Long Time No Hear, Magnificent Wohlfahrtia! Morphological and Molecular Evidence of Almost Forgotten Flesh Fly in Serbia and Western Balkans.

The "beautiful viviparous fly", Wohlfahrtia magnifica, may have a magnificent appearance due to its striking morphology; however, it is a potentially deadly agent of obligate traumatic myiasis in humans and animals, with a serious impact on welfare and economics. The fly is found across the Palearctic realm, including the Western Balkan region, with reports from former Yugoslavian countries from the first half of the 20th century. In this paper, a recent case of wohlfahrtiosis recorded in Northern Serbia is evidenced using morphological and molecular techniques. Larvae were collected from two adult sheep with severe hoof myiasis and two young sheep with genital and interdigital myiasis. Morphological identification was performed for adults bred from the infested vulva and third-stage larvae (L3) collected from the hoof wounds, supported with barcoding sequences of the COI gene obtained from larval pairs from the hoof wounds of older and the genitalia of younger sheep. W. magnifica was identified according to the appearance of male fly terminalia and the morphology of L3, which was confirmed after the comparison of representative sequences of the COI gene (deposited in GenBank™ under accession numbers MT027108-MT027114) to those available in GenBank™. This finding represents the first reported case of wohlfahrtiosis in the Western Balkans in 80 years, highlighting the need to re-inform relevant stakeholders to achieve adequate disease control.

The Occurrence and Diversity of Tick-Borne Pathogens in Small Mammals from Serbia.

Background: Despite abundance of small mammals in Serbia, there is no information on their role in the epidemiology of tick-borne diseases (TBDs). This retrospective study aimed to identify different tick-borne pathogens (TBPs) in small mammals in Serbia collected during 2011. Materials and Methods: A total of 179 small mammals were collected from seven different localities in Serbia. The five localities belong to the capital city of Serbia-Belgrade: recreational areas-Ada Ciganlija, Titov gaj, and Kosutnjak as well as mountainous suburban areas used for hiking-Avala and Kosmaj. The locality Veliko Gradiste is a tourist place in northeastern Serbia, whereas the locality Milosev Do is a remote area in western Serbia with minor human impact on the environment. Results: The results of the presented retrospective study are the first findings of Rickettsia helvetica, Rickettsia monacensis, Neoehrlichia mikurensis, Borrelia afzelii, Borrelia miyamotoi, Babesia microti, Hepatozoon canis, and Coxiella burnetii in small mammals in Serbia. The presence of R. helvetica was confirmed in two Apodemus flavicollis, the presence of one of the following pathogens, R. monacensis, B. afzelii, H. canis, Ba. microti, and N. mikurensis was confirmed in one A. flavicollis each, whereas the presence of B. miyamotoi was confirmed in one Apodemus agrarius. Coinfection with B. afzelii and Ba. microti was confirmed in one A. flavicollis. DNA of C. burnetii was detected in 3 of 18 pools. Conclusions: The results confirm that detected pathogens circulate in the sylvatic cycle in Serbia and point to small mammals as potential reservoir hosts for the detected TBPs. Further large-scale studies on contemporary samples are needed to clarify the exact role of particular small mammal species in the epidemiology of TBDs caused by the detected pathogens.

Evolutionary dynamics and geographical dispersal of Borrelia lusitaniae.

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis.

Ixodid ticks and zoonotic tick-borne pathogens of the Western Balkans.

Ixodid ticks are distributed across all countries of the Western Balkans, with a high diversity of species. Many of these species serve as vectors of pathogens of veterinary and medical importance. Given the scattered data from Western Balkan countries, we have conducted a comprehensive review of available literature, including some historical data, with the aim to compile information about all recorded tick species and associated zoonotic pathogens in this region. Based on the collected data, the tick fauna of the Western Balkans encompasses 32 tick species belonging to five genera: Ixodes, Haemaphysalis, Dermacentor, Rhipicephalus and Hyalomma. A range of pathogens responsible for human diseases has also been documented, including viruses, bacteria and parasites. In this review, we emphasize the necessity for integrated surveillance and reporting, urging authorities to foster research by providing financial support. Additionally, international and interdisciplinary collaborations should be encouraged that include the exchange of expertise, experiences and resources. The present collaborative effort can effectively address gaps in our knowledge of ticks and tick-borne diseases.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

Autochthonous infection with Ehrlichia Canis and Hepatozoon Canis in dogs from Serbia.

