Rhipicephalus linnaei (Audouin, 1826) recognised as the “tropical lineage” of the brown dog tick Rhipicephalus sanguineus sensu lato: Neotype designation,redescription, and establishment of morphological and molecular reference

We re-describe the adult stages of Rhipicephalus linnaei (Audouin, 1826), and characterise its diagnostic molecular traits. A male R. linnaei collected in Esna City, Luxor Governorate, Egypt is designated as the neotype. Rhipicephalus linnaei is re-established as a valid tick name and removed from the synonymy list of Rhipicephalus sanguineus (Latreille, 1806). Rhipicephalus linnaei is most similar to R. sanguineus and Rhipicephalus camicasi Morel, Mouchet & Rodhain, 1976 because they share similar elongated comma-like spiracula that are narrowly visible dorsally, and the dorsal prolongation is narrower than the width of the adjacent festoon. The male of R. camicasi is distinguished from R. linnaei by the non-tapering caudal widening of the spiracula. The male of R. sanguineus is distinguished from R. linnaei by shorter extension that does not taper into a long narrow extension of the spiracula. The genital pore atrium of female R. linnaei is broadly U-shaped, while it is a narrower U-shape in R. sanguineus. The remaining species within the R. sanguineus species complex – Rhipicephalus sulcatus Neumann, 1908, Rhipicephalus turanicus Pomerantsev, 1940, Rhipicephalus guilhoni Morel & Vassilades, 1963, Rhipicephalus secundus Feldman-Muhsam, 1952 and Rhipicephalus afranicus Bakkes, 2020, all exhibit spiracula with the dorsal prolongation as wide as the adjacent festoon. The DNA sequence of R. linnaei is most closely related to R. guilhoni. The phylogenetic analysis of mitogenome (mtDNA) sequences including assembled mtDNA from whole genome sequencing of the neotype supports R. linnaei as a well-defined taxon when compared with DNA sequences of other species of the R. sanguineus species complex, in particular: R. sanguineus, R. turanicus, R. secundus and R. camicasi. Molecularly, R. linnaei belongs to the so-called R. sanguineus s.l. "tropical lineage" distributed globally including the Americas, Africa, Europe, Asia and is the only species from R. sanguineus species complex in Australia.     

Pathogens in ticks collected in Israel: II. Bacteria and protozoa found in Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus

Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus are very prevalent in Israel and are known to be vectors of human and animal diseases. The aim of this study was to identify the pathogens found in questing ticks and such parasitizing domestic and wild animals. Ticks were collected from 16 localities in Israel with the flagging technique and by examining dogs, hedgehogs, a badger and a tortoise. Bacterial and protozoal pathogens were analyzed by PCR and sequencing. Overall, 374 R. sanguineus s.l. specimens were collected, out of which 142 by flagging and 132 from six dogs. Rickettsia africae, Rickettsia massiliae, Rickettsia conorii subsp. israelensis, and Anaplasma sp. were identified in ticks collected by flagging, Rickettsia aeschlimannii was found only in specimens collected from dogs, while Ehrlichia sp., Coxiella burnetii, Hepatozoon canis and Leishmania infantum were recorded in ticks collected by flagging and from dogs. Out of 226 specimens of R. turanicus, 124 were collected by flagging, while additional 33 from eight dogs, 64 from seven southern white-breasted hedgehogs (Erinaceus concolor), two from a European badger (Meles meles) and one from a Greek tortoise (Testudo graeca). Out of 65 R. sanguineus s.l. pools 17 (26.2%) had pathogens, while seven of them were positive for one pathogen, and 10 for two pathogens. In 43 R. turanicus pools, R. aeschlimannii R. africae, Rickettsia barbariae, R. massiliae, Anaplasma sp., Ehrlichia sp. and C. burnetii, as well as Babesia microti, B. vogeli, Hepatozoon felis, and L. infantum was detected, while Listeria monocytogenes, Bartonella sp. and Toxoplasma gondii were negative in all R. sanguineus s.l. and R. turanicus pools examined. In conclusion, Babesia microti is reported for the first time in Israel, R. africae, R. aeschlimannii, C. burnetii and L. infantum are reported for the first time in R. sanguineus s.l. and R. turanicus, while H. felis is reported for the first time from R. turanicus in the country.     

Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks.     

Vector competence of Rhipicephalus sanguineus sensu stricto for Anaplasma platys

Anaplasma platys is a Gram-negative, obligate intracellular bacteria that causes canine infectious cyclic thrombocytopenia in dogs. The brown dog tick Rhipicephalus sanguineus sensu lato is presumed to be the vector of A. platys based on the overlap in distribution of R. sanguineus and A. platys infections, detection of A. platys DNA in both flat and engorged field-collected R. sanguineus, and the fact that dogs are primary hosts of both brown dog ticks and A. platys. However, the only study evaluating the vector competence of R. sanguineus for A. platys under controlled laboratory conditions reported an apparent inability of ticks to acquire or maintain the pathogen. In 2016, engorged female Rhipicephalus sanguineus sensu stricto ticks were collected off dogs to start a laboratory tick colony. After one generation of colony maintenance on tick-naïve and pathogen-free New Zealand White rabbits, a rabbit used to feed F1 adults seroconverted to Anaplasma phagocytophilum antigen. PCR and subsequent DNA sequencing identified the presence of A. platys in both the adult ticks fed on this rabbit and their resulting F2 progenies. Retrospective testing of all previous (P and F1) life stages of this colony demonstrated that the infection originated from one field-collected A. platys-infected female whose progeny was propagated in the laboratory and produced the PCR-positive F1 adults. Over the following (F2-F4) generations, the prevalence of A. platys in this colony reached 90–100 % indicating highly efficient transovarial and horizontal transmission, as well as transstadial maintenance, of this pathogen by R. sanguineus s.s.     

The brown dog tick Rhipicephalus sanguineus sensu Roberts, 1965 across Australia: Morphological and molecular identification of R. sanguineus s.l. tropical lineage

The brown dog tick Rhipicephalus sanguineus (Latreille, 1806) is the most widely distributed tick species globally. Throughout the world there are at least two divergent lineages on dogs that are traditionally grouped into what was known as R. sanguineus. The species R. sanguineus was recently redescribed using a neotype reported from countries with a temperate climate. The second lineage distributed in countries with primarily tropical climates is currently designated R. sanguineus s.l. tropical lineage. Here, we present a comprehensive genetic evaluation of Australian brown dog ticks from across the continent that complements the morphological study of R. sanguineus sensu Roberts (1965). A total of 294 ticks were collected from dogs around Australia ― including New South Wales, Queensland, the Northern Territory and Western Australia ― for morphological identification. All ticks were morphologically identified as R. sanguineus sensu Roberts (1965). DNA was isolated from a single leg from morphologically characterised individuals from New South Wales (n = 14), Queensland (n = 18), Northern Territory (n = 7) and Western Australia (n = 13), together with ticks from Fiji (n = 1) and the Seychelles (n = 1) for comparison with Australian ticks. The study revealed three cox1 haplotypes clustered only with R. sanguineus s.l. tropical lineage’. An updated distribution of R. sanguineus sensu Roberts (1965) is compared to the 1965 distribution. In the Australian context, R. sanguineus s.l. has appeared in north-western New South Wales but remains absent from coastal New South Wales. Despite both temperate and tropical climates being present in Australia, only R. sanguineus s.l. tropical lineage was found. The evidence does not support the presence of the strictly defined brown dog tick, R. sanguineus by Nava et al. (2018) in Australia, because the examined ticks are genetically and morphologically distinct. We recommend using the term brown dog tick, R. sanguineus sensu Roberts (1965) for specimens from Australia.     

