage Divergences Within Alberta Populations of Dermacentor albipictus (Acari: Ixodidae) Do Not Indicate Distinct Species

The winter tick Dermacentor albipictus (Packard) has a single-host life cycle that allows it to reach severe infestation levels on ungulates, particularly moose. Genotypic variation within these and related ticks has been a source of taxonomic confusion, although the continuity in their morphology and life history has generally been interpreted as indicating the existence of a single species. To further investigate this variation, we sequenced regions of two mitochondrial DNA (mtDNA) genes (COI and 16S rDNA), two nuclear genes (lysozyme and ITS-2), and two bacterial markers from Francisella-like endosymbionts found in these ticks (eubacterial mtDNA 16S rRNA and a homolog of Francisella tularensis [Dorofe’ev] 17-kDa lipoprotein). We sampled 42 D. albipictus individuals from whitetail and mule deer culled from three populations in east-central Alberta, as well as four D. albipictus and two Dermacentor variabilis (Say) from other locations. We then compared DNA sequence variation between the genes and related this to variation in the morphology of spiracle plates. Both mtDNA regions indicated two deeply diverged lineages (mean difference of 7.1% for COI and 4.5% for 16S) that would normally be considered diagnostic of distinct species in DNA barcoding studies. However, very little divergence was revealed by nuclear gene sequences, bacterial endosymbionts, and morphometric analyses, and any variation that did occur in these markers was not congruent with mtDNA divergences. We conclude that the sampled populations in Alberta represent a single species, D. albipictus, and reiterate the importance of integrative approaches in species delimitation.

Deep Mitochondrial DNA Lineage Divergences Within Alberta Populations of <I>Dermacentor albipictus</I> (Acari: Ixodidae) Do Not Indicate Distinct Species

The winter tick Dermacentor albipictus (Packard) has a single-host life cycle that allows it to reach severe infestation levels on ungulates, particularly moose. Genotypic variation within these and related ticks has been a source of taxonomic confusion, although the continuity in their morphology and life history has generally been interpreted as indicating the existence of a single species. To further investigate this variation, we sequenced regions of two mitochondrial DNA (mtDNA) genes (COI and 16S rDNA),two nuclear genes (lysozyme and ITS-2), and two bacterial markers from Francisella-like endosymbionts found in these ticks (eubacterial mtDNA 16S rRNA and a homolog of Francisella tularensis [Dorofe'ev] 17-kDa lipoprotein). We sampled 42 D. albipictus individuals from whitetail and mule deer culled from three populations in east-central Alberta, as well as four D. albipictus and two Dermacentor variabilis (Say) from other locations. We then compared DNA sequence variation between the genes and related this to variation in the morphology of spiracle plates. Both mtDNA regions indicated two deeply diverged lineages (mean difference of 7.1% for COI and 4.5% for 16S) that would normally be considered diagnostic of distinct species in DNA barcoding studies. However, very little divergence was revealed by nuclear gene sequences, bacterial endosymbionts, and morphometric analyses, and any variation that did occur in these markers was not congruent with mtDNA divergences. We conclude that the sampled populations in Alberta represent a single species, D. albipictus, and reiterate the importance of integrative approaches in species delimitation.

Prevalence of Coxiella burnetii and Rickettsia spp. in ticks and rodents in southern Germany

Coxiella burnetii, the causative agent of Q fever, and Rickettsia spp. are bacterial pathogens that can be transmitted by ticks of the genus Dermacentor (i.e., Dermacentor marginatus and D. reticulatus). In Germany, the occurrence of these ticks is currently limited to few areas. However, due to increasing temperatures, these vectors will likely extend their distribution in the future, and C. burnetii and Rickettsia spp. might spread with them. To assess the prospective risk of human infections by these agents, it is important to know their current distribution. We collected 666 adult Dermacentor spp. and 119 rodents, mainly Microtus arvalis, in 3 Q fever endemic areas in southern Germany. Ticks and rodent organ pools were screened by PCR for C. burnetii and Rickettsia spp. No evidence of C. burnetii infections could be found in ticks or rodents, suggesting that these animals do not play an essential role in the epidemiology of Q fever in Germany. Rickettsia raoultii and R. slovaca could be detected in 30.3% and 0.75% of all examined ticks, respectively. In contrast, no rickettsia infections could be found in any rodent samples. Both rickettsia species can cause tick-borne lymphadenopathy (TIBOLA), a usually mild human disease. Because of the possible transmission of these rickettsiae to humans, TIBOLA should be considered in the differential diagnosis of tick-borne diseases. Our data show that a spread of these rickettsiae is possible in Germany and that more studies on the distribution of these agents are necessary.

