European Tick Research Articles

Vectorial dynamics underpinning current and future tick-borne virus emergence in Europe.

Tick-borne diseases pose a growing threat to human and animal health in Europe, with tick-borne encephalitis virus (TBEV) and Crimean-Congo haemorrhagic fever virus (CCHFV), vectored by Ixodes ricinus and Hyalomma marginatum, respectively, emerging as primary public health concerns. The ability of ticks to transmit pathogens to multiple hosts and maintain infections across life stages makes them highly efficient vectors. However, many aspects of tick ecology and vectorial capacity remain understudied. This review examines key factors contributing to the vectorial competence of European ticks and their associated viruses. We first explore the influence of climate change on vector and disease ecology, using TBEV and CCHFV as case studies. We then analyse the role of the tick antiviral response in shaping vector competence. By integrating these elements, this review aims to enhance our understanding of tick-borne viral diseases and support the development of public health strategies, particularly through the One Health framework, to mitigate their impact in Europe.

Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland.

Toxoplasma gondii occurs in a wide range of intermediate hosts, whose blood may be a meal for different tick species. A few studies have examined the role of ticks in the life cycle of T. gondii. This one includes the largest number and all stages of Ixodes ricinus collected from the widest area, covering seven recreational localities within a forest biotope in Northern Poland. This study aimed to determine the prevalence of T. gondii DNA in 2144 collected questing ticks to establish whether they may be involved in T. gondii life cycle. The additional goal was to genotype the detected T. gondii, as knowledge about its genotypes occurring in European ticks is insufficient. A further purpose was to detect coinfection with T. gondii and Borreliaceae in the collected ticks, as all of them have previously been tested for the presence of bacteria DNA. Nested PCR and sequencing of the obtained B1 gene fragment were conducted. T. gondii DNA was detected in 0.9% of all ticks (1.1% of nymphs and 0.7% of larvae). The presence of T. gondii in unfed larvae and nymphs may indicate the possibility of its vertical transmission. The prevalence of T. gondii DNA in ticks collected from individual sites was focal (0-4.3%) and seems to depend on local climatic conditions. Among all examined ticks, 0.3% were coinfected with T. gondii and Borreliella spp., vs. 0.6% of specimens with a single T. gondii infection. The obtained B1 sequences showed the greatest similarity (99.71-100%) to the sequence representing type III.

Anthropogenic electromagnetic radiation alters the transcription levels of the genes encoding the SIFamide and myoinhibitory peptide and their receptors in Ixodes ricinus synganglion

The research of the influences of man-made electromagnetic fields on tick physiology has been very sparse and long neglected since the pioneer studies published in 1996 and 2000. Once multiple behavioral tests confirmed an attraction and possible perception of electromagnetic fields in ticks, a new interest in this topic erupted in recent years. In this study, qRT-PCR is utilized to determine the changes in the mRNA transcript levels of neuropeptides SIFamide and myoinhibitory peptide (mip and sifa) and their representative receptors (mip-r1 and sifa-r1) in the synganglia of the tick Ixodes ricinus irradiated by 900 MHz radiofrequency electromagnetic field. It was determined that 40 V/m intensity has a significant suppressory effect on the transcript levels of all genes after at least 60 minutes of constant exposure in both sexes. Commonly occurring intensity of radiation in urban areas (2 V/m) produced an elevation in mRNA levels after various timespans in every gene. A significant decrease of transcript abundances was detected in females after one hour of exposure to 2 V/m. Results of this study widen the knowledge of EMF-induced alterations in the neurophysiology of I. ricinus, the most commonly distributed hard tick in Europe.

Successful diagnosis and treatment of Borrelia miyamotoi in a patient with joint and muscle pains, ME/CFS and cognitive dysfunction following tick bites: a case report

Abstract Introduction Diagnosing Borrelia miyamotoi disease (BMD) presents challenges due to its overlap with Lyme disease (LD) symptoms and the lack of reliable laboratory diagnostics. This case study demonstrates the successful use of phage-based PCR (phb-PCR) in identifying B. miyamotoi in a patient with multiple tick bites. A 46-year-old female presented with joint and muscle pain, chronic fatigue, and cognitive impairment after being bitten by ticks in Europe. Standard diagnostic tests, including Enzyme-linked immunosorbent assay (ELISA), immunoblot for LD, and antibody tests for Bartonella, Anaplasma, and autoimmune conditions, all returned negative results. However, phb-PCR identified the presence of B. miyamotoi. The patient was treated with intravenous ceftriaxone, oral azithromycin, and intravenous vitamin and mineral therapy, resulting in significant improvement in symptoms, including reduced pain, improved cognitive function, and decreased fatigue. This case emphasises the importance of direct diagnostic methods like phb-PCR for accurately identifying BMD, especially when conventional serological tests fail. Clinicians should consider testing for B. miyamotoi in cases of complex tick-borne diseases for timely and effective management.

