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This study presents a comprehensive investigation into the evolutionary trajectories of Rhipicephalus ticks (Ixodidae) through the interpretation of molecular phylogenetics, elucidating their chromatographic spectrum. The use of advanced chromatographic tools in this study explored the dynamics chemical profiling, providing valuable insights into the evolutionary history and ecological adaptations. Prevalence of Rhipicephalus ticks was 4.5% in sheep and 3.9% in goats. The ITS2 sequence of the Rhipicephalus sanguineus (OK642408) and Rhipicephalus microplus (OK642409) form a distinct clade with sequences from other countries. The 16S rRNA sequences of R. sanguineus (OK560870) clustered with sequences form three lineages, tropical, temperate, and south-eastern. The Cox I gene-identified Rhipicephalus turanicus (OK623472) and R. microplus (OK623463) form separate clades with sequences. The HPLC chromatogram of tick samples reveals a diverse array of identified hydrocarbons, explained the complex chemical composition of their exoskeletons. This analytical approach provides valuable insights into the specific hydrocarbon profiles, allowing for potential applications in species differentiation, ecological studies, and a deeper understanding of the functional roles played by hydrocarbon compounds in tick physiology. The findings revealed the potential of applying molecular phylogenetics tools with chromatography not only to enhance our understanding of tick evolution but also to inform strategies for disease control and management in regions where Rhipicephalus ticks (Ixodidae) are endemic. RESEARCH HIGHLIGHTS: Chemical mapping utilizing advanced chromatographic techniques. Scanning microscopic insights high-resolution scanning tool to observe structural and morphological features of ticks at a molecular level. Molecular phylogeny data elucidate the evolutionary relationships among tick species., (© 2024 Wiley Periodicals LLC.)

We describe all the life stages of Rhipicephalus hibericus n. sp., provide the types, and present molecular support for a new species of the Rhipicephalus sanguineus sensu lato group, present in southwestern Europe, that has been historically confused with Rhipicephalus turanicus Pomerantzev, 1940. A new name is proposed for this taxon because it was impossible to ascribe to types of already described species in the group, deposited for more than 100 years in natural history institutions. The males have a dorsum showing deep and coarse punctations (absent in Rhipicephalus sanguineus sensu stricto) and adanal plates with large punctations (absent in R. sanguineus s.s.); the tail of the spiracular plate is as wide as the closest festoon (half the width in R. sanguineus s.s.). Females have large punctations in dorsal fields, a wide spiracular plate, and a "V" shaped genital opening; such a combination of characters cannot be found in other species of the group. Immatures are described from specimens collected on hosts (Rodentia and Eulipotyphla). Both larvae and nymphs are markedly smaller than R. sanguineus s.s. Nymphs display long, backward-projected auriculae; larvae are almost half the size of R. sanguineus s.s. The new species can hybridize with R. sanguineus s.s. in laboratory colonies producing an unfertile F2, laying brown and dry eggs that did not hatch. Phylogenetic analysis of partial coxI gene sequences placed R. hibericus in a well-supported clade with other sequences of R. sanguineus s.l. from Portugal, as a sister clade of R. sanguineus s.s. The new species does not belong to the R. turanicus group of species. Both 12S and 16S partial gene sequences were not as precise in the correct phylogenetic placement of R. hibericus, in part probably due to the existence of erroneously identified sequences in GenBank©. This description, together with the previous reinstatement of Rhipicephalus secundus and Rhipicephalus rutilus, and the description of the neotypes of R. sanguineus s.s. should help researchers to adequately identify their collections. Our findings demonstrate that R. turanicus is absent in southwestern Europe. Old collections should be re-examined to provide the actual range of the new species., Competing Interests: Declaration of competing interest Authors declare no conflict of interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background: Ticks are obligate hematophagous ectoparasites involved in transmitting viruses of public health importance. The objective of this work was to identify the Jingmen tick virus in hard ticks from the Colombian Caribbean, an arbovirus of importance for public health., Methods: Ticks were collected in rural areas of Córdoba and Cesar, Colombia. Taxonomic identification of ticks was carried out, and pools of 13 individuals were formed. RNA extraction was performed. Library preparation was performed with the MGIEasy kit, and next-generation sequencing (NGS) with MGI equipment. Bioinformatic analyses and taxonomic assignments were performed using the Galaxy platform, and phylogenetic analyses were done using IQ-TREE2., Results: A total of 766 ticks were collected, of which 87.33% (669/766) were Rhipicephalus microplus, 5.4% (42/766) Dermacentor nitens, 4.2% (32/766) Rhipicephalus linnaei, and 3.0% (23/766) Amblyomma dissimile. Complete and partial segments 1, 2, 3, and 4 of Jingmen tick virus (JMTV) were detected in the metatranscriptome of the species R. microplus, D. nitens, and A. dissimile. The JMTVs detected are phylogenetically related to JMTVs detected in Aedes albopictus in France, JMTVs detected in R. microplus in Trinidad and Tobago, JMTVs in R. microplus and A. variegatum in the French Antilles, and JMTVs detected in R. microplus in Colombia. Interestingly, our sequences clustered closely with JMTV detected in humans from Kosovo., Conclusions: JMTV was detected in R. microplus, D. nitens, and A. dissimile. JMTV could pose a risk to humans. Therefore, it is vital to establish epidemiological surveillance measures to better understand the possible role of JMTV in tropical diseases., (© 2024. The Author(s).)

Ticks are a public health threat due to their tendency to spread pathogens that affect humans and animals. With reports of Rhipicephalus (Boophilus) microplus invasion in neighbouring countries, there is the risk of this species invading Ghana through livestock trade. Previous identification of tick species in Ghana has been based on morphological identification, which can be ineffective, especially with damaged tick specimens or engorged nymphs. This study focused on the Kassena-Nankana District, which serves as a trade route for cattle into Ghana, to determine the presence of R. microplus. Three genera of ticks were identified as Amblyomma (70.9%), Hyalomma (21.3%) and Rhipicephalus (7.8%). The engorged nymphs that could not be identified morphologically were analyzed using primers that target the mitochondrial 16S rRNA gene. This study reports the first record of R. (B.) microplus in Ghana. Furthermore, R. microplus constituted 54.8% of the Boophilus species collected in this study. This finding is an addition to the diverse tick species previously collected in Ghana, most of which are of veterinary and public health importance. With reports of acaricide resistance in R. microplus and its role in spreading infectious pathogens, the detection of this species in Ghana cannot be overlooked. Nationwide surveillance will be essential to ascertain its distribution, its effects on cattle production, and the control measures adopted., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: The ixodid tick genera Rhipicephalus and Haemaphysalis contain several species of medical and/or veterinary importance, but their diversity in some regions of the world remains under-explored. For instance, very few modern studies have been performed on the taxonomy of these genera on the Arabian Peninsula., Methods: In this study, we trapped small mammals in the 'Asir Mountains of south-western Saudi Arabia and collected tick specimens for morphological examination and molecular barcoding, targeting three mitochondrial loci: cox1, 16S rRNA and 12S rRNA., Results: We obtained a total of 733 ticks (608 Haemaphysalis spp. and 125 Rhipicephalus spp.) from 75 small mammal hosts belonging to six species. All tick specimens were immature except for nine adults recovered from a hedgehog (Paraechinus aethiopicus). Morphologically, the Rhipicephalus ticks resembled R. camicasi, but the Haemaphysalis ticks showed differences in palp morphology compared with species previously described from Saudi Arabia. Phylogenetic analysis and automatic barcode gap discovery identified a novel clade of Rhipicephalus sp. representing most of the nymphs. This was most closely related to R. leporis, R. guilhoni and R. linnaei. The adult ticks and a small proportion of nymphs clustered with R. camicasi sequences from a previous study. Finally, the Haemaphysalis nymphs formed two distinct clades that were clearly separated from all reference sequences but closest to some African species., Conclusions: This apparent high level of tick diversity observed in a single study site of only ~ 170 km 2 , on a relatively small number of hosts, highlights the potential for the discovery of new tick species on the Arabian Peninsula., (© 2021. The Author(s).)