Potential role of ticks as vectors of bluetongue virus

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus.

The application of lambda-cyhalothrin in tick control

In recent years, in urban areas of Novi Sad, unique ecological conditions, specific floristic and faunistic composition and poor habits of citizens in sense of public health, facilitate the development and maintenance of ticks. Regarding the importance of ticks as vectors of severe human and animal diseases, complex and detailed studies are conducted with an aim to find the most efficient methods for tick control. Two tick species, Ixodes ricinus and Dermacentor marginatus, were identified during a 3-year period on the territory of Municipality of Novi Sad. During 2006, the efficacy of the pyrethroid lambda-cyhalothrin in tick control varied from 60.7 to 100%. The highest efficacy recorded in 2007 was 92.3%. The efficacy of lambda-cyhalothrin in 2008 varied from 39.1 to 100%. Lambda-cyhalothrin showed high efficacy in tick control at localities which were improved before the application (mowed, litter removed, abundance control and euthanasia of abandoned cats and dogs). The results of this research indicate that lambda-cyhalothrin has a toxic effect on ticks and could be used as efficient acaricide for tick control, although its efficacy depends on formulation, terrain features and methods of application.

Detection of a new Arsenophonus-type bacterium in Canadian populations of the Rocky Mountain wood tick, Dermacentor andersoni

Ticks of the genus Dermacentor are important vectors of human and animal pathogens in North America. They also carry a variety of endosymbiotic (i.e. non-pathogenic) bacteria. The American dog tick, D. variabilis, is known to be infected with gammaproteobacteria of the genus Arsenophonus. However, there have been no previous reports of Arsenophonus-type bacteria in the Rocky Mountain wood tick, D. andersoni, a species that is sympatric with D. variabilis in the western parts of its distributional range. In this study, the presence of Arsenophonus-type bacteria was determined by PCR and DNA sequencing for 338 D. andersoni and 448 D. variabilis adults from western Canada. Fifty-one (15%) of the D. andersoni were found to be infected with Arsenophonus, whereas only a single D. variabilis was infected. The prevalence of Arsenophonus in D. andersoni varied among localities (0-27%). The 16S rDNA sequences of Arsenophonus in Canadian D. andersoni and D. variabilis were identical to one another, but the results of a phylogenetic analysis showed that they were genetically distinct from, and may represent a different species to, the Arsenophonus in D. variabilis and Amblyomma americanum in eastern USA.

Spatial and temporal factors affecting parasite genotypes encountered by hosts: Empirical data from American dog ticks ( Dermacentor variabilis) parasitising raccoons ( Procyon lotor)

The American dog tick ( Dermacentor variabilis) is an important vector of numerous pathogens of humans and animals. In this study, we analysed population genetic patterns in D. variabilis at scales of the host individual (infrapopulation) and population (component population) to elucidate fine-scale spatial and temporal factors influencing transmission dynamics. We genotyped D. variabilis collected from raccoons ( Procyon lotor) trapped in two habitat patches (located in Indiana, USA) which were spatially proximate (5.9 km) and limited in size (10.48 Ha and 25.47 Ha, respectively). Despite the fine spatial sampling scale, our analyses revealed significant genetic differentiation amongst component populations and infrapopulations (within each component population), indicating a non-random pattern of encountering tick genotypes by raccoons at both scales evaluated. We found evidence for male-biased dispersal in the ticks themselves (in one component population) and an age-bias in spatial scales at which raccoons encountered ticks in the environment. At the scale of the component population, our analyses revealed that raccoons encountered ticks from a limited number of D. variabilis family groups, likely due to high reproductive variance amongst individual ticks. Finally, we found evidence for a temporal effect with raccoons encountering ticks in the environment as “clumps” of related individuals. While the genetic structure of parasite populations are increasingly being investigated at small spatial scales (e.g. the infrapopulation), our data reveal that genetic structuring can originate at scales below that of the infrapopulation, due to the interaction between temporal and biological factors affecting the encounter of parasites by individual hosts. Ultimately, our data indicate that genetic structure in parasites must be viewed as a consequence of both spatial and temporal variance in host–parasite interactions, which in turn are driven by demographic factors related to both the host and parasite.