Ticks in the metropolitan area of Berlin,Germany

A high-resolution city map showing the geographic distribution of 12 tick species (Acari: Argasidae, Ixodidae) that have been recorded from the metropolitan area of Berlin, Germany is presented. A total of 237 tick locations was mapped. These include ten ixodid tick species: Dermacentor reticulatus, Haemaphysalis concinna, Hyalomma rufipes, Ixodes ricinus, Ixodes canisuga, Ixodes hexagonus, Ixodes arboricola, Ixodes frontalis, Ixodes trianguliceps and Rhipicephalus sanguineus sensu lato. The two tick species Hy. rufipes and R. sanguineus s.l. are not endemic to Berlin. Hyalomma rufipes ticks are introduced in Europe with migratory birds from Africa every spring. Rhipicephalus sanguineus s.l. are introduced to Central Europe with dogs that had travelled to or were imported from countries where this tick is endemic. In Germany, they are able to develop and reproduce inside heated buildings. Occurrences of two soft tick species, the pigeon tick Argas reflexus and the short-legged bat tick Carios vespertilionis were also mapped. Other tick species that are likely to be endemic to Berlin and its environs, but for which documented findings or geographical coordinates are lacking, are mentioned. These include the long-legged bat tick I. vespertilionis and the marten tick I. rugicollis documented in Brandenburg, the federal state surrounding Berlin. It can be assumed that if appropriate field studies are carried out, these tick species will also be found in the metropolitan area of Berlin. The high-resolution mapping of all tick species found in a city (like Berlin) forms the basis for further investigations into the impact of climate change and changing land use on ticks and tick-borne diseases, precisely in those habitats where most people will live in the future.     

Cercopithifilaria spp. in ticks of companion animals from Asia: new putative hosts and vectors

Cercopithifilaria bainae, Cercopithifilaria grassi, and Cercopithifilaria sp. II sensu Otranto et al., 2013 tick borne filarioids are typically found in dogs. Among them, Cercopithifilaria bainae has a worldwide distribution according to the occurrence of its tick vector, Rhipicephalus sanguineus sensu lato (s.l.). Nevertheless, in Asian countries, despite the wide presence of this tick species, data on Cercopithifilaria spp. are scant. Therefore, this study aimed to assess the occurrence of these dermal filarioids in ixodid ticks collected on dogs and cats from Asian countries, providing a better epidemiological picture on their distribution in this continent. Ticks (n = 687) of the species Rhipicephalus sanguineus s. l. (n = 667), Rhipicephalus haemaphysaloides (n = 8), Haemaphysalis longicornis (n = 7), Haemaphysalis campanulata (n = 1), Haemaphysalis wellingtoni (n = 2), Haemaphysalis hystricis (n = 1), and Ixodes sp. (n = 1) were collected on dogs and cats under the frame of previous studies in China, India, Indonesia, Malaysia, Singapore, Taiwan, Thailand, the Philippines and Vietnam. Tick samples were molecularly screened for Cercopithifilaria spp. by conventional PCR and real-time PCR using two pair of primers targeting partial sequences of cytochrome c oxidase 1 gene. Overall, Cercophitifilaria spp. DNA was detected in 9.5% (n = 65/687) of the tick specimens tested, with C. bainae being the most prevalent species (8.9%), followed by C. grassii (0.6%). Most Cercophitifilaria spp. positive ticks were collected on dogs (92.3%; 60/65); whereas ticks collected on cats represented 7.7% of the positive specimens. In addition, Cercopithifilaria spp. were mostly detected in R. sanguineus s.l. ticks (96.9%; 63/65), followed by Rhipicephalus haemaphysaloides (3.1%; 2/65). Data herein presented demonstrate the occurrence of dermal tick borne filarioids of the genus Cercopithifilaria in several Asian countries, with C. bainae being the most prevalent species. We also report for the first time the molecular detection of C. bainae in R. sanguineus s.l. ticks collected on cats, as well as in R. haemaphysaloides ticks, suggesting that the biological cycle of this filarioid species may involve other intermediate and definitive hosts than R. sanguineus s.l. and dogs. However, confirmatory studies on the role of other tick species and domestic cats on the biology of C. bainae are advocated.     

