[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.

Molecular detection of Anaplasma infections in ixodid ticks from the Qinghai-Tibet Plateau.

Anaplasma species are tick-transmitted obligate intracellular bacteria that infect many wild and domestic animals and humans. The prevalence of Anaplasma spp. in ixodid ticks of Qinghai Province is poorly understood. In this study, a total of 1104 questing adult ticks were investigated for the infection of Anaplasma species. As a result, we demonstrated the total infection rates of 3.1, 11.1, 5.6, and 4.5% for A. phagocytophilum, A. bovis, A. ovis and A. capra, respectively. All of the tick samples were negative for A. marginale. The positive rates of A. phagocytophilum, A. ovis and A. capra in different tick species were significantly different. The positive rates of A. capra and A. bovis in the male ticks were significantly higher than that in the female ticks. Sequence analysis of A. ovis showed 99.5-100% identity to the previous reported isolates. The sequences of A. phagocytophilum had 100% identity to strains Ap-SHX21, JC3-3 and ZAM dog-181 from sheep, Mongolian gazelles, and dogs. Two genotypes of A. capra were found based on 16S rRNA, citrate synthase (gltA) gene and heat shock protein (groEL) gene analysis. In conclusion, A. bovis, A. ovis, A. phagocytophilum, and A. capra were present in the ticks in Qinghai Province. Anaplasma infection is associated with tick species, gender and distribution. These data will be helpful for understanding prevalence status of Anaplasma infections in ticks in Qinghai-Tibet Plateau.

Molecular epidemiological surveillance to assess emergence and re-emergence of tick-borne infections in tick samples from China evaluated by nested PCRs.

An investigation was performed to detect eight pathogens in ticks collected from grass tips or animals in the southern, central and northeast regions of China. DNA samples extracted from ticks were collected from ten different locations in eight provinces of China and subjected to screening for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Ehrlichia spp., Rickettsia spp., Babesia/Theileria spp., Ehrlichia ruminantium, Coxiella burnetii, and Francisella tularensis, using nested PCR assays and sequencing analysis. The results indicated that Borrelia spp., Rickettsia spp., and Babesia/Theileria spp. were detected in all of the investigated provinces. Ehrlichia spp. was also found in all of the surveyed areas, except Guangxi, Luobei and Tonghe counties in Heilongjiang province. The average prevalence of these pathogens was 18.4% (95% CI=12.8-42.5), 60.3% (95% CI=18.2-65.3), 26.0% (95% CI=25.8-65.1), and 28.7% (95% CI=5.6-35.2), respectively. A sequencing analysis of the pCS20 gene of E. ruminantium revealed an E. ruminantium-like organism (1/849, 0.1%, 95% CI=0-0.3) in one tick DNA sample extracted from Rhipicephalus (Boophilus) microplus in Hunan. In addition, Borrelia americana in Ixodes persulcatus, Babesia occultans in Haemaphysalis qinghaiensis and both Rhipicephalus sanguineus and an Ehrlichia muris-like organism in R. (B.) microplus was detected, possibly for the first time in China. Four DNA sequences closely related to Borrelia carolinensis and/or Borrelia bissettii from Haemaphysalis longicornis, Candidatus Rickettsia principis from H. qinghaiensis, and I. persulcatus and Ehrlichia canis (named E. canis-like) from Haemaphysalis bispinosa were also detected in this work.

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.