Based on physiography, climate, land use and soil type, Pakistan is divided into 10 AEZs [41 ]. However, the fodder availability and climatic conditions mainly govern the type of livestock species kept across different AEZs. The bovine population is mainly distributed in two provinces (Punjab and Sindh) of Pakistan. Ticks (n = 774) were collected from clinically healthy cattle (n = 242) and water buffaloes (n = 200) from 30 villages located in six districts of Punjab and Sindh from September to November 2017. These districts are located in five different AEZs and include Bahawalpur (sandy desert), Okara (northern irrigated plain), Jhelum and Layyah (arid; two districts were selected to cover diversity within this zone) districts in Punjab and Sukkur (southern irrigated plain) and Thatta (Indus delta) districts in Sindh (Fig. 1).

Map of Pakistan showing the districts (grey-coloured areas) included in this study. The names of districts include Jhelum (1), Okara (2), Layyah (3), Bahawalpur (4), Sukkur (5) and Thatta (6). Abbreviations: KPK, Khyber Pakhtunkhwa; FATA, Federally Administered Tribal Areas; AJ & K, Azad Jammu and Kashmir

Tick specimens from each animal were stored in separate tubes containing 70% ethanol. Subsequently, each tick was morphologically characterized under a dissecting microscope (Olympus SZ40, Japan) using dichotomous keys [42 (link), 43 ]. Following morphological identification, ticks of the same species from the same animal were pooled in one tube. This resulted in a total of 234 tubes where 131 of those contained ticks from cattle whereas 103 were from buffaloes. DNA was extracted from one tick per tube as per the protocol described previously [Ghafar et al., 2019b, unpublished]. Morphological characterization of ticks was validated using PCR by amplifying cytochrome c oxidase subunit 1 (cox1) gene, 16S rRNA gene, and the second internal transcribed spacer and these results have been submitted for publication previously [Ghafar et al., 2019b, unpublished].
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