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The genus Hyalomma includes the most frequent tick species infesting livestock in North Africa, one of these species, Hyalomma scupense (syn. H. detritum) is particularly important due to its role in the transmission of tropical theileriosis to cattle (Theileria annulata infection). We have cloned and characterized the orthologs of the Bm86 gene from H. scupense strains collected over Tunisia in 2006 and 2009. The recombinant protein rHd86 was expressed in Pichia pastoris for vaccination purpose using a transcript from the 2006 strain. The rHd86 was then purified from the yeast culture supernatant by a filtration and a size exclusion process. It was recognized by specific anti-Bm86 antisera. An important extent of inter-specific diversity ranging from 35 to 40% was recorded between Hd86 and Bm86/Bm95 proteins whilst a very limited level of intra-specific diversity (1.7%) occurred between the Hd86 vaccine candidate protein and its homologues from H. scupense strains collected in 2009. These results emphasise the need for assessing the efficacy against H. scupense and others important cattle Hyalomma species in Tunisia of our Hd86 vaccine candidate alongside with a Bm86 vaccine.