Signatures in in vitro infection of NSC-34 mouse neurons and their cell nucleus with Rickettsia helvetica

BackgroundRickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34.ResultsNSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron.ConclusionsThe findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

Year-round tick exposure of dogs and cats in Germany and Austria: results from a tick collection study

BackgroundTicks and tick-borne diseases play a major role in companion animal health. Additionally, the European tick fauna is changing, for instance due to the spread of Dermacentor reticulatus, displaying a higher likelihood of winter activity than Ixodes ricinus. Therefore, we investigated current tick infestations in dogs and cats in Germany and in parts of Austria and the seasonal infestation risk.MethodsOverall, 219 veterinary practices were invited to collect ticks from cats and dogs on a monthly basis. Ticks were morphologically identified and female I. ricinus specimens were measured to estimate attachment duration.ResultsIn total, 19,514 ticks, 17,789 (91.2%) from Germany and 1506 (7.7%) from Austria, were received between March 2020 and October 2021, with 10,287 specimens (52.7%) detached from dogs, 8005 from cats (41.0%) and 1222 from other species (6.3%). In Germany, the most common tick species collected from dogs were I. ricinus (78.0%) and D. reticulatus (18.8%), while cats mainly harboured I. ricinus (91.3%) and I. hexagonus (5.5%) and only few D. reticulatus (0.6%). In Austria, collected I. ricinus reached similar proportions in dogs (90.4%) and cats (95.3%), followed by D. reticulatus in both dogs (5.2%) and cats (1.5%), with I. hexagonus (0.9%) collected only marginally from cats. The average infestation intensity amounted to 1.62 ticks/dog and 1.88 ticks/cat. The single to multiple infestation ratio was 79.1% to 20.9% in dogs and 69.0% to 31.0% in cats, with cats being significantly more often multiple infested than dogs, while the proportion of mixed-species infestations was 2.0% for both dogs and cats. The average attachment duration of female I. ricinus specimens amounted to 78.76 h for dogs and 82.73 h for cats. Furthermore, year-round tick exposure was confirmed, with 108 D. reticulatus and 70 I. ricinus received on average per month during December 2020 to February 2021.ConclusionsThe study shows a year-round tick infestation risk, with activity of both D. reticulatus and I. ricinus during winter, and confirms the widespread occurrence of D. reticulatus in Germany. Additionally, long average attachment durations and frequent multiple infestations underline the need for adequate year-round tick control, even during the winter months.Graphical

Diversity of Hepatozoon species in wild mammals and ticks in Europe

BackgroundHepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe.MethodsSamples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene.ResultsOf the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozooncanis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H.martis in 125 (3 Artiodactyla, 122 Carnivora), H.sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H.ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H.canis (n = 22).ConclusionsHepatozooncanis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H.martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H.canis in ticks other than R.sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.Graphical

Parasitized or non-parasitized, why? A study of factors influencing tick burden in roe deer neonates.

Ixodes ricinus, the most common species of tick in Europe, is known to transmit major pathogens to animals and humans such as Babesia spp. or Borrelia spp. Its abundance and distribution have been steadily increasing in Europe during recent decades, due to global environmental changes. Indeed, as ticks spend most of their life in the environment, their activity and life cycle are highly dependent on environmental conditions and therefore, on climate or habitat changes. Simultaneously, wild ungulates have expanded their range and increased dramatically in abundance worldwide, in particular roe deer (Capreolus capreolus), allowing tick populations to grow and spread. Currently, tick infestation on newborn wild ungulates is poorly documented. However, newborn ungulates are considered more sensitive to tick bites and pathogen transmission because of their immature immune systems. Thus, improving knowledge about the factors influencing tick infestation on newborns is essential to better understand their health risks. This study was conducted at Trois-Fontaines Forest, Champagne-Ardenne, France (1992–2018). Based on a long-term monitoring of roe deer fawns, we used a novel Bayesian model of the infestation of fawns to identify which biotic or abiotic factors were likely to modify the level of infestation by ticks of 965 fawns over time. We show that tick burden increased faster during the first days of life of the fawns and became constant when fawns were five days old and more, which could be explained by the depletion of questing ticks or the turnover of ticks feeding on fawns. Moreover, despite the known positive influence of humidity on tick activity, the tick burdens were weakly related to this parameter. Our results demonstrate that tick infestation was highly variable among years, particularly between 2000–2009. We hypothesize that this results from a modification of habitat caused by Hurricane Lothar.

