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Cloning and Sequencing of the Gene Encoding Variant-Specific Surface Antigen from Giardia lamblia

Yajie LI ; Meijun TENG ; Yongzhi LUN ; Da LI ; Yongqing ZHANG

Chinese Journal of Parasitology and Parasitic Diseases.2008;26(3):197-202.

Objective To clone and sequence variant-specific surface antigen gene from Giardia lamblia isolate SUCH/89/BTMRI/2(C2) derived from human in China. Methods Total genomic DNA of G.lamblia was extracted and a full-length variant-specific surface antigen gene fragment was amplified by pelymerase chain reaction (PCR). The PCR product was cloned into pMD19-T simple-vector, transformed into an Escherichia coli JM109 strain and then sequenced. The sequence analysis for cloned fragment was finished by Vector NTI 9.0 software for the homology of Giardia variantspecific surface antigen gene to that of sequences publishend in GenBank. Results The full-length variant-specific surface antigen gone fragment from G. lamblia was found to be 2 142 bp, encoding a 713 amino acid polypeptide and contained a single open reading frame (ORF). The deduced polypeptide sequence was rich in cysteine (11.8 mol%), most of which occurred with in 29 copies of the 4-amino acid CXXC motif, one GGCY-tetrapeptide motifs and three NXS consensus N-linked glycosylation sites. This polypeptide was also rich in threonine (10.2 mol%), glycine (12.1 mol%) and alanine (10.1 mol%). Like other previously identified VSPs, it contained a highly conserved hydropbebic Cterminal region. The homology of G. lamblia SUCH/89/BTMRI/2(C2) variant-specific surface antigen gene to that of sequence (TSA417) published in GenBank was 99% both at the nueleotide and the amino acid levels. Conclusion The full length variant-specific surface antigen gene from the isolate of G. lamblia has the common characteristics with other previously identified VSPs.

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Protective Immunity and Antibody Response of Rats Infected with Trichinella spiralis

Fushi QUAN ; Haengsook LEE ; Myungsook CHUNG ; Sungweon CHO ; Kyounghwan JOO ; Joonsang LEE ; Hanjong RIM

Chinese Journal of Parasitology and Parasitic Diseases.2008;26(2):119-123.

Objective To study the protective immunity and antibody(IgG,IgG1 and IgG2a)response against adult and larva infection of T.spiralis Korean isolate in rats.Methods Fony-six rats were randomly divided into 7 groups.Group A(A1,A2,10 rats)was used for the determination of protective efficacy from adult stage infection,group B (B1,B2,14 rats)was for the protective efficacy from muscle larva stage infection,group C(C1,C2,17 rats)was for challenge control,and group D(5 rats)served as normal control.Rats in groups A,B and C were infected with 1000 T.spiralis muscle larvae,and the infected rats were treated with flubendazole(20 mg/ks,10 d)at day 7(A1,A2) and at day 30(B1,B2).Rats in groups A and B were re-infected with 500 T.spiralis muscle larvae at day 10 after treatment.Rats in groups A1 and B1 were killed at day 7 and day 30 to inspect the reduction of adult worms in the intestines.Rats in groups A2 and B2 were killed at day 30 to detect the reduction of muscle larvae in diaphragms.Rats in groups C and D were killed at the same time,and all rats were bled at the same time.Specific anti-Trichinella IgG,IsG1 and IgG2a were detected by ELISA.Results Adult stage infection induced protective efficacy by 100% against adult stage and by 99.96% against larva stage.Larval stage infection induced protective efficacy by 99.92% against adult stage and 99.89% against muscle larvae.Anti-muscle stage larval ES Ag(IgG 3.0,IgG1 2.2,IgG2a 0.8)and anti-adult crude Ag antibodies(IgG 1.9,IgG1 0.8,IgG2a 0.3) significantly increased in the muscle larval stage infection compared to normal control(IgG 0.5,IgG1 0.1,IgG2a 0.1)and adult stage infection(IgG 0.5,IgG1 0.09,IgG2a 0.09) (P<0.01).Higher specific IgG1 antibody(IgG1 2.2) in larva stage infection was shown than specific IgG2a antibody response(IgG2a 0.8)(P<0.01).Conclusion Protective immunity against both adult and larva worms has been induced from adult and muscle larva stage infections of T.spiralis.

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Effect of Artemether on the Tegument of Adult Schistosoma haematobium Recovered from Mice

Shuhua XIAO ; Tanner MARCEL ; Binggui SHEN ; Utzinger JüRG ; Chollet JACQUES

Chinese Journal of Parasitology and Parasitic Diseases.2006;24(6):425-432.

Objective To assess the effect of artemether on the tegument of adult Schistosoma haematobium harbored in mice. Methods Ten mice were infected subcutaneously with 100-120 S. haematobium cercariae each. At day 81 post-infection, 8 mice were treated orally with 400 mg/kg artemether. Mice were sacrificed at 1, 3, 7 and 14 days post-treatment, and schistosomes were collected by the perfusion technique, fixed and examined under a scanning electron microscope. Schistosomes obtained from the 2 untreated mice served as a control. Results Twenty-four hours post-treatment, tubercles on the tegument of male worms showed lesions, characterized by enlargement, collapse and partial peeling off from the border with the tegument. In both male and female worms, the tegument showed focal or extensive swelling, fusion, vacuolization, erosion, peeling, and destruction of sensory structures. Three days post-treatment,tegumental alterations further aggravated; particularly severe damage was the swelling or collapse of the oral sucker observed in both sexes. In addition, extensive swelling, erosion and peeling of tegumental ridges and destruction of discoidlike sensory structures were seen in female worms. Seven to 14 days post-treatment, moderate-to-severe damage was still evident in some worms, whereas other worms surviving the treatment showed apparent recovery in most parts of their tegument. Conclusion Artemether causes extensive and severe tegumental damage in adult S. haematobium.

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Ultrastructural Alterations of Adult Schistosoma haematobium Harbored in Mice Following Artemether Administration

Shuhua XIAO ; Utzinger JüRG ; Binggui SHEN ; Tanner MARCEL ; Chollet JACQUES

Chinese Journal of Parasitology and Parasitic Diseases.2006;24(5):321-328.

Objective To perform a temporal examination of ultrastructural alterations in adult Schistosoma haematobium due to artemether Methods Eight mice infected with 100-120 S. haematobium cercariae for 81 days were treated intragastrically with 400 mg/kg artemether. At 24 hours, 3, 7 and 14 days post-treatment, groups of 2 mice were sacrificed and schistosomes collected by the perfusion technique. Worm samples were fixed and examined by transmission electron microscopy. Schistosomes were also obtained from 2 untreