Ear Tick Research Articles

Linking spatial distribution of Rhipicephalus appendiculatus to climatic variables important for the successful biocontrol by Metarhizium anisopliae in Eastern Africa

Cattle production is constantly threatened by diseases like East Coast fever, also known as theileriosis, caused by the protozoan parasite Theileria parva which is transmitted by ticks such as the brown ear tick, Rhipicephalus appendiculatus. To reduce the extensive use of chemical acaricides, fungal-based microbial control agents such as Metarhizium anisopliae have been tested and show promising results against R. appendiculatus both in field and in semi-field experiments in Africa. However, no known endeavors to link the spatial distribution of R. appendiculatus to climatic variables important for the successful application of M. anisopliae in selected East African countries exists. This work therefore aims to improve the successful application of M. anisopliae against R. appendiculatus by designing a temperature-dependent model for the efficacy of M. anisopliae against three developmental stages (larvae, nymphs, adults) of R. appendiculatus. Afterward a spatial prediction of potential areas where this entomopathogenic fungus might cause a significant epizootic in R. appendiculatus population in three selected countries (Kenya, Tanzania, Uganda) in Eastern Africa were generated. This can help to determine whether the temperature and rainfall at a local or regional scale might give good conditions for application of M. anisopliae and successful microbial control of R. appendiculatus.

From Diagnosis to Treatment of Human Otoacariasis: Demographic and Clinical Characteristics of Patients.

Otoacariasis is the presence of ticks and mites in the ear canals of humans or animals, and particularly common in rural areas. This study aimed to present the clinical characteristics of patients that presented with ticks in their ear canal. The study was conducted with a total of 425 patients with 527 ticks in their ear canal at the Muş Malazgirt State Hospital Ear-Nose-Throat Clinic between June 2019 and June 2020. The removed ticks were examined at the parasitology laboratory of Van Yüzüncü Yıl University. Of the 425 cases included in the study, 72% (n=306) were female and 28% (n=119) were male (mean age 40±20, minimum-maximum: 4 months-81 years). A total of 527 ticks were removed in the one-year period. Three-hundred-and-fifty-one patients had adult or nymph ticks, and 74 patients had a larval form of the tick. Of the patients with adult or nymph tick, foreign body sensation was the dominant symptom in 68.7% (n=242), whereas pain was the dominant symptom in 62% (n=46) of those with larval tick. In the comparison between groups, foreign body sensation was statistically significantly higher in the adult tick group, and pain was higher in the larval tick group (p<0.001). There were no systemic diseases related to the ticks in any of the cases. Ticks in the ear is endemic in Eastern Anatolia and poses a public health problem. Tick infestations could be minimized with various precautions and educating the general public on preventive methods. Our study is the largest series in the literature on cases with ear ticks.

Essential oils of Tagetes minuta and Tithonia diversifolia affect host location behaviour and on-host attachment site preference of the brown ear tick, Rhipicephalus appendiculatus in the semi-field studies

Effects of essential oils of Tagetes minuta and Tithonia diversifolia (Asteraceae) on orientation behaviour and on-host attachment site preference of newly hatched adult Rhipicephalus appendiculatus were evaluated. Host animals were treated at the ear pinna (by smearing the oil directly on the ear or suspending a tube containing the oil on the ear pinna) and legs + tail in semi-field plots. The legs + tail sites of the essential oil application showed the lowest mean percentage of ticks observed on host body (16.5 ± 1.9% and 26.0 ± 2.8%) and the highest mean percentage reduction of attaching ticks (76.5 ± 3.9% and 67.0 ± 0.8%) for essential oils of Tagetes minuta and Tithonia diversifolia, respectively. The control animals had the highest mean percentage of ticks observed (93.0 ± 2.1%). The ear tube resulted in the highest mean percentage of ticks on the host (47.5 ± 5.1% and 55.8 ± 5.1%) and a lowest mean percentage reduction of attaching ticks (44.8 ± 5.1% and 36.5 ± 7.4%) for the essential oils of Tagetes minuta and Tithonia diversifolia, respectively. For both the essential oils, legs + tail sites of essential oil application, followed by ear smear and then ear tube, had significant effects on orientation to the host and attachment site preference of adult R. appendiculatus on the host, in that order. We, therefore, recommend the ear smear site for treating hosts with essential oils whose performance may improve upon increasing concentration and formulating them in a carrier material that stabilizes the active ingredients.

