Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India.

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

A mutation associated with resistance to synthetic pyrethroids is widespread in US populations of the tropical lineage of Rhipicephalus sanguineus s.l.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), is an important vector for Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever. Current public health prevention and control efforts to protect people involve preventing tick infestations on domestic animals and in and around houses. Primary prevention tools rely on acaricides, often synthetic pyrethroids (SPs); resistance to this chemical class is widespread in ticks and other arthropods. Rhipicephalus sanguineus s.l. is a complex that likely contains multiple unique species and although the distribution of this complex is global, there are differences in morphology, ecology, and perhaps vector competence among these major lineages. Two major lineages within Rh. sanguineus s.l., commonly referred to as temperate and tropical, have been documented from multiple locations in North America, but are thought to occupy different ecological niches. To evaluate potential acaricide resistance and better define the distributions of the tropical and temperate lineages throughout the US and in northern Mexico, we employed a highly multiplexed amplicon sequencing approach to characterize sequence diversity at: 1) three loci within the voltage-gated sodium channel (VGSC) gene, which contains numerous genetic mutations associated with resistance to SPs; 2) a region of the gamma-aminobutyric acid-gated chloride channel gene (GABA-Cl) containing several mutations associated with dieldrin/fipronil resistance in other species; and 3) three mitochondrial genes (COI, 12S, and 16S). We utilized a geographically diverse set of Rh sanguineus s.l. collected from domestic pets in the US in 2013 and a smaller set of ticks collected from canines in Baja California, Mexico in 2021. We determined that a single nucleotide polymorphism (T2134C) in domain III segment 6 of the VGSC, which has previously been associated with SP resistance in Rh. sanguineus s.l., was widespread and abundant in tropical lineage ticks (>50 %) but absent from the temperate lineage, suggesting that resistance to SPs may be common in the tropical lineage. We found evidence of multiple copies of GABA-Cl in ticks from both lineages, with some copies containing mutations associated with fipronil resistance in other species, but the effects of these patterns on fipronil resistance in Rh. sanguineus s.l. are currently unknown. The tropical lineage was abundant and geographically widespread, accounting for 79 % of analyzed ticks and present at 13/14 collection sites. The temperate and tropical lineages co-occurred in four US states, and as far north as New York. None of the ticks we examined were positive for Rickettsia rickettsii or Rickettsia massiliae.

Effect of an altitudinal gradient on the morphology, molecular identification and distribution of Rhipicephalus linnaei in Veracruz, Mexico.

Studies of morphological and genetic variation in vector populations across environmental gradients can help researchers to estimate species' responses to climate change scenarios and the potential risk of disease-causing pathogen expansion, which impacts negatively on human health. In this study, we analysed the effect of altitudinal gradients on the phenotypic response of the hard tick of medical and veterinary importance, Rhipicephalus sanguineus sensu lato (s.l.). Specimens of R. sanguineus s.l. were collected from host animals in one of Mexico's regions with high climatic heterogeneity (Veracruz), and geometric morphometric theory was employed to assess the response of three morphological characters to the altitudinal gradient. Additionally, genetic similarity data were provided, and ecological niche models were used to project the climatic distribution in the region. Our results demonstrate that the shape and size of ticks respond to altitude. Molecular identification indicate that all analysed samples correspond to the tropical lineage recently named Rhipicephalus linnaei. According to ecological niche models, the mean annual temperature contributes significantly to the spatial distribution of this tick species, with areas of higher suitability in the mountainous region. These changes in morphological structure and the presence of ticks at higher altitudinal gradients suggest that R. linnaei has a high potential for adaptation. Due to the variability of ecosystems in the state of Veracruz, our results could be valuable in assessing the response of this tick in a changing environment, aiding in predicting future scenarios in the distribution and abundance of this species.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

Evidence on the prevalence of emerging and re-emerging tick- and flea-borne rickettsial agents in acute encephalitis syndrome endemic areas of northeast Uttar Pradesh, India.