BACKGROUND: The epidemiological status concerning many canine tick-borne diseases (TBDs) in Serbia is still insufficiently known. OBJECTIVES: Our study aimed to investigate the presence of tick-borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. METHODS: Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April-October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. RESULTS: The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3-month-old female Rottweiler puppy, an 8-year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4-year-old mixed breed male dog was co-infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. CONCLUSIONS: The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.

High Prevalence and Genetic Variability of Hepatozoon canis in Grey Wolf (Canis lupus L. 1758) Population in Serbia.

Wild canids are globally recognised as hosts and reservoirs of a large number of ecto- and endoparasites. Data that reveal the importance of the grey wolf (Canis lupus L.1758) in the spread of hepatozoonosis are very scarce. There are a large number of different potential host species that can be infected by Hepatozoon canis, but the most common are domestic and wild carnivores, such as dogs, jackals, foxes, and wolves. In this study, the epidemiological significance of the grey wolf as a host for the pathogen was analysed for the first time in Serbia, as well as the genetic variability of H. canis. The presence of H. canis in wolf spleens has been demonstrated using molecular methods. A total of 107 wolf spleen samples from 30 localities in Serbia were analysed. The presence of H. canis was confirmed in 62 (57.94%) individuals from 26 out of 30 localities. According to the analysis, the sampled H. canis sequences were found to be characterised by a certain heterogeneity. Based on five mutated nucleotide sites in the sequences, H. canis could be divided into five sequence types, S1 to S5. The five sequence types can potentially circulate in grey wolf populations as well as among other domestic and wild canids. This study is the first confirmation of the presence of H. canis in grey wolf populations in Serbia. Considering that the role of this vector-borne disease is poorly researched in wild carnivores, it is very important to indicate the role of this species in the circulation of this pathogen in natural ecosystems.

Knowledge, Attitudes, and Practices on Tick-Borne Encephalitis Virus and Tick-Borne Diseases within Professionally Tick-Exposed Persons, Health Care Workers, and General Population in Serbia: A Questionnaire-Based Study.

This study assessed the level of knowledge, attitudes, and practices (KAP) regarding tick-borne encephalitis virus (TBEV) and tick-borne diseases (TBDs) among different groups of people in Serbia. Professionally tick-exposed persons (PTEPs), health care workers (HCWs), and the general population (GP) were subjected to an anonymous, voluntary, online questionnaire using Microsoft Forms. A total of 663 questionnaire responses were collected (February-March 2021), while 642 were included in the analysis. The significant difference in knowledge in TBDs existed between GP and PTEPs, and HCWs (p < 0.001). The perception of risk-to-tick exposure and TBDs was generally high (42.4 (95% CI: 33.6-51.2) within GP, 44.9 (95% CI: 35.8-53.9) within PTEPs and 46.2 (95% CI: 38.0-54.5) within HCWs), while fear was low (13.7 (95% CI: 7.9-19.5) within GP, 12.6 (95% CI: 7.3-19.9) within PTEPs, and 13.5 (95% CI: 7.4-19.5) within HCWs). Protective practices differed across groups (F (2639) = 12.920, p < 0.001, η2 = 0.039), with both PTEPs (t = 3.621, Cohen d = 0.332, p < 0.001) and HCWs (t = 4.644, Cohen d = 0.468, p < 0.001) adhering to more protective practices than the GP, without differences between PTEPs and HCWs (t = 1.256, Cohen d = 0.137, p = 0.421). Further education about TBDs in Serbia is required and critical points were identified in this study.

