[Effect of Toxoplasma gondii prugniaud strain infection in pregnant mice on the learning ability of the F1 generation].

OBJECTIVE: To study the effect of Toxoplasma gondii prugniaud strain infection on female reproductive toxicity in mice and learning ability of their F1 generation. METHODS: Thirteen ICR mice were each infected intragastrically with 10 T. gondii cysts on the 15th day of pregnancy (late stage pregnancy). 12 mice were treated with physiological saline as control. The time from conception to birth and the number of offspring were recorded. Three mice from each group were sacrificed when pregnant 20 d, placentas from the sacrificed and output stillbirth mice were examined by using histopathology and immunohistochemistry. DNA extraction was performed from placenta tissue, and then T. gondii B1 gene was amplified by PCR. The F1 generation mice from experiment group and control group were tested by Morris water maze test. Statistical analysis on learning and memory ability was made by SPSS 13.0 software. RESULTS: The time from conception to birth in experiment group [(19.2 +/- 1.751)d] was shorter than that in control group [(21.0 +/- 1.732)d] (P < 0.05). No significant difference was found in the number of offspring between experiment group (70) and control group (85) (P > 0.05). Microscopic examination with HE staining showed multiple T. gondii among placental villi, the increase of the number of Hofbauer cells, blood sinus expansion and hyperemia, and visible nucleated erythrocytes. Immunohistochemically, T. gondii antigen was detected in placenta tissue. T. gondii B1 gene was detected in placenta tissue (194 bp). On the third and fourth day of the Morris water maze test, the latency of experiment group [(29.92 +/- 4.28) s, (27.69 +/- 6.23) s] was longer than that of the control [(24.07 +/- 5.32) s, (22.25 +/- 7.94) s] (P < 0.05). In the spatial probe test, the distance across the platform quadrant of experiment group [(384.66 +/- 41.33) cm] was shorter than that of the control [(426.12 +/- 46.48) cm] (P < 0.05). CONCLUSION: T. gondii Prugniaud strain infection in late stage pregnancy of mice may induce reproductive toxicity and affect the learning and memory capability of the F1 generation.

[Dynamic pathologic changes in pigs infected with Taenia solium].

Four three-way crossed hybrid pigs were infected each with 80,000 Taenia solium eggs. One pig was sacrificed respectively at 40, 80, 120, and 150 d post-infection, and tissue samples containing cysticerci were collected for pathological sections. The results showed that in 40-80 days, cysticerci in the liver were surrounded by membrane formed with the hyperplastic fibrous connective tissue and inflammatory cells. On 120th day, a large number of the cysticerci were dead or calcified. On 150th day, all cysticerci died or calcified. On 40th, day, the inflammatory cells infiltrated around the cysticerci in the tongue and skeletal muscle, and the inflammatory responses near the scolex were more serious than that near cyst wall. In the 80-150 days, the inflammatory cells showed band like distribution. The pathological response in cardiac muscle was similar to that of the skeletal muscle and the tongue, but with more inflammatory cells. On 40th day, there were a few inflammatory cells infiltrating around cysticerci in the brain, in the 80-150 days, a large number of inflammatory cells aggregated around the cysticerci. The inflammatory response in the tissues progressed with the duration of infection and the inflammation in the liver was significantly stronger than that in other tissues.

[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.

[Total protein analysis by two-dimensional electrophoresis in cysticerci of Taenia solium and Taenia asiatica].

Two 20-day-old three-way crossed hybrid pigs were infected with 80000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by Image-Master 2D Platinum 6.0 software. The results showed that there were (236 +/- 12) and (231 +/- 14) protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus.

[Histopathology changes in mice infected with Toxoplasma gondii Prugniaud strain].

OBJECTIVE: To observe the symptoms and dynamic changes of histopathology in the organs from ICR mice infected by Toxoplasma gondii Prugniaud strain. METHODS: Thirty ICR mice were infected intraperitoneally with cysts, 10 cysts per mouse. 16 mice were injected with PBS. Incidence of the mice was observed. Three mice from the infected group and two mice from the control group were sacrificed, and the liver, spleen, lung, brain, kidney and heart were collected for pathology and immunohistochemistry examinations on the day 5, 10, 15, 20, 25, 30, 60 and 90 post-infection. RESULTS: The infected mice began to fall ill at 6 d post-infection, symptoms including decreased appetite, pilomotor fur, sloth, shakes and diarrhea, with a mortality rate of 20%. From 5 d to 20 d post-infection, microscopic examination for HE stain-slides showed the destroyed liver structure, cellular edema, ballooning change, focal necrosis, sinus hepatic expansion and hyperemia, and inflammatory infiltration. Splenic corpuscles demolished and disappeared, red pulp widened and white pulp atrophied, splenic sinusoid extended with hyperemia. Lungs showed destruction of the structure and pathological changes of interstitial pneumonia. The pathological changes began to alleviate until recovery after 20 d post-infection. In the brain, neuronal degeneration and necrosis were found at 10 d post-infection. Some neuroglial cell tubercle, blood vessel sleeve cuffing, inflammatory cell infiltration on cavitas subarachnoidealis and cysts were observed from 15 d to 90 d. Granulation tissue was seen at 90 d post-infection. By immunohistochemistry test, internal organs showed toxoplasma antigen at 5 d post-infection, and the positive reaction was remarkable at 10 d post-infection, then began to taper until negative. Toxoplasma antigen was revealed in the brain from 10 d to 90 d post-infection. CONCLUSION: Non-specific clinical manifestation and the degeneration, necrosis and inflammatory cell infiltration in poly-organs appear in earlier period of toxoplasma tachyzoite infection in the ICR mice, followed by the co-existing phenomenon of non-specific infection with cysts in the brain.