Rhipicephalus are a species-diverse genus of ticks, mainly distributed in the Afrotropics with some species in the Palearctic and Oriental regions. Current taxonomic consensus comprise nine informal species groups/lineages based on immature morphology. This work integrates biogeographic, ecological and molecular lines of evidence to better understand Rhipicephalus evolution. Phylogenetic analysis based on four genes (12S, 16S, 28S-D2 and COI) recovered five distinct clades with nine descendant clades that are generally congruent with current taxonomy, with some exceptions. Historical biogeography is inferred from molecular divergence times, ancestral distribution areas, host-use and climate niches of four phylogenetically significant bioclimatic variables (isothermality, annual, seasonal and diurnal temperature range). Novel hosts enabled host-linked dispersal events into new environments, and ticks exploited new hosts through nested predator-prey connections in food webs. Diversification was further induced by climate niche partitioning along gradients in temperature range during off-host periods. Ancestral climate niche estimates corroborated dispersal events by indicating hypothetical ancestors moved into environments with different annual and seasonal temperature ranges along latitudinal gradients. Host size for immature and adult life stages was important for dispersal and subsequent diversification rates. Clades that utilise large, mobile hosts (ungulates and carnivores) early in development have wider geographic ranges but slower diversification rates, and those utilising small, less mobile hosts (rodents, lagomorphs and afroinsectivores) early in development have smaller ranges but higher diversification rates. These findings suggest diversification is driven by a complex set of factors linked to both host-associations (host size, ranges and mobility) and climate niche partitioning along annual and seasonal temperature range gradients that vary with latitude. Moreover, competitive interactions can reinforce these processes and drive speciation. Off-host periods facilitate adaptive radiation by enabling host switches along nested predator-prey connections in food webs, but at the cost of environmental exposure that partitions niches among dispersing progenitors, disrupting geneflow and driving diversification. As such, the evolution and ecological niches of Rhipicephalus are characterised by trade-offs between on- and off-host periods, and these trade-offs interact with nested predator-prey connections in food webs, host-use at different life stages, as well as gradients in annual and seasonal temperature ranges to drive adaptive radiation and speciation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Birds are major hosts for many tick species (Acari: Ixodidae, Argasidae), and their role is especially important in transporting ticks over large distances along their seasonal migratory routes. Accordingly, most studies across Europe focus on the importance of avian hosts in tick dispersal, and less emphasis is laid on resident birds and their role in supporting tick life cycles. Eurasian eagle owls (Bubo bubo) exemplify the latter, but all the few studies on their tick infestation were carried out in Western Europe and even those did not involve a large sample size and did not assess infestation prevalence in natural habitats., Methods: In this study, 320 ixodid ticks were collected from nestlings of Eurasian eagle owls during the period 2018-2020 in Bulgaria in south-eastern Europe. These ticks were analysed morphologically, and selected specimens molecularly based on cytochrome c oxidase subunit I (cox1) gene. The effects of environmental and habitat-related conditions and of the species of prey eaten by eagle owls on tick infestation were also evaluated., Results: The majority of ticks were identified as adults of Rhipicephalus turanicus (n = 296). In addition, 15 Hyalomma marginatum (three males, 11 nymphs and a larva), one female of Haemaphysalis erinacei and of Ha. punctata, and a nymph of Ixodes ricinus were found. Among R. turanicus, two distinct morphotypes were observed, but they do not form a monophyletic clade in the phylogenetic tree based on the mitochondrial gene cox1. We found a positive correlation between the total number of ticks on nestlings from a particular nest and the number of medium-sized to large prey mammals brought to the nestling owls. Also, the most important predictor for tick abundance was the effect of the extent of arable land (negative), while forests and grasslands contributed less, with no effect observed in case of urbanized areas and watercourses., Conclusions: The intensity of tick infestation can be high on nestling Eurasian eagle owls (mean intensity 16.59 ticks/nestling). In this study, five different tick species were recorded, among which R. turanicus dominated. Two male morphotypes of this tick species were found, but their morphological differences were not reflected by genetic diversity or phylogenetic clustering. The most important factor determining tick abundance was the land-use structure.

Background: Some tick species are invasive and of high consequence to public and veterinary health. Socioeconomic development of rural parts of the USA was enabled partly through the eradication by 1943 of cattle fever ticks (CFT, Rhipicephalus (Boophilus) annulatus and R. (B.) microplus). The southern cattle fever ticks (SCFT, R. (B.) microplus) remain a real and present threat to the USA animal agriculture because they are established in Mexico. Livestock-wildlife interactions in the Permanent Quarantine Zone (PQZ) established by the century-old Cattle Fever Tick Eradication Programme (CFTEP) in south Texas endanger its operations., Methods: We describe a spatially-explicit, individual-based model that simulates interactions between cattle, white-tailed deer (WTD, Odocoileus virginianus), and nilgai (Boselaphus tragocamelus) to assess the risk for SCFT infestations across the pathogenic landscape in the PQZ and beyond. We also investigate the potential role of nilgai in sustaining SCFT populations by simulating various hypothetical infestation and eradication scenarios., Results: All infestation scenarios resulted in a phase transition from a relatively small proportion of the ranch infested to almost the entire ranch infested coinciding with the typical period of autumn increases in off-host tick larvae. Results of eradication scenarios suggest that elimination of all on-host ticks on cattle, WTD, or nilgai would have virtually no effect on the proportion of the ranch infested or on the proportions of different tick habitat types infested; the entire ranch would remain infested. If all on-host ticks were eliminated on cattle and WTD, WTD and nilgai, or cattle and nilgai, the proportions of the ranch infested occasionally would drop to 0.6, 0.6 and 0.2, respectively. Differences in proportions of the ranch infested from year to year were due to primarily to differences in winter weather conditions, whereas infestation differences among tick habitat types were due primarily to habitat use preferences of hosts., Conclusions: Infestations in nilgai augment SCFT refugia enabled by WTD and promote pest persistence across the landscape and cattle parasitism. Our study documented the utility of enhanced biosurveillance using simulation tools to mitigate risk and enhance operations of area-wide tick management programmes like the CFTEP through integrated tactics for SCFT suppression.

Rhipicephalus turanicus ticks are widely distributed across the Palearctic and Afrotropics. These two continental populations display differences in morphological characters that raise the question of a potential species boundary. However, the taxonomic status of these morphologically divergent lineages is uncertain because R. turanicus from Cyprus and Zambia have been shown to interbreed and produce fertile hybrids. We employ integrative taxonomy that considers data from mtDNA sequences (12S and 16S rDNA), geographic distribution, traditional (qualitative) morphology, as well as shape outlines of female spiracles and male adanal plates measured in a geometric morphometric framework (quantitative morphology) to resolve this taxonomic issue. Molecular lines of evidence (12S and 16S rDNA) support taxonomic separation between ticks sampled in the Afrotropics and the Palearctic. This is corroborated by qualitative and quantitative morphology. Within the Palearctic, two sub-lineages were recovered based on sequence data that loosely correspond to southern Europe and the Middle East/Asia. One new species, Rhipicephalus afranicus n. sp. is described from South Africa with a geographic distribution that extends into eastern Africa. This leaves R. turanicus sensu lato comprised of two lineages located in southern Europe and the Middle East/Asia. The type locality for R. turanicus is in Uzbekistan, thus the Middle East/Asia lineage is considered R. turanicus sensu stricto. Detailed descriptions are provided for R. afranicus n. sp. and R. turanicus sensu stricto together with high resolution images. Speciation is attributed to recent Sahara desert expansion that formed a natural barrier to dispersal approximately 5-7 million years ago. However, reproductive potential between these two species suggests that divergence time and mode of speciation were not sufficient for the development of reproductive isolation. We suggest speciation was complicated by divergence and population reintegration events driven by oscillating climatic conditions contributing to reticulate evolution and maintenance of compatibility between reproductive mechanisms. This study represents an integrative (iterative) approach to delimiting Rhipicephalus spp., and provides the first application of shape outlines for female spiracles and male adanal plates measured in a geometric morphometric framework, applied to testing species boundaries between ticks., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Equine piroplasmosis, caused by the protozoan pathogens Theileria equi or Babesia caballi , is endemic in many (sub-)tropical regions worldwide, including Southern Europe. In Germany, it mainly plays a role as an imported parasitosis. Nevetherless, tick species with known vector potential for equine piroplasms occur in Germany. Thus, a long-term establishment of autochthonous cycles after introduction via infected equids or ticks cannot be excluded. In the present case, 2 horses developed clinical T. equi infection after returning from a trail riding trip to southern France. During treatment, a feeding tick was discovered on one of the horses, which was identified as a Rhipicephalus bursa specimen. This tick species cannot establish in Germany under the current climatic conditions; nevertheless, this discovery illustrates the potential of introducing infected ticks to Germany. The 2 horses were treated with imidocarb (3 mg/kg i. m.; 3 treatments in 3-day intervals) and no adverse drug effects were recorded. To prevent establishment of autochthonous infection cycles in Germany as well as a chronic manifestation of piroplasmosis, treatment should aim at eliminating the parasites despite possible adverse effects of imidocarb., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

