Study On Biological Characteristics And Functional Genes Of Taenia Pisiformis

Taenia pisiformis is a globally distributed and common intestinal tapeworm. The adult of Taenia pisiformis establish and mature in the small intestine of canines and felines such as dogs and foxes. The cysticercosis usually parasitizes the liver capsule, greater omentum and mesentery in rabbits. China is the world's largest producer of rabbits. Taenia pisiformis larvae have been found in 24 provinces of China, which have become one of the most common parasites, severely affecting rabbit breeding.Here, the biological characteristics, synonymous codon usage and functional genes of Taenia pisiformis were studied. The main results of researches are summarized as following:1. Biological characteristics of Taenia pisiformisIn order to make further investigation on the biological characteristics of Taenia pisiformis, five dogs were infected experimentally with Cysticercus pisiformis. The numbers of gravid prolottides evacuated per day were recorded. Fifty-five gravid proglottides were collected randomly to observe the numbers and sizes of eggs in each gravid proglottid. Micro-examination was conducted to observe the incubation of eggs activated by 1% sodium hypochlorite solution. The mature Cysticercus pisiformis were isolated from the rabbits sacrificed on day 60 post-infection and counted. The results showed that the five dogs firstly evacuated gravid progottides on day 50 post-infection (PI), 43 PI,47 PI,39 PI and 45 PI, respectively. The largest number of gravid proglottides evacuated per day was 158. The number of eggs evacuated harbored in each per gravid proglottid ranged from 60 to 26480, and the average number was 3372. The size of eggs was (41.32±4.53) ?m× (36.23±4.92)?m on average. Egg hatch process lasted about 4 to 5 minutes. Cysticercus pisiformis mainly parasitized on great omentum and mesentery. This study should provide the important basic information for Cysticercus pisiformis of rabbits.2. Synonymous codon usage patterns in Taenia pisiformis and 43 parasitic platyhelminth mitochondrial genomesSynonymous codon usage of Taenia pisiformis was examined based on 8118 reconstructed annotations of transcriptomic sequences. The mean value of GC content for the reconstructed genes was 49.48%. Twenty-four codons were determined as "optimal codons". All translational optimal codons (except CGU) ended with G or C. The genes positions on the primary strand were strongly positively correlated with GC content at the third codon position of individual genes. The gene expression level assessed by CAI value, hydrophobicity and aromaticity in encoded proteins were significantly correlated with the GC content at the third codon positions and with the effective number of codons (ENC), respectively. We inferred that the gene expression level, the hydrophobicity and aromaticity of the encoded proteins also influence the codon usage in Taenia pisiformis.Among the mitochondrial genomes of 19 trematode species, the GC content of third codon positions varied from 0.151 to 0.592, with a mean of 0.295 ± 0.116. In cestodes, the mean GC content of third codon positions was 0.254 ± 0.044. A comparison of the nucleotide composition at fourfold synonymous sites revealed that, on average, there was a greater abundance of codons ending on U (51.9%) or A (22.7%) than on C (6.3%) or G (19.14%). Twenty-two codons, including UUU, UUA and UUG, were frequently used. In the ENC-plot, most of points were distributed well below or around the expected ENC curve. In addition to compositional constraints, the degree of hydrophobicity and the aromatic amino acids also influenced codon usage in the mitochondrial genomes of these 43 parasitic platyhelminth species.3. Cloning, expression of Tp-UBC2 gene and functions of recombinant Tp-UBC2 from Taenia pisiformis oncosphereThe full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTp-UBC2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against Taenia pisiformis larvae infection. The open reading frame of the Tp-UBC2 gene was 444bp and encoded a 16.63kDa protein. There were 79.3% reductions (P< 0.01) in recovery of larvae compared with that in the control group. Anti-rTp-UBC2-specific antibodies from immunized rabbits had significantly higher levels of IgG (P< 0.01) when compared with control groups, but no significant differences in IgA levels were observed between the two groups (P> 0.05). Dot-ELISA assay showed a high sensitivity (83.3?97.6%) and specificity (88.8?98.4%) to detect Taenia pisiformis larvae infections.4. Cloning, expression of Tp1 gene and construction of Dot-ELISA diagnostic method for rabbit Taenia pisiformis larvaeThe complete coding region of Tp1 gene was amplified from the cDNA of activated oncospheres by RACE-PCR and RT-PCR. The expression vector of pET32a-Tp1 was successfully constructed and expressed in Escherichia coli BL21(DE3) with about 45.8kDa. The positive serum of rabbit Taenia pisiformis larvae could specifically bound to Tp1 recombinant protein through Western Blotting. This purified recombinant fusion protein, rTp1, was probed by Dot-ELISA with sera from rabbits infected with Taenia pisiformis larvae and with other parasites. Results showed that the Dot-ELISA assay had both high sensitivity (92.9?97.6%) and specificity (95.2?98.4%) to detect Taenia pisiformis larvae infections.5. Cloning, expression of Tp-dUTPase gene from Taenia pisiformis oncosphere and construction of Dot-ELISA diagnostic method for rabbit Taenia pisiformis larvaeThe complete coding region of Tp-dUTPase gene was amplified from the cDNA of activated oncospheres by RT-PCR. The expression vector of pET32a-Tp-dUTPase was successfully constructed and expressed in Escherichia coli BL21(DE3) with about 36.20kDa. The positive serum of rabbit Taenia pisiformis larvae could specifically bound to Tp-dUTPase recombinant protein through Western Blotting. This purified recombinant fusion protein, rTp-dUTPase, was probed by Dot-ELISA with sera from rabbits infected with Taenia pisiformis larvae and with other parasites. Results showed that the Dot-ELISA assay had both high sensitivity (85.7?92.9%) and specificity (86.4?88.0%) to detect Taenia pisiformis larvae infections.6. Bioinformatics analysis of Tp-TSP2 gene from Taenia pisiformis oncosphereThe complete coding region of Tp-TSP2 gene was amplified from the cDNA of activated oncospheres by RT-PCR. The expression vector of pET32a-Tp-TSP2 was successfully constructed. A search of the public databases revealed that Tp-TSP2 shared 92.23% similarity with the nucleotide sequence of TSP2 from Echinococcus multilocularis. Some bioinformatics analysis, such as amino acid and atomic compositions, isoelectric point and secondary structure predictions, was performed. The results showed that the cloned open reading frame comprised of 621bp. After the bioinformatics analysis, we found that the Tp-TSP2 gene encoded 206 amino acids with molecular weight 52.40kDa.