Outbreaks of acute encephalitis syndrome (AES) with unknown aetiology are reported every year in Gorakhpur district, Uttar Pradesh, India, and Orientia tsutsugamushi, the rickettsial pathogen, responsible for scrub typhus has been attributed as the primary cause of AES problem. However, information on the prevalence of other rickettsial infections is lacking. Hence, this study was carried out to assess any occurrence of tick- and flea-borne rickettsial agents in villages reporting AES cases in this district. In total, 825 peridomestic small mammals were trapped, by setting 9254 Sherman traps in four villages with a trap success rate of 8.9%. The Asian house shrew, Suncus murinus, constituted the predominant animal species (56.2%) and contributed to the maximum number (87.37%) of ectoparasites. In total, 1552 ectoparasites comprising two species of ticks and one species each of flea and louse were retrieved from the trapped rodents/shrews. Rhipicephalus sanguineus, the brown dog tick, was the predominant species retrieved from the trapped rodents/shrews, and the overall infestation rate was 1.75 per animal. In total, 4428 ectoparasites comprising five tick species, three louse species and one flea species were collected from 1798 domestic animals screened. Rhipicephalus microplus was the predominant tick species collected from the domestic animals. The cat flea, Ctenocephalides felis, constituted 1.5% of the total ectoparasites. Of all the ectoparasite samples (5980) from domestic animals and rodents, tested as 1211 pools through real-time PCR assays, 64 pools were positive for 23S rRNA gene of rickettsial agents. The PCR-positive samples were subjected to multi-locus sequence typing (MLST). In BLAST and phylogenetic analysis, the ectoparasites were found to harbour Rickettsia asembonensis (n = 9), Rickettsia conorii (n = 3), Rickettsia massiliae (n = 29) and Candidatus Rickettsia senegalensis (n = 1). A total of 22 pools were detected to have multiple rickettsial agents. The prevalence of fleas and high abundance of tick vectors with natural infections of rickettsial agents indicates the risk of transmission of tick- and flea-borne rickettsial diseases in rural villages of Gorakhpur. Further epidemiological studies are required to confirm the transmission of these agents to humans.

Seroprevalence and transmission of severe fever with thrombocytopenia syndrome virus in a coastal endemic area in Southeastern China.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerged tick-borne viral zoonosis and widely prevalent in China, Japan and South Korea. Most reported SFTS cases have been identified in mountainous and hilly areas, with a few in island areas. In this study, we conducted a systematic investigation about natural infection of SFTS virus (SFTSV) among humans, animals and ticks in a coastal endemic prefecture, containing island, plains and mountain settings, in Zhejiang Province, Southeastern China. From July 2020 to June 2021, 1117 participants completed a survey with questionnaire interview and serum testing. Meanwhile, 862 serum samples of domestic animals, 275 spleen tissue samples of wild animals and 829 ticks representing five species (predominantly Haemaphysalis longicornis and Rhipicephalus sanguineus sensu lato) were collected. The seroprevalence of anti-SFTSV total antibody and IgM antibody among the participants was 4.8 % (54/1117) and 0.6 % (7/1117), respectively. Multivariate logistic regression analysis indicated that living in the island area (OR=2.66; 95 %CI: 1.04-6.80; P = 0.041) was significantly associated with seropositivity of total antibody to SFTSV. Furthermore, a higher seroprevalence was observed in domestic animals (36.1 %), while the SFTSV-RNA infection rate was 0.4 % in wild animals and the minimum infection rate (MIR) was 0.8 % for all tick species combined. The only tick species infected with SFTSV was H. longicornis. The prevalence of SFTSV infection in the island area, manifested by anti-SFTSV total antibody (P = 0.012) and IgM antibody (P = 0.004) among humans, anti-SFTSV total antibody (P<0.001) among domestic animals, and SFTSV-RNA among ticks (P = 0.022), was significantly higher than that in the mountainous area and the plain area. Furthermore, phylogenetic analysis showed that SFTSV sequences obtained from ticks in the island area were clustered with reported strains in Japan and South Korea. These results suggest that islands in the study area might be an important natural focus of SFTSV.

Characterization of the complete mitochondrial genomes of five hard ticks and phylogenetic implications.

