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Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109–452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology. [ABSTRACT FROM AUTHOR]

Babesia gibsoni (B. gibsoni), an intracellular apicomplexan protozoan, poses great threat to canine health. Currently, little information is available about the B. gibsoni (WH58) endemic to Wuhan, China. Here, the mitochondrial (mt) genome of B. gibsoni (WH58) was amplified by five pairs of primers and sequenced and annotated by alignment with the reported mt genome sequences of Babesia canis (B. canis, KC207822), Babesia orientalis (KF218819), Babesia bovis (AB499088), and Theileria equi (AB499091). The evolutionary relationships were analyzed with the amino acid sequences of cytochrome c oxidase I (cox1) and cytochrome b (cob) genes in apicomplexan parasite species. Additionally, the mt genomes of Babesia, Theileria, and Plasmodium spp. were compared in size, host infection, form, distribution, and direction of the protein-coding genes. The full size of the mt genome of B. gibsoni (WH58) was 5865 bp with a linear form, containing terminal-inverted repeats on both ends, six large subunit ribosomal RNA fragments, and three protein-coding genes: cox1, cob, and cytochrome c oxidase III (cox3). Babesia, Theileria, and Plasmodium spp. had a similar mt genome size of about 6000 bp. The mt genomes of parasites that cause canine babesiosis showed a slightly smaller size than the other species. Moreover, Babesia microti (R1 strain) was about 11,100 bp in size, which was twice larger than that of the other species. The mt form was linear for Babesia and Theileria spp. but circular for Plasmodium falciparum and Plasmodium knowlesi. Additionally, all the species contained the three protein-coding genes of cox1, cox3, and cob except Toxoplasma gondii (RH strain) which only contained the cox1 and cob genes. The phylogenetic analysis indicated that B. gibsoni (WH58) was more identical to B. gibsoni (AB499087), B. canis (KC207822), and Babesia rossi (KC207823) and most divergent from Babesia conradae in Babesia spp. Despite the highest similarity to B. gibsoni (AB499087) reported in Japan, B. gibsoni (WH58) showed notable differences in the sequence of nucleotides and amino acids and the property in virulence to host and in vitro cultivation. This study compared the mt genomes of the two B. gibsoni isolates and other parasites in the phylum Apicomplexa and provided new insights into their differences and evolutionary relationships. [ABSTRACT FROM AUTHOR]