Exploring the Coinfection and Genetic Diversity of Multiple Tick-Borne Pathogens in Livestock Population of Punjab, Pakistan

Tick-borne diseases affecting domestic animals and humans have increased globally in recent years. Pakistan, in particular, faces a significant economic threat from ticks, where two specific species, Rhipicephalus microplus and Hyalomma anatolicum, act as vectors for various pathogens such as piroplasma, Anaplasma, Ehrlichia, and Rickettsia that pose a significant burden on livestock production in the country. To better understand the risk that tick-borne pathogens (TBPs) pose to livestock in Pakistan, we conducted a cross-sectional study of the occurrence, diversity, and coinfection of these pathogens in small and large ruminants owned by small farms as well as in ticks collected from these animals. We collected blood samples from 224 cattle, 224 buffalo, 69 goats, and 56 sheep, gathered from 112 farms located in seven districts of Punjab, one of Pakistan's largest province. In addition, we collected a total of 476 ticks attached to these animals. Based on the identification of tick species through morphology and sequence analysis of the 16S rRNA and cytochrome c oxidase subunit 1 (cox1) gene, we confirmed that the most commonly collected tick species were Rh. microplus (38.65% of all individuals), H. anatolicum (31.93%), and Rh. decoloratus (8.40%). Notable pathogens detected in the collected ticks included Theileria annulata (18.4% prevalence), Anaplasma ovis (15.79%), A. centrale (13.16%), and Rickettsia slovaca (13.16%). In blood samples, the most frequently detected pathogens were T. annulata (n = 8), Babesia bovis (n = 7), A. centrale (n = 6), and B. bigemina (n = 5). In some cases, both cattle and buffaloes were found to be coinfected with B. bovis, T. annulata, and A. centrale. These findings provide valuable insights into the circulation of TBPs in livestock and highlight the need for further research on the epidemiological risk that these pathogens pose to ruminants in Pakistan.