Ticks are blood-sucking ectoparasites with significant medical and veterinary importance, capable of transmitting bacteria, protozoa, fungi, and viruses that cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of five hard tick species and analyzed features of their gene contents and genome organizations. The complete mt genomes of Haemaphysalis verticalis, H. flava, H. longicornis, Rhipicephalus sanguineus and Hyalomma asiaticum were 14855 bp, 14689 bp, 14693 bp, 14715 bp and 14722 bp in size, respectively. Their gene contents and arrangements are the same as those of most species of metastriate Ixodida, but distinct from species of genus Ixodes. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes with two different computational algorithms (Bayesian inference and maximum likelihood) revealed the monophylies of the genera Rhipicephalus, Ixodes and Amblyomma, however, rejected the monophyly of the genus Haemaphysalis. To our knowledge, this is the first report of the complete mt genome of H. verticalis. These datasets provide useful mtDNA markers for further studies of the identification and classification of hard ticks.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei.

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally. R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with ß-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 µM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 µM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Infection and transovarial transmission of severe fever with thrombocytopenia syndrome virus in Rhipicephalus sanguineus in Hainan Island, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) RNA level increased in female ticks after injection with SFTSV. Furthermore, SFTSV RNA was detected in the eggs and larvae that originated from the virus-infected female ticks.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy.

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.

Tropical and Temperate Lineages of Rhipicephalus sanguineus s.l. Ticks (Acari: Ixodidae) Host Different Strains of Coxiella-like Endosymbionts.

Nonpathogenic bacteria likely play important roles in the biology and vector competence of ticks and other arthropods. Coxiella, a gram-negative gammaproteobacterium, is one of the most commonly reported maternally inherited endosymbionts in ticks and has been associated with over 40 tick species. Species-specific Coxiella-like endosymbionts (CLEs) have been reported in the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), throughout the world, while recent research suggests low Coxiella diversity among tick species. We investigated CLE diversity among R. sanguineus s.l. ticks across Arizona. We detected 37 recurrent sequence variants (SVs) of the symbiont, indicating greater diversity in these symbiotic bacteria than previously reported. However, two SVs accounted for the vast majority of 16S rRNA amplicon reads. These two dominant CLEs were both closely related to Candidatus C. mudrowiae, an identified symbiont of Rhipicephalus turanicus ticks. One strain strongly associated with the tropical lineage of R. sanguineus s.l. while the other was found almost exclusively in the temperate lineage, supporting the conclusion that CLEs are primarily vertically transmitted. However, occasional mismatches between tick lineage and symbiont SV indicate that horizontal symbiont transfer may occur, perhaps via cofeeding of ticks from different lineages on the same dog. This study advances our understanding of CLE diversity in Rh. sanguineus s.l.

Predicting the northward expansion of tropical lineage Rhipicephalus sanguineus sensu lato ticks in the United States and its implications for medical and veterinary health.

The tropical lineage within the Rhipicephalus sanguineus species complex is cause for growing concern in the U.S. based on its prominent role in creating and perpetuating multiple recently identified outbreaks of Rocky Mountain spotted fever in the southwestern United States and northern Mexico. This lineage is undergoing a northward range expansion in the United States, necessitating the need for enhanced surveillance for Rh. sanguineus. To inform more focused surveillance efforts we use species distribution models (SDMs) to predict current (2015-2019) and future (2021-2040) habitat for the tropical lineage. Models using the MaxEnt algorithm were informed using geolocations of ticks genetically confirmed to be of the tropical lineage, for which data on 23 climatic and ecological variables were extracted. Models predicted that suitability was optimal where temperatures are relatively warm and stable, and there is minimal precipitation. This translated into habitat being predicted along much of the coast of southern states including California, Texas, Louisiana, and Florida. Although the endophilic nature of tropical Rh. sanguineus somewhat violates the assumptions of SDMs, our models correctly predicted known locations of this tick and provide a starting point for increased surveillance efforts. Furthermore, we highlight the importance of using molecular methods to distinguish between ticks in the Rh. sanguineus species complex.

Rickettsia massiliae circulation in sheep and attached Rhipicephalus sanguineus in Central Portugal.