[Identification of Taenia solium with abnormal number of scolex hooklets].

OBJECTIVE: To identify 3 suspected adults Taenia solium with abnormal number of hooklets on scolex collected from 3 patients of Dali in Yunnan Province. METHODS: Tapeworms were observed with unaided eyes. Morphology of the scolices and gravid proglottids was observed under microscope. DNA of gravid proglottids of the 3 adult tapeworms was extracted. T. solium mitochondrial cytochrome c oxidase subunit 1 gene (cox1) fragment and the full coxz1 gene were amplified by PCR. The cox1 gene of one isolate was sequenced. Eggs were hatched and oncospheres were inoculated into mice subcutaneously. Each mouse was subcutaneously injected with 1 mg dexamethasone once daily. Sixty days after infection, all mice were sacrificed and the morphology of cysticerci was observed. Two macaque monkeys were fed with eggs (2.5 x 10(5) per monkey). Euthanasia and autopsy were performed on day 47. Morphology of cysticerci were observed by light and scanning electron microscopy, and pathological changes of livers were observed. RESULTS: The number of hooklets on scolices of the three tapeworms was 0, 4 and 10, respectively, and lateral uterine branches in gravid proglottids were 7-12. PCR results of co1l gene fragment with species-specific primer for T. solium were all positive. The complete sequence of cox1 gene had 99.8% identity to the reported T. solium sequences. Cysticerci were obtained from hypoderm of mouse, muscles and hearts of monkey. Four suckers and 26-28 hooklets ranged in two rows around rostellum on scolex were microscopically observed. Milia-like lesions were found in monkey liver. Histological examination showed that there was fibrous connective tissue hyperplasia and eosinophil infiltration around lesion, and parasites were found in some cysts. CONCLUSION: The three tapeworms with abnormal number of hooklets have all been identified as T. solium. The larvae can infect macaque and lead to muscle and liver cysticercosis.

[Research progress on molecular identification and biologic behavior of Taenia saginata in Western China].

This article reviews the epidemiological investigation of taeniasis saginata in 10 counties of 7 provinces/ autonomous regions in western China. The morphological observation of adult worms, molecular identification of 10 isolates of the worms, experimental infection on pigs and cattle with Taenia saginata and T. asiatica, observation on development and biology behavior of cysticercus, and pathological changes in the intermediate host pig and cattle revealed that T. asiatica is a new species, instead of a subspecies of T. saginata.

[Screening, identification and bioinformatic analysis of Taenia solium cysticercus].

OBJECTIVE: To screen and identify specific antigens of Taenia solium cysticercus, and predict the function of target proteins using bioinformatics method. METHODS: Patients infected with Taenia solium were dewormed by decoction arecae and pumpkin seeds to collect worms, and eggs were then prepared. Six three-way crossed hybrid pigs were randomly divided into experimental group and control group, and each experimental pig was infected with 80,000 T. solium eggs. Serum samples were collected at 40 days after infection. The total protein of T. solium cysticercus was separated by two-dimensional electrophoresis, and Western blotting was performed to find out distinct antigens. Proteins from the two groups were identified by ESI-Trap MS. Query in NCBI database was made to confirm function of the proteins. RESULTS: 207+/-9 spots were detected through Coomassie brilliant blue-stained gels with Mr 14,400-94,000 and pI 3.0-10.0. Western blotting showed 7 specific antigen spots with pool sera of infected pigs. Four of the 7 antigens with known functions were respectively ascribed to cytoskeletal actin-2 (adult-specific), tropomyosin (cysticercus-specific), AF239799-1 annexin (cysticercus-specific) and actin-1 (cysticercus-specific). CONCLUSIONS: Three specific antigens of Taenia solium cysticercus have been identified.

[Analysis of Toxoplasma gondii specific antigens by two dimensional electrophoresis and western blotting].

The total protein of Toxoplasma gondii RH strain tachyzoites was separated by two-dimensional electrophoresis (2-DE), and Western blotting was performed to find out distinct antigens. 209 spots were detected through Coomassie brilliant blue-stained small gels (7x8 cm, pH 3-10). Western blotting showed 17 specific antigen spots with sera of the experimental group and two nonspecific spots with the control sera.

[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.

[The random amplified polymorphic DNA identification of 9 Taenia saginata isolates from four provinces].

