In the present study, we assembled and annotated the mitochondrial genomes of six ovine Babesia isolates and performed a mitochondrial genomic analysis with published mitochondrial genomes of apicomplexan parasites. The mitochondrial genomes of six ovine Babesia is highly similar to most of Babesia spp. in respect of genome size, high A + T content, genome form, and gene content. However, the mitochondrial genomes of ovine Babesia are smaller than B. microti, B. rodhaini and Theileria equi, and larger than Toxoplasma gondii [19, 20, 42]. The tRNA genes were not found in the mitochondrial genomes of six ovine Babesia, which is consistent with other apicomplexan parasites. We speculate that they may be directly encoded by the nuclear genome. The order and transcriptional direction of three protein-encoding genes are the same in B. bovis, B. bigemina, B. gibsoni , but different from B. microti, T. equi, T. orientalis and Plasmodium falciparum [19, 20].
In the reported mitochondrial genomes of piroplasma, B. microti and B. rodhaini have a dual flip-flop inversion system, range from 184–1082 bp in length . However, one pair of TIR was found at the mitochondrial genomes of other Babesia spp. and Theileria spp., range from 25–1563 bp in length [17-19, 21, 31]. These findings indicated that the number and size of TIRs are one of the main causes of different mitochondrial genome sizes. We also found that different numbers of LSUs are responsible for the size of the mitochondrial genomes. The TIRs are considered to play a crucial role in the replication and stabilization of the linear mitochondrial genomes . In the published mitochondrial genome of apicomplexan parasites, the sequences of the coding genes and LSU are basically the same. Therefore, we speculated that differences in lengths and sequences of TIRs may be responsible for divergence in host-specific and in vitro culture characteristics of protozoa.
The difference between the first-generation and second-generation sequencing data is mainly caused by the nucleotide substitutions, deletions and insertions, which is caused by the different sequencing techniques, assembly and annotation software used. And settings of a few parameters are different, which is one of the reasons for the difference between the two sets of data. The results of Illumina sequencing method are more prone to errors. The Sanger approach is more accurate and thus more appropriate for further studies.
Mitochondria are essential organelles and play an important role in the energy metabolism and the growth and development of apicomplexan parasites . Cytochrome bc1 is an integral membrane protein complex, which is essential to cellular respiration. The highly conserved of the cob gene binding to inhibitors is the molecular basis for the effect of the drugs on yeast, fungi and parasites . The drug binding residues of cob gene of six ovine Babesia are completely consistent. In the conserved PEWY region of the cob gene, Phe278 (yeast number of cob) is present in most organism, whereas T. gondii and B. taurus at this position are Tyr and Ala, respectively. Studies have reported that the L275F mutation in yeast has no effect on enzyme activity, but IC50 has increased ten-fold . Compared with the wild type of yeast, the mutation Y279S indicated a B40-fold increased IC50 (B1.7 mM) for atovaquone , and Y268S in the P. falciparum numbering system results in a 3,000-fold loss of atovaquone sensitivity. In addition, the mutations M133I, L271V and Y268N of cob gene of P. berghei confer resistant to atovaquone . Therefore, we conclude that the mutations of cob gene are largely responsible for the efficacy of drugs (target protein is bc1 complex) in apicomplexan parasites.
Currently, atovaquone and ELQs have been reported for the treatment of human babesiosis and malarial by modifying of the drug target by disruption of cytochrome bc1 complex [25, 27, 28, 39, 41]. In 2019, a B. motasi-like parasite has been detected in human blood in Korea , which suggests that B. motasi may be potentially zoonotic. Therefore, we should investigate the infection of B. motasi in human in China, evaluate the zoonotic potential of B. motasi, and the effect of inhibitors binding to cytochrome bc1 complex.
In this study, the taxonomical relationships of Babesia infective to sheep and goats are consistent with the reported phylogenetic analyses based on cob, cox1, cox3, nuclear small subunit (SSU) and internal transcribed spacer (ITS) [5, 6, 8, 31, 46]. The ovine Babesia are divided into two species: Babesia sp. and B. motasi. B. motasi further fell into two small clades, named BmLT/TZ and BmNX/HB. With the expect of B. conradae, piroplasm infective to the same host fell into one clade. These findings indicated that the mitochondrial genome data are valuable for the population genetic, phylogenetic and molecular epidemiological studies.