Rickettsiosis is considered an emerging/re-emerging vector-borne disease that causes significant public health threats. Ticks are reservoirs and vectors of Rickettsia having a significant role in the transmission of rickettsiae. In Portugal, little is known about tick-borne Rickettsia species in sheep. Therefore, this study aimed to investigate rickettsiae infection in ticks and their sheep host from 27 farms in four districts of central Portugal, to clarify the role of the sheep host in the circulation of this zoonotic agent. Between March and May 2021, EDTA blood samples (n = 100) of healthy grazing sheep and their ticks (n = 100, one tick per animal) were collected during a herd health program in central Portugal. Obtained ticks were identified as Rhipicephalus sanguineus sensu lato by PCR targeting a partial sequence of 16S rRNA gene followed by sequence analysis. Rhipicephalus sanguineus s.l. and host sheep blood were tested for the presence of Rickettsia spp. by PCR targeting a partial sequence of ompB and ompA genes. From a total of 100 paired R. sanguineus s.l. and host sheep, Rickettsia massiliae was detected in 62 ticks and 35 grazing sheep blood samples, collected in central Portugal, 2021. All 35 positive sheep had attached positive R. sanguineus s.l., with matching nucleotidic sequences. These findings suggest that sheep may develop rickettsiemia and are likely capable of transmitting and amplifying the infection to uninfected ticks maintaining rickettsiae in circulation in the domestic cycle.

Severe Rhipicephalus sanguineus tick infestation in a home from rural area of Nanchang in China.

On October 13, 2021, a tick infestation occurred in a home in rural area of Nanchang city, China, and we were asked to inspect the tick infestation. Ticks were collected in the largest number on courtyard door jambs, followed by living room and bedroom door jambs. Ticks were identified morphologically as Rhipicephalus sanguineus adults. The 16S rRNA analysis effectively distinguished the ticks in this study from other Rhipicephalus species, including R. sanguineus south-east, temperate and tropical lineages and identified genetically as R. sanguineus south China lineage. Tick samples were subjected to conventional PCR analysis and detected negative for the presence of tick-borne pathogens. Our findings indicate that there was low transmission risk of tick-borne pathogens to humans in the tick-infested home. Further studies are needed to proactively investigate the tick species in Nanchang, and determine the presence of tick-borne pathogens for assessing their threat to human health in the region.

Molecular detection and genetic characterization of Coxiella-like endosymbionts in dogs and ticks infesting dogs in Northeast India.

An epidemiological study was performed to determine the role of dogs and ticks infesting dogs in the transmission of Q fever in humans and animals from April 2019 to March 2020 in the northeastern hill states of India. In total, 245 pet and stray dogs irrespective of age or sex were sampled, without specific inclusion or exclusion criteria. In total, 478 ticks belonging to three species were detected, namely Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum. The DNA extracted from blood and tick samples was assayed for molecular characterization of Coxiella burnetii targeting the 16S rRNA and superoxide dismutase (SOD) genes. Amplified PCR products were purified, cloned and custom sequenced. PCR assay showed 3.3% (8/245) of the dogs were positive for Coxiella-like bacteria. Coxiella-like bacterial DNA was detected in adult fully engorged females of R. sanguineus (7.7%, 13/168), R. (B.) microplus (3.3%, 4/123) and H. anatolicum (1.9%, 1/54). Coxiella-like bacterial DNA lacked in adult male or nymphal stage. The infection rate did not vary significantly between seasons, nor according to sex or age of the host. Six nucleotide sequences of 16S rRNA and SOD genes are discussed.

Genetic Variation in Rhipicephalus sanguineus s.l. Ticks across Arizona.

Rhipicephalus sanguineus s.l. (Latreille, 1806), the brown dog tick, is the most widely distributed tick species in the world. The two dominant lineages, a temperate group and a tropical group, are recognized as important disease vectors for both dogs and humans. The temperate and tropical lineages overlap in range in some regions of the world, including the southwestern United States, where recent outbreaks of Rocky Mountain spotted fever are linked to R. sanguineus s.l. While it is unclear to what extent they may differ in their capacity to transmit pathogens, finer-scale resolution of temperate and tropical lineage distribution may provide insight into the ecology of these two tick groups and the epidemiology of R. sanguineus s.l.-vectored diseases. Using diagnostic polymerase chain reaction assays, we examined the geospatial trends in R. sanguineus s.l. lineages throughout Arizona. We found the temperate and tropical lineages were well delineated, with some overlap in the eastern part of the state. In one county, tropical and temperate ticks were collected on the same dog host, demonstrating that the two lineages are living in sympatry in some instances and may co-feed on the same host.

