Sample collection from horses

All procedures performed in this study involving animals were in strict compliance with the ethical standards of Ahmadu Bello University, Zaria, Nigeria, ABUCAUC/2020/34. Blood samples were collected from 300 horses ranging from 2.5 to 18 years old from three States in northwestern Nigeria (Fig. 1), namely Jigawa (Maigatari: 12° 48′ 18.15′′ N, 9° 26′ 52.13″ E) Kaduna (Zaria 11° 2′ 57.18′′ N, 7° 41′ 56.39″ E; Igabi 10° 48′ 23.53′′ N, 7° 42′ 55.82″ E), and Katsina (Mai’adua 13° 10′ 45.00′′ N, 8° 13′ 48.98″ E). Samples from horses at livestock border markets (Maigatari, Kaduna state; Mai’adua, Katsina state) and samples from 8 horses imported from Argentina (Polo pony) were collected to determine if imported horses are infected with pathogens that cause equine piroplasmosis. Samples from resident horses, including polo horses with permission by owners, were selected to determine the presence of equine piroplasmosis (Igabi, Kaduna state, polo ponies; Zaria, Kaduna state; polo ponies and Dubar horse procession). Table ​Table11 describes samples collected from horses in Nigeria.

Geographic location of the study sites in Nigeria. The map was modified from Wikipedia

Description of horse samples collected in Nigeria

Samples were collected in a vacutainer from the external jugular vein of randomly chosen animals and transported to the Department of Veterinary Medicine, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria. One hundred fifty microliters of blood was applied to Whatman FTA cards (GE Healthcare companies, USA). The application began at the center of the circle and moved out spirally towards the edge to result in a uniform distribution and the opportunity to isolate DNA from repeated sample punches. After applying the blood, the card was stored at room temperature for 6 h to dry. The dried card was sealed in a re-sealable bag with a pack of desiccant and stored in a cool, dry, and dark place until analysis.