New Cell Lines Derived from European Tick Species.

Tick cell lines are important tools for research on ticks and the pathogens they transmit. Here, we report the establishment of ten new cell lines from European ticks of the genera Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain. For each cell line, the method used to generate the primary culture, a morphological description of the cells and species confirmation by sequencing of the partial 16S rRNA gene are presented. Further molecular analysis of the two new Ixodes ricinus cell lines and three existing cell lines of the same species revealed genetic variation between cell lines derived from ticks collected in the same or nearby locations. Collectively, these new cell lines will support research into a wide range of viral, bacterial and protozoal tick-borne diseases prevalent in Europe.

THE TICKS <i>HYALOMMA RUFIPES</i> KOCH, 1844 IMPORTATION BY MIGRATORY BIRDS TO THE SOUTH OF RUSSIA - VECTORS OF CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS: EPIDEMIOLOGICAL ASPECT

Koch, 1844 (Acari, Ixodidae) is the main vector of the Crimean-Congo haemorrhagic fever (CCHF) virus in southern and eastern Africa. In the spring migrations of birds, immature stages of this species are carried out beyond the range of the parasite and are often found in various countries of Europe. Finds of adult ticks in Europe are single. In June 2018, in the north of the Stavropol Territory, one male of with characteristic features was removed from a cow. Prior to this, single registrations of this species were known on the territory of the Astrakhan Region, Dagestan and the Stavropol Territory in the middle of the last century.

Epizootiological aspects of natural nidality of Ixodes tick-borne borreliosis in the Moscow region (Russian Federation)

Background and Aim:At present, tick-borne borreliosis is the most common infectious disease transmitted by ticks in Europe, Asia, and North America. This study aimed to examine the epizootiological aspects of the natural nidality of tick-borne borreliosis in Moscow region (the Russian Federation).Materials and Methods:A total of 2,537 ticks representing two species were collected, namely, Ixodes ricinus and Dermacentor reticulatus. The activity, number of ticks, and Borrelia infestation rates were investigated during a high season, that is, from early spring to mid-autumn.Results:In May, amount of I. ricinus spp. was found 2.5 times more than those representing D. reticulatus spp. (p≤0.01). In June, August, and September, the amount of I. ricinus was 9.0 (p≤0.0001), 2.0 (p≤0.05), and 5.0 times higher, respectively, compared to D. reticulatus. In the first 10 days of April, the amount of D. reticulatus was 3 times higher than that of I. ricinus (p≤0.02); in the next 10 days, their amounts were equal (p≥0.05) and in the last 10 days the amount of I. ricinus exceeded that of D. reticulatus (p≤0.05) by 1.5 times. In general, Borrelia afzelii, and Borrelia garinii, were detected. In addition, the naturally occurring tick-borne borreliosis pesthole was revealed in the Moscow region.Conclusion:Borrelia infection rates for ticks comprise 30%. An increase in Borrelia tick infestation was detected within the vicinity of populated areas. The amount of ticks directly depends on the temperature (20°C-25°C) and moisture (from 50%) values.

Cross-reactivity between tick and wasp venom can contribute to frequent wasp sensitization in patients with the α-Gal syndrome.

Backgroundα‐Gal syndrome (AGS) is a food allergy with severe delayed allergic reactions, mediated by IgE‐reactivity to galactose‐α1,3‐galactose (α‐Gal). AGS is strongly associated with tick bites. An increased incidence of venom sensitization has been found in AGS patients. Here, we evaluated the frequency of wasp sensitization in Swedish AGS patients and the possible cross‐reactivity between wasp venom and tick proteins.MethodsSera from 136 Swedish AGS patients and 29 wasp‐positive non‐AGS control sera were analyzed for IgE‐reactivity against wasp venom (Vespula spp.), the European tick Ixodes ricinus (Streptavidin ImmunoCAP), α‐Gal and total IgE by ImmunoCAP. The presence of α‐Gal on wasp venom proteins (Vespula vulgaris) was investigated by western blot (WB), and possible cross‐reactivity between wasp venom and tick proteins by enzyme‐linked immunosorbent assay and WB. Involvement of cross‐reactive carbohydrate domains (CCDs) was also assessed.ResultsWasp sensitization was present in 54% of AGS patients, although the IgE levels were low. Wasp sensitized patients had higher IgE levels to α‐Gal and total IgE levels compared to non‐wasp sensitized AGS patients. α‐Gal was not detected in wasp venom, but cross‐reactivity between wasp and tick proteins was demonstrated which was not dependent on CCDs. The same cross‐reactivity was also observed in the control sera. Furthermore, 17 putative cross‐reactive peptides were identified using an in silico approach.ConclusionsFor the first time, cross‐reactivity between wasp venom and tick proteins has been described. This may be a reason why the majority of Swedish AGS patients, who have all been tick bitten, are also sensitized against wasp.