An acute severe ehrlichiosis in a dog experimentally infected with a new virulent strain of Ehrlichia canis

Canine monocytic ehrlichiosis (CME) is caused by Ehrlichia canis, an obligate intracellular bacterium with tropism for monocytes and macrophages. It is transmitted by the ticks Rhipicephalus sanguineus and Dermacentor variabilis. CME has been reported throughout the world, with a higher frequency in tropical and subtropical regions. The disease may be manifested by fever, depression, dyspnoea, anorexia, haemorrhages, oedema and weight loss accompanied with laboratory findings of thrombocytopenia, leukopenia, anaemia, and hypergammaglobulinaemia [1–5]. It has been reported that experimental infection of dogs with a cell culture-passaged type strain of E. canis Oklahoma induces mild clinical signs and mild pathological alterations in dogs [4]. Therefore, an acute severe experimental canine infection model is needed to study the pathological and immunological pathways induced by virulent E. canis. The purpose of the present study was to describe the experimental infection of a dog with a virulent strain of E. canis (New Mexico) by analysing haematological and pathological findings.

Comparative Efficacy of BioUD to Other Commercially Available Arthropod Repellents against the Ticks Amblyomma americanum and Dermacentor variabilis on Cotton Cloth

BioUD is an arthropod repellent that contains the active ingredient 2-undecanone originally derived from wild tomato plants. Repellency of BioUD was compared with five commercially available arthropod repellents against the ticks Amblyomma americanum (L.) and Dermacentor variabilis Say in two-choice bioassays on treated versus untreated cotton cheesecloth. Overall mean percentage repellency against both species was greatest for and did not differ significantly between BioUD (7.75% 2-undecanone) and products containing 98.1% DEET, 19.6% IR3535, and 30% oil of lemon eucalyptus. Products containing 5% and 15% Picaridin and 0.5% permethrin were also repellent compared with untreated controls but to a lesser degree than BioUD. The four most active repellents at the same concentrations used before were directly compared in head-to-head bioassays on cotton cheesecloth. BioUD provided significantly greater overall mean percentage repellency than IR3535 for A. americanum and D. variabilis. BioUD was significantly more repellent than oil of lemon eucalyptus for A. americanum but did not differ significantly in repellency against D. variabilis. No statistically significant difference in overall mean percentage repellency was found between BioUD and DEET for A. americanum or D. variabilis. In a 7-week time course bioassay, BioUD applied to cotton cheesecloth and held at room temperature provided 5 weeks of > 90% repellency against A. americanum.

New defensins from hard and soft ticks: Similarities, differences, and phylogenetic analyses

Despite the importance of ticks as vectors of disease very little is known about their immune system. Antimicrobial peptides, including defensins (phylogenetically ancient antibacterial peptides) are major components of innate immunity in ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites. With the aim of studying the evolution of the genes involved in tick defense, we identified the preprodefensin genes from four Ornithodoros tick species (O. papillipes: isoforms A, B, and D; O. tartakovskyi and O. puertoricensis: isoforms A and B; O. rostratus: isoform A) and from two Dermacentor tick species (D. reticulatus and D. marginatus: one isoform) not previously described. Phylogenetic analyses revealed that Ornithodoros defensin isoforms (A, B, C, and D) form 4 separate clades, while hard tick defensins are divided into several branches based on particular tick species.

Molecular characterization of hard ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238–1819 bp, and the lengths of ITS-2 were 137–1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0–1.5%, nucleotide differences between hard tick species ranged 2–25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks.

Neem-tree ( Azadirachta indica Juss.) extract as a feed additive against the American dog tick ( Dermacentor variabilis) in sheep ( Ovis aries)

