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Graphical abstractResearch highlights► Natural populations of Theileria annulata show extensive diversity. ► Evidence of panmixia with linkage disequilibrium detected in some localities. ► Individual cattle are infected with a number of genotypes. ► Multiplicity of infection positively correlates with age of host.

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BackgroundBuparvaquone (BW 720C) is the major hydroxynaphtoquinone active against tropical theileriosis (Theileria annulata infection). Previous studies showed that buparvaquone, similarly to others hydroxynaphtoquinone, probably acts by binding to cytochrome b (cyt b) inhibiting the electron transport chain in the parasite. Several observations suggested that T. annulata is becoming resistant to buparvaquone in many endemic regions (Tunisia, Turkey and Iran), which may hinder the development of bovine livestock in these areas.Methodology/Principal FindingsIn the present study we sought to determine whether point mutations in T. annulata cytochrome b gene could be associated to buparvaquone resistance. A total of 28 clones were studied in this work, 19 of which were obtained from 3 resistant isolates (ST2/12, ST2/13 and ST2/19) collected at different time after treatment, from a field treatment failure and nine clones isolated from 4 sensitive stocks of T. annulata (Beja, Battan, Jed4 and Sousse). The cytochrome b gene was amplified and sequenced. We identified five point mutations at the protein sequences (114, 129, 253, 262 and 347) specific for the clones isolated from resistant stocks. Two of them affecting 68% (13/19) of resistant clones, are present in the drug-binding site Q02 region at the position 253 in three resistant clones and at the position 262 in 11 out of 19 resistant clones. These two mutations substitute a neutral and hydrophobic amino acids by polar and hydrophilic ones which could interfere with the drug binding capabilities. When we compared our sequences to the Iranian ones, the phylogenetic tree analyses show the presence of a geographical sub-structuring in the population of T. annulata.Conclusions/SignificanceTaken together, our results suggest that the cytochrome b gene may be used as a tool to discriminate between different T. annulata genotypes and also as a genetic marker to characterize resistant isolates of T. annulata.

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Four Theileria annulata cell lines were characterised at low passage levels using two polymorphic markers and then used to infect calves. Their virulence seemed to be related to the number of genotypes present within the cell line. In all, 3 of the 4 cell lines were cultured up to passage 100 or 200 and inoculated into calves. Their characterisation using the same markers indicated that the attenuation was related to a reduction in the parasite polymorphism down to a single genotype. The immunogenicity of the three attenuated cell lines was assessed in calves using two types of challenge. Optimal protection was observed against homologous challenges. The level of immunity to heterologous challenges appeared to decrease with attenuation and seemed to depend on the cell line used.

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