Earliest record of the spinose ear tick, Otobius megnini (Dugès) (Ixodida: Argasidae), from the Paisley Caves site (Oregon, USA), with implications for prehistoric human ectoparasitism

The discovery of five individual specimens of the spinose ear tick, Otobius megnini (Dugès), in late Pleistocene through middle Holocene deposits of the Paisley Caves site highlights an interesting aspect of prehistoric life not ordinarily visible through the analysis of traditional archaeological artifacts. A review of the literature provides ample evidence to postulate how the tick remains got into the cave deposits, and also implies the possibility of host-switching behavior in this tick and explores the manner by which ancient people dealt with ectoparasites. Furthermore, these remains mark the first record of the species in both archaeological and paleontological contexts.

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.

Structure-activity studies on analogues of 4-methylguaiacol, a cattle anal odour constituent repellent to the brown ear tick (Rhipicephalus appendiculatus)

Previously, 4-methylguaiacol, a major constituent of cattle anal odour, was found to have a high repellency on Rhipicephalus appendiculatus. In the present study, 10 structural analogues of the phenol were tested for repellency against R. appendiculatus in order to assess the effects of (i) absence or presence of the 4-alkyl group of varying length, (ii) inclusion of a double bond in the 4-alkyl chain, (iii) linking the two phenolic oxygen in a methylenedioxy bridge, (iv) replacement of the OCH3 with CH3 and inclusion of another CH3 at position 6, and (v) presence of an additional OCH3 group at position 6. The analogues comprised of 2-methoxyphenol (guaiacol), 4-ethyl-2-methoxyphenol, 4-propyl-2-methoxyphenol, 4-allyl-2-methoxyphenol (eugenol), 3,4-methylenedioxytoluene, 2,4-dimethylphenol, 4-ethyl-2-methylphenol, 2,4,6-trimethylphenol, 4-propyl-2,6-dimethoxy-phenol and 4-allyl-2,6-dimethoxyphenol, which were compared at different concentrations in a two-choice climbing assay set up. Each analogue showed either increased or reduced repellency compared with 4- methylguaiacol. The structural feature that was associated with the highest repellency was 4-propyl moiety in the guaiacol unit (RD75 = 0.031 for 4-propyl-2-methoxyphenol; that of 4-methylguaiacol = 0.564). Effects of blending selected analogues with high repellency were also compared. However, none of the blends showed incremental increases in repellency compared with that of 4-propyl-2-methoxyphenol. We are currently evaluating the effects of controlled release of this compound at different sites on cattle on the behavior and success of R. appendiculatus to locate their predilection for feeding sites.

Molecular evidence of &lt;i&gt;Babesia&lt;/i&gt; infections in Spinose ear tick, &lt;i&gt;Otobius&lt;/i&gt; &lt;i&gt;megnini&lt;/i&gt; infesting stabled horses in Nuwara Eliya racecourse: A case study

Spinose ear tick, Otobius megnini (Family Argasidae) is a one-host soft tick that parasitizes domesticated animals and occasionally humans. It causes otoacariasis or parasitic otitis in humans and animals and also known to carry infectious agents. Intra aural infestations of O. megnini is a serious health problem in the well-groomed race horses in Nuwara Eliya. Otobius megnini collected from the ear canal of stabled horses in Nuwara Eliya racecourse were tested for three possible infections, Rickettsia, Theileria and Babesia. Genomic DNA was extracted from 22 ticks collected from 11 horses and then pathogen-specific DNA was amplified using PCR. Ticks from two horses (one thoroughbred and one stallion) tested PCR positive for Babesia but not for other infections. None of the horses however, showed any clinical symptoms of babesiosis. This is the first record of Babesia infections in O. megnini. However, the presence of Babesia DNA in O. megnini doesn’t qualify it as the vector of equine babesiosis, horse blood has to be tested for the presence of parasite DNA or antibodies.

