Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

Sequence analysis of Theileria annulata surface protein in Chinese isolates.

OBJECTIVE: To study the TaSP polymorphism in three Chinese isolates of Theileria annulata. METHODS: The isolates from Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region were cultured in RPMI 1640 medium. TaSP gene was amplified from genomic DNA extracted from schizonts using polymerase chain reaction (PCR) and sequenced. Its amino acid sequence comparison was carried out with Clustal W2 multiple sequence alignment program. Molecular component and motif prediction were performed with online servers. RESULTS: The comparison of TaSP amino acid sequences of the three isolates showed that the central region (aa position 38-161) predicted to be the highly immunogenetic domain was polymorphic both in size and amino acid sequence, while the N-terminal (first 37 aa) and C-terminal (last 154 aa) parts were strongly conserved. Phylogenetic analysis and percentage identity revealed that the Chinese isolates were closely related to the isolates from Turkey, but quite different from those of India, Morocco and Tunisia. More importantly, variability was noticed among Chinese isolates, which caused both the location and number's differences of motif (casein kinase II phosphorylation sites) among three TaSP sequences. CONCLUSION: TaSP polymorphism exists in the Chinese isolates of T. annulata.

[Morphological comparison of two species of Theileria in sheep].

Two splenectomized sheep were infected respectively with Theileria luwenshuni and T. uilenbergi, 2 species identified by PCR. When piroplasms were found in blood smears from ears of the sheep, morphological observation on the Theileria spp. was carried out by optical microscopy. By Giemsa staining, the cytoplasm exhibited in slight blue and nucleus in purple. The two Theileria species displayed various shapes, but pyriform-shaped, round-shaped and needle-shaped parasites appeared in every stage of the infection. In fact, there is no significant morphological difference between the two species.

[Prokaryotic expression of Bm86 gene of Boophilus microplus and optimization of the expression condition].

A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates, and subcloned into the prokaryotic plasmid pGEX-4T-1. The recombined plasmid was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time. SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST (Mr 94 000) after being induced with IPTG. High level expression of Bm86-GST was found at 1 mmol/L IPTG condition a fter incubation for 8 h at 37 degree C, and the expression level of the recombinant Bm86-GST reached up to 29% of total E coli proteins Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B. microplus positive serum.