Exploring immunogenicity of tick salivary AV422 protein in persons exposed to ticks: prospects for utilization.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

In vitro efficacy of antibiotics against different Borrelia isolates.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125-2 mg/L; doxycycline, 0.125-1 mg/L, ceftriaxone, 0.016-0.063 mg/L; cefuroxime, 0.063-1 mg/L; azithromycin, 0.0017-0.11 mg/L; amikacin 32-512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK-H and BSK-II media stored for prolonged periods.

Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidious Borrelia species - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth of Borrelia would be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 °C for 2.5 years and BSK-II stored at -20 °C for 11 years). Growth of different Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana strains was assessed during 2 weeks of incubation at 33 °C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 °C (median final cell number of 1.5 × 106 /mL) than in BSK-II at -20 °C (median final cell number of 7.75 × 106 /mL) and in fresh BSK-H media (median final cell number of 8.95 × 106 /mL). Duration of storage of media had no impact on Borrelia morphology and motility. Our results indicate that temperature of -20 °C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth of Borrelia and may be employed for cultivation.

A short-term and long-term relationship between occurrence of acute canine babesiosis and meteorological parameters in Belgrade, Serbia.

Belgrade, the capital of the Republic of Serbia, is an endemic location for canine babesiosis caused by Babesia canis. This tick-borne disease occurs seasonally in regions with moderate continental climate. However, recent clinical data show that: 1) some cases of canine babesiosis have been recorded during the winter, and 2) canine babesiosis is spreading to the northern parts of Europe with a colder climate, which is a region previously free of this disease. Our study investigates the occurrence of canine babesiosis in different seasons over 2013-2016, and explores a short-term link between meteorological parameters and number of diagnosed cases of canine babesiosis in Belgrade. It also looks into possible long-term correlations that span one year before the onset of the disease. Based on 872 recorded cases over four years, our results show a bimodal seasonal distribution of canine babesiosis, with a pronounced peak in the spring, and a less conspicuous one in the autumn. Throughout the year, even over the coldest and warmest periods, there is a broad range of temperatures and relative humidities when the disease is recorded. Over one year prior to the spring and autumn onset of the disease, we found a noticeable impact of temperature and relative humidity, and to a lesser extent, of atmospheric pressure and cloud cover, on the number of diagnosed cases. These findings imply short-term and long-term relationships between occurrence of acute canine babesiosis and certain meteorological parameters, and they open further questions that need to be investigated in order to understand the epidemiology of this disease.

First evidence of tick-borne protozoan pathogens, Babesia sp. and Hepatozoon canis, in red foxes (vulpes vulpes) in Serbia.

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Coinfection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes.

Golden jackals (Canis aureus) as hosts for ticks and tick-borne pathogens in Serbia.

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens. In a study conducted during a three-year period (01/2010-02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses. The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected. From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

Contributions to the phylogeny of Ixodes (Pholeoixodes) canisuga, I. (Ph.) kaiseri, I. (Ph.) hexagonus and a simple pictorial key for the identification of their females.

BACKGROUND: In Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers. RESULTS: Between 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2-100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5-100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5-100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes. CONCLUSIONS: A simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies.

Assessment of using recombinant Ixodes ricinus AV422 saliva protein for confirmation of tick bites in hunting dogs as naturally infested hosts.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.

East and west separation of Rhipicephalus sanguineus mitochondrial lineages in the Mediterranean Basin.