Characterisation of cattle anal odour constituents associated with the repellency of Rhipicephalus appendiculatus.

Adults of the Brown Ear Tick (Rhipicephalus appendiculatus) have a predilection for feeding inside and around the ears of cattle and other hosts. A previous study has shown that the tick locates the host ears by 'push-pull' deployment of a repellent blend emitted at the anal region and an attractant blend emitted at the ears. Interestingly, the two odours play reverse roles with Rhipicephalus evertsi, which prefer to feed around the anal region. The present study was undertaken to characterize the major constituents of the cattle anal odour and to evaluate their repellence to R. appendiculatus. The anal odour was trapped with reverse-phase C18-bonded silica, Porapak Q and Super Q placed in an oven bag attached at the anal region of the cattle for 6h. The adsorbents were then removed and extracted with dichloromethane, and the extracted compounds analyzed by linked gas chromatography-mass spectrometry (GC-MS). The major constituents of the odour were o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethylbenzene, 2,6,6-trimethyl-[1S(1α,β,5α)]bicycloheptanes, 5-ethoxydihydro-2(3H)-furanone, 3-methylene-2-pentanone, 5-methyl-2-phenyl-1H-indole, and 3-pentanone. The repellency of the available compounds (o-xylene, 4-hydroxy-4-methyl-2-pentanone, 4-methyl-2-methoxyphenol, ethyl benzene, 3-methylene-2-pentanone, and 3-pentanone) and blends was evaluated using a dual choice tick climbing assay at different doses. The anal odour showed repellence with RD75 of 0.39. Of the compounds tested, 4-methyl-2-methoxyphenol was found to be most repellent (RD75 = 0.56) and 3-pentanone least repellent (RD75 = 622.7). The blend of the six constituents showed RD75 of 0.34, comparable to that of the crude anal odour blend. A series of subtractive bioassays with one constituent of the 6-component blend missing was also carried out. Subtraction of 3-methylpentanone gave the most repellent blend (RD75 = 0.097), whereas subtraction of 4-methylguaiacol gave the least repellent blend (RD75 = 160.7) consistent with the high individual activity of this phenol. The study lays down useful groundwork for on-host deployment of controlled-release of a selected repellent or blend to disrupt the tick's ability to locate its preferred feeding site.

Epidemiology and treatment of spinose ear ticks on a California dairy

While not widely reported on dairies, spinose ear ticks may be more prevalent in the Southwest US dairy industry than previously thought. The dairy where this research was conducted had a serious infestation and the owner was looking for guidance in the control or eradication of this parasite due to the irritation the ticks were causing the cows. The dairy owner felt that the ticks were causing a decrease in milk production and an increased culling risk. The objective of this research was to analyze various treatment options to determine the most effective method to control spinose ear ticks through 4 studies. Study 1 examined the efficacy of acaricides to determine the most effective treatment option. Study 2 focused on locating where the adult ticks were living and reproducing within the freestall bedding. Study 3 analyzed how soon cows became infested after being moved to the freestall barn from the dry lots. Study 4 looked at the efficacy of the CyLence Ultra® ear tags in relation to infestation severity.

Otobius megnini(Dugès) on a Dog from the North-Central Part of Mexico

The purpose of the study was to report the presence of the soft tick Otobius megnini (Duges) parasitizing a dog. Three specimens were collected during a routine consultation, and by morphological examination, identified as the ear tick. Physicians and public health authorities should be aware of the tick as part of differential diagnosis when earache is manifested by patients in close contact with animals and the posible incrimination of the parasite in harboring and transmitting pathogens.

Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.