Morphological identification and molecular characterization of economically important ticks (Acari: Ixodidae) from North and North-Western Egypt.

Ticks represent a major source of growing economic and public health concern, especially in the tropics and sub-tropics. Towards evidencing ticks' invasion in the North and North-Western parts of Egypt, the present study aimed to investigate the morpho-molecular aspects of those ectoparasites using stereomicroscopy, scanning electron microscopy (SEM) and sequencing of the mitochondrial 16S ribosomal DNA gene (rDNA) and nuclear second internal transcribed spacer (ITS2). Our analysis confirmed the presence and well-distribution of Rhipicephalus sanguineus s.l. infesting dogs and sheep (Alexandria governorate), Rhipicephalus annulatus infesting cattle (Beheira governorate), and Hyalomma dromedarii infesting camels (Marsa Matruh governorate) from North/North-Western Egypt. 16S rDNA and ITS2 sequences of the ticks were amplified using universal and gene-specific sets of primers, sequenced and analyzed. Lengths of amplified 16S rDNA sequences in all examined tick species were found to be similar in size (approximately 460 bp); however, they differed in base pair constitutions, whereas ITS2 lengths were 1,500 bp, 1,550 bp, and 1,800 bp for Rh. annulatus, Rh. sanguineus s.l., and Hy. dromedarii, respectively. Phylogenetically, based on the 16S rDNA results, Rh. sanguineus s.l. ticks clustered with the southeastern Europe lineage from Romania and Greece, Rh. annulatus ticks were similar to Turkish populations, and Hy. dromedarii were close to the isolates from Tunisia. Similarly, based on ITS2 sequences, Rh. sanguineus s.l. from dogs were showing 99% similarity to Nigerian populations; however, those collected from sheep were closer to Iranian populations with 4.1% nucleotide divergence between the two populations of different hosts. Rh. annulatus ticks were identical to a population from Romania, whereas Hy. dromedarii was close by 99.7% similarity to a population from Kenya. This is the first study reporting nucleotide sequences of 16S rDNA and ITS2 in integration with morphological identification of ticks from this part of Egypt.

Molecular characterisation of Rhipicephalus sanguineus sensu lato ticks from domestic dogs in Nigeria.

Rhipicephalus sanguineus is the most widely reported tick in the world. Molecular characterisation is important to verify its taxonomic status in the different parts of the world. In this study, we provide information on the molecular characterisation of R. sanguineus tick of dogs collected from Nigeria. Ticks were collected from 62 of 93 sampled dogs. The collected ticks were subjected to morphological identification with the aid of appropriate entomological keys. Deoxyribonucleic acid (DNA) was extracted from the most prevalent tick species (R. sanguineus) and was subjected to further molecular characterisation protocols. The partial mitochondrial 16S rRNA gene sequences (∼300 bp) were obtained from representative specimens. Data were statistically analysed using the chi-square (χ2 ) test. Phylogenetic analysis was performed including different lineages of R. sanguineus (sl) from Africa, Asia, Europe and America, and other species belonging to the R. sanguineus 'tropical lineage' (R. linnaei) as well as Rhipicephalus turanicus and Ixodes ricinus. Results of this study showed that R. sanguineus was the most abundant ticks of dogs with a prevalence of 61.8% (68/110; 95% CI = 52.5-70.54), followed by Amblyomma variegatum (20.0%) and Haemaphysalis leachi (18.2%). The molecular analysis shows that they are genetically different from the temperate strains but closely related to those from other West African countries. There is a need to establish the vector competence of this common Nigerian dog tick.

Reestablishment of Rhipicephalus secundus Feldman-Muhsam, 1952 (Acari: Ixodidae).