BACKGROUND: Rhipicephalus sanguineus belongs to a complex of hard tick species with high veterinary-medical significance. Recently, new phylogenetic units have been discovered within R. sanguineus, which therefore needs taxonomic revision. The present study was initiated to provide new information on the phylogeography of relevant haplotypes from less studied regions of Europe and Africa. With this aim, molecular-phylogenetic analyses of two mitochondrial markers were performed on 50 ticks collected in Hungary, the Balkans, countries along the Mediterranean Sea, Kenya and Ivory Coast. RESULTS: In the "temperate lineage" of R. sanguineus, based on cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes, Rhipicephalus sp. I was only found in the eastern part of the Mediterranean Basin (with relatively homogenous haplotypes), whereas Rhipicephalus sp. II occurred in the middle-to-western part of this region (with phylogenetically dichotomous haplotypes). Ticks identified as R. leporis (based on morphology and cox1 gene) were found in Kenya and Ivory Coast. These clustered phylogenetically within R. sanguineus (s.l.) ("tropical lineage"). CONCLUSIONS: In the Mediterranean Basin two mitochondrial lineages of R. sanguineus, i.e. Rhipicephalus sp. I and Rhipicephalus sp. II exist, which show different geographical distribution. Therefore, data from this study confirm limited gene flow between Rhipicephalus sp. I and Rhipicephalus sp. II, but more evidence (analyses of nuclear markers, extensive morphological and biological comparison etc.) are necessary to infer if they belong to different species or not. The phylogenetic relationships of eastern and western African ticks, which align with R. leporis, need to be studied further within R. sanguineus (s.l.) ("tropical lineage").

Ixodes ricinus immunogenic saliva protein, homologue to Amblyomma americanum AV422: Determining its potential for use in tick bite confirmation.

Tick bites often go unnoticed, so specific reliable tests are needed to confirm them for prompt diagnosis and treatment of tick-borne diseases. One of the promising candidates for developing such a test is AV422, a tick saliva protein that has been conserved across tick genera. In this study, we demonstrate the potential of the AV422 homologue from Ixodes ricinus to be used for tick bite detection for both Prostriata and Metastriata. We expressed recombinant (r) I. ricinus (Ir) AV422 in E. coli and subjected it to Western blot analysis using rat antibodies to saliva proteins of both I. ricinus (Prostriata) and Dermacentor reticulatus (Metastriata) larvae. Our data demonstrate that rIrAV422 specifically bound to antibodies from sera of rats used for both I. ricinus and D. reticulatus larvae feeding, but not to antibodies from control serum, emphasizing its specificity since tick bites were the sole cause of sera reactivity.

High degree of mitochondrial gene heterogeneity in the bat tick species Ixodes vespertilionis, I. ariadnae and I. simplex from Eurasia.

BACKGROUND: Phylogeographical studies allow precise genetic comparison of specimens, which were collected over large geographical ranges and belong to the same or closely related animal species. These methods have also been used to compare ticks of veterinary-medical importance. However, relevant data are missing in the case of ixodid ticks of bats, despite (1) the vast geographical range of both Ixodes vespertilionis and Ixodes simplex, and (2) the considerable uncertainty in their taxonomy, which is currently unresolvable by morphological clues. METHODS: In the present study 21 ticks were selected from collections or were freshly removed from bats or cave walls in six European and four Asian countries. The DNA was extracted and PCRs were performed to amplify part of the cytochrome oxidase I (COI), 16S and 12S rDNA genes, followed by sequencing for identification and molecular-phylogenetic comparison. RESULTS: No morphological differences were observed between Ixodes vespertilionis specimens from Spain and from other parts of Europe, but corresponding genotypes had only 94.6 % COI sequence identity. An I. vespertilionis specimen collected in Vietnam was different both morphologically and genetically (i.e. with only 84.1 % COI sequence identity in comparison with I. vespertilionis from Europe). Two ticks (collected in Vietnam and in Japan) formed a monophyletic clade and shared morphological features with I. ariadnae, recently described and hitherto only reported in Europe. In addition, two Asiatic specimens of I. simplex were shown to differ markedly from European genotypes of the same species. Phylogenetic relationships of ticks showed similar clustering patterns with those of their associated bat host species. CONCLUSIONS: Although all three ixodid bat tick species evaluated in the present study appear to be widespread in Eurasia, they exhibit pronounced genetic differences. Data of this study also reflect that I. vespertilionis may represent a species complex.