Control of ticks and tick-borne pathogens is a priority for human and animal health. Livestock-holders extensively rely on acaricide applications for tick control. Different groups of acaricides including cypermethrin and amitraz have been consistently used in Pakistan. There has been a gap in understanding the susceptibility or resistance of Rhipicephalus microplus, the most prevalent tick in Pakistan, to acaricides. The present study aimed to molecularly characterize cypermethrin and amitraz targeted genes such as voltage-gated sodium channel (VGSC) and octopamine tyramine (OCT/Tyr) of R. microplus ticks in Khyber Pakhtunkhwa (KP), Pakistan to monitor the acaricides resistance. Tick specimens were collected from cattle and buffaloes in northern (Chitral, Shangla, Swat, Dir, and Buner), central (Peshawar, Mardan, Charsadda, Swabi, and Nowshera), and southern districts (Kohat, Karak, Lakki Marwat, Tank, and Dera Ismail Khan) of KP, Pakistan. Different concentrations of commercially available cypermethrin (10%) and amitraz (12.5%) were prepared for in vitro larval immersion tests (LIT). In LIT, the average mortality rate of immersed larvae was recorded that was increased gradually with an increase in the concentration of specific acaricide. The larvae's highest mortality rates (94.5% and 79.5%) were observed at 100-ppm of cypermethrin and amitraz, respectively. A subset of 82 R. microplus ticks was subjected to extract genomic DNA, followed by PCR to amplify partial fragments of VGSC (domain-II) and OCT/Tyr genes. The BLAST results of the consensus sequence of VGSC gene (domain-II) showed 100% identity with the acaricides susceptible tick sequence from the United States (reference sequence). Obtained identical sequences of OCT/Tyr genes showed maximum identity (94-100%) with the identical sequences reported from Australia (reference sequence), India, Brazil, Philippines, USA, South Africa, and China. Thirteen single nucleotide polymorphisms (10 synonymous and three non-synonymous) were observed at various positions of partial OCT/Tyr gene fragments. The SNP at position A-22-C (T-8-P) in OCT/Tyr gene has been linked to amitraz resistance in R. microplus ticks. Molecular analysis and LIT bioassay's findings indicate the availability of resistant R. microplus ticks in the KP region. To our understanding, this is the first preliminary study to monitor cypermethrin and amitraz resistance via molecular profiling of cypermethrin and amitraz targeted genes (VGSC and OCT/Tyr) in combination with in vitro bioassays (LIT) in R. microplus ticks from Pakistan.
Knowledge of molecular identification of tick-borne pathogens in camels in Saudi Arabia is very limited; few molecular epidemiological studies have been under taken. This study was to detect Anaplasma spp. and Piroplasma spp. in camels from Asir Province, Saudi Arabia. A total of 150 blood samples were collected from camels in Asir Province and investigated by polymerase chain reaction (PCR) that targeted 18S rRNA and 23S rRNA to detect the DNA of Piroplasma spp. and Anaplasma spp., respectively. The positive samples for 23S rRNA were assayed again by PCR targeting the 16S rRNA. All the blood samples were free from Piroplasma spp. infection. Three camels (2%) were found to be positive for Anaplasma infection through use of PCR that targeted the 23S rRNA gene. There were no significant differences between ages or sexes in the camels that tested positive for Anaplasma. All positive Anaplasma infections were recorded in camels that were infested by ticks. Two Anaplasma sequences for the16S rRNA gene were deposited in GenBank with accession numbers MN882724 and MN882725. They recorded 99.16% and 99.34% similarities (respectively) with KF843825.1 (Candidatus Anaplasma camelii reported in Unizah, Saudi Arabia). Phylogenetic analyses revealed that the two sequences recorded in this study were close to each other; both were located in one cluster with Candidatus Anaplasma camelii isolates that were recorded before in the adjacent areas of Unizah in Saudi Arabia and Iran. In conclusion: two new Anaplasma genotypes close to Candidatus Anaplasma camelii were found in camels in Asir Province, Saudi Arabia for the first time. The camels in this province were found to be free of Piroplasma infection.
Rhipicephalus microplus tick highly affects the veterinary sector throughout the world. Different tick control methods have been adopted, and the identification of tick-derived highly immunogenic sequences for the development of an anti-tick vaccine has emerged as a successful alternate. This study aimed to characterize immunogenic sequences from R. microplus ticks prevalent in Pakistan. Ticks collected in the field were morphologically identified and subjected to DNA and RNA extraction. Ticks were molecularly identified based on the partial mitochondrial cytochrome C oxidase subunit (cox) sequence and screened for piroplasms (Theileria/Babesia spp.), Rickettsia spp., and Anaplasma spp. PCR-based pathogens-free R. microplus-derived cDNA was used for the amplification of full-length cysteine protease inhibitor (cystatin 2b), cathepsin L-like cysteine proteinase (cathepsin-L), glutathione S-transferase (GST), ferritin 1, 60S acidic ribosomal protein (P0), aquaporin 2, ATAQ, and R. microplus 05 antigen (Rm05Uy) coding sequences. The cox sequence revealed 100% identity with the nucleotide sequences of Pakistan's formerly reported R. microplus, and full-length immunogenic sequences revealed maximum identities to the most similar sequences reported from India, China, Cuba, USA, Brazil, Egypt, Mexico, Israel, and Uruguay. Low nonsynonymous polymorphisms were observed in ATAQ (1.5%), cathepsin-L (0.6%), and aquaporin 2 (0.4%) sequences compared to the homologous sequences from Mexico, India, and the USA, respectively. Based on the cox sequence, R. microplus was phylogenetically assembled in clade C, which includes R. microplus from Pakistan, Myanmar, Malaysia, Thailand, Bangladesh, and India. In the phylogenetic trees, the cystatin 2b, cathepsin-L, ferritin 1, and aquaporin 2 sequences were clustered with the most similar available sequences of R. microplus, P0 with R. microplus, R. sanguineus and R. haemaphysaloides, and GST, ATAQ, and Rm05Uy with R. microplus and R. annulatus. This is the first report on the molecular characterization of clade C R. microplus-derived immunogenic sequences.
Haemaphysalis ticks are globally distributed with the greatest diversity in the Oriental region. This study aimed to primarily provide information on the morphology, host record, and preliminary phylogenetic position of a poorly known tick Haemaphysalis danieli. Herds comprised of goats and sheep were examined for this tick species in Upper Dir, Khyber Pakhtunkhwa, Pakistan. A total of 127 ticks, including males (n = 15, 11.8%) and females (n = 112, 88.2%), were collected, and morphologically identified as H. danieli. The morphological identification was confirmed through the 16S rDNA and cytochrome c oxidase (cox1) sequences. Phylogenetic analysis inferred based on 16S rDNA and cox1 showed a close evolutionary relationship of H. danieli with a conspecific from China and an undetermined Haemaphysalis sp. from China and Anatolia. A total of 32/223 (14.3%) goats in two different herds were the only host infested by H. danieli. The earliest study provided the morphological description of H. danieli male, host record, and phylogenetic position. The information provided herein could assist in minimizing the knowledge gap regarding the systematic and taxonomy of Haemaphysalis species.