Genetic characterisation of the Theileria annulata cytochrome b locus and its impact on buparvaquone resistance in bovine

Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in T. annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 12 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis. [Abstract copyright: Copyright © 2022. Published by Elsevier Ltd.

Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage

International audienceLive attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata–transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/cC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata–infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis. Citation: Echebli N, Mhadhbi M, Chaussepied M, Vayssettes C, Di Santo JP, et al. (2014) Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage. PLoS Negl Trop Dis 8(11): e3183