Exploring the genetic diversity of ticks, molecular detection of multiple tick-borne pathogens in blood and ticks, and their co-infection from small and large ruminants of Punjab, Pakistan

In Recent year, ticks and tick-borne diseases are continuously increasing all over the world. In Pakistan, ticks possess huge economic threat especially R. microplus and H. anatolicum which are the vectors for piroplasmosis, anaplasmosis, ehrlichiosis, and rickettsiosis. Nevertheless, there have been no studies undertaken to detect the coinfections of multiple pathogens present in blood and ticks in Pakistan. To fill this gap, we conducted this study with objectives of (i) to provide insight into tick species found on cattle, buffalo, sheep and goats of Punjab, Pakistan, (ii) to identify the pathogens in ticks and small and large ruminants (iii) to check coinfection of pathogens circulating in ticks and blood of small and large ruminants (iv) to detect the simultaneous presence of the pathogen in ticks and blood of tick-host (small and large ruminants). In this study blood samples of cattle (n=224), buffalo (n=224), goat (69) and sheep (n=56) were collected from 112 farms and 476 ticks from infested animals. Tick species were identified by morphology and by sequence analysis of the 16S RNA and cytochrome C oxidase subunit 1 (cox1) gene. Ticks and blood samples from small and large ruminants were subjected to detect the tick-borne pathogens, including Babesia, Theileria (18S RNA), Anaplasma, Ehrlichia (16S RNA), Borrelia (16S RNA) and Rickettsia spp. (16S RNA and gltA). Out of 112 Farms, 43 (37.5%; 95% CI 29.5-48.0) farms had tick infestation, majority of the ticks were R. microplus (38.65%), H. anatolicum (31.93%), R. decoloratus (8.40%). From ticks, major pathogens; T. annulata (18.4%2), A. ovis (15.79%), A. centrale (13.16%), R. slovaca (13.16%) were detected and from blood major pathogens; T. annulata (n=8), B. bovis (n=7), A. centrale (n=6), B. bigemina (n=5) were detected. The most common coinfection was of R. slovaca, and R. massiliae, followed by A. centrale, and T. annulata. While, in case of the blood, coinfection of B. bovis, T. annulata 4/573 followed by A. centrale, B. bovis 2/573 reported. Coinfection of piroplasmosis and anaplasmosis were simultaneously detected from the eight ticks and blood of the tick host animals. These findings shed the light on tick borne pathogens circulations in livestock and suggest the further research on the vector competence of most common tick species in Pakistan.