Ants as predators of the Spinose Ear Tick, Otobius megnini (Dugès) in Sri Lanka

Many pathogens, parasitoids and predators have been documented as natural enemies of ticks, but their impact on tick populations are rarely evaluated. Here, we report the predatory behaviour of ants on the spinose ear tick, Otobius megnini. Ticks were collected from the ear canal of stabled horses in Nuwara Eliya and were brought to the laboratory. Eggs, unfed and engorged larvae, engorged nymphs and adults were naturally exposed to ants under laboratory conditions and the predatory behaviour of the different species and their feeding preferences were observed. Five ant species were found feeding on different life stages of O. megnini including eggs, larvae (fed/unfed) and adults; ants did not feed on the nymphal stages. Ant species were identified as Tapinoma melanocephalum, two species of Monomorium, one species of Pheidole and one species of Crematogaster. The predatory preference differed among the five ant species, with T. melanocephalum being identified as the best predator as it fed on eggs and adults, the non-parasitic stages of O. megnini. Different strategies have been used to control the infestations of O. megnini in the stabled horses but none of them were successful. Although we cannot extrapolate our empirical findings to a natural context, observations suggest that these ant species may be potential bio-suppressors of this tick species.

English

The effects of 2 Neem derivatives, Neem oil (NO) and Neem Seed Powder (NSP), on various physiological parameters of the brown ear tick, Rhipicephalus appendiculatus, the vector for Theileria parva. Neem oil was applied on rabbit ears using a fine brush, whereas NSP was mixed with rabbit pellets at various concentrations and fed on goats on which various instars of the tick were allowed to feed. Tick larvae smeared with Neem oil (NO) while attached to rabbit ears exhibited significant mortalities. The 10% NO induced a mortality of 40% compared to less than 1% observed in Peanut oil (PO) control. Furthermore, engorgement weights were significantly reduced (0.1 mg NO vs 4 mg PO). Their corresponding moulting percentages were 1 and 71% in NO and PO, respectively. Adult female ticks exhibited reduced engorgement weights and egg mass. Larvae able to attach on goats maintained on 100% NSP were only 11% compared to 66% for control goats fed on rabbit pellets (RP). The corresponding nymphal attachments were 24% NSP and 79% RP. The number and weights of eggs produced by adult ticks fed on goats maintained on NSP also dropped significantly. The hatchability of eggs produced by these ticks was also significantly reduced. Key words: East Coast Fever, economic losses, Neem derivatives, Rhipicephalus appendiculatus, Theileria parva, tick control.

Ants as predators of the Spinose Ear Tick, Otobius megnini (Dugès) in Sri Lanka

Ants as predators of the Spinose Ear Tick, Otobius megnini (Dugès) in Sri Lanka

Seasonal dynamics of spinose ear tick Otobius megnini associated with horse otoacariasis in Sri Lanka

Otobius megnini is a one host, nidicolous soft tick (Family Argasidae) whose larvae and nymphs parasitize the external ear canal of many wild and domestic animals and occasionally humans. The present study was conducted to determine the seasonal dynamics of O. megnini infesting stabled horses in Nuwara Eliya racecourses. Ticks were sampled biweekly for two years from May 2013 to May 2015 from the ear canal of seven thoroughbred male horses. Weather data: temperature, rainfall and humidity for the study period were obtained from the Meteorological Department, Colombo. A total of 23, 287 ticks of O. megnini were collected from the ear canal of horses and all of which were immature stages comprising 41.4% larvae and 59.6% nymphs. Larval counts were high during warmer months of the year with minimum numbers recording September–November. High larval abundance may be due to the high hatching rate of eggs at high temperatures. Larval abundance was significantly affected by maximum temperature, minimum relative humidity and average relative humidity (Multiple regression; r2=0.5, F=3.7, p=0.01). Population peaks for nymphs occurred in May, October and January. Nymph counts were low in March and April. Weather conditions did not have any correlation with the abundance of nymphs. Rainfall had no effect on both larvae and nymph counts. Even though control measures have been taken, O. megnini infestation is a serious problem for the stable owners especially because these horses are used in racing and other recreational purposes. Further, there is a high risk of spreading this tick within the country due to the presence of suitable hosts and the adaptability of the tick to survive in changing climatic conditions. Information on the seasonal dynamics of the tick population is important for employing control measures.

Extensive polymorphism of Ra86 genes in field populations of Rhipicephalus appendiculatus from Kenya.

