Molecular Detection of Anaplasma marginale and Theileria annulata in Buffaloes by Using Duplex PCR from Sheikhupura, Jhang, and Rawalpindi Districts of Punjab, Pakistan.

Tick and tick-borne pathogens are among the major threats to animal health and production causing huge economic losses in the form of morbidity and mortality throughout the world. Due to the scarcity of epidemiological data and appropriate diagnostic methods, the study was carried out to detect Anaplasma marginale and Theileria annulata from buffaloes of Sheikhupura, Jhang, and Rawalpindi districts by using duplex PCR. A total of 1,152 blood samples of buffaloes were screened for epidemiological investigations. DNA extracted from blood samples was used for the amplification of the msp1ß gene and cytochrome b gene by using single and duplex PCR. The prevalence of these pathogens and associated risk factors were observed through the multiple logistic regression method. The overall prevalence of a single infection of A. marginale was found to be 14.26% and for T. annulata it was 15.28%. The mixed infection, through duplex PCR in the buffalo population, was observed as 12.92%. Different associated risk factors were evaluated to assess their association with the prevalence of these pathogens. Generally, the prevalence was significantly higher in female buffaloes (P=0.0001) during summer season (P=0.0001). Sequencing and phylogenetic analysis of A. marginale showed that Sequence Pak-4* was closely similar to sequences CP006847.1 and appeared in the same clade. Sequence Pak-4* was ascended to Pak-3* isolate and descendant to MK792344.1 Pakistani isolate. On the other hand, phylogenetic analysis of T. annulata showed that the Pak-2* sequence appeared to be descendants of Indian isolate MH78945.1. In the future, this study will be helpful for large-scale analysis of multiple species by using duplex and multiplex PCR and also for improving the control of associated tick-borne diseases in endemic regions through vaccine development. [ABSTRACT FROM AUTHOR]