Lan He

Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China.

The presence and prevalence of tick-borne haemoparasites in water buffalo from the Hubei province, south China was investigated using the reverse line blot (RLB) hybridization assay and phylogenetic analysis of the parasite 18S rRNA gene. Theileria buffeli (19.1%) was the most frequently found species in all of the locations, followed by Babesia orientalis (8.9%), Babesia bovis (1.0%) and Babesia bigemina (0.7%). Only 12 (3.9%) of the samples had mixed infections. Eleven samples with single infections were selected for further characterization using 18S rRNA gene sequence analysis. Phylogenetic analysis showed that the eight T. buffeli 18S rRNA gene sequences obtained grouped into four clusters, of which three grouped with the known T. buffeli types B and D. The remaining five grouped separately from the previously describe T. buffeli types, constituting new T. buffeli types. The two B. bigemina 18S rRNA gene sequences obtained grouped closely with B. bigemina Kunming; this serves as the first report of B. bigemina in the Hubei province. The B. orientalis Daye 18S rRNA gene sequence obtained grouped closely with the previously reported B. orientalis Wuhan strain and with Babesia sp. Kashi 1 and Kashi 2.

Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii.

To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-gamma expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P<0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P<0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P>0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.

Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70.

The heat shock protein 70 (hsp70) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis infected buffalo sera. The nucleotide sequence of the cDNA was 2192bp with an open reading frame (ORF) of 1944bp encoding a polypeptide of 648 amino acid residues. Phylogenetic analysis of the 1944bp sequence together with 30 inter-erythrocytic protozoa hsp70 nucleotide sequences available from GenBank was performed. The results showed that B. orientalis was occurred within the Babesia clade, and most closely related to B. ovis and B. bovis. Similar topologies were obtained from trees based on apicomplexa parasite 18S rRNA sequence. Meanwhile, the BoHsp70 gene was cloned into pET-32a and subsequently expressed in Escherichia coli Rosetta strain as a Trx-fusion protein. The recombinant hsp70 of B. orientalis (rBoHsp70) was purified and evaluated as an antigen in the western blot. The serum from B. orientalis infected buffalo recognized the 92kDa rBoHsp70 expressed in E. coli Rosetta (DE3) by western blotting. The rabbit antiserum against rBoHsp70 recognized a specific 70kDa band in lysates of B. orientalis infected buffalo erythrocytes. These results suggested that hsp70 gene was well conserved among inter-erythrocytic protozoa and the BoHsp70 might be a diagnostic and candidate vaccine antigen.

Loop-mediated isothermal amplification (LAMP) detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758) in China

Loop-mediated isothermal amplification (LAMP) is a rapid method with high specificity and efficiency under isothermal condition using a set of four specifically designed primers that recognize six distinct sequences on the target gene. In this study, a LAMP method was developed for specific detection of Babesia orientalis in water buffalo (Bubalus babalis, Linnaeus, 1758). Four primers were designed from the V4 hypervariable region of the 18S rRNA gene of B. orientalis. Blood samples were collected from B. orientalis experimentally infected water buffalo as well as from 165 water buffalo from eight different regions of the Hubei province, south China. Genomic DNA was extracted, subjected to the LAMP assay and compared with results obtained using a previously described semi-nested PCR. The LAMP assay proofed to be B. orientalis specific and more sensitive than the semi-nested PCR. While previously B. orientalis had not been reported north of the Yangtse River, our results show that B. orientalis has spread to the north of the river. This could pose a serious threat to the water buffalo industry.

Loop-mediated isothermal amplification (LAMP) assay for detection of Theileria sergenti infection targeting the p33 gene.

Theileria is a widespread, intraerythrocytic tick-borne protozoan parasite. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method offering rapid, accurate and cost-effective diagnosis of infectious diseases. In this study, the LAMP method was developed for detecting Theileria sergenti. Four primers were designed from six distinct regions of the target gene, a 33-kDa major piroplasm surface protein (p33) gene in T. sergenti. The specificity assay showed it was specific for T. sergenti whilst the LAMP was able to detect a parasitemia level of 0.000002% which was more sensitive than conventional PCR. 154 field samples from water buffalo and 159 field samples from cattle were analyzed using the LAMP method. About 60.4% (96/159) of cattle samples were positive by LAMP, compared to 30.0% (46/154) of water buffalo samples that were positive. Compared with conventional PCR, the LAMP method exhibited higher detection abilities than conventional PCR. All the results indicated that the LAMP assay is a simple and convenient diagnostic tool for theileriosis.

DEVELOPMENT AND EVALUATION OF REAL-TIME PCR ASSAY FOR THE DETECTION OF BABESIA ORIENTALIS IN WATER BUFFALO (BUBALUS BUBALIS, LINNAEUS, 1758)

Abstract Babesia orientalis is the causative agent of babesiosis in water buffalo (Bubalus babalis, Linnaeus, 1758). In this study, a TaqMan real-time PCR assay was developed for quantitative detection of B. orientalis in water buffalo. Hybridization probe and oligonucleotide primers were designed based on the v4 region of 18S rRNA gene. Detection limit was determined at 2 parasites. Blood samples were collected from experimentally infected water buffalo, as well as from 180 field samples, which were collected from 4 different geographical locations to the north and south of the Yangtse River. The parasite was detected by real-time PCR on day 2 until day 39 post-infection, while reverse line blot (RLB) was on day 6 until day 36 in experimentally infected water buffalo. For the results of 180 field samples, statistical analysis showed no significant difference in relative effectiveness of real-time PCR and RLB. The analysis also indicated that there was no difference in the prevalence of B. orientalis between the regions of south and north of the Yangtse River by both the real-time PCR assay and RLB detection. These results indicated that the parasite infection has spread to the north of the Yangtse River.

Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection.

A sensitive and inexpensive quantification method with online extraction using a short C-18 column for sulfonamide residues in honey by high performance liquid chromatography with fluorescence detector was developed and validated. In sample preparation, acid hydrolysis was used to break the N-glycoside bond between the honey sugar and sulfonamide drugs and derivatization of sulfonamide residues with fluorescamine was conducted at pH 3.5 using a citrate buffer (0.5M) in the honey matrix. The chromatography was carried out on Zorbax Extended C-18 (250mm×4.6mm; 5μm) column, using a mixture of acetonitrile and an acetate buffer (pH 4.50, 20mM) as a mobile phase. A Zorbax Extended C-18 (12mm×4.6mm; 5μm) column was used for online extraction of fifteen sulfonamide residues from honey sample with the help of a two position valve. The limit of quantification of sulfonamide residues in honey was less than 3ngg(-1), and the percentage recovery of study compounds in spiked honey sample was from 80% for sulfacetamide to 100% of sulfachloropyridazine. The developed method has excellent linearity for all studied sulfonamides with a correlation coefficient 0.993.

Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China.

Abstract: The sika deer (Cervus nippon) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia/Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.

Characterization and annotation of Babesia orientalis apicoplast genome.

BackgroundBabesia orientalis is an obligate intraerythrocytic protozoan parasite of the buffalo (Bubalus bubalis, Linnaeus, 1758) transmitted by the tick Rhipicephalus heamaphysaloides. It is the causative agent of water buffalo babesiosis, one of the most important pathogens of water buffalo in central and southern China. As a member of the phylum Apicomplexa, B. orientalis possesses a relatively independent and alga originated organelle the apicoplast. Apicoplasts in other apicomplexa parasites are involved in the biosynthesis of haem, fatty acids, iron-sulphur clusters and isoprenoids. Some of these metabolic pathways were shown to be essential for parasite survival, therefore can serve as potential drug targets.Methods30 pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported apicoplast genomes of Babesia bovis and Theileria parva. Conventional PCRs was performed to obtain overlapped fragments to cover the whole apicoplast genome. Then the apicoplast genome of B.orientalis was sequenced, assembled and aligned with reported apicoplast genomes of B. bovis and T. parva. The obtained apicoplast genome was annotated by using Artemis and comparing with published apicomplexan apicoplast genomes. The SSU and LSU nucleotide sequences generated were used in a phylogenetic analysis using the maximum likelihood implemented in MAGE 6.0.ResultsWe have obtained and analyzed the complete genome sequence of the B. orientalis apicoplast. It consisted of a 33.2 kb circular DNA (78.9 % A + T). The apicoplast genome unidirectionally encodes one large and one small subunit ribosomal RNAs, 24 tRNA genes, 4 DNA-dependent RNA polymerase beta subunits (rpoB, rpoC1, rpoC2a and rpoC2b), 17 ribosomal proteins, one EF-Tu elongation factor, 2 Clp protease chaperones, and 14 hypothetical proteins. In addition, it includes two copies of the clpC gene. The structure and organization of the B. orientalis apicoplast genome are most similar to those of the B. bovis apicoplast.ConclusionsThis is the first report of the complete sequence of the B. orientalis apicoplast genome. This information should be useful in the development of safe and efficient treatment against buffalo babesiosis.

Mitochondrial genome of Babesia orientalis, apicomplexan parasite of water buffalo (Bubalus babalis, Linnaeus, 1758) endemic in China

BackgroundApicomplexan parasites of the genus Babesia, Theileria and Plasmodium are very closely related organisms. Interestingly, their mitochondrial (mt) genomes are highly divergent. Among Babesia, Babesia orientalis is a new species recently identified and specifically epidemic to the southern part of China, causing severe disease to water buffalo. However, no information on the mt genome of B. orientalis was available.MethodsFour pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported mt genomes of B. bovis, B. bigemina, and T. parva. The entire mt genome was amplified by four sets of PCR. The obtained mt genome was annotated by aligning with published apicomplexan mt genomes and Artemis software v11. Phylogenetic analysis was performed by using cox1 and cob amino acid sequences.ResultsThe complete mt genome of B. orientalis (Wuhan strain) was sequenced and characterized. The entire mt genome is 5996 bp in length with a linear form, containing three protein-coding genes including cytochrome c oxidase I (cox1), cytochrome b (cob) and cytochrome c oxidase III (cox3) and six rRNA large subunit gene fragments. The gene arrangement in B. orientalis mt genome is similar to those of B. bovis, B. gibsoni and Theileria parva, but different from those of T. orientalis, T. equi and Plasmodium falciparum. Comparative analysis indicated that cox1 and cob genes were more conserved than cox3. Phylogenetic analysis based on amino acid sequences of cox1, cob and cox1 + cob, respectively, revealed that B. orientalis fell into Babesia clade with the closest relationship to B. bovis.ConclusionsThe availability of the entire mt genome sequences of B. orientalis provides valuable information for future phylogenetic, population genetics and molecular epidemiological studies of apicomplexan parasites.