A major risk factor for the spread of livestock diseases and their vectors is the uncontrolled transboundary movement of live animals for trade and grazing. Such movements constrain effective control of tick-transmitted pathogens, including Theileria parva. Only limited studies have been undertaken to identify ticks and tick-borne diseases (TTBDs) affecting cattle in central African countries, including Cameroon. We hereby report the collection of baseline data on the prevalence of T. parva in Cameroon through a countrywide cross-sectional survey, conducted in 2016, involving collection of blood samples from cattle from 63 sites across the five agro-ecological zones (AEZs) of the country. ELISA-based surveillance of infected cattle was performed on 479 randomly selected samples and revealed specific antibodies to T. parva in 22.7% and T. mutans in 41.1% of cattle. Screening of 1,340 representative DNA samples for the presence of T. parva identified 25 (1.86%) positives using a p104 antigen gene-based nested PCR assay. The positives were distributed across agro-ecological zones I, II, III and V. None of the p104 positive cattle exhibited clinical symptoms of East Coast fever (ECF). Using reverse line blot (RLB), 58 (4.3%) and 1,139 (85%) of the samples reacted with the T. parva and T. mutans oligonucleotide probes, respectively. This represents the first report of T. parva from Cameroon. Surprisingly, no Rhipicephalus appendiculatus ticks, the main vector of T. parva, were identified in a parallel study involving comprehensive morphological and molecular survey of tick species present in the country. Only two of the 25 p104 positive cattle were PCR-positive for the CD8+ T-cell target schizont-expressed antigen gene Tp1. Cloning and sequencing of Tp1 amplicons revealed sequence identity with the reference T. parva Muguga. This new finding raises serious concerns of a potential spread of ECF into the central African region., (© 2020 Blackwell Verlag GmbH.)

The morphological diversity of African ticks of the genus Rhipicephalus and subgenus Boophilus have been studied in detail. However, their taxonomy remains poorly resolved with limited molecular studies performed to improve inter-species discrimination. Herein, ribosomal cytochrome c oxidase I (COI), 12S ribosomal DNA (12S rDNA) and nuclear ribosomal DNA internal transcriber spacer 2 (ITS2) were analyzed in Rhipicephalus tick populations in Kenya. While the morphological and molecular criteria separated R. e. evertsi, R. pulchellus and R. appendiculatus from other members of the genus, except the morphologically similar sibling species R. zambeziensis, this was not the case for other tick populations. COI sequences of Rhipicephalus ticks from Ruma National Park (RNP) in Southwestern Kenya, that were morphologically similar to R. praetextatus/R. simus, a formed distinct clade and barcode gap group. 12S rDNA haplotypes of this population were 99% identical to a GenBank accession of R. muhsamae which is thought to be endemic in West and Central Africa. However, the ITS2 locus indicated that the RNP samples were genetically closest to ticks identified morphologically as R. praetextatus. The COI and 12S rDNA haplotype sequences of R. praetextatus clustered closely with R. simus reference sequences though the two species occurred in distinct barcode gap groups. Our results suggest that the R. simus/R. praetextatus/R. muhsamae comprise a closely related tick species complex found across sub-Saharan Africa and includes the yet to be described RNP population. More studies on the biology, ecology and genomics of all life stages of tick species in the complex may clarify their taxonomic status. A continent-wide study that combines morphology, DNA marker sequencing and emerging methods, such as mass spectrometry and whole-genome resequencing may reveal the diversity and distribution of taxa within the genus Rhipicephalus in sub-Saharan Africa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rhipicephalus microplus and R. decoloratus are one-host ticks that preferentially feed on cattle. They are capable of transmitting various tick-borne pathogens which may be detrimental to the agricultural and livestock industry in South Africa. Previous studies have shown that R. microplus forms five lineages in the R. microplus complex, segregating into different geographical areas based on mitochondrial markers. This study examined the phylogenetic relationship within and between R. microplus and R. decoloratus using the nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit I (COI) genes. The results showed that the nuclear ITS2 marker is informative for interspecific variation but lacks the resolution for intraspecific variation. Analysis of the mitochondrial COI gene revealed that R. microplus ticks from South Africa grouped into a clade comprised of ticks from Asia and South America. The population structure of these two tick species was also investigated using novel microsatellite markers. Population structure analyses revealed that both the R. microplus and R. decoloratus populations presented with two genetic clusters. Rhipicephalus microplus ticks from the Kwa-Zulu Natal (KZN) province belonged to cluster 1, and those from the Eastern Cape (EC) province predominantly grouped into cluster 2. No observable population structure was noted for R. decoloratus. The overlap of genetic clusters in both species could be attributed to inbreeding between the regions by unrestricted movement of cattle across provinces. Such movement promotes tick mobility, gene flow and the homogenisation of tick populations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Many tick species have great morphological similarity and are thus grouped into species complexes. Molecular methods are therefore useful in the classification and identification of ticks. However, little is known about the genetic diversity of hard ticks in China, especially at the subspecies level. Tengchong is one of the epidemic foci of tick-borne diseases in China, but the tick species inhabiting the local area are still unknown., Methods: Eighteen villages in Tengchong County, China, were selected for sampling carried out from September to October 2014. Infesting hard ticks were removed from the body surface of domestic animals and questing ticks were collected from grazing fields. After morphological identification, molecular characteristics of each tick species were analyzed based on both 16S rRNA and cytochrome c oxidase subunit 1 (cox1) gene fragments., Results: Six tick species were identified based on morphology: Rhipicephalus microplus, R. haemaphysaloides, Ixodes ovatus, Haemaphysalis longicornis, H. shimoga and H. kitaokai. Phylogenetic analysis using the cox1 gene revealed that R. microplus ticks from the present study belong to clade C. For tick samples of both R. haemaphysaloides and I. ovatus, three phylogenetic groups were recognized, and the intergroup genetic distances exceeded the usual tick species boundaries. Haemaphysalis longicornis ticks were clustered into two separate clades based on the cox1 gene. For ticks from both H. shimoga and H. kitaokai, two phylogenetic groups were recognized based on the phylogenetic analysis of the 16S rRNA gene, and the intergroup genetic distances also exceeded the known boundaries for closely related tick species., Conclusions: According to molecular analyses, new species or subspecies closely related to R. haemaphysaloides, I. ovatus, H. shimoga and H. kitaokai probably exist in the China-Myanmar border Tengchong County, or these ticks form species complexes with highly divergent mitochondrial lineages. Morphological comparisons are warranted to further confirm the taxonomic status of these tick species.