Tick Cell Culture Analysis of Growth Dynamics and Cellular Tropism of Rickettsia buchneri, an Endosymbiont of the Blacklegged Tick, Ixodes scapularis.

Simple SummaryThe blacklegged tick, Ixodes scapularis, a species of significant medical and veterinary importance, harbors an endosymbiont, Rickettsia buchneri. This bacterium is largely restricted to the ovaries, but all life stages can harbor different numbers or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. We characterized the cells using microscopy. The doubling time of wildtype R. buchneri and a transformant expressing green fluorescent protein was determined to be >7 days when measured by quantitative PCR. Quantification based on fluorescence indicated that 11 days were needed to double the amount of green fluorescent protein. Two rRNA probes were tested using rickettsiae grown in vitro and adapted to localize R. buchneri in different organs of field-collected female I. scapularis ticks. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. The sequestration of rickettsia in ticks and the slow growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.The blacklegged tick, Ixodes scapularis, a species of significant importance to human and animal health, harbors an endosymbiont Rickettsia buchneri sensu stricto. The symbiont is largely restricted to the ovaries, but all life stages can harbor various quantities or lack R. buchneri entirely. The endosymbiont is cultivable in cell lines isolated from embryos of Ixodes ticks. Rickettsia buchneri most readily grows and is maintained in the cell line IRE11 from the European tick, Ixodes ricinus. The line was characterized by light and electron microscopy and used to analyze the growth dynamics of wildtype and GFPuv-expressing R. buchneri. qPCR indicated that the genome copy doubling time in IRE11 was >7 days. Measurements of fluorescence using a plate reader indicated that the amount of green fluorescent protein doubled every 11 days. Two 23S rRNA probes were tested via RNA FISH on rickettsiae grown in vitro and adapted to evaluate the tissue tropism of R. buchneri in field-collected female I. scapularis. We observed strong positive signals of R. buchneri in the ovaries and surrounding the nucleus of the developing oocytes. Tissue tropism in I. scapularis and in vitro growth dynamics strengthen the contemporary understanding of R. buchneri as a transovarially transmitted, non-pathogenic endosymbiont.

Symbiont dynamics during the blood meal of Ixodes ricinus nymphs differ according to their sex

Ticks harbour rich and diverse microbiota and, among the microorganisms associated with them, endosymbionts are the subject of a growing interest due to their crucial role in the biology of their arthropod host. Midichloria mitochondrii is the main endosymbiont of the European tick Ixodes ricinus and is found in abundance in all I. ricinus females, while at a much lower density in males, where it is even absent in 56 % of the individuals. This endosymbiont is also known to increase in numbers after the blood meal of larvae, nymphs or females. Because of this difference in the prevalence of M. mitochondrii between the two sexes, surveying the density of these bacteria in nymphs that will become either females or males could help to understand the behaviour of Midichloria in its arthropod host. To this aim, we have set up an experimental design by building 3 groups of unfed nymphs based on their scutum and hypostome lengths. After engorgement, weighing and moulting of a subset of the nymphs, a significant difference in sex-ratio among the 3 groups was observed. In parallel, Midichloria load in individual nymphs was quantified by qPCR both before and after engorgement.No difference in either body mass or Midichloria load was observed at the unfed stage, but following engorgement, both features were significantly different between each size group.Our results demonstrate that symbiont dynamics during nymphal engorgement is different between the two sexes, resulting in a significantly higher Midichloria load in nymphs that will become females. The consequences of those findings on our understanding of the interplay between the endosymbiont and its arthropod host are discussed.

