Assessing genetic diversity and coinfection of tickborne pathogens in small and large ruminants of Punjab, Pakistan

Objective: Tick-borne diseases affecting domestic animals and humans have increased globally in recent years. Pakistan 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. These tick-borne pathogens, responsible for endemic diseases in Pakistan, have never been studied in ticks and blood of tick-infested animals simultaneously. This study aims to use molecular techniques to study the impact of tick-borne pathogens on livestock animals in Pakistan, exploring the diversity and co-infection of tick-borne pathogens in both small and large ruminants and analyzing e ticks collected from the animals.

Methods: To better understand the risk that tick-borne pathogens pose to livestock in Pakistan, we conducted a cross-sectional study of the occurrence, diversity, and co-infection of these pathogens in small and large ruminants owned by small farms and in ticks collected from these animals. We collected blood samples from 224 cattle, 224 buffalo, 69 goats, and 56 sheep from 112 farms in the seven Punjab districts, one of Pakistan's largest provinces. Additionally, we collected a total of 476 ticks attached to these animals. Ticks and blood samples were processed for pathogens identification, such as piroplasma, Anaplasma, Ehrlichia, and Rickettsia and their coinfection using conventional and microfluidic PCR.

Results: Based upon 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 R. microplus (38.65% of all individuals), H. anatolicum (31.93%) and R. decoloratus (8.40%). Notable pathogens detected in the collected ticks included Theileria annulata (18.4%), 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 co-infected with B. bovis, T. annulata, and A. central.

Conclusions: Overall, 13 tick-borne pathogens were identified from ticks and blood samples of animals, encompassing the variability in their distribution among districts of Pakistan. Co-infection of multiple pathogens were observed in both small and large ruminants, highlighting the complexity of pathogen associations. These findings contribute to our understanding of the epidemiology and distribution of tick-borne diseases, which can aid in developing effective control and prevention strategies in veterinary medicine.