Constituting a glutathione S-transferase-cocktail vaccine against tick infestation

Cocktail vaccines are proposed as an attractive way to increase protection efficacy against specific tick species. Furthermore, such vaccines made with different tick antigens have the potential of cross-protecting against a broad range of tick species. However, there are still limitations to the selection of immunogen candidates. Acknowledging that glutathione S-transferases (GSTs) have been exploited as vaccines against ticks and other parasites, this study aimed to analyze a GST-cocktail vaccine as a potential broad-spectrum tick vaccine. To constitute the GST-cocktail vaccine, five tick species of economic importance for livestock industry were studied (Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus microplus, Amblyomma variegatum, and Haemaphysalis longicornis). Tick GST ORF sequences were cloned, and the recombinant GSTs were produced in Escherichia coli. rGSTs were purified and inoculated into rabbits, and the immunological response was characterized. The humoral response against rGST-Rd and rGST-Av showed a stronger cross-reactivity against heterologous rGSTs compared to rGST-Hl, rGST-Ra, and rGST-Rm. Therefore, rGST-Rd and rGST-Av were selected for constituting an experimental rGST-cocktail vaccine. Vaccination experiment in rabbits showed that rGST-cocktail caused 35% reduction in female numbers in a Rhipicephalus sanguineus infestation. This study brings forward an approach to selecting immunogens for cocktail vaccines, and the results highlight rGST–Rd and rGST-Av as potentially useful tools for the development of a broad-spectrum tick vaccine.     

Ticks and tick-borne rickettsiae from dogs in El Salvador,with report of the human pathogen Rickettsia parkeri

Twelve tick species have been reported in El Salvador; however, information regarding ticks infesting domestic dogs is lacking, and pathogenic tick-borne Rickettsia species have never been reported in El Salvador. This work evaluated ticks infesting 230 dogs from ten municipalities in El Salvador from July 2019 to August 2020. A total of 1,264 ticks were collected and identified into five species: Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus microplus, Amblyomma mixtum, Amblyomma ovale, and Amblyoma cf. parvum. The tick R. sanguineus s.l. was the most frequent species in all localities (81.3% of sampled dogs), followed by Amblyomma mixtum (13.0%), Amblyomma ovale (10.9%) and Amblyomma cf. parvum (10.4%). The overall mean intensity of tick infestation was 5.5 ticks/dog. The highest specific mean intensity value was for R. sanguineus s.l. (4.8 ticks/dog), varying from 1.6 to 2.7 ticks/dog for the three Amblyomma species. From a random sample of 288 tick specimens tested molecularly for the presence of rickettsial agents, three spotted fever group Rickettsia were detected: Rickettsia amblyommatis in 90% (36/40) A. mixtum, 46% (11/24) A. cf. parvum, 4% (7/186) R. sanguineus s.l., and 17% Amblyomma spp.; Rickettsia parkeri strain Atlantic rainforest in 4% (1/25) A. ovale; and an unnamed rickettsia agent, designated as ‘Rickettsia sp. ES-A.cf.parvum’, in 4% (1/24) A. cf. parvum. Our finding of R. parkeri strain Atlantic rainforest in A. ovale is highly relevant because this agent has been associated to spotted fever illness in other Latin American countries, where A. ovale is implicated as its main vector. These findings suggest that spotted fever cases caused by R. parkeri strain Atlantic rainforest could be occurring in El Salvador.     

Relationships between the Rhipicephalus sanguineus complex ecology and mediterranean spotted fever epidemiology in France

The authors examine the epidemiologic features of Meditteranean spotted fever in France in light of the bioecological pecularities of each of the three known member of the Rhipicephalus sanguineus tick group (R. sanguineus, R. turanicus, R. pusillus). The results show that R. sanguineus is the main vector. Certain aspects of this tick species are of interest: affinity for man, close contact with humans for a long periods, peak of tick population (preimaginal stages) at the same time as the peak of the disease. The largest populations of R. sanguineus are noted in the endemic zone of human rickettsiosis. The fact that immature stages are more prevalent during the hot season and these forms' ability to bite humans is important and may suggest a role for them in the epidemiology of the disease. The sporadic isolation of this species outside the endemic zone may explain the occurrence of isolated cases of the disease in these areas. We cannot currently exclude vector roles for the two other species, which can parasitize humans, though none of our data supports this hypothesis.     

