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Mitochondrial genomes provide new insights that help elucidating biological features, genetic evolution, and classification of protozoans. Theileria uilenbergi (T. uilenbergi), transmitted by Haemaphysalis qinghaiensis and H. longicornis, is considered as highly pathogenic to sheep and goats in China. This study reports and outlines features of its mitochondrial genome. The T. uilenbergi mitochondrial genome is a linear monomeric molecule of 6.0 kb length, which encodes three protein-coding genes named cytochrome c oxidase I (cox1), cytochrome b (cob), and cytochrome c oxidase III (cox3), as well as six large subunit (LSU) rRNA gene fragments, and ends in terminal inverted repeats (TIRs). The array structure and organization of the mitochondrial genome of T. uilenbergi is identical to that of T. parva. Phylogenetic analysis based on the amino acid sequences of cox1, cob, and cox3 genes suggests that T. uilenbergi is distantly related to the group of transforming Theileria species such as T. parva. This study contributes to a comprehensive understanding of the phylogeny and evolution of the mitochondrial genome of piroplasms and provides useful information of diagnostic marker for T. uilenbergi.

We developed a SYBR green I–based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23–0.89% and 0.23–1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary significance. The assay detected BEFV RNA as early as 1 d post-infection (dpi) and up to 7–8 dpi in the blood samples of experimentally infected cattle. The most stable reference gene, peptidylprolyl isomerase A ( PPIA), was selected for the quantification of BEFV. Viral RNA loads reached peak level at 3–5 dpi and then decreased rapidly through 7–8 dpi. Our assay provides a reliable approach for the detection of BEFV in the early infection stage and for use in the profiling of BEFV kinetics in vivo.

Tropical theileriosis, caused by Theileria annulata, is distributed worldwide and causes great economic losses in dairy. The reliable diagnostic method is critical for prevention and control of the disease. In this study, a sporozoite and macroschizont gene 2 (spm2) protein from T. annulata was used to develop an indirect ELISA for tropical theileriosis. Specificity test showed that there were no cross-reactions with antibodies raised against other bovine piroplasm species using ELISA or western blotting. The specificity and sensitivity were 98.4% and 98.7%, respectively, with a threshold of 35.5% of the specific mean antibody rate (AbR). Furthermore, a total of 196 field sera samples collected from Xinjiang and Gansu provinces were detected by the spm2 ELISA and IFA. The results obtained with the spm2 ELISA and IFA in this study had the moderate agreement. The average positive rates of T. annulata sera samples detected in the present study were close to the prevalence of previous reports in these endemic areas. This indicated that the Spm2 ELISA could be used as a reliable diagnostic tool for serological survey of T. annulata infection in areas where Theileria parva is not present.

In this study, we report the complete genome sequence of bovine ephemeral fever virus (BEFV) JT02L, which has been used in our laboratory, in mainland China, for more than a decade. The genome is 14941 nucleotide (nt), comprising a leader sequence of 50 nt, nucleoprotein (N) gene of 1328 nt, phosphoprotein (P) gene of 858 nt, matrix protein (M) gene of 691 nt, glycoprotein (G) gene of 1897 nt, non-structural glycoprotein (GNS) gene of 1785 nt, α1α2 gene of 638 nt, β gene of 460 nt, γ gene of 400 nt, large multi-functional enzyme (L) gene of 6470 nt and a trailer sequence of 73 nt. Individual genes are separated by intergenic regions (IGRs) of 26, 44, 47, 51, 37, 39, 68 and -21 nt respectively. The overall organization is similar to an Australian BEFV isolate BB7721 but demonstrates some distinctive features including longer α3 and β open reading frames, intact termination/polyadenylation (TTP) sequence downstream of the β open reading frame and a longer β-γ IGR integrated with a 38 nt AT-rich fragment. To our knowledge, this is the first report describing the complete genome of a BEFV strain of East Asian lineage, which may facilitate studies on genomic diversity among geographic strains of BEFV in China and the world.

A putative novel TBC1 domain family member 13 (HqTBC1D13) from Haemaphysalis qinghaiensis was cloned using rapid amplification of the cDNA ends (RACE), the HqTBC1D13 cDNA is 1702 bp in length and encodes 396 amino acid residues with predicted molecular weight of 46.09 kDa. The TBC-domain containing protein has a catalytic ‘arginine finger’ analogous to those of Ras and Rho family GAPs, which is critical determinants of GAP activity. The amino acid sequences of TBC domain were evolutionarily highly conserved across species. The partial coding sequence of HqTBC1D13 with the predicted molecular weight of 37.2 kDa was expressed and purified in the PGEX-4T-1 vector. Real-time RT PCR analysis showed that the HqTBC1D13 was extensively expressed in the tested organs (salivary glands, midguts, ovaries and cuticles), and its transcriptional levels in salivary glands were significantly up-regulate induced by blood-feeding. The recombinant HqTBC1D13 protein vaccination in the rabbit model resulted in the extension of the duration of feeding and the reduction of 37% female engorgement and 14.8% oviposition compared to the control group. These results indicated that the HqTBC1D13 in ticks could be invovled in the regulation of feeding and oviposition.