Identification of genetic variations linked to buparvaquone resistance in Theileria annulata infecting dairy cattle in India

Abstract

Buparvaquone (BPQ) is used for the treatment of bovine theileriosis, a tickborne disease caused by parasites of the Theileria genus. Studies on T. annulata have linked the mechanism of BPQ resistance predominantly to genetic variations in the parasite cytochrome b (cytb) gene. In addition, cryptic mechanisms of resistance involving the parasite peptidyl-prolyl isomerase (pin1) and dihydroorotate dehydrogenase (dhodh) genes require assessment. In India, where bovine theileriosis is endemic, and BPQ is widely used for treatment, it is necessary to establish the prevalence of genetic variations linked to BPQ resistance. In this study, multiplexed PCR amplification and nanopore sequencing method was used for genotyping the complete gene loci of the three target genes. Analysis of 454 T. annulata field samples collected from seven different states of India revealed the presence of previously reported BPQ resistance associated variations S129G, A146T and P253S in cytb gene and A53P in pin1 gene. The A146T and I203V variations in cytb were found to be prevalent and mostly co-occurring, and their role in BPQ resistance needs further evaluation. This study has revealed the presence of previously reported BPQ resistance-linked mutations in cytb and pin1 genes in T. annulata infecting dairy cattle in India and establishes an Oxford nanopore sequencing method suitable for large-scale surveillance of genetic variation in Theileria parasites from field samples.

Fig 1. A, Schematic representation of coding region of T. annulata cytb, dhodh, and pin1 genes (red colour) including flanking sequences (grey colour) is shown along with a table listing the PCR primer used in the study.

PCR amplification (B, C) and nanopore sequencing (D, E) of cytb (C), dhodh (D), pin1 (P) and all three genes multiplexed (M) from T. annulata species. DNA templates used for PCR and sequencing were obtained from Ana2014 strain (B, D) and a representative field sample (C, E). Lane L in B and C, 1 kb DNA marker ladder. Nucleotide-level mapping of nanopore sequence data for multiplexed PCR amplicons was plotted in real-time using the RAMPART program.

Fig 2. A, Schematic representation of T. annulata CYTb protein annotated with vertical-coloured lines indicating the conserved residues in functional domains shown by horizontal shading (details taken from conserved domain database.

https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The black-filled arrowheads shown at the top are the previously reported variations identified from parasites exhibiting resistance to BPQ. The grey-filled arrowheads represent the variations reported from India [24]. The open and red-filled arrowheads shown at the bottom are the variations identified in this study from field samples compared to the T. annulata reference sequence. The two highly prevalent and co-occurring variations A146T and I203V are highlighted by red-filled arrowheads. B, List of the number of field samples analyzed from each state (column 2; grey highlight) and number of samples with different Tacytb mutations. Data for the two most prevalent variations is shown in red. MH, Maharashtra; GJ, Gujarat; JH, Jharkhand; OD, Odisha; BH, Bihar; UP, Uttar Pradesh; PB, Punjab. C, Co-occurrence of A146T and I203V mutations in Tacytb gene. State-wise sample number is shown in column 2, and number of samples with either one or both mutations is given. State names are abbreviated as in B. D, Multiple sequence alignment of CYTb protein sequences of T. annulata Ankara reference strain (TaAnk), T. annulata Ana2014 Indian isolate (TaAna2014) and P. falciparum 3D7 strain (Pf3D7). Positions of the previously reported mutations linked to BPQ resistance in T. annulata are highlighted in cyan on all three sequences. The positions with variations detected in field samples are shown in bold black fonts and the variant residues observed in these positions are shown in red font above the alignment. The A146T and I203V variation is highlighted in green and the corresponding position in the TaAna2014 sequence is shown in red font. The purple highlighted residues in Pf3D7 cytb are mutated in parasites exhibiting atovaquone resistance.

Fig 3. A, Schematic representation of T. annulata DHODH protein annotated with vertical-coloured lines indicating the conserved residues in functional domains shown by horizontal shading (details taken from conserved domain database.

https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The black-filled arrowhead indicates the C124 residue which corresponds to the C276F mutation in P. falciparum dhodh which confers atovaquone resistance. The open arrowheads shown at the bottom are the variations identified in this study. B, Prevalence of T. annulata dhodh mutations in field samples from different states of India (name of state abbreviated as in Fig 2B; the number of samples analyzed from each state is given within parentheses). C, Multiple sequence alignment of DHODH protein sequences of T. annulata Ankara reference strain (TaAnk), T. annulata Ana2014 Indian isolate (TaAna2014) and P. falciparum 3D7 strain (Pf3D7). The cyan highlighted residue in the Pf3D7 sequence indicates the C276 position which is mutated to confer atovaquone resistance. The corresponding residue in the T. annulata is a leucine (L125) and so the neighboring cysteine residue (C124) is considered; C124 and L125 are highlighted in cyan in TaAnk and TaAna2014 sequences. The residues shown in bold black font are altered in the field samples analyzed in this study. The variant residues observed in these positions are shown in red font above the alignment.

Fig 4. A, Schematic representation of T. annulata PIN1 protein annotated with functional domain shown by horizontal shading (details taken from conserved domain database.

https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The black-filled arrowhead shown at the top identifies the previously reported A53P variation linked to BPQ resistance. The open and red-filled arrowheads shown at the bottom are the variations identified from field sample analysis in this study. B, Prevalence of T. annulata pin1 mutations in field samples from different states of India (name of state abbreviated as in Fig 4B; the number of samples analyzed from each state is given within parentheses). The A53P mutation identified from the field sample is highlighted in green. C, Multiple sequence alignment of PIN1 protein sequences of T. annulata Ankara reference strain (TaAnk), T. annulata Ana2014 Indian isolate (TaAna2014) and T. parva Muguga strain (Tpar). The A53P mutation is highlighted in cyan. The residues shown in bold black font are altered in the field samples analyzed in this study. The variant residues observed in these positions are shown in red font. A frameshift mutation detected at position G99 is indicated with * in red font.