Animals ; DNA, Helminth ; isolation & purification ; Genome, Helminth ; Polymerase Chain Reaction ; methods ; Schistosoma japonicum ; genetics

No abstract available.
Animals ; *Base Sequence ; DNA, Helminth/*genetics ; DNA, Mitochondrial/*genetics ; *Databases, Nucleic Acid ; Electron Transport Complex IV/*genetics ; Helminth Proteins/*genetics

BACKGROUNDCystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.

METHODSDNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.

RESULTSWe cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.

CONCLUSIONSWe have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.

Animals ; Blotting, Western ; Computational Biology ; DNA, Helminth ; genetics ; Echinococcus granulosus ; enzymology ; genetics ; Genome, Helminth ; genetics ; Helminth Proteins ; genetics ; metabolism ; Polymerase Chain Reaction

OBJECTIVEIn order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain).

METHODSProbe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals.

RESULTSThe result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity.

CONCLUSIONThe genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.

Animals ; China ; DNA, Helminth ; genetics ; Genes, Helminth ; genetics ; Genetic Techniques ; Polymerase Chain Reaction ; methods ; Schistosoma japonicum ; genetics ; Sensitivity and Specificity

OBJECTIVE: To clone and characterize new genes of Schistosoma japonicum, Sj, and to provide efficient vaccine candidates. METHODS: Sj adult cDNA library was screened with rabbit sera raised against male worm soluble antigen. The inserted cDNA fragments from the positively selected clones were amplified with PCR and further sequenced, as well as characterized through internet NCBI GenBank software. RESULTS: Eleven positive clones were obtained and two were verified by GenBank as new, including a novel gene designated as Sj-P8 (GenBank accession No. AF517843) and a new partial cDNA of Sj myosin (GenBank accession No. AY770506). The two new genes encoded a transmembrane protein of 75 amino acids and a myosin protein fragment of 212 amino acids respectively. CONCLUSION The newly obtained genes may provide useful information for the research on Sj vaccine.
Amino Acid Sequence ; Animals ; Antigens, Helminth ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Helminth ; genetics ; Gene Library ; Genes, Helminth ; genetics ; Male ; Molecular Sequence Data ; Rabbits ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccines, Synthetic

In attempt to investigate histone fractions and non-histones of parasites, nuclei were isolated from Fasciola hepatica by the procedure of Pogo et al. (1966). Histone fractions H1, H2a, H2b, H3 and H4 were prepared from isolated nuclei by the procedure of Johns (1964 and l967). The five histone fractions found in most tissues were also present in the Fasciola hepatica histones. These histone fractions were characterized by amino acid analysis and by polyacrylamide disc gel electrophoresis. Non-histone proteins were extracted from isolated Fasciola hepatica nuclei and separated by SDS-polyacrylamide gel electrophoresis. The results of the experiment were summarized as follows: The yield of whole histone recovered was 2.47 mg per 1 g of Fasciola hepatica. The yield of DNA was 1.02 mg per gm of tissues. Consequently the DNA to histone ratio was 1:2.44. The relative amounts of five fractions, i.e., Hl, H2a, H2b, H3 and H4 were 19.96 percent, 26.48 percent, 29.60 percent, 12.56 percent and 14.37 percent, respectively. Amino acid analysis of the individual histone fractions showed that the over-all compositions were similar but not identical to those of corresponding fraction from calf thymus. It was found that histone H2b fraction of Fasciola hepatica contained detectable amounts of epsilon-N-monomethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. In SDS-polyacrylamide disc gel, it showed that 17 protein bands of nuclear acidic protein can be identified visually.
parasitology-helminth-trematoda ; Fasciola hepatica ; histone ; DNA ; biochemistry ; amino acid ; epsilon-N-monomethyllysine