Exploration of binary protein\u2013protein interactions between tick-borne flaviviruses and Ixodes ricinus

BackgroundLouping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells.MethodsTo investigate virus-vector protein–protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV.ResultsFor each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways.ConclusionWe provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.Graphical

Ecotyping of Anaplasma phagocytophilum from Wild Ungulates and Ticks Shows Circulation of Zoonotic Strains in Northeastern Italy.

Simple SummaryTick-borne infectious diseases represent a rising threat both for human and animal health, since they are emerging worldwide. Among the bacterial infections, Anaplasma phagocytophilum has been largely neglected in Europe. Despite its diffusion in ticks and animals, the ecoepidemiology of its genetic variants is not well understood. The latest studies identify four ecotypes of Anaplasma phagocytophilum in Europe, and only ecotype I has shown zoonotic potential. The aim of the present study was to investigate the genetic variants of Anaplasma phagocytophilum in wild ungulates, the leading reservoir species, and in feeding ticks, the main vector of infection. The analyzed samples were collected in northeastern Italy, the same area where the first Italian human cases of anaplasmosis in the country were reported. Using biomolecular tools and phylogenetic analysis, ecotypes I and II were detected in both ticks (Ixodes ricinus species) and wild ungulates. Specifically, ecotype II was mainly detected in roe deer and related ticks; and ecotype I, the potentially zoonotic variant, was detected in Ixodes ricinus ticks and also in roe deer, red deer, chamois, mouflon, and wild boar. These findings reveal not only the wide diffusion of Anaplasma phagocytophilum, but also the presence of zoonotic variants.Anaplasma phagocytophilum (A. phagocytophilum) is a tick-borne pathogen causing disease in both humans and animals. Human granulocytic anaplasmosis (HGA) is an emerging disease, but despite the remarkable prevalence in European ticks and wild animals, human infection appears underdiagnosed. Several genetic variants are circulating in Europe, including the zoonotic ecotype I. This study investigated A. phagocytophilum occurrence in wild ungulates and their ectoparasites in an area where HGA has been reported. Blood samples from wild ungulates and ectoparasites were screened by biomolecular methods targeting the mps2 gene. The groEL gene was amplified and sequenced to perform genetic characterization and phylogenetic analysis. A total of 188 blood samples were collected from different wild ungulates species showing an overall prevalence of 63.8% (88.7% in wild ruminants and 3.6% in wild boars). The prevalence of A. phagocytophilum DNA in ticks (manly Ixodes ricinus), and keds collected from wild ruminants was high, reflecting the high infection rates obtained in their hosts. Among ticks collected from wild boars (Hyalomma marginatum and Dermacentor marginatus) no DNA was detected. Phylogenetic analysis demonstrated the presence of ecotype I and II. To date, this is the first Italian report of ecotype I in alpine chamois, mouflon, and wild boar species. These findings suggest their role in HGA epidemiology, and the high prevalence detected in this study highlights that this human tick-borne disease deserves further attention.

Prevalence and monitoring of ixodid ticks in the western region of Ukraine

Ixodes ricinus is the most common tick in Europe, distributed almost throughout the continent, except the northern regions. Dermacentor reticulatus is a widespread species of tick in Europe and Western Asia. The study included monitoring the distribution density and seasonal activity of ticks D. reticulatus and I. ricinus in the natural environment during 2018-2019. A total of 1592 D. reticulatus ticks and 712 I. ricinus ticks were collected in Ternopil, Ivano-Frankivsk and Lviv oblasts. There was a tendency to increasing of the number of ticks of both species in 2019 compared to 2018. The largest number of ticks was collected in autumn 2019, during the autumn peak of activity, which amounted to 710 adult ticks. Thus, the average density of ticks of both species collected in the Ternopil region was 40 ticks/1000 m2, in Ivano-Frankivsk – 32 and 45 in Lviv regions in 2018 and 62, 46 and 63 ticks/1000 m2 in 2019, respectively. In the spring of 2018, almost twice as many adult ticks of D. reticulatus were detected as in the fall in all areas. With regard to I. ricinus ticks, we did not observe a statistically significant difference in seasonal activity. In the spring and autumn, two peaks of ticks’ activity were registered in all regions and the average number of ticks in the spring in the meadows was 20 ticks/1000 m2, and on the wooded area – 39 ticks/1000 m2, and in autumn – 17 and 41 ticks/ 1000 m2 respectively. During both years and in all three oblasts, females prevailed over males during all the period. Although the density of ticks of both species in all areas was quite high, but the ticks were unevenly distributed. The largest number of D. reticulatus ticks was found in the Lviv region in 2019, which ranged from 46 to 119 ticks/1000 m2. Low and medium density of adult ticks D. reticulatus and I. ricinus, from 11 to 77 ticks/1000 m2 was observed in Ivano-Frankivsk region in 2018. Throughout the study period, the seasonal activity of D. reticulatus and I. ricinus ticks was significantly affected by air temperature, which fluctuated during collection of ticks.

