Molecular Profiling of Ticks and Associated Pathogens: First Report of <i>Rickettsia sibirica</i> , <i>Rickettsia slovaca</i> , and <i>Babesia microti</i> in Ticks From Pakistan

Global distribution of ticks and their associated tick-borne pathogens (TBPs) presents substantial health concerns for both humans and animals. The present study aimed to investigate the distribution, morpho-molecular identification, and associated TBPs of diverse tick species collected from Khyber Pakhtunkhwa and Punjab provinces of Pakistan. Morphological identifed ticks were molecularly confirmed via cytochrome oxidase I (<i>COI</i>) and <i>16S rRNA</i> genes, which showed 15 different tick species. Among them, we found the highest prevalence rate of <i>Rhipicephalus</i> (<i>Rh</i>) <i>microplus</i> (404/1803; 22.41%), while the lowest prevalent tick species were <i>Haemaphysalis</i> (<i>Hae</i>) <i>montgomeryi</i> (44/1803; 2.44%). Similarly, the highest tick load was found on cattle (544/186; 2.92), while least was found on goats (272/164; 1.66). Various TBPs, including <i>Rickettsia</i> (<i>R</i>) spp. (via <i>gltA</i>, <i>sca4</i>, <i>ompA</i>, <i>ompB</i> genes), <i>Anaplasma</i> (<i>A</i>) sp. (via <i>16S rRNA</i> gene), <i>Coxiella burnetii</i> (via <i>IS1111</i> gene), Piroplasm and <i>Hepatozoon</i> (<i>H</i>) spp. (via <i>18S rRNA</i> gene) were screened. The <i>Rh. microplus</i> tick species showed highest positivity rate (11.63%) for various TBPs, whereas <i>Hae. sulcata</i> ticks were recorded as TBPs free in current study. Among the detected TBPs, <i>Coxiella burnetii</i> was most prevalent (1.72%), followed by <i>A. phagocytophilum</i> & <i>R. slovaca</i> (1.44% each), <i>R. sibirica</i> (0.78%), <i>R. raoultii</i> (0.61%), <i>H. canis</i> (0.55%), <i>R. conorii</i> subsp. <i>raoultii</i> (0.50%), <i>Hepatozoon</i> sp., (0.44%), <i>Theileria</i> (<i>Th</i>) <i>uilenbergi</i> (0.39%), <i>Babesia</i> (<i>B</i>) <i>microti</i> (0.33%), and <i>Th. luwenshuni</i> (0.28%). This study provides the first report of TBPs, including <i>R. slovaca</i>, <i>R. sibirica</i>, and <i>B. microti</i>, in ticks from Pakistan. Phylogenetic analysis was performed based on the aforementioned genetic markers, in which ticks and their associated TBPs formed distinct clades with their corresponding isolates. This research work provides a deep insight regarding the characterization of different ticks and their associated TBPs in Pakistan and, significantly, it presents evidence of potential zoonotic threats to both animal and public health due to these newly detected pathogens.