Bursaphelenchus rainulfi isolated from dead pine trees in Zhejiang, China, is described and illustrated. It also provided some molecular characters of the Chinese population, including the PCR-RFLP and sequences of ITS region and D2-D3 expansion region of the large subunit (LSU) rRNA gene. Both the morphological characters and ITS-RFLP patterns match with the original description. The phylogenetic trees based on the 13 sequences of D2-D3 expansion region of the LSU rRNA gene and ITS region of Bursaphelenchus species were constructed, respectively, with the results showing the similar clades. The phylogenetic relationship based on the molecular data is similar to that with morphological characters. This is the first report of the species on pine wood in eastern China.
Animals ; Biological Evolution ; China ; DNA, Helminth ; genetics ; Nematoda ; anatomy & histology ; genetics ; Phylogeny ; Pinus ; parasitology ; Species Specificity

OBJECTIVETo identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.

METHODSThe egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.

RESULTSEighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.

CONCLUSIONThe cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.

Animals ; Antigens, Helminth ; genetics ; Base Sequence ; China ; DNA, Complementary ; analysis ; Ovum ; Schistosoma japonicum ; genetics ; immunology

OBJECTIVE: To obtain the coding genes related to Schistosoma japonicum (Sj) cercariae 66 to approximately 68 kD antigens,and to provide antigens for diagnosis and vaccine of schistosomiasis. METHODS: Sj cercariae cDNA library was screened using the monospecific anti-sera of rabbit against soluble cercariae 66 to approximately 68 kD antigens as probes.The inserted cDNA fragments of the positive clones were amplified with PCR and identified by agarose gel electrophoresis. Four strong positive clones were further sequenced and analyzed through the internet NCBI/BLAST software. RESULTS: Twenty-one positive clones were obtained, 10 of which revealed a single band (0.5 to approximately 3.0 kb).The 4 strong positive clones showed high identity to SJCHGC05187,SJCHGC05173,SJCHGC06989, and SJCHGC01894 at the nucleotide level. CONCLUSION Four coding genes related with Sj antigens are obtained.
Animals ; Antibodies, Helminth ; immunology ; Antigens, Helminth ; immunology ; Cercaria ; genetics ; immunology ; DNA, Complementary ; genetics ; Gene Library ; Immune Sera ; immunology ; Schistosoma japonicum ; genetics ; immunology

OBJECTIVETo prepare Sj14-3-3 DNA vaccine and observe its immunoprotection against Schistosoma japonicum in mice.

METHODSThe Sj14-3-3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into eukaryotic expression vector pBK. The recombinant plasmid pBK-Sj14-3-3 was extracted, purified and inoculated into BALB/c mice by intramuscular injection. Mice were attacked by Schistosoma japonicum cercariae and then killed. Adult worm and egg were counted, respectively. Diameter of the egg granulomas in the liver of infected mice was measured.

RESULTSElectrophoresis on 1% agarose gel showed that the product of RT-PCR and the inserted fragment of recombinant plasmid digested with EcoR I and Xho I had the same size, about 765 bp, confirming the latter was the 14-3-3 encoding gene by nucleotide sequencing. Adult worm load declined by 27%, average egg load of per gram (EPG) of the liver tissues by 79%, average egg production per couple of adult worm (EPWP) by 51%, and mean diameter of egg granulomas by 29% in vaccinated mice.

CONCLUSIONThe recombinant plasmid pBK-Sj14-3-3 was successfully constructed, which had some immunoprotection against Schistosoma japonicum in infected mice, indicating its potential to be vaccine candidate molecule of Schistosoma japonicum.

14-3-3 Proteins ; genetics ; immunology ; Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; genetics ; immunology ; Cloning, Molecular ; DNA, Helminth ; genetics ; Female ; Helminth Proteins ; genetics ; immunology ; Membrane Proteins ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Parasite Egg Count ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Schistosoma japonicum ; genetics ; immunology ; Schistosomiasis japonica ; immunology ; prevention & control ; Vaccines, DNA ; immunology