Transovarial transmission of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus under field conditions extrapolated from DNA detection in questing larvae

BackgroundIxodes ricinus constitutes the main European vector tick for the Lyme borreliosis pathogen Borrelia burgdorferi (sensu lato), the relapsing fever borrelia Borrelia miyamotoi, as well as Anaplasma phagocytophilum and several Rickettsia species. Under laboratory conditions, a transovarial transmission to the next tick generation is described for Rickettsia spp. and Borrelia spp., especially regarding B. miyamotoi, whereas the efficiency of transovarial transfer under field conditions is largely unstudied.MethodsIn order to better estimate the potential infection risk by tick larvae for humans and animals, 1500 I. ricinus larvae from 50 collected “nests” (larvae adhering to the flag in a clumped manner) were individually examined for Borrelia, Rickettsia and A. phagocytophilum DNA using quantitative real-time PCR (qPCR).ResultsThirty-nine of 50 nests each (78.0%, 95% CI: 64.0–88.5%) were positive for Borrelia spp. and Rickettsia spp. DNA, and in three nests (6.0%, 95% CI: 1.3–16.5%) A. phagocytophilum DNA was detected. Overall, DNA from at least one pathogen could be detected in 90.0% (45/50, 95% CI: 78.2–96.7%) of the nests. Of the 1500 larvae, 137 were positive for Borrelia spp. DNA (9.1%, 95% CI: 7.7–10.7%), 341 for Rickettsia spp. DNA (22.7%, 95% CI: 20.6–24.9%) and three for A. phagocytophilum DNA (0.2%, 95% CI: 0–0.6%). Quantity of Borrelia spp. and Anaplasma spp. DNA in positive larvae was low, with 2.7 × 100Borrelia 5S-23S gene copies and 2.4 × 101A. phagocytophilum msp2/p44 gene copies detected on average, while Rickettsia-positive samples contained on average 5.4 × 102gltA gene copies. Coinfections were found in 66.0% (33/50, 95% CI: 51.2–78.8%) of the nests and 8.6% (38/443, 95% CI: 6.1–11.6%) of positive larvae. In fact, larvae had a significantly higher probability of being infected with Borrelia spp. or Rickettsia spp. when both pathogens were present in the nest.ConclusionsThis study provides evidence for transovarial transmission of Rickettsia spp. and Borrelia spp. in I. ricinus under field conditions, possibly facilitating pathogen persistence in the ecosystem and reducing the dependence on the presence of suitable reservoir hosts. Further studies are needed to prove transovarial transmission and to explain the surprisingly high proportion of nests containing Rickettsia and/or Borrelia DNA-positive larvae compared to infection rates in adult ticks commonly reported in other studies.

Successful Infection of Domestic Pigs by Ingestion of the European Soft Tick O. Erraticus That Fed on African Swine Fever Virus Infected Pig.

African swine fever is a highly lethal hemorrhagic fever of Suidae, threatening pig production globally. Suidae can be infected by different ways like ingestion of contaminated feed, direct contact with infected animals or fomites, and biting by infected soft tick bites. As already described, European soft ticks (Ornithodoros erraticus and Ornithodoros verrucosus) were not able to transmit African swine fever virus by biting pigs although these ticks maintained the infectious virus during several months; therefore, the possibility for pigs to become infected through the ingestion of infected ticks was questioned but not already explored. To determine if such oral ingestion is an alternative pathway of transmission, O. erraticus ticks were infected by blood-feeding on a viremic pig infected with the European African swine fever virus strain Georgia2007/1, then frozen at zero and two months post-engorgement, then after, were embedded in the food to pigs. Pig infection was successful, with superior efficiency with ticks frozen just after the infectious blood meal. These results confirmed the potential role of O. erraticus ticks as an ASFV reservoir and demonstrated the efficiency of non-conventional pathways of transmission.

Molecular discrimination of Hyalomma tick species serving as reservoirs and vectors for Crimean-Congo hemorrhagic fever virus in sub-Saharan Africa

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies.