[Study on hepatocyte apoptosis of domestic pigs experimentally infected with Taenia asiatica and Taenia saginata].

OBJECTIVE: To investigate apoptosis in liver tissue of the domestic pigs infected with eggs of Taenia asiatica and Taenia saginata. METHODS: The adult worms of T. asiatica and T. saginata were collected and identified from the taeniasis patients in Dunyun and Congjiang districts, Guizhou province. Eggs were collected from gravid proglottids and prepared by washing and centrifugation. Nineteen 20-day hybrid domestic pigs (Duroc-Yorkshire-Landrace strain) were randomly divided into T. asiatica group (6 pigs), T. saginata group (8 pigs) and control group (5 pigs). Each animal of experimental groups was infected with 1.5 x 10(5) eggs by stomach injection. On day 15, 32, 46 and 74 after infection, animals were sacrificed and liver samples were collected for further experiments. The liver tissues were sliced for glass slides and prepared for ultrathin sections. The apoptosis of hepatocytes was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling. The morphological features of liver tissue were observed under transmission electron microscope. RESULTS: The infection rate of two experiment groups reached 100%. Better developed cysticerci were found in liver of T. asiatica group than that of T. saginata group, but the liver pathological changes caused by cysticerci were similar. On day 15 and 32 after infection, hydropic degeneration, obvious vacuolization and some balloon-like degeneration were found in hepatocytes, and focal hepatic necrosis was observed. On day 46, spotty necrosis occurred in some local liver tissues. On day 74, main damages were granulomatous reactions surrounding cysticercus and focal liver fibrosis. On day 46, apoptosis index in T. asiatica group [(15.07 +/- 3.42)6%] and T. saginata group [(17.13 +/- 1.62)5%] was considerably higher than that in the control [(9.53 +/- 1.06)%] (P < 0.05). On day 74, apoptosis index in T. asiatica group [(27.33 +/- 0.92)5%] and T. saginata group [(34.20 +/- 0.73)%] was higher than that in the control [(13.60 +/- 2.26)%] (P < 0.05), and the apoptosis index in T. saginata group was significantly higher than that of T. asiatica group (P < 0.05). Simultaneously, morphological characteristics of apoptosis were clearly observed in hepatocytes in two experimental groups, showing shrunken, wrinkled and deformed nucleus with consolidation of chromosomes and appearance of apoptotic body. CONCLUSION: The hepatocyte apoptosis of domestic pig is induced in the middle and late stages of infection by the cysticerci of T. asiatica and T. saginata, indicating that hepatic apoptosis might be related to focal liver fibrosis of the host caused by the cysticerci.

[Prokaryotic expression, identification and histo-localization of the Taenia asiatica enolase gene].

OBJECTIVE: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. METHODS: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. RESULTS: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. CONCLUSION: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.

[Informatics analysis of malate dehydrogenase from Taenia saginata asiatica].

Tools from bioinformatics websites such as NCBI, ExPaSy were used for the analysis. The malate dehydrogenase full-length gene from Taenia saginata asiatica was 1 212 bp in length, with a coding region of 30-1 028 bp and coding 332 amino acids. It was a complete and full-length gene compared with the homologues in GenBank. The protein showed no transmembrane region, with stable physical-chemical characteristics. Three major linear epitopes located aa95-aa100, aa322-aa327 and aa117-aa122, with certain distance from each other on the surface of spatial structure of malate dehydrogenase (MDH). The last one was the linear epitope of Taenia. This cytosolic malate dehydrogenase gene is a potential antigen for diagnosis.

[Cloning and prokaryotic expression of malate dehydrogenase gene of Taenia saginata asiatica and immunogenicity analysis of the recombinant protein].

OBJECTIVE: To clone and express the lactate dehydrogenase (LDH) gene of Taenia saginata asiatica and analyze the immunogenicity of the recombinant protein. METHODS: By screening the full length cDNA plasmid library, the coding region of LDH was amplified with PCR, and cloned into the prokaryotic expression vector pET-30a (+), then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. RESULTS: PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blotting analysis of LDH recombinant protein testified that the recombinant protein could be recognized by sera of the Taenia saginata asiatica infected swine and the patient. CONCLUSIONS: The LDH gene of Taenia saginata asiatica has been cloned and expressed, and the purified protein has been confirmed with immunogenicity.

[Morphological observation on the adult worms of Taenia saginata in western China].

OBJECTIVE: To investigate the morphological characteristics of the adult worms of Taenia saginata from four areas of Western China. METHODS: 42, 41, 7 and 18 integral worms of Taenia saginata were collected from Duyun and Congjiang of Guizhou Province, Wushi of Xinjiang, and Lhasa of Tibet respectively. The length of worms was measured and the segments were counted. The specimens of scolex, mature and gravid proglottids of the worms were stained, measured and photographed. RESULTS: The mean length of the worms from Duyun, Congjiang, Wushi and Lasa was (1.81+/-0.69) m, (3.84+/-1.32) m, (2.76+/-0.86) m and (3.72+/-1.12) m, and with (574.64+/-189.33), (913.84+/-317.41), (971.29+/-168.30) and (940.38+/-368.26) proglottids, respectively. The mean ratio of the distance between two lateral excretory vessels and the length of vitellarium of the mature proglottids was (1.71+/-0.13), (2.23+/-0.06), (2.03+/-0.21), (2.31+/-0.15) respectively. All the 3 parameters of the worms from Duyun were significantly less than those from other 3 areas (P<0.05). Rudimentary rostellum was found obviously in 3 of 10 scolices of the worms from Duyun. CONCLUSION: The morphological characteristics of the adult worms from Duyun are similar to that of Taenia saginata asiatica, while those of the worms from Congjiang, Wushi and Lhasa are alike to those of Taenia saginata saginata.

[Epidemiological investigation of Taenia saginata asiatica in Duyun, Guizhou and detection of amino acids and elements of adult worms].

OBJECTIVE: To study epidemiological factors of taeniasis and to detect amino acid and element components of adult worms in Duyun of Guizhou Province. METHODS: 1. Traditional methods were used for epidemiological investigation. 2. Automatic amino acid analyzer and bioassay were applied for the detection. RESULTS: Among 70 persons with clinical symptoms, 25 patients (24 men and 1 woman) were found to have adult taenia worms in their faeces after taking Areca catechu L. and other drugs. Sixteen amino acids and 12 elements were determined in adult worms. CONCLUSION: Duyun area in Guizhou is a highly endemic area of taeniasis. The pathogenic parasite is identified as Taenia saginata asiatica. Its clinical symptoms are similar to that of Taenia saginata saginata.