Different infestation patterns by ixodid ticks were studied in three sheep breeds in Tunisia: Barbarine, Queue Fine de l'Ouest and their cross-bred animals. During one year, 700 sheep were monitored and examined for tick infestation. A total of 722 ticks were collected from sheep ears. The most frequent tick species was by far Rhipicephalus sanguineus sensu lato (99%) and there were few specimens of Rhipicephalus bursa (1%) (p < 0.001). Overall infestation prevalence was estimated at 10.4%. The lowest infestation prevalence was in Barbarine sheep (7.3%), followed by Queue Fine de l'Ouest (16.7%) and the highest prevalence was in cross-bred sheep (19.1%) (p < 0.001). Mean overall infestation intensity was 1.6 ticks/sheep: lowest in Barbarine (1.4), followed by Queue Fine de l'Ouest (1.7) and cross-bred sheep (1.8). Similarly, abundance was lowest in Barbarine sheep (0.1), and was 0.3 in Queue Fine de l'Ouest and cross-bred animals. The results demonstrated a reduced infestation, possibly due to reduced attractiveness and/or increased resistance to tick infestation, of the Barbarine breed compared with the other two breeds. Further behavioural, genetic and molecular studies are needed to explain the mechanisms for the lower infestation indicators., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called "temperate" and "tropical" lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal)., Methods: Ticks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted., Results: Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically., Conclusions: The temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks.

Ticks of the Boophilus subgenus, classified in the genus Rhipicephalus, are widespread in subtropical and tropical regions, but knowledge on their distribution in Asia is fragmentary. The most important representatives belong to the Rhipicephalus (Boophilus) microplus complex, which is composed of species that share many morphological similarities and are therefore difficult to distinguish. In this study, adult Rhipicephalus (Boophilus) ticks from Bangladesh, Myanmar and Pakistan were subjected to phylogenetic and Scanning Electron Microscopy (SEM) analyses. Phylogenetic analyses of cytochrome oxidase 1 (cox1), internal transcribed spacer 2 (ITS2) and 12S rRNA gene sequences confirmed that the R. microplus complex consists of at least five taxa: R. annulatus, R. australis, and R. microplus clades A-C. Ticks from Bangladesh, Myanmar and Pakistan were assigned to R. microplus clade C. SEM images revealed a wide range of variation in the morphology of the clade C adults, including morphological features previously identified as critical for distinguishing R. microplus from R. australis, which is illustrative for the complications in identifying species within the R. microplus complex using morphology only. Further morphological, genomic and crossbreeding studies are required to elucidate the species status of R. microplus clades A-C., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

A multigene phylogeny including 24 Rhipicephalus species from the Afrotropical and Mediterranean regions, based on mitochondrial DNA genes (COI, 12S and 16S), was constructed based on Bayesian inference and maximum likelihood estimations. The phylogenetic reconstruction revealed 31 Rhipicephalus clades, which include the first molecular records of Rhipicephalus duttoni (Neumann), and Rhipicephalus senegalensis (Koch). Our results support the R. pulchellus, R. evertsi and R. pravus complexes as more phylogenetically close to Rhipicephalus (Boophilus) than to the remaining Rhipicephalus clades, suggesting two main monophyletic groups within the genus. Additionally, the phenotypic resembling R. sanguineus s.l. and Rhipicephalus turanicus (Pomerantsev) are here represented by nine clades, of which none of the R. turanicus assemblages appeared as distributed in the Iberian Peninsula. These results not only indicate that both species include more cryptic diversity than the already reported, but also suggest that R. turanicus distribution is less extended than previously anticipated. This analysis allowed to improve species identification by exposing cryptic species and reinforced mtDNA markers suitability for intra/inter-species clarification analyses. Incorporating new species molecular records to improve phylogenetic clarification can significantly improve ticks' identification methods which will have epidemiologic implications on public health.

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53-4.84, 3.57-4.92, 3.57-4.07, 3.57-4.39 and 3.18-4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.

Ticks are considered to be second only to mosquitoes as vectors of diseases. In recent years, severe fever with thrombocytopenia syndrome, a new emerging tick-borne disease has been detected in many areas of China, including Shandong Province, Eastern China. Here, we report the tick species diversity based on surveys between 2014 and 2016 covering 16 locations in seven cities of Shandong. Based on DNA barcoding, 1859 ticks belonging to three species were identified: Haemaphysalis longicornis, Rhipicephalus turanicus and Haemaphysalis verticalis. Samples of the same species clustered together in a neighbor-joining phylogenetic tree, with intraspecific distances between 0 and 3.0% and interspecific distances ranged between 15.5 and 24.3%. Goats and dogs were the major hosts of ticks and H. longicornis was regarded as predominant tick species of Shandong. In order to reduce tick populations and prevent tick-borne diseases, effective control measures should be implemented on human and domestic animals, respectively.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different age groups. Tick species were identified morphologically, and the identity of R. appendiculatus was confirmed by DNA barcoding. Overall, the T. parva infection rate in R. appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T. parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T. parva. This is the first molecular confirmation of T. parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T. parva was determined as N⁰06.17.792, about 242 Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed., (© 2016 Blackwell Verlag GmbH.)

Background: The taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal., Methods: A total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens., Results: The ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.) CONCLUSIONS: These results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this.

The filamentous fungus Metarhizium anisopliae is an arthropod pathogen used in the biological control of pests in agriculture and livestock. The effect of the fungus M. anisopliae on ticks has been shown in experiments in vitro. The aim of the present study was to compare the susceptibility of different field isolates of Rhipicephalus microplus to M. anisopliae. A total of 67 field isolates were evaluated. Rhipicephalus microplus larvae were immersed in an M. anisopliae suspension (10 8 conidia/mL) for 5min. The median lethal times (LT 50 ) ranged from 2.6 to 24.9days. Mortality observed at the 15th day after treatment ranged from 26.3 to 100% in the tested samples. The effect of M. anisopliae on tick isolates was not associated with their susceptibility to any acaricide tested, animal stocking rate, cattle breed, rotational grazing, cultivated pasture, production system, presence of wet areas or biome. The integration of livestock and agriculture practices in the farm and the frequency of acaricide treatment seem to be associated with tick susceptibility to M. anisopliae. These results demonstrated that field populations of R. microplus show a high variation in their susceptibility to M. anisopliae., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The Rhipicephalus (Boophilus) microplus complex currently consists of five taxa, namely R. australis, R. annulatus, R. (B.) microplus clade A sensu, R. microplus clade B sensu, and R. (B.) microplus clade C sensu. Mitochondrial DNA-based methods help taxonomists when they are facing the morpho-taxonomic problem of distinguishing members of the R. (B.) microplus complex. The purpose of this study was to perform molecular characterization of ticks in all five regions of Brazil and infer their phylogenetic relationships. Molecular analysis characterized 10 haplotypes of the COX-1 gene. Molecular network analysis revealed that haplotype H-2 was the most dispersed of the studied populations (n = 11). Haplotype H-3 (n = 2) had the greatest genetic differentiation when compared to other Brazilian populations. A Bayesian phylogenetic tree of the COX-1 gene obtained strong support. In addition, it was observed that the population of R. (B.) microplus haplotype H-3 exhibited diverging branches among the other Brazilian populations in the study. The study concludes that the different regions of Brazil have R. (B.) microplus tick populations with distinct haplotypes.

Background: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively., Results: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation., Conclusions: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

The Rhipicephalus (Boophilus) microplus tick is responsible for considerable economic losses in Brazil, causing leather damage, weight loss and reduced milk production in cattle and results in the transmission of pathogens. Currently, the main method for controlling this tick is using acaricides, but their indiscriminate use is one of the major causes of resistance dissemination. In this study, the adult immersion test (AIT) was used to evaluate resistance in ticks from 28 properties located in five different states (Mato Grosso do Sul, Mato Grosso, Ceará, São Paulo, e Minas Gerais) and the Distrito Federal (DF) of Brazil. The resistance was found in 47.64% of the repetitions demonstrating an efficacy of less than 90% in various locations throughout the country. The larvae packet test was used to evaluate samples from ten properties in four states (Mato Grosso do Sul, Mato Grosso, Minas Gerais and Espírito Santo). Spray products belonging to the main classes of acaricides, including combination formulations, were used in both types of test. New cases of resistance were found on properties within the states of Ceará, Espírito Santo and Mato Grosso, where such resistance was not previously reported.