Tick species infesting cattle in the central region of Burkina Faso: Presence of Rhipicephalus microplus less than ten years after its first identification in the Southwest part of the country

Ticks are among the most important vectors for medical and veterinary importance in African countries, representing thus an obstacle for development and improvement of livestock production. The central part of Burkina Faso is an arid region which has a low diversity of tick species and in which Rhipicephalus microplus has been absent. This study aimed to examine ticks infesting cattle within this region, focusing on the peripheral area of Ouagadougou. Tick sampling was undertaken monthly on 30 selected cattle in the state breeding station of Loumbila, from June 2017 to February 2018. Morphological identification was carried out based on previous established keys and catalogue. A total of 1,076 ticks belonging to four genera and seven species were collected. Amblyomma variegatum (50.5%) was the most abundant species followed by Hyalomma rufipes (27.4%), Hyalomma truncatum (13.7%), Rhipicephalus decoloratus (4.5%), Hyalomma impressum (2.4%), Rhipicephalus microplus (1.5%), and Rhipicephalus sanguineus sensu lato (0.1%). The results suggested a decrease of A. variegatum and Hyalomma spp. load compared to data collected in 1996 from the same study area.Presence of R. microplus and R. decoloratus has been highlighted in the studied area for the first time. This finding highlights the relevance of regular investigations on tick species infesting livestock utilizing collaboration with different stakeholders.     

DNA barcoding of hard ticks (Ixodidae), notes on distribution of vector species and new faunal record for Croatia

Molecular methods are increasingly being utilized for accurate identification of ticks (Acari: Ixodidae), especially in cases of morphologically highly similar species. In this study, we performed molecular research of the tick fauna in Croatia using DNA barcoding method. Ticks were sampled in three biogeographical regions and thirteen species were recorded: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma marginatum, Ixodes frontalis, Ixodes hexagonus, Ixodes kaiseri, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus. Ixodes kaiseri is for the first time recorded in the fauna of Croatia. Of the thirteen hard tick species analyzed in this study, pathogens from different groups (bacteria, protozoa and viruses) have been detected in eight species in Croatia so far. For the important vector species R. sanguineus s.s., new distributional data for Croatia are given. The standard COI barcoding region was amplified, and the sequences were analyzed by species delimitation methods together with the sequences of conspecific and congeneric species from the public BOLD database. Our specimens of H. punctata represent a new, genetically distinct MOTU. A brief overview of the available public DNA barcoding data for Ixodidae is presented, highlighting the need for an integrative approach for the clarification of the taxonomic status of problematic Ixodid taxa. The results provide a basis for the establishment of a molecular data platform for the Ixodidae of the Croatian fauna.     

Mediterranean spotted fever: A preliminary tick field study

We studied the prevalence of rickettsia-like organisms infection in different tick species (3154 adults) collected in several areas of the Ebro valley (Spain). The Gimenez stain showed a prevalence of rickettsia-like organisms in 38.6% of the studied ticks. Using the immunofluorescence assay, only two species, Rhipicephalus sanguineus (16.4%) and R. pusillus (19.9%) have shown organisms compatible with spotted fever group rickettsiae.     

Lesser protein degradation machinery correlates with higher BM86 tick vaccine efficacy in Rhipicephalus annulatus when compared to Rhipicephalus microplus

Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody–antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody–BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody–antigen interactions and higher vaccine efficacy. These results have important implications for tick vaccine research, indicating that not only genetic differences, but also physiological factors may influence tick vaccine efficacy.     

Distribution of ticks in Bosnia and Herzegovina

Ticks are one of the important groups of parasites, whose populations have increased in Europe in the last 20 years and, as vectors of many emerging diseases, are a major threat to human and animal health. Bosnia and Herzegovina has both climate and environmental conditions favorable for ticks. Given this risk, the aim of this study was to identify tick species currently present in this country and to compare the findings with previous studies, in order to update information on their spatial distribution and other parameters of occurrence.From March 2017 to April 2020, ticks were collected in 42 sampling areas (municipalities) throughout the country. Prespecified identification keys were used for identifying the ticks to the species level.A total of 6193 tick specimens from the Ixodidae family were collected from animal hosts or from vegetation. Ixodes ricinus was the most dominant tick species found. However, our results indicate a decrease in the relative contribution of this tick species to the overall abundance of tick populations. Dermacentor marginatus, which almost doubled in abundance compared to earlier studies, is the second most common tick species. We further confirmed the presence of Dermacentor reticulatus, Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato, Hyalomma marginatum and Haemaphysalis punctata. Ixodes hexagonus, Ixodes canisuga and Haemaphysalis concinna, which were previously reported at low abundance were not found amongst our specimens.     