OBJECTIVE: To make molecular identification for 9 isolates of Taenia saginata from 4 provinces. METHODS: Genomic DNA was extracted from the segments of adult tapeworms collected from Taoyuan of Taiwan (TW1), Duyun of Guizhou (DY1, DY2), Congjiang of Guizhou (CJ1, CJ2, CJ3, CJ4), Dali of Yunnan (DL1) and Wushi of Xinjiang (XJ1) respectively. PCRs were carried out with 13 random primers. A phylogenetic tree of different geographical strains was constructed. RESULTS: 331 DNA fragments were amplified. The number of DNA fragments amplified by single primer was between 3 and 28. The average number of amplified DNA fragments by the 13 primers was 14.15. The average number of fragments from the 9 isolates of T. saginata was 14.08. Phylogenetic tree revealed that there were two branches in the tree, DY1, DY2, DL1 and TW1 occupied one branch, while CJ1, CJ2, CJ3, CJ4 and XJ1 occupied the other one. CONCLUSIONS: By the RAPD analysis, the isolates DY1, DY2, DL1 and TW1 belong to Taenia saginata asiatica, and the isolates CJ1, CJ2, CJ3, CJ4 and XJ1 belong to T. saginata saginata.

[Effect of gamma-interferon on hepatic granuloma formation in mice infected by cysticerci of Taenia saginata asiatica].

Immunohistochemical streptavidin biotin-peroxidase complex method was used to investigate the effect of gamma-interferon (IFN-gamma) on the hepatic granuloma formation and liver fibrosis in mice infected with Taenia saginata in Duyun area of Guizhou Province. The results reveal contrary relation between the level of IFN-gamma in the liver and the degree of liver fibrosis (p<0.01). The injection of IFN-gamma considerably decreased (p<0.01) the area and size of granuloma (p<0.01).

[Changes of elements in Culex quinque fasciatus larvae after infected by Lagenidium giganteum (Oomycetes: Lagenidiales)].

OBJECTIVE: To analyze the quantitative change of 18 elements in Culex quinque fasciatus Larvae infected by Lagenidium giganteum (Oomycetes: lagenidiales), and to explore possible mechanism of mosquito-killing by the fungus. METHODS: Elements in the mosquito larvae at different developmental stages from infected and non-infected (control) groups were detected with automatic bioassay machine. RESULTS: At the early stage of infection, content of Ca and Cu in the infected group was markedly lower while the content of Co and Cd were higher than the control. In 24 hrs after infection, the content of Ca, Mg, Na, P, Fe, Zn, Cu, Mn and Cr was significantly lower while that of K, Mo, Co, Ni, Al, Cd and Pb were higher than those in non-infected larvae. No marked difference was found for the content of Sr and B between the two groups. In 48 hrs after infection, considerable difference was found in 17 elements between infected and noninfected larvae, with only one exception of Sr. CONCLUSION: Many elements which play an important role in maintaining the vitality of the mosquito larvae decreased and some of the heavy metal elements accumulated after the infection of Lagenidium giganteum. This might be a part of the killing mechanism to Culex quinque fasciatus larvae.

[Identification of Taenia saginata by mtCO I in four areas of Yunnan and Guizhou provinces].

OBJECTIVE: To identify the types of Taenia saginata isolated from Dali of Yunnan Province, and from Duyun and Congjiang of Guizhou Province. METHODS: Genomic DNA was isolated, and the mitochondrial cytochrome C oxidase subunit I (mtCO I) genes were amplified by polymerase chain reaction(PCR) and analyzed by PHYLIP software package. RESULTS: The mtCO I gene sequences of Lanping sample were identical to that of T. saginata asiatica known in Taiwan, and the samples obtained from Dali and Duyun showed the same mtCO I gene sequences, while the sample from Congjiang had the same mtCO I gene sequences as T. saginata. The homology between these two groups of gene sequences was 97.44%, while the homology of amino acid sequences reached to 99.16%. The constructed phylogenetic tree revealed that the relationship between T. saginata asiatica and T. saginata is closer, both are distant relative to T. solium and other species of cestodes. CONCLUSION: The Taenia prevalent in Lanping, Dali and Duyun is identified as T. saginata asiatica, while that isolated in Congjiang is the typical T. saginata.

[Change of lipid, esterase and lipase in mosquito larvae infected with Lagenidium giganteum].

OBJECTIVE: To explore possible physiological and biochemical mechanisms of Lagenidium giganteum infection in killing mosquito larvae. METHODS: The content of lipid and the activities of esterase and lipase between the normal and infected mosquito larvae were observed with histochemical method. The results were photomicrograph and analysed by image analysis using computer. RESULTS: In 24 hrs after infection, the content of lipid in the infected Culex quinquefasciatus larvae was lower while the activities of esterase and lipase in the larvae were higher than the control. In 48 hrs and 78 hrs after infection, the content of lipid in the infected larvae of C. quinquefasciatus and Ae. albopictus were significantly lower while the activities of esterase and lipase were significantly higher than those of control. CONCLUSION: The content of lipid in the mosquito larvae decreased while the activities of esterase and lipase increased after the infection of Lagenidium giganteum. The disorder of lipid metabolism might be part of the killing mechanisms to C. quinquefasciatus and Ae. albopictus larvae.

[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.