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Vector-borne pathogens are the subject of several investigations due to the zoonotic concern of some of them. However, limited data are available about the simultaneous presence of these pathogens in cats and their ectoparasites. The aim of the present study was to define the species of ectoparasites found on cats as well as to investigate vector-borne pathogens in cats and their ectoparasites in southern Italy., Methods: Blood from 42 cats and fleas or flea pools (n = 28) and ticks (n = 73) collected from them were investigated by quantitative PCR for the detection of vector-borne pathogens. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania infantum, Bartonella henselae, Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Babesia microti, Ehrlichia canis and Anaplasma phagocytophilum antigens., Results: Only one flea species (Ctenocephalides felis) and four tick species belonging to the genera Rhipicephalus and Ixodes were identified on cats from southern Italy. Molecular evidence of Bartonella spp., Rickettsia spp., hemoplasmas, Babesia vogeli and L. infantum was found in ectoparasites (fleas and/or ticks) while DNA from Hepatozoon felis and Ehrlichia/Anaplasma spp. was not detected. Likewise, DNAs from Bartonella, hemoplasma and Leishmania were the only pathogens amplified from feline blood samples. Cats had also antibodies against all the investigated pathogens with the exception of Rickettsia typhi. Agreement between serological and molecular results in individual cats and their ectoparasites was not found. The only exception was for Bartonella with a fair to moderate agreement between individual cats and their ectoparasites. Bartonella clarridgeiae was the species most frequently found in cats and their fleas followed by B. henselae., Conclusions: In conclusion, cats harboring ticks and fleas are frequently exposed to vector-borne pathogens. Furthermore, ticks and fleas harbored by cats frequently carry pathogens of zoonotic concern therefore appropriate feline ectoparasiticide preventative treatments should be used in cats.

Ticks and tick-borne diseases are among the main causes of economic loss in the South African cattle industry through high morbidity and mortality rates. Concerns of the general public regarding chemical residues may tarnish their perceptions of food safety and environmental health when the husbandry of cattle includes frequent use of acaricides to manage ticks. The primary objective of this study was to identify single nucleotide polymorphism (SNP) markers associated with host resistance to ticks in South African Nguni cattle. Tick count data were collected monthly from 586 Nguni cattle reared in four herds under natural grazing conditions over a period of two years. The counts were recorded for six species of ticks attached in eight anatomical locations on the animals and were summed by species and anatomical location. This gave rise to 63 measured phenotypes or traits, with results for 12 of these traits being reported here. Tick count (x) data were transformed using log10(x+1) and the resulting values were examined for normality. DNA was extracted from hair and blood samples and was genotyped using the Illumina BovineSNP50 assay. After quality control (call rate >90%, minor allele frequency >0.02), 40,436 SNPs were retained for analysis. Genetic parameters were estimated and association analysis for tick resistance was carried out using two approaches: a genome-wide association (GWA) analysis using the GenABEL package and a regional heritability mapping (RHM) analysis. The Bonferroni genome-wide (P<0.05) corrected significance threshold was 1.24×10(-6), with 2.47×10(-5) as the suggestive significance threshold (P<0.10) (i.e., one false positive per genome scan) in the GWA analysis. Likelihood ratio test (LRT) thresholds for genome-wide and suggestive significance were 13.5 and 9.15 for the RHM analysis. Six ixodid tick species were identified, with Amblyomma hebraeum (the vector for Heartwater disease) being the dominant species. Heritability estimates (h(2)) from the fitted animal and sire models ranged from 0.02±0.00 to 0.17±0.04 for the transformed tick count data. Several genomic regions harbouring quantitative trait loci (QTL) were identified for different tick count traits by both the GWA and RHM approaches. Three genome-wide significant regions on chromosomes 7, 10 and 19 were identified for total tick count on the head, total body A. hebraeum tick count and total A. hebraeum on the perineum region, respectively. Additional regions significant at the suggestive level were identified on chromosomes 1, 3, 6, 7, 8, 10, 11, 12, 14, 15, 17, 19 and 26 for several of the traits. The GWA approach identified more genomic regions than did the RHM approach. The chromosomal regions identified here as harbouring QTL underlying variation in tick burden form the basis for further analyses to identify specific candidate genes and polymorphisms related to cattle tick resistance and provide the potential for marker-assisted selection in Nguni cattle., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: The variation of tick abundance on ruminants had received little attention in West Africa before Rhipicephalus (Boophilus) microplus started to invade this region in the early 2000s. Ten years later, R. microplus was suspected to have replaced the native ticks. In addition to testing this hypothesis, this study investigated the interactions between native and invasive ticks and the relative role of climatic and geographical variables in the variations of tick community composition (beta diversity) on cattle herds., Methods: A one-year-long survey was performed in Benin and Burkina Faso during which adult ticks were collected from 144 steers from 12 localities in four different areas once a month. Morphological features were used to assign the collected ticks to different species (A. variegatum, R. annulatus, R. decoloratus, R. microplus and R. geigyi). Beta diversity analyses and generalized linear models allowed characterizing the geographical variations in species assemblage and the effect of co-infestation patterns on the seasonal variations in the abundance and incidence rates of each taxon., Results: About 68% (22,491/32,148) of all the adult ticks collected in one year were R. microplus. The most heterogeneously distributed taxa were Hyalomma spp and R. microplus and the lowest specific diversity was found in Central Burkina Faso. Although climatic variables did not provide any additional information on the variation in species assemblages compared with the sampling geography, adult tick abundance tended to peak during the late (Boophilus subgenus) or early (other taxa) rainy season. In most taxon-per-locality analyses, the abundance and incidence rate of a given tick taxon significantly increased when the host was co-infested by other taxa. The comparison with previous estimates (when possible) did not support the hypothesis that R. microplus invasion led to a decrease in native tick species abundance., Conclusions: The co-infestation patterns among native and invasive tick species are key factors for the determination of the community structure and the infestation dynamics of each tick taxon in West African cattle.

Background: Rickettsia spp. belonging to the spotted fever group (SFG) cause infections in humans, domestic animals and wildlife. At least five SFG rickettsial species have been reported in China, but the occurrence of Rickettsia aeschlimannii and R. massiliae in ticks has not been characterized to date., Findings: A total of 114 adult ticks were collected from sheep in Yining County, Xinjiang Uygur Autonomous Region, in northwest China. The ticks were identified from morphological and molecular characteristics. All samples were examined by polymerase chain reaction (PCR), and six genetic markers were used to determine the Rickettsia spp. in the ticks. The ticks collected were identified as Rhipicephalus turanicus. Three different lineages of Rh. turanicus from Yining County were discovered on phylogenetic analysis of 16S rDNA and cox1. Twenty-one of the 114 samples (18.42%) were positive for rickettsial agents. Phylogenetic analysis based on six genetic sequences showed that three rickettsial species were present, namely: R. aeschlimannii (19.05%, 4/21), R. massiliae (19.05%, 4/21) and R. sibirica variant (61.90%, 13/21), which is clustered in the clade of R. sibirica subsp. sibirica., Conclusions: This is the first description of R. aeschlimannii and R. massiliae in China. R. massiliae, R. aeschlimannii and R. sibirica variant co-circulate in the region of the China-Kazakhstan border, in northwest China. Rickettsial agents in ticks of the genus Rhipicephalus from migrant birds, transported livestock, wildlife and human beings should be investigated further in the region of the China-Central Asian border.