Cloning and characterization of Rhipicephalus appendiculatus voraxinα and its effect as anti-tick vaccine

Male tick-derived voraxinα and voraxinβ, a pair of testicular proteins, are transferred to female via copulation to stimulate female blood feeding in the tick Amblyomma hebraeum (A. hebraeum). Immunized animals with recombinant (r-)voraxinα and voraxinβ have been shown as highly resistant to the tick infestation. In this study, we describe the cloning and characterization of voraxinα homologue from the tick Rhipicephalus appendiculatus (R. appendiculatus), the major vector for East Coast fever in Eastern Africa. The sequence analysis of the R. appendiculatus voraxinα indicated that the deduced amino acid sequence had high similarity with voraxinα of the tick A. hebraeum and Dermacentor variabilis, suggesting that voraxinα is conserved in different tick genera. Quantitative RT-PCR and Western blotting analysis showed that male voraxinα was predominantly expressed in testis and its expression was induced by blood feeding. R. appendiculatus voraxinα was not secreted into the host during tick feeding and was detected in mated female hemolymph as measured by Western blotting. Preliminary vaccination of rabbits with r-voraxinα elicited the humoral immunity and conferred protective immunity against female ticks, resulting in the reduced fed weight. These results suggest that r-voraxinα could be a good candidate as anti-tick vaccine.     

Crimean-Congo hemorrhagic fever virus in ticks collected from livestock in Albania

Albania is a Balkan country endemic for Crimean-Congo hemorrhagic fever (CCHF). It was shown previously that CCHF virus (CCHFV) sequences from Albanian patients cluster into Europe 1 clade. Aim of the present study was to test for CCHFV ticks collected in several regions of Albania, and to determine the genetic lineage(s) of the CCHFV strains in relation with their geographic distribution. A total of 726 ticks (366 Hyalomma marginatum, 349 Rhipicephalus bursa and 11 Rhipicephalus sanguineus) collected from livestock during 2007–2014 were included in the study. Thirty of 215 (13.9%) tick pools were positive for CCHFV. Lineage Europe 1 was detected in H. marginatum ticks collected in the endemic region of Albania, while lineage Europe 2 was detected mainly in R. bursa ticks in various regions of the country. Both genetic lineages were detected in the CCHF endemic area (northeastern Albania), while only Europe 2 lineage was detected in the south of the country. A higher genetic diversity was seen among Europe 2 than Europe 1 Albanian sequences (mean distance 3.7% versus 1%), suggesting a longer evolution of AP92-like strains (Europe 2) in their tick hosts. The present study shows that besides CCHFV lineage Europe 1, lineage Europe 2 is also present in Albania. Combined with results from recent studies, it is concluded that lineage Europe 2 is widely spread in the Balkans and Turkey, and is associated mainly with R. bursa ticks (at least in this region). Its pathogenicity and impact to the public health remain to be elucidated.     

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.     

Characterization of ferritin 2 for the control of tick infestations

Ixodes ricinus is one the most abundant tick species in Europe and these ticks transmit pathogens causing human and animal diseases. The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, a limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, the tick iron metabolism pathway was targeted in an effort to identify new tick protective antigens. Recombinant I. ricinus (IrFER2) and Rhipicephalus microplus (RmFER2) ferritin 2 proteins were expressed in Escherichia coli and used to immunize rabbits and cattle, respectively. Vaccination with IrFER2 reduced I. ricinus tick numbers, weight and fertility in rabbits with an overall vaccine efficacy (E) of 98%. Control of cattle tick, R. microplus and Rhipicephalus annulatus infestations was obtained in vaccinated cattle with overall E of 64% and 72%, respectively. Notably, the efficacy of the RmFER2 vaccine was similar to that obtained with Bm86 against R. microplus. These collective results demonstrated the feasibility of using ferritin 2 to develop vaccines for the control of tick infestations.