Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone

Abstract

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydın region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.

A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone

“…Tacytb and Tapin1 sequences of wild type TaC12 were 100% identical to the reference Ankara gene sequences. The mutation found in Tacytb following BPQ exposure was located within the predicted ubiquinone biding site of cytochrome B (Q 01 ) that includes residues 116 to 144 of TaCytB (PiroplasmaDB ID: Tap370b08.q2ca38.03c) [ 28 ]. The ATG to ATT change led to a non-synonymous amino acid codon substitution of methionine to isoleucine at codon 128 (M128I).…”

“…BPQ resistance in the field is associated with clonal selection of specific parasite genotypes [ 28 ]. We therefore used a cloned T .…”

“…The boundaries of the sites are assigned based on the pairwise alignment of TaCytB and PfCytB sequences and following the assignment by [ 8 ]. We have mapped the locations of all reported BPQ mutation sites [ 13 – 15 , 28 , 31 ] on our model as shown in S2B Fig . As shown in S2B Fig , the majority of mutations are observed within or near the Q 01 and Q 02 sites, but at least one mutation (V227M) outside these sites has also been reported.…”

“…DNA sequencing chromatograms were visualized in Chromas software. Cytb primers and pin1 primers were those described previously [ 17 , 28 ].…”

“…However, these molecules are far from entering the market leaving BPQ as the only choice for field veterinarians to treat clinical theileriosis. Unfortunately, one study has shown tick transmissibility of BPQ-resistant T. annulata parasites [27] and another detailed investigation found increasing prevalence of cytochrome B mutations associated with BPQ resistance in the field [28]. The Hacilarilioglu et al study reported that BPQ selection pressure results in reduced clonal diversity of parasite genotypes within the host.…”