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Giemsa-stained blood smears were examined microscopically with immersion oil at 1000x magnification for the presence of piroplasms. 2014b) were used as negative and positive controls respectively.

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1982): Genomic DNA purification kit (Promega, Madison, USA) according to the manufacturer’s instructions. A nested PCR detecting specific Theileria DNA of the 18S r RNA gene was performed using Thei F1 and Thei R2 primers for the primary PCR and Thei F2, Thei R2 primers for the secondary PCR (Heidarpour Bami et al. The discrimination of three Theileria species (Theileria annulata, T. ovis) was done by RFLP analysis of amplicons with restriction enzyme: Hpa II (Fermentas, Lithuania) (Heidarpour Bami et al. The enzymatic digestion mixture consisting of 10 μL PCR amplicons, 10x buffer, and restriction enzyme (10 U) was then incubated at 37 °C for 2 h. ovis either alone or in association with other pathogens is to be investigated.

Restriction digests were separated by electrophoresis in 2% agarose and visualised in a UV transilluminator. The study was financially supported by Laboratoire d’épidémiologie des infections enzootiques des herbivores en Tunisie: application à la lutte (Ministère de l’enseignement supérieuret de la recherche scientifique, Tunisia) and the Deutsche Forschungsgemeinschaft project ‘Molecular epidemiology network for promotion and support of delivery of live vaccines against Theileria parva and Theileria annulata infection in eastern and northern Africa’ (SE862/2-1).

The molecular prevalence by Babesia ovis was significantly higher in females than in males (p Based on their dentition, sheep were ranked into two age groups: less than and more than 2 years of age.

Blood samples were collected in EDTA tubes from each animal and stored at -20 °C until used.

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