Commercial vaccines based on recombinant forms of the Bm86 tick gut antigen are used to control the southern cattle tick, Rhipicephalus microplus, a 1-host species, in Australia and Latin America. We describe herein sequence polymorphism in genes encoding Ra86 homologues of Bm86 in the brown ear tick, Rhipicephalus appendiculatus, isolated from four Kenyan field populations and one laboratory colony. Sequencing of 19 Ra86 sequences defined two alleles differentiated by indels, encoding 693 amino acids (aa) and 654 aa respectively, from the Muguga laboratory reference strain. Ra86 sequences were also determined from gut cDNA from four field populations of R. appendiculatus collected in different livestock production systems in Kenya. Analysis of approximately 20 Ra86 sequences from each of the four field sites in central and Western Kenya; Makuyu, Kiambu, Kakamega and Uasin Gishu, revealed three additional size types differentiated by 39–49 amino acid indels resulting in a total of 5 indel-defined genotypes. The 693 aa type 5 was isolated only from the laboratory tick stock; genotypes 1, 2 and 3 were identified in ticks from the four Kenyan field sites and appeared to be derivatives of the shorter RA86 genotype found in Muguga laboratory stock genotype 4. By contrast no large indels have yet been observed between R. microplus sequences from Australia, South America or Africa.Evidence that selection contributes to the observed sequence variation was provided by analysis of ratio of synonymous and non-synonymous substitutions and application of the selective neutrality and neutral evolution tests to the primary data. Phylogenetic analysis clustered sequences from all Ra86 size types and Bm86, into four major clades based on amino acid substitutions, but there was no evidence that these groupings correlated with geographical separation of R. appendiculatus populations

Paralog analyses reveal gene duplication events and genes under positive selection in Ixodes scapularis and other ixodid ticks.

BackgroundHard ticks (family Ixodidae) are obligatory hematophagous ectoparasites of worldwide medical and veterinary importance. The haploid genomes of multiple species of ixodid ticks exceed 1 Gbp, prompting questions regarding gene, segmental and whole genome duplication in this phyletic group. The availability of the genome assembly for the black legged tick, Ixodes scapularis, and transcriptome datasets for multiple species of ticks offers an opportunity to assess the contribution of gene duplication to the genome. Here we developed a bioinformatics pipeline to identify and analyze duplicated genes (paralogs) using gene models from the prostriate tick, I. scapularis IscaW1.1 annotation and expressed sequence tags (ESTs) from I. scapularis and the metastriate ticks, Rhipicephalus microplus (southern cattle tick), R. appendiculatus (brown ear tick) and Amblyomma variegatum (tropical bont tick).ResultsApproximately 1-2 % of I. scapularis gene models and 2-14 % of ESTs from the four species represent duplicated genes. The ratio of non-synonymous to synonymous nucleotide substitution rates suggests ~ 25 % of duplicated genes are under positive selection pressure in each species. Analyses of synonymous substitution rates provide evidence for two duplication events in I. scapularis and R. microplus involving several hundred genes. Conservative molecular clock estimates based on synonymous substitution rates for species of Anopheles mosquitoes and the fruit fly, Drosophila melanogaster, suggest these events occurred within the last 50 MYA. Mapping of paralogs to the I. scapularis genome assembly supports tandem, or possibly segmental duplication events.ConclusionsThe present study marks the first genome-level analyses of gene duplication for the Ixodidae and provides insights into mechanisms shaping genome evolution in this group. At least two duplication events involving hundreds of genes may have occurred independently in the lineages prostriata and metastriata, with tandem and segmental duplication the most likely mechanisms for paralog generation. Duplicated genes under positive selection pressure may be linked to emerging functions in the tick and represent important candidates for further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2350-2) contains supplementary material, which is available to authorized users.

