Search

Multiple ticks (Acari: Ixodoidea) carrying Rickettsiales bacteria have significant importance for both human and animal health. Thus, the purpose of this work was to genetically analyze tick species and their associated Rickettsiales bacteria in animal hosts. In order to achieve these objectives, various animals (including camels, cattle, goats, sheep, dogs, and mice) were inspected in four districts (Mardan, Peshawar, Kohat, and Karak) of Khyber Pakhtunkhwa to collect ticks, while blood samples were collected from all the symptomatic and asymptomatic cattle in all four districts. A total of 234 ticks were obtained from 86 out of 143 (60.14%) host animals, which were morphologically identified as Rhipicephalus turanicus , Rhipicephalus microplus , Haemaphysalis cornupunctata , and Hyalomma asiaticum . Among these, their representative ticks (126/234, 53.85%) were processed for molecular confirmation using cytochrome c oxidase ( cox1 ) gene. Obtained cox1 sequences of four different tick species showed 99.72%-100% maximum identity with their corresponding species reported from Pakistan, China, India, and Kazakhstan and clustered phylogenetically. This study presented the first genetic report of Hy. asiaticum ticks in Pakistan. Moreover, genetically confirmed tick species were molecularly analyzed by PCR for detection of Rickettsiales DNA using partial fragments of 16S rDNA , 190-kDa outer membrane protein A ( ompA ), and 120-kDa outer membrane protein B ( ompB ) genes. In addition, blood samples were analyzed to identify Rickettsiales bacteria using the aforementioned genes. Rickettsiales bacteria were found in 24/126 (19.05%) ticks and 4/16 (25.00%) in symptomatic cattle's blood. The obtained ompA and ompB sequences from Hy. asiaticum ticks showed 99.73%-99.87% with Candidatus Rickettsia shennongii and unidentified Rickettsia sp., whereas the obtained 16S rDNA sequences from cattle's blood and ticks ( Hae. cornupunctata ) showed 99.67% highest identity with Anaplasma phagocytophilum . The 16S rDNA sequence of Rickettsiales DNA from Rh. turanicus ticks showed 100% identity with Ehrlichia canis and unidentified Ehrlichia sp. Obtained sequences of Rickettsiales bacteria were grouped along with their respective species in phylogenetic trees, which were previously reported in Greece, Cuba, Iraq, Turkey, Pakistan, South Korea, and China (mainland and Taiwan). This extensive study explores the wide range of damaging ticks and their corresponding tick-borne bacteria in the area, suggesting a possible danger to both livestock and human communities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Obaid, Shehla, Guan, Rashid and Shams.)

Despite high diversity in the Oriental region, ticks of the genus Haemaphysalis have been neglected regarding their genetic data and vector potential. This study aimed to genetically characterize three species of the genus Haemaphysalis : Haemaphysalis cornupunctata , Haemaphysalis kashmirensis, and Haemaphysalis montgomeryi infesting goats and sheep, and Rickettsia spp. associated with these tick species in the Hindu Kush Himalayan range of Pakistan. Altogether, 834 ticks were collected by examining 120 hosts including goats (64/120, 53.3%) and sheep (56/120, 46.6%), in which 86 (71.6%) hosts were found to be tick-infested. The morphologically identified ticks were subjected to DNA extraction and PCR for the amplification of partial 16S rDNA and cox fragments. Rickettsia spp. associated with the collected ticks were detected through the amplification of gltA , ompA and ompB partial fragments. The 16S rDNA of H. cornupunctata and H. montgomeryi showed a maximum identity of 100% with the sequences of the same species, whereas the 16S rDNA of H. kashmirensis showed the highest identity of 93-95% with Haemaphysalis sulcata . The cox sequence of H. montgomeryi displayed 100% identity with the same species. In comparison, the cox sequences of H. cornupunctata and H. kashmirensis showed maximum identities of 87.65-89.22% with Haemaphysalis punctata and 89.34% with H. sulcata , respectively. The gltA sequence of Rickettsia sp. from H. kashmirensis showed the highest identity of 97.89% with Rickettsia conorii subsp. raoultii , while the ompA and ompB fragments from the same DNA samples revealed 100% and 98.16% identity with Rickettsia sp. and " Candidatus Rickettsia longicornii", respectively. Another gltA sequence amplified from H. montgomeryi ticks showed 100% identity with Rickettsia hoogstraalii , while the attempts to amplify ompA and ompB for R. hoogstraalii were unsuccessful. In the phylogenetic tree, the 16S rDNA of H. cornupunctata clustered with the corresponding species while its cox clustered with H. punctata . Both 16S rDNA and cox sequences of H. kashmirensis clustered with H. sulcata . The gltA sequence of Rickettsia sp. was clustered individually in the spotted fever (SF) group of Rickettsia , while the gltA sequence of R. hoogstraalii was clustered with the same species in the transition group of Rickettsia . In the SF group, the rickettsial ompA and ompB sequence clustered with undetermined Rickettsia sp. and " Candidatus Rickettsia longicornii", respectively. This is the earliest study regarding the genetic characterization of H. kashmirensis . This study indicated that ticks belong to the genus Haemaphysalis have the potential of harboring and/or transmitting Rickettsia spp. in the region.