Background: Limited information is available about the species of ticks infesting the cat and the pathogens that they harbor. The aims of the present study were to identify the species of ticks removed from cats living in Sicily and Calabria (Italy) and to detect DNA of vector-borne pathogens in the same ticks., Findings: Morphological identification of 132 adult ticks collected throughout the year from cats was carried out. Real-time PCRs for Hepatozoon felis, Piroplasmid, Ehrlichia/Anaplasma spp., Rickettsia spp., Bartonella spp., Mycoplasma spp. and Leishmania infantum were performed from each individual tick. Ticks belonging to Rhipicephalus (R. sanguineus sensu lato, R. pusillus) and Ixodes (I. ricinus, I. ventalloi) genera were identified. Ixodes ventalloi was the most frequently found tick species (47 %). The positivity rate to at least one pathogen was 14.4 % (19/132 ticks). Leishmania infantum, Rickettsia spp. (R. monacensis and R. helvetica), Bartonella spp. (B. clarridgeiae), Piroplasmid (Babesia vogeli), and Ehrlichia/Anaplasma spp. (E. canis) DNAs were amplified in 8.3, 5.3, 1.5, 0.75 and 0.75 % of ticks, respectively. Hepatozoon felis, Anaplasma spp. and hemotropic Mycoplasma spp. DNAs were not detected. Four (21.1 %) out of nineteen positive ticks were co-infected., Conclusions: This study provides novel data about ticks infesting cats and the DNA of pathogens that they harbor. In Southern Italy, anti-tick prophylaxis should be implemented throughout the year in cats without neglecting winter time.

Rhipicephalus rossicus (Ixodida: Ixodidae) is a three-host tick with a broad host spectrum that includes wild animals, pets, livestock and humans. Despite its local abundance in certain areas, most of the available information on R. rossicus was published decades ago, mainly by former soviet authors. Its distribution largely overlaps the Eurasian steppe. However, its range may be more extensive than is currently known because this species may have been misidentified as Rhipicephalus sanguineus, principally in areas where the latter species is present. Although R. rossicus has been occasionally reported to feed on people, little attention has been given to its medical importance. It has been shown to have a vectorial role in the transmission of Francisella tularensis, Crimean-Congo haemorrhagic fever virus and West Nile virus. However, the vectorial importance of R. rossicus may be significantly greater, mainly as the closely related species R. sanguineus s.l. is known to transmit a very wide spectrum of pathogens. The probably underestimated vectorial role of R. rossicus may represent a hidden public health threat., (© 2015 The Royal Entomological Society.)

Background: The morphotaxonomy of Rhipicephalus microplus complex has been challenged in the last few years and prompted many biologists to adopt a DNA-based method for distinguishing the members of this group. In the present study, we used a mitochondrial DNA analysis to characterise the genetic assemblages, population structure and dispersal pattern of R. microplus from Southeast Asia, the region where the species originated., Methods: A phylogeographic analysis inferred from the 16S rRNA and cytochrome oxidase subunit I (COI) genes was performed with five populations of R. microplus collected from cattle in Malaysia. Malaysian R. microplus sequences were compared with existing COI and 16S rRNA haplotypes reported globally in NCBI GenBank., Results: A total of seven and 12 unique haplotypes were recovered by the 16S rRNA and COI genes, respectively. The concatenated sequences of both 16S rRNA and COI revealed 18 haplotypes. Haplotype network and phylogenetic analyses based on COI+16S rRNA sequences revealed four genetically divergent groups among Malaysian R. microplus. The significantly low genetic differentiation and high gene flow among Malaysian R. microplus populations supports the occurrence of genetic admixture. In a broader context, the 16S rRNA phylogenetic tree assigned all isolates of Malaysian R. microplus into the previously described African/the Americas assemblage. However, the COI phylogenetic tree provides higher resolution of R. microplus with the identification of three main assemblages: clade A sensu Burger et al. (2014) comprises ticks from Southeast Asia, the Americas and China; clade B sensu Burger et al. (2014) is restricted to ticks that originated from China; and clade C sensu Low et al. (2015) is a new genetic assemblage discovered in this study comprising ticks from India and Malaysia., Conclusions: We conclude that the R. microplus complex consisting of at least five taxa: R. australis, R. annulatus, R. microplus clade A sensu Burger et al. (2014), R. microplus clade B sensu Burger et al. (2014) and the new taxon, R. microplus clade C sensu Low et al. (2015). The use of COI as the standard genetic marker in discerning the genetic assemblages of R. microplus from a broad range of biogeographical regions is proposed.

Eperythrozoonosis is a small ruminant disease caused by the bacterium Mycoplasma ovis (formerly known as Eperythrozoon ovis). Whilst acute infection in sheep may result in an anaemia and ill thrift syndrome, most animals do not develop clinical signs. Molecular methods were used to compare and evaluate the prevalence of infection with M. ovis in sheep and goats in Tunisia. A total of 739 whole blood samples from 573 sheep and 166 goats were tested for the M. ovis 16S rRNA gene using PCR. The overall prevalence was 6.28% ± 0.019 (36/573). Only sheep were infected with M. ovis (p &lt; 0.001), and the prevalence was significantly higher in central Tunisia (29.2%) compared with other regions (p &lt; 0.05). The prevalence revealed significant differences according to breed and bioclimatic zones (p &lt; 0.001). Furthermore, the prevalence in young sheep (35/330; 10.6%) was higher than in adults (1/243; 0.41%) (p &lt; 0.001). Only sheep of the Barbarine breed were infected, with a prevalence of 11.8% (p &lt; 0.001). This is the first molecular study and genetic characterisation of M. ovis in North African sheep breeds.

Besides mosquitoes, ticks are regarded as the primary source of vector-borne infectious diseases. Indeed, a wide variety of severe infectious human diseases, including those involving viruses, are transmitted by ticks in many parts of the world. To date, there are no published reports on the use of next-generation sequencing for studying viral diversity in ticks or discovering new viruses in these arthropods from China. Here, Ion-torrent sequencing was used to investigate the presence of viruses in three Rhipicephalus spp. tick pools (NY-11, NY-13, and MM-13) collected from the Menglian district of Yunnan, China. The sequencing run resulted in 3,641,088, 3,106,733, and 3,871,851 reads in each tick pool after trimming. Reads and assembled contiguous sequences (contigs) were subject to basic local alignment search tool analysis against the GenBank database. Large numbers of reads and contigs related to known viral sequences corresponding to a broad range of viral families were identified. Some of the sequences originated from viruses that have not been described previously in ticks. Our findings will facilitate better understanding of the tick virome, and add to our current knowledge of disease-causing viruses in ticks living under natural conditions.

In most parts of the world, ticks are rapidly developing resistance to commonly used acaricides thus rendering control difficult. This constraint is further compounded by the introduction of new species in areas where they did not exist before. Such is the case with the introduction into and rapid spread of Rhipicephalus (Boophilus) microplus in some countries of West Africa. With the looming threat of its further spread in the region, the objective of the present study was to update knowledge on cattle ticks in Cameroon. Among 19,189 ticks collected monthly from 60 animals in 5 herds from March 2012 to February 2013, Rh. (B.) decoloratus was the most abundant species with a relative prevalence of 62.2%, followed by Amblyomma variegatum (28.4%), Rh. (B.) annulatus (0.2%), Rh. (B.) geigyi (0.03%), other Rhipicephalus spp. (8.4%) and Hyalomma spp. (0.3%). Rh. (B.) decoloratus and A. variegatum were also the most widely distributed in space. Infestation rate was generally high, with average tick count/animal of about 80 during peak periods. Tick distribution and abundance in the different sites was as varied as the underlying factors, among which the most important were management systems and climatic factors. The effects of rainfall and temperature were confounded by other factors and difficult to evaluate. However, it appears tick development depends among other factors, on a humidity threshold, above which there is not much more effect. Rh. microplus was not found during this study, but more extensive tick collections have to be done to confirm this. In conclusion, cattle tick infestation in Cameroon remains an important cause for concern. Farmers need assistance in the use and management of acaricides in order to increase their efficiency and reduce the development of resistance. Although Rh. microplus was not found, its introduction from other West African countries is imminent if adequate measures, especially in the control and limitation of animal movements, are not taken., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation with R. microplus and R. decoloratus, a common vector species on local livestock, occurs widely in the northern and eastern parts of the country. An alternative to chemical control methods is sought in the form of a tick vaccine to control these tick species. However, sequence information and transcriptional data for R. decoloratus is currently lacking. Therefore, this study aimed at identifying genes that are shared between midgut tissues of feeding adult female R. microplus and R. decoloratus ticks. In this regard, a custom oligonucleotide microarray comprising of 13,477 R. microplus sequences was used for transcriptional profiling and 2476 genes were found to be shared between these Rhipicephalus species. In addition, 136 transcripts were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly overrepresented in R. microplus and R. decoloratus. This study is the first transcriptional profiling of R. decoloratus and is an additional resource that can be evaluated further in future studies for possible tick control., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Seventy species of ticks are known from Australia: 14 soft ticks (family Argasidae) and 56 hard ticks (family Ixodidae). Sixteen of the 70 ticks in Australia may feed on humans and domestic animals (Barker and Walker 2014). The other 54 species of ticks in Australia feed only on wild mammals, reptiles and birds. At least 12 of the species of ticks in Australian also occur in Papua New Guinea. We use an image-matching system much like the image-matching systems of field guides to birds and flowers to identify Ixodes holocyclus (paralysis tick), Ixodes cornuatus (southern paralysis tick) and Rhipicephalus (Boophilus) australis (Australian cattle tick). Our species accounts have reviews of the literature on I. holocyclus (paralysis tick) from the first paper on the biology of an Australian tick by Bancroft (1884), on paralysis of dogs by I. holocyclus, to papers published recently, and of I. cornuatus (southern paralysis tick) and Rhipicephalus (Boophilus) australis (Australian cattle tick). We comment on four controversial questions in the evolutionary biology of ticks: (i) were labyrinthodont amphibians in Australia in the Devonian the first hosts of soft, hard and nuttalliellid ticks?; (ii) are the nuttalliellid ticks the sister-group to the hard ticks or the soft ticks?; (iii) is Nuttalliella namaqua the missing link between the soft and hard ticks?; and (iv) the evidence for a lineage of large bodied parasitiform mites (ticks plus the holothyrid mites plus the opiliocarid mites)., (Copyright © 2014. Published by Elsevier Ltd.)

Changes in the distribution of tick species are among the major causes for the increase in prevalence of zoonotic diseases worldwide, with tick-borne diseases' prevalence showing an emerging pattern. One of these ticks, Rhipicephalus rossicus, which is reported occasionally from humans, seems to be particularly interesting because of its demonstrated vectorial role for zoonotic pathogens like Francisella tularensis, Coxiella burnetii, or CCHF and West Nile viruses. Here we report a case of dominant occurrence of R. rossicus on household dogs in the wetlands of Eastern Europe (Romania). Ticks were collected from dogs in 5 distinct locations, with 1068 ticks of 6 species found. R. rossicus had a dominant occurrence in dogs in all but one location, accounting for 87.1% of all ticks (32.3-95.3% in different locations). Until this study, Rhipicephalus sanguineus was considered as the only important tick species on dogs in south-temperate regions of Europe, as well in Romania. The dominant presence of R. rossicus in dogs, its vectorial competence and broad host spectrum (including humans), make this tick species an important candidate for further analysis and highlight the paucity of our knowledge on disease vectors in this region of Europe., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cattle ticks of the subgenus Rhipicephalus (Boophilus) are major agricultural pests worldwide, causing billions of dollars in losses annually. Rhipicephalus (Boophilus) annulatus and R. microplus are the most well-known and widespread species, and a third species, R. australis, was recently reinstated for 'R. microplus' from Australia and parts of Southeast Asia. We use mitochondrial genome sequences to address the phylogenetic relationships among the species of the subgenus Boophilus. We sequenced the complete or partial mitochondrial genomes of R. annulatus, R. australis, R. kohlsi, R. geigyi, and of three geographically disparate specimens of R. microplus from Brazil, Cambodia and China. Phylogenetic analyses of mitochondrial genomes, as well as cox1 and 16S rRNA sequences, reveals a species complex of R. annulatus, R. australis, and two clades of R. microplus, which we call the R. microplus complex. We show that cattle ticks morphologically identified as R. microplus from Southern China and Northern India (R. microplus clade B) are more closely related to R. annulatus than other specimens of R. microplus s.s. from Asia, South America and Africa (R. microplus clade A). Our analysis suggests that ticks reported as R. microplus from Southern China and Northern India are a cryptic species. This highlights the need for further molecular, morphological and crossbreeding studies of the R. microplus complex, with emphasis on specimens from China and India. We found that cox1 and, to a lesser extent, 16S rRNA were far more successful in resolving the phylogenetic relationships within the R. microplus complex than 12S rRNA or the nuclear marker ITS2. We suggest that future molecular studies of the R. microplus complex should focus on cox1, supplemented by 16S rRNA, and develop nuclear markers alternative to ITS2 to complement the mitochondrial data., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rhipicephalus microplus is an important ectoparasite that is responsible for transmission of anaplasmosis and babesiosis to cattle. Tissue kallikrein inhibitors might play an important role in R. microplus eggs. In the present work, we purified and characterized, a tissue kallikrein inhibitor presents in R. microplus eggs (RmKK), a protein which contains two Kunitz domain in tandem. Purified inhibitor was confirmed by amino terminal determination and its dissociation constant (Ki) for bovine trypsin and porcine pancreatic kallikrein were 0.6 nM and 91.5 nM, respectively. Using a cDNA library from R. microplus midgut, we cloned the cDNA fragment encoding mature RmKK and expressed the protein in Pichia pastoris system. Recombinant RmKK was purified by ion exchange chromatography and presented molecular mass of 16.3 kDa by MALDI-TOF analysis. Moreover, RmKK showed a tight binding inhibition for serine proteases as bovine trypsin (Ki=0.2 nM) and porcine pancreatic kallikrein (PPK) (Ki=300 nM). We performed, for the first time, the characterization of a tissue kallikrein inhibitor presents in R. microplus eggs, which the transcript is produced in the adult female gut. BmKK seems to be the strongest PPK inhibitor among all BmTIs present in the eggs and larvae (Andreotti et al., 2001; Sasaki et al., 2004). This data suggests that BmKK may participate in the development of tick egg and larvae phase., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Identification of cross-protective tick vaccine antigens is a challenging area of veterinary research. To address this challenge, a recently identified candidate tick protective antigen, Subolesin (SUB), was targeted in this research. The conservation of subolesin ortholog of Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus across different Indian strains was 98.1-99.4% (within species), while at the amino acid level SUB sequence homology was ≥53.2% (between tick species). Recombinant R. (B.) microplus SUB (rBmSu) was produced in Escherichia coli and characterized. Cross-bred cattle male calves (N=10) were immunized with three doses of 100 μg each of the rBmSu emulsified in 10% Montanide 888 at monthly intervals on days 0, 30 and 60. The control group was injected with PBS in 10% Montanide 888. For the first tick challenge, calves were infested with larvae of R. (B.) microplus generated from 100mg eggs 2 weeks after last immunization (day 75). The immunization resulted in 16.3%, 8.0%, 9.4%, and 26.1% reduction in female tick numbers (DT), weight (DW), oviposition (DO) and egg fertility (DF), respectively, when compared to controls. In the subsequent challenge on day 105, DT, DW, DO and DF were reduced by 9.0%, 4.1%, 8.6%, and 24.2%, respectively, when compared to controls. The vaccine efficacy (E) was equal to 44.0% and 37.2% after the first and second challenges, respectively. The results showed a positive correlation between antibody titers for both total IgG and IgG1 and E in the second but not in the first tick challenge. These results suggested the possibility of developing a SUB-based vaccine for control of cattle tick infestations under Indian conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Abstract' Background: For >100 years cattle production in the southern United States has been threatened by cattle fever. It is caused by an invasive parasite-vector complex that includes the protozoan hemoparasites Babesia bovis and B. bigemina, which are transmitted among domestic cattle via Rhipicephalus tick vectors of the subgenus Boophilus. In 1906 an eradication effort was started and by 1943 Boophilus ticks had been confined to a narrow tick eradication quarantine area (TEQA) along the Texas-Mexico border. However, a dramatic increase in tick infestations in areas outside the TEQA over the last decade suggests these tick vectors may be poised to re-invade the southern United States. We investigated historical and potential future distributions of climatic habitats of cattle fever ticks to assess the potential for a range expansion., Methods: We built robust spatial predictions of habitat suitability for the vector species Rhipicephalus (Boophilus) microplus and R. (B.) annulatus across the southern United States for three time periods: 1906, present day (2012), and 2050. We used analysis of molecular variance (AMOVA) to identify persistent tick occurrences and analysis of bias in the climate proximate to these occurrences to identify key environmental parameters associated with the ecology of both species. We then used ecological niche modeling algorithms GARP and Maxent to construct models that related known occurrences of ticks in the TEQA during 2001-2011 with geospatial data layers that summarized important climate parameters at all three time periods., Results: We identified persistent tick infestations and specific climate parameters that appear to be drivers of ecological niches of the two tick species. Spatial models projected onto climate data representative of climate in 1906 reproduced historical pre-eradication tick distributions. Present-day predictions, although constrained to areas near the TEQA, extrapolated well onto climate projections for 2050., Conclusions: Our models indicate the potential for range expansion of climate suitable for survival of R. microplus and R. annulatus in the southern United States by mid-century, which increases the risk of reintroduction of these ticks and cattle tick fever into major cattle producing areas.

Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in many organisms. In arthropods these proteins are involved in innate immune system, morphogenesis and development. In mammals serpins regulate pathways that are essential to life such as blood coagulation, fibrinolysis, inflammation and complement activation, some of which are considered the host's first line of defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use serpins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNA sequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1-18 (R. microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpins are transcribed during feeding, suggesting their participation in tick physiology regulation. We speculate that the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis. Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by putative signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealed that R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptive study enlarges the knowledge on the molecular biology of R. microplus, an important tick species., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To investigate and identify the ticks prevalent in the North East part of India, scanning electron microscope (SEM) and DNA sequence of nuclear second internal transcribed spacer (ITS2) and mitochondrial 16S ribosomal DNA (rDNA) were used. Based on the morphological and molecular analysis, the ticks infesting cattle of North East India were found to be Rhipicephalus (Boophilus) microplus and Haemaphysalis bispinosa. ITS2 and 16S rDNA sequence from R. (B.) microplus and H. bispinosa were amplified using universal and gene specific primers, sequenced and analysed. The length of the amplified ITS2 sequence of R. (B.) microplus and H. bispinosa, were found to be approximately 1,500 and 1,700 bp, respectively. The length of the 16S rDNA sequences in both the ticks was found to be similar in size, but they differ in their base pair constitutions. This is the first report of the nucleotide sequences of ITS2 and 16S rDNA of H. bispinosa. Phylogenetic analysis revealed that H. bispinosa is a close relative of H. longicornis. A polymerase chain reaction-restriction fragment length polymorphism diagnostic tool was developed based on HindIII digestion of ITS2 in order to facilitate the identification of these two species which cannot be distinguished once it is fully-fed. Present study describes the use of SEM and 16S rDNA/ITS2 based molecular analysis in identification and differentiation of fully fed tick species.

Mitochondrial sequences of four mitochondrial markers: 12S rRNA, 16S rRNA, cytochrome c oxidase subunit I (COX1) and cytochrome b (CytB) from four Rhipicephalus species were analyzed to establish genetic relationships and enable molecular identification. Field-collected samples from the species Rhipicephalus annulatus, Rhipicephalus bursa, Rhipicephalus sanguineus and Rhipicephalus turanicus were amplified by PCR and compared with GenBank™ annotated sequences. PCR products were obtained using primers that were designed to amplify orthologous sequences from different tick species and genera. The average intra-species sequence identity was 98.5-99.5%, while the average inter-species identity was 86.5-89.6%, reflecting a ≈ 10% decrease in the identity, when different species are compared. The "closest" two species, in terms of sequence identity, were R. sanguineus and R. turanicus, while the "least close" ones were R. annulatus and R. sanguineus. Molecular identification of each species was accomplished by a combined restriction analysis of 12S, COX1 and CytB markers, obviating the need for field sample sequencing. The restriction mapping data suggest that by using several markers, each with a unique digestion pattern, the identity of a given sample could be determined at the species level. It is anticipated that with the accumulation of more information on additional species and markers, molecular identification will become a standard approach for tick classification, complementing morphological taxonomy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cuticular fatty acids (CFA) are important constituents of the arthropod exoskeleton, serving as structural and defense components, and participating in intra-species communication. Here we describe for the first time a comparative analysis of the CFA profiles of three tick species of the genus Rhipicephalus: R. annulatus, R. bursa and R. sanguineus. CFA profiles were determined for R. bursa and R. sanguineus grown both on rabbit or calf, and for R. annulatus grown on calf. CFA composition was compared for each species before and after ethanol treatment, for different hosts of each species, and between the different species. Our data suggest that adsorption of the host's fatty acids changes the apparent CFA composition. Ethanol treatment efficiently removed the unbound fatty acids from the ticks and revealed the actual composition. Comparison between ticks grown on rabbit versus calf showed significant difference in the relative abundance of fatty acids C14 and 9,12-C18:2 for R. bursa, and a difference in the relative abundance of C14 for R. sanguineus. Comparison of the CFA between the three species revealed significant differences in the abundance of fatty acids C16, 9,12-C18:2, 9-C18:1, C18 and C20. Our results show that while the host had a minor effect on CFA composition within each species, significant differences were observed in the CFA profiles of different species. We suggest that CFA profiles may be used to distinguish between related species. CFA analysis can also be used in studies of communication and defense mechanisms in ticks and other arthropods.

A tick population was monitored on 30 camels (Camelus dromedarius) over one year in Kairouan region, Central Tunisia. A total of 1630 ticks was collected and identified resulting in an estimate of different parasitological indicators. The ticks belonged to 2 genera and 5 species: Hyalomma impeltatum (53%) and Hyalomma dromedarii (45%) were the dominant species followed by Hyalomma excavatum (1%), Hyalomma marginatum (0.5%), and Rhipicephalus turanicus (0.5%) (p<0.001). Mean infestation prevalence was 90.6%; all the animals were infested by at least one tick from May to September. The highest mean prevalence was observed in H. impeltatum (60%), the lowest was reported in R. turanicus (0.03%) (p<0.05). Mean overall intensity of infestation was 4.4 ticks/animal. The highest mean intensity was observed in H. impeltatum (2.7 ticks/animal). Overall mean abundance of ticks was 4.4 ticks/animal. Different abiotic factors, namely monthly mean minimum and monthly mean maximum temperatures and the number of sunny days were positively correlated with overall monthly tick burdens which were in turn negatively correlated with the monthly mean relative humidity. This is the first study on camel tick dynamics in Tunisia., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The invasive pantropical blue tick, Rhipicephalus microplus, has recently been collected from cattle in Namibia. A cross-sectional study aimed at recording the geographic distribution of Rhipicephalus decoloratus and establishing whether R. microplus is present in Namibia was conducted towards the end of summer (March-April) 2013. Ticks were collected from cattle on 18 privately owned farms across a large geographical scale. Ticks were collected from three to five cattle per farm and species belonging to the genera Hyalomma and Rhipicephalus were recovered. Rhipicephalus decoloratus was present on all farms and R. microplus was recorded on four of the farms. The small numbers of R. microplus compared to R. decoloratus collected in the mixed infestations, suggests that the introduction events were recent.