Investigation of Hyalomma turanicum and Hyalomma asiaticum in Pakistan, with notes on the detection of tickborne Rickettsiales

There is limited information on the occurrence of <i>Hyalomma turanicum</i> and <i>Hyalomma asiaticum</i> ticks, as well as associated <i>Rickettsia, Anaplasma,</i> and <i>Ehrlichia</i> species in Pakistan. Addressing this knowledge gap, the current study aimed at morphomolecular confirmation of these ticks and molecular assessment of associated Rickettsiales bacteria (<i>Rickettsia, Anaplasma</i>, and <i>Ehrlichia</i> spp.) in Balochistan, Pakistan. A total of 314 ticks were collected from 74 of 117 (63.2%) hosts, including 41 of 74 (55.4%) goats and 33 of 74 (44.5%) sheep. Subsequently, a subset of microscopically identified ticks was subjected to DNA extraction and PCR to amplify 16S rDNA and <i>cox1</i> fragments. Additionally, <i>gltA</i>, <i>ompA,</i> and <i>ompB</i> fragments were targeted for <i>Rickettsia</i> spp. and 16S rDNA fragments for both <i>Anaplasma</i> and <i>Ehrlichia</i> spp. The 16S rDNA and <i>cox1</i> sequences of <i>Hy. turanicum</i> demonstrated 100% identity with those of the same species previously reported from Pakistan. The 16S rDNA and <i>cox1</i> sequences of <i>Hy. asiaticum</i> exhibited 99.52 and 100% identities, respectively, with corresponding species reported from China, Kazakhstan, and Turkey. The <i>gltA, ompA,</i> and <i>ompB</i> fragments associated with <i>Hy</i>. <i>turanicum</i> showed 100% identities with <i>Rickettsia aeschlimannii</i> reported from Egypt, Italy, Kazakhstan, Kenya, Pakistan, and Senegal. The 16S rDNA sequences of <i>Anaplasma</i> sp. and <i>Ehrlichia</i> sp. associated with both <i>Hy</i>. <i>asiaticum</i> and <i>Hy</i>. <i>turanicum</i> exhibited 99.67 and 100% identities with unknown <i>Anaplasma</i> sp. and <i>Ehrlichia</i> sp. reported from Morocco and Pakistan, respectively. In the 16S rDNA and <i>cox1</i> phylogenetic trees of ticks, <i>Hy</i>. <i>turanicum</i> and <i>Hy</i>. <i>asiaticum</i> from the current study clustered with their respective species. Similarly, in <i>gltA, ompA,</i> and <i>ompB</i> phylogenetic trees of <i>Rickettsia</i>, <i>R. aeschlimannii</i> of the present study clustered with the same species, whereas <i>Anaplasma</i> sp. and <i>Ehrlichia</i> sp. of this study clustered with undetermined <i>Anaplasma</i> spp. and <i>Ehrlichia</i> spp. in the 16S rDNA phylogenetic tree of Anaplasmataceae. Among the DNA samples from the screened ticks, a coinfection rate of <i>R. aeschlimannii</i>, <i>Anaplasma</i> sp., and <i>Ehrlichia</i> sp. (2 of 80, 2.5%) was observed in <i>Hy. turanicum</i>, whereas individual infection rates were noted as follows: <i>R. aeschlimannii</i> (8 of 80, 10%), <i>Anaplasma</i> sp. (5 of 80, 6.3%), and <i>Ehrlichia</i> sp. (5 of 80, 6.3%). This study marks the first record of molecular characterization of <i>Hy</i>. <i>turanicum</i> and <i>Hy</i>. <i>asiaticum</i> as well as the detection of associated <i>R. aeschlimannii</i>, <i>Anaplasma</i> sp., and <i>Ehrlichia</i> sp. in Balochistan, Pakistan.