Rhipicephalus secundus is reestablished as a valid tick name within the Rhipicephalus sanguineus group and removed from the synonymy list of Rhipicephalus turanicus. Morphological re-description of both male and female of R. secundus and the analysis of its phylogenetic position based on mitochondrial DNA sequences are presented. The morphological re-description was made with tick specimens collected on goat in Israel. The phylogenetic analyses showed that R. secundus belong to a different clade from those formed by R. turanicus sensu stricto (s.s.) and R sanguineus s.s., and by other taxa from the R. sanguineus group. Rhipicephalus secundus is morphologically related to R. turanicus, but the scutal punctation pattern of both male and female allows the morphological differentiation between R. secundus and R. turanicus, punctations being clearly more numerous and larger in the latter. Both male and female of R. secundus can be differentiated from those of R. sanguineus s.s. by the shape of the spiracular plate. In males, the dorsal prolongation of the spiracular plate is equal to the breadth of the adjacent festoon in R. secundus, while it is narrower than the breadth of the adjacent festoon in R. sanguineus s.s. The dorsal prolongation of the spiracular plate in the female of R. secundus is wider than in the female of R. sanguineus s.s. The genital apertures of the females of R. secundus and R. sanguineus are both U-shaped, but in R. sanguineus s.s. it is broader than in R. secundus. Considering the results obtained in this study, it can be stated that R. secundus is present at least in Israel, Palestinian Territories, Turkey, Albania and southern Italy, but it is necessary to carry out additional studies to determine the geographical range and host usage of this species.

Investigation on haplotypes of ixodid ticks and retrospective finding of Borrelia miyamotoi in bank vole (Myodes glareolus) in Switzerland.

The current status of tick species, important tick-borne bacteria and protozoan parasites is well-documented in Switzerland. However, reports on the genetic diversity and geographical relationships of tick species in this country appear to be in part lacking or outdated. Thus, the aim of this study was to collect ticks from various host species in southern Switzerland, to compare them in a geographical context and to screen in these samples rare tick-borne pathogens hitherto not reported or having low prevalence in Switzerland. In 2019-2020 altogether 177 ixodid ticks were collected from the vegetation, as well as from humans (n = 17), dogs (n = 23), cats (n = 41), red deer (n = 8), a European rabbit and a European hedgehog at 25 locations in three cantons of south Switzerland. Tick species were identified morphologically, followed by DNA extraction and comparison of mitochondrial haplotypes with molecular-phylogenetic methods. Tick DNA extracts, as well as sixty-two rodent liver or spleen tissue DNA extracts (representing six species) available from 2005 to 2006 were screened for trypanosomes, Occidentia massiliensis and Borrelia miyamotoi. Morphologically, three tick species were identified: Ixodes ricinus (n = 170), Rhipicephalus sanguineus sensu lato (n = 6) and I. hexagonus (n = 1). In contrast to companion animals (dogs, cats) immature ticks (larvae and nymphs) predominated on humans, which was a highly significant association (P < 0.0001). Molecular comparison of the cytochrome c oxidase subunit I (cox1) gene with GenBank data established the species as R. sanguineus sensu stricto and confirmed I. hexagonus, both showing 99.8-100% sequence identity to conspecific ticks from northern Italy. Seventy-nine specimens morphologically identified as I. ricinus revealed high 16S rRNA gene haplotype diversity and represented two phylogenetic groups. Two I. ricinus haplotypes from Switzerland belonged to the same haplogroup with I. inopinatus from Spain, Germany and Austria as well as with I. ricinus reported from a broad geographical range of Europe (including Italy, the Netherlands, Poland, Latvia and Sweden). All 141 tick DNA extracts (from five R. sanguineus s.l., 135 I. ricinus and one I. hexagonus) and 62 rodent tissue DNA extracts were negative for trypanosomes and O. massiliensis. However, B. miyamotoi was identified in a bank vole (Myodes glareolus) and three ticks by sequencing. From Switzerland, this is the first report of tick haplotypes that are phylogenetically closely related to I. inopinatus. However, based on their morphology, both specimens are considered as I. ricinus. These results highlight the importance that the identification of I. inopinatus should be based on coherent morphologic and molecular properties. This is also the first report of rodent-borne B. miyamotoi in Switzerland. Taking into account the year of collection (2005), in a chronological order this might be the first indication of B. miyamotoi in any rodent species in Europe.