Acaricides can be conveyed to ticks via the blood of their hosts. As fruit and kernel extracts from the Meliaceae family, and, in particular the tetranortriterpenoid azadirachtin (AZA) inhibits tick egg production and embryogenesis in the Ixodidae ticks, we investigated the effects of Neem Azal ®, an extract containing 43% AZA, given as a feed additive to lambs artificially infested with engorging adult Dermacentor vairiabilis ticks. After tick attachment, the lambs were allotted to three dietary treatments: AZA0 (control, n = 10), AZA0.3 ( n = 5), and AZA0.6 ( n = 5), with feed containing 0%, 0.3%, and 0.6% AZA on DM basis, respectively. In half of the AZA0 lambs, ticks were sprayed on day 4 after attachment with an ethanol:water:soap emulsion containing 0.6% AZA (AZA0S). In spite of its very pungent odor, the neem extract was well accepted by all but one lamb. No differences were found between treatment groups in liver enzymes in blood, and there was no indication of toxicity. The plasma AZA concentrations after 7 and 14 days of feeding AZA were (4.81 and 4.35 μg/mL) for the AZA0.6 and (3.32 and 1.88 μg/mL) for the AZA0.3 treatments, respectively ( P < 0.0001). Treatments were not lethal to ticks, but tick weights at detachment were 0.64, 0.56, 0.48, and 0.37 g for ticks from the AZA0, AZA0.3, AZA0S, and AZA0.6 treatments ( P < 0.04), respectively, suggesting that blood AZA impaired blood-feeding. The highest mortality rate after detachment was for AZA0.6 ( P < 0.09). As AZA affects embryo development and ticks at the molting stages, we expect that following treatments of hosts for longer periods, one-host ticks will be more affected than the three-host tick D. variabilis.

Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238-1819 bp, and the lengths of ITS-2 were 137-1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0-1.5%, nucleotide differences between hard tick species ranged 2-25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks.

Utilisation of rodent species by larvae and nymphs of hard ticks (Ixodidae) in two habitats in NE Poland

The impact of host identity and habitat type on the density of hard ticks (Ixodes ricinus and Dermacentor reticulatus) infections on rodents in forest and abandoned field habitats in NE Poland was investigated. Ixodes ricinus was most abundant in the forest system, but D. reticulatus, although rarer, was most abundant in the field system. Environmental humidity and the much lower density of rodents probably limited the abundance of I. ricinus larvae in the field system, although this tick was still common on Microtus oeconomus from around small ponds. Nymphs of I. ricinus were comparatively rare in the forest, probably because of infection of non-rodent hosts. Dermacentor reticulatus nymphs on the other hand were very much more common in the ears of Microtus species than would have been predicted based on larval densities. The impact of habitat change (in this case successional change following field abandonment) on tick densities is emphasised, and the role of Apodemus as an epidemiological bridge, linking woodland and field habitats, is highlighted.

Demonstration of Borrelia burgdorferi sensu stricto infection in ticks from the northeast of Mexico

Borrelia burgdorferi sensu lato infection has been confirmed in clinical cases in the northeast of Mexico; however, the bacterium has not been identified as infecting the tick vector Ixodes, Amblyomma and Dermacentor ticks were collected from mammals and plants in northeastern Mexico and examined for Borrelia. Eighteen of 214 ticks were PCR-positive for the fla and 16S rRNA genes and 15 for the ospA gene. Southern blotting with a fla probe and sequencing of ospA genes confirmed infection with B. burgdorferi sensu stricto. These findings, together with reports of indigenous cases, fulfil the criteria that allow northeastern Mexico to be considered as a zone endemic for Lyme disease.

Allochronic seasonal peak activities of Dermacentor and Haemaphysalis spp. under continental climate in Hungary

Hard ticks (Acari: Ixodidae) were collected from the vegetation at monthly intervals in 2008 on six places of three different biotopes in Hungary. Except for Haemaphysalis concinna and H. punctata, predominance of females was observed in the questing population. From among the species with biphasic activity period, metastriate ticks ( Dermacentor spp. and H. inermis) had higher prevalence of host-seeking males in the autumn than in the spring, as opposed to prostriate Ixodes ricinus. In comparison with the permanently mild weather experienced at the beginning of 2007, sharply rising temperatures during consecutive winter days in January and February of 2008 appeared to be more efficient in triggering a 1–2 month earlier spring peak activity of hard ticks, except for I. ricinus. Regarding seasonality, D. marginatus was most numerous in February and March, whereas D. reticulatus in September and October; H. punctata showed peak activity in March–April, H. concinna in May, and H. inermis in November–December. Within these genera such a temporal difference (allochrony) between seasonal peak activities of sympatric species under continental climate is described for the first time.