Multi-locus genotyping reveals absence of genetic structure in field populations of the brown ear tick (Rhipicephalus appendiculatus) in Kenya

Rhipicephalus appendiculatus is an important tick vector of several pathogens and parasitizes domestic and wild animals across eastern and southern Africa. However, its inherent genetic variation and population structure is poorly understood. To investigate whether mammalian host species, geographic separation and resulting reproductive isolation, or a combination of these, define the genetic structure of R. appendiculatus, we analyzed multi-locus genotype data from 392 individuals from 10 geographic locations in Kenya generated in an earlier study. These ticks were associated with three types of mammalian host situations; (1) cattle grazing systems, (2) cattle and wildlife co-grazing systems (3) wildlife grazing systems without livestock. We also analyzed data from 460 individuals from 10 populations maintained as closed laboratory stocks and 117 individuals from five other species in the genus Rhipicephalus. The pattern of genotypes observed indicated low levels of genetic differentiation between the ten field populations (FST=0.014±0.002) and a lack of genetic divergence corresponding to the degree of separation of the geographic sampling locations. There was also no clear association of particular tick genotypes with specific host species. This is consistent with tick dispersal over large geographic ranges and lack of host specificity. In contrast, the 10 laboratory populations (FST=0.248±0.015) and the five other species of Rhipicephalus (FST=0.368±0.032) were strongly differentiated into distinct genetic groups. Some laboratory bred populations diverged markedly from their field counterparts in spite of originally being sampled from the same geographic locations. Our results demonstrate a lack of defined population genetic differentiation in field populations of the generalist R. appendiculatus in Kenya, which may be a result of the frequent anthropogenic movement of livestock and mobility of its several wildlife hosts between different locations.

Spatial and temporal distributions of the Spinose Ear Tick,Otobius megnini, within animal shelters at Fossil Rim Wildlife Center

Spinose ear ticks, Otobius megnini, are monoxenous parasites that feed within the ear of ungulates, subjecting them to debilitating conditions. Little is known about the spatial dispersion of spinose ticks in animal shelters or the temporal variation in their abundance. Therefore, the objectives of this study were to: 1) determine the temporal distribution of spinose ear tick abundance within animal shelters, 2) determine the spatial distribution of larvae and adults within animal shelters, and 3) examine the effects of climatic variables on tick abundance. No temporal trend was identified for larval or adult ticks. Larval abundance was highest in quadrats located against the shelter wall while no spatial difference was found in adult abundance. Mean larval tick abundance was significantly correlated with mean temperature while mean adult tick abundance was significantly correlated with mean relative humidity.

First phylogenetic analysis of a Crimean-Congo hemorrhagic fever virus genome in naturally infected Rhipicephalus appendiculatus ticks (Acari: Ixodidae).

Crimean-Congo haemorrhagic fever (CCHF) is a potentially fatal systemic viral disease in many parts of the world, including Iran. The nationwide incidence of human CCHF in endemic areas was 870 confirmed cases with 126 deaths (case fatality rate, CFR = 17.6 %) in the decade leading to 2012. The detection of the CCHF virus (CCHFV) genome in tick vectors is of fundamental importance for identifying these ticks as potential reservoirs of CCHFV infection. From May to October 2013, following detection of four new clinical cases resulting in two deaths in the city of Mashhad (northeast Iran), hard ticks were recovered from infested livestock in 40 villages in Khorasan-Razavi province and examined by the microscopic method for species identification. About a quarter of the ticks were then subjected to reverse-transcription polymerase chain reaction (RT-PCR) to detect the CCHFV genome. The PCR products were then sequenced, and their phylogenetic lineages were determined. A total of 407 hard ticks were captured, representing seven different species in two distinct genera. Members of the genus Hyalomma were widely distributed in all but two of the villages studied, and this was also the most frequent (83.3 %) tick genus. Of 105 adult ticks subjected to RT-PCR, four (3.8 %) ticks were found positive for the CCHFV genome. One brown ear tick, Rhipicephalus appendiculatus, was found to be naturally infected for the first time anywhere in the world. Ticks of Hyalomma asiaticum, Hyalomma marginatum, and Rhipicephalus turanicus were also found to be naturally infected with CCHFV. CCHFV found in these four different tick species were clustered in the same lineage with the Matin and SR3 strains from Pakistan and some other strains from Iran, indicating that these tick species were naturally infected with genetically closely related CCHFV in the region. The presence of CCHFV infection in four different hard tick species was confirmed using RT-PCR in northeast Iran. Part of this infection was attributed to Rh. appendiculatus, which is thus a potential new natural vector of CCHFV in Iran. It is also confirmed by phylogenetic analysis that CCHFV in this region is genetically closely related, even in the different tick species.