RPS8--a new informative DNA marker for phylogeny of Babesia and Theileria parasites in China.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. We compared the intron-exon structure and DNA sequences of the RPS8 gene from Babesia and Theileria spp. isolates in China. Similar to 18S rDNA, the 40S ribosomal protein S8 gene, RPS8, including both coding and non-coding regions is a useful and novel genetic marker for defining species boundaries and for inferring phylogenies because it tends to have little intra-specific variation but considerable inter-specific difference. However, more samples are needed to verify the usefulness of the RPS8 (coding and non-coding regions) gene as a marker for the phylogenetic position and detection of most Babesia and Theileria species, particularly for some closely related species.

First report on the occurrence of Rickettsia slovaca and Rickettsia raoultii in Dermacentor silvarum in China.

BACKGROUND: Rickettsioses are among both the longest known and most recently recognized infectious diseases. Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China. METHODS: In an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened by polymerase chain reaction based on the outer membrane protein A gene. RESULTS: 22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%). CONCLUSIONS: This study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens for rickettsial disease in this region will define the incidence of infection.

[An indirect ELISA for the detection of Babesia caballi in equine animals].

OBJECTIVE: To clone and express BC48 gene of Babesia caballi, and to establish an indirect ELISA for the diagnosis of B. caballi in equine animals. METHOD: The genomic DNA of B. caballi was extracted from the infected donkey blood. BC48 gene was amplified by PCR. The PCR product was cloned into expression plasmid pET28a, and expressed in E. coli BL21 with IPTG induction. The recombinant protein was purified by Ni-NTA affinity chro-matography and was used as a diagnostic antigen to establish an indirect ELISA. The reaction conditions of the indirect ELISA were optimized. Specificity and sensitivity of this method were evaluated. RESULT: BC48 gene of B. caballi was 1 272 bp. The recombinant protein was expressed in E. coli BL21 as a soluble protein with a molecular weight of about M, 46 000 under induction of IPTG. The concentration of purified protein was 12.98 mg/ml. The best conditions were obtained for the ELISA when the antigen concentration was 65 microg/ml with the serum dilution of 1:80. The protein specifically reacted with serum from donkey infected by B. caballi, but did not react with serum from donkey infected by Theileria equi (B. equi). Both ELISA and microscopy were applied to examine 17 donkeys in the field, 3 were positive by ELISA and 2 were found parasite-positive, respectively. CONCLUSION: The indirect ELISA method may be used to detect B. caballi infection in equine animals.

[Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

OBJECTIVE: To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. METHODS: Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. RESULTS: The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. CONCLUSION: The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

[Construction of cDNA expression library of salivary gland from Boophilus microplus].

Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.