Population Genetic Structure,Transcriptome, And Functional Genes Of Taenia Pisiformis

The larval of the tapeworm Taenia pisiformis (Cestoda: Taeniidae) is the causative agent of cysticercosis, and widely distributed in the world. Cysticercosis usually parasitizes in the liver capsule, the gastric omentum majus and the mesentery of rabbits, while adult T. pisiformis establish and mature in the small intestine of dogs and foxes. China is the largest producer of rabbits, and the T. pisiformis has become one of the most common parasites, which severely affecting rabbit breeding. It mainly causes emaciation, weakens resistance against other diseases of the host, and what is worse may cause death. There are no obvious clinical symptoms of Taenia pisiformis cysticercosis. So far, the pharmacotherapy is generally useful for controlling cysticercosis, but which may cause drug resistance and drug residues. Furthermor, the cost of pharmacotherapy is relatively high, and which were harmful for human health. Most current reports mainly pay more attention to the epidemiology, pathogenicity, prevention and treatment of T. pisiformis. However, the study of prenatal diagnosis and immune prevention of T. pisiformis do not have reported Therefore, it is essential for the study of the population genetic structure, transcriptome, and functional genes of T. pisiformis. The main research work is as follows:1. Population genetic diversity based on cox2and nad4genes in Taenia pisiformis from SichuanIn order to determine the level of genetic variation and population genetic diversity of T. pisiformis of rabbit from seven different regions (Ya’an, Panzhihua, Leshan, Guangyuan, Luzhou, Guang’an, and Aba) in Sichuan, China, the partial sequences of the mitochondrial cytochrome oxidase subunit2(cox2,468bp) and NADH dehydrogenase subunit4(nad4,1,077bp) gene of23isolates of T. pisiformis were obtained by PCR. The analysis results showed that there were21variable sites in cox2sequences, and23variable sites in nad4sequences. The sequence divergence of cox2and nad4were0-3.0%and0-2.9%, respectively. Five haplotypes with0.451of Hd value and0.00536of Pi value was presented in cox2nucleotide sequences, and seven haplotypes with0.625of Hd value and0.00538of Pi value was presented in nad4nucleotide sequences. NJ/MP trees showed that there was no direct correlation between phylogeny of seven populations and geographical regions. The results indicate that the lower genetic variation and heredity diversity presented in seven geographical populations among intrapopulation and intraspecific T. pisiformis.2. Genetic population structure analysis of Taenia pisiformis from Sichuanby using the mitochondrial cytochrome b geneThe genetic population structure of T. pisiformis from eight separate regions in Sichuan province were analyzed based on the sequences of mitochondrial cytochrome b (cytb). The fragments of cytb (922bp) gene were amplified from53isolates in eight districts of T. pisiformis.12haplotypes were found in the53cytb sequences. Molecular genetic variation intra-districts included98.43%of intra-district and1.57%of inter-districts. Lower genetic diversity was presented in district, and genetic differentiation of inter-district was not obvious. There was no correlation between genetic differentiation, gene flow and geographic distribution. Eight districts did not form apparent geographical clusters, but the clustering of their haplotypes was obvious in NJ and MrBayes trees. Thus, eight districts may come from a larger population.The analysis of population dynamics presented that size of populations may not experienced expansion in process of transmission history. And mutational time adjacent haplotype began with late pleistocene.3. Genetic population structure of Taenia pisiformis based on mitochondrial coxl and nadl genes61isolates of T. pisiformis from eight separated regions in Sichuan province, China, were used for determining their genetic population structure.based on thepartial sequences of the mitochondrial cytochrome c oxidase subunit I (coxl,1,427bp) and NADH dehydrogenase1(nadl,738bp). Five haplotypes were found, with molecular genetic variation of intra-district as the main source. There was no genetic variation in Luzhou; the other districts had the lower genetic diversity. No correlation between genetic differentiation, phylogeny and geographic distribution was observed, and mutational time adjacent haplotype begun with late pleistocene. Eight districts may belong to a larger population. And the analysis of population dynamics presented that size of populations may not experenced expansion in process of transmission history.4. Transcriptomic analysis of Taenia pisiformisIn present study,2.67million sequencing clean reads and72,957unigenes of Taenia pisiformis were obtained using the RNA-seq technique (Illumina sequencing). Based on a sequence similarity with known proteins, a total of26,012unigenes (no redundancy) were identified after quality control procedures via the alignment of four databases. Overall,15,920unigenes were mapped to203Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Functional distribution characteristics were gained through comparing four cestode species (72,957unigenes of T. pisiformis,30,700ESTs of T. solium,1,058ESTs of between Echinococcus granulosus and E. multilocularis]), with the cluster of orthologous groups (COG) and gene ontology (GO) functional classification systems. Furthermore, the conserved common genes in these four cestode species were obtained and aligned by the KEGG database. The identification of conserved genes may provide novel approaches for potential drug targets and vaccinations against cestode infections. The results of the study can provide the basic data for study of functional genomics, immunity and gene expression profiles of cestode species.5. Cloning, expression of TpFABP gene and construction of Dot-ELISA diagnostic method for Taenia pisiformis cysticercosisThe complete coding region of TpFABP gene was amplified from cDNA of cysticeri, using specific pair primers by TsFABP gene of T. solium. This sequence contain an open reading frame encoding a putative protein of133amino acids. The expression vetcor of pET32a/TpFABP was successfully constructed and obtained the36.1kDa fusion protein after IPTG induced.. The positive serum of rabbit Taenia pisiformis cysticercosis could specifically bound to TpFABP recombinant protein through Western Blotting. And TpFABP recombinant protein was expressed in perinuclear cytoplasm of tapeworm and cystic wall of cysticeri by immunohistochemistry. Furthermore dot-ELISA diagnostic method of TpFABP successfully constructed, which had higher sensibility (93.22%) and specificity (94.21%) in169rabbits serum for T. pisiformis cysticercosis. 6. Cloning, expression of Tpl8gene and immune efficacy analysis of rabbit Taenia pisiformis cysticercosisTp18gene was selected from transcriptome of adult T. pisiformis. The Tp18cDNA fragments were amplified by RACE t from the activated oncospheres. Then the Tp18gene was subcloned into pET32a vector. The vetcor of pET32a/Tpl8was transformed into Escherichia coli BL21and obtain the Tp18fusion protein after IPTG induced. The expression product was analyzed by SDS-PAGE and Western blotting. An open reading frame of Tp18gene was378bp. The recombinant Tp18protein had an approximate molecular mass of33kDa, and had postival signals with the serum from rabbit naturally infected with T. pisiformis eggs. The Reduction in numbers of metacestodes with the Tp18recombinant protein was95.59%and97.38%. Meanwhile, the specific antibody was IgG for recombinant Tp18protein.7. Cloning, expression of TpcC1gene and construction of Dot-ELISA diagnostic method for rabbit Taenia pisiformis cysticercosisThe complete coding region of TpcC1gene was amplified from the cDNA of activated oncospheres by RACE technology, and the primers were designed by TpcC1unigene from transcriptome dataset of adult T. pisiformis. The expression vetcor of pET32a/TpcCl was successfully constructed and expressed in E.coli BL21(DE3) with about58.3kDa fusion TpcCl protein. The positive serum of rabbit T. pisiformis cysticercosis could specifically bound to TpcCl recombinant protein through Western blotting. Furthermore Dot-ELISA diagnositic method for T. pisiformis cysticercosis successfully constructed with higher sensibility (94.55%) and specificity (96.49%) in169rabbits serum using TpcC1recombinant protein.