Emerging arthropod-borne diseases of companion animals in Europe

Vector-borne diseases are caused by parasites, bacteria or viruses transmitted by the bite of hematophagous arthropods (mainly ticks and mosquitoes). The past few years have seen the emergence of new diseases, or re-emergence of existing ones, usually with changes in their epidemiology (i.e. geographical distribution, prevalence, and pathogenicity). The frequency of some vector-borne diseases of pets is increasing in Europe, i.e. canine babesiosis, granulocytic anaplasmosis, canine monocytic ehrlichiosis, thrombocytic anaplasmosis, and leishmaniosis. Except for the last, these diseases are transmitted by ticks. Both the distribution and abundance of the three main tick species, Rhipicephalus sanguineus, Dermacentor reticulatus and Ixodes ricinus are changing. The conditions for such changes involve primarily human factors, such as travel with pets, changes in human habitats, social and leisure activities, but climate changes also have a direct impact on arthropod vectors (abundance, geographical distribution, and vectorial capacity). Besides the most known diseases, attention should be kept on tick-borne encephalitis, which seems to be increasing in western Europe, as well as flea-borne diseases like the flea-transmitted rickettsiosis. Here, after consideration of the main reasons for changes in tick vector ecology, an overview of each “emerging” vector-borne diseases of pets is presented.

Detection of a NovelFrancisellainDermacentor reticulatus: A Need for Careful Evaluation of PCR-Based Identification ofFrancisella tularensisin Eurasian Ticks

Francisella tularensis, the causative agent of tularemia, has been detected in ixodid ticks in some regions of North America, Europe, and Asia. In the present study, 245 Dermacentor reticulatus, 211 Ixodes ricinus, and 194 Haemaphysalis concinna adults from Hungary were tested for the presence of F. tularensis by polymerase chain reaction (PCR) assays based on 16S ribosomal RNA (16S rDNA) and T-cell epitope of a Francisella membrane protein (TUL4). No Francisella-specific amplification products were detected in I. ricinus and H. concinna ticks. Francisella DNA was identified using PCR assays based on 16S rDNA and TUL4 gene in D. reticulatus with similar prevalence (minimum 1.2%) as demonstrated in earlier European and Asian studies detecting F. tularensis in D. reticulatus. However, the 16S rDNA and TUL4 gene sequences of the Francisella-like agent occurring in D. reticulatus differed from the homologous sequences of Francisella spp. deposited in GenBank. Phylogenetic reconstructions showed that the new genotype detected in D. reticulatus was closely related to Francisella-like endosymbionts of North American Dermacentor ticks. Although further studies are needed on the relationship of this bacterium with ticks, the results highlight the need for careful evaluation of PCR-based identification in European and Asian laboratories that screen ixodid ticks for F. tularensis.

Development and characterization of 12 polymorphic microsatellite loci in the American dog tick (Dermacentor variabilis)

Twelve polymorphic microsatellite loci were developed for the American dog tick (Dermacentor variabilis), an important vector of infectious diseases in humans and animals. Four multiplexed panels comprising the loci were developed and 45 ticks collected from two raccoons (Procyon lotor) were genotyped. The number of alleles per locus ranged from nine to 30, and single locus heterozygosities ranged from 0.18 to 0.93. Data generated using these markers will further our understanding of factors affecting gene flow in D. variabilis, thus helping to elucidate the transmission dynamics of diseases associated with this vector.

Biogeography of Tick-Borne Bhanja Virus (Bunyaviridae) in Europe

Bhanja virus (BHAV) is pathogenic for young domestic ruminants and also for humans, causing fever and affections of the central nervous system. This generally neglected arbovirus of the family Bunyaviridae is transmitted by metastriate ticks of the genera Haemaphysalis, Dermacentor, Hyalomma, Rhipicephalus, Boophilus, and Amblyomma. Geographic distribution of BHAV covers southern and Central Asia, Africa, and southern (partially also central) Europe. Comparative biogeographic study of eight known natural foci of BHAV infections in Europe (in Italy, Croatia, Bulgaria, Slovakia) has revealed their common features. (1) submediterranean climatic pattern with dry growing season and wet mild winter (or microlimatically similar conditions, e.g., limestone karst areas in central Europe), (2) xerothermic woodland-grassland ecosystem, with plant alliances Quercetalia pubescentis, Festucetalia valesiacae, and Brometalia erecti, involving pastoral areas, (3) presence of at least one of the tick species Haemaphysalis punctata, Dermacentor marginatus, Rhipicephalus bursa, and/or Hyalomma marginatum, and (4) presence of ≥60% of the 180 BHAV bioindicator (157 plant, 4 ixodid tick, and 19 vertebrate spp.). On that basis, Greece, France (southern, including Corsica), Albania, Spain, Hungary, European Turkey, Ukraine (southern), Switzerland (southern), Austria (southeastern), Germany (southern), Moldova, and European Russia (southern) have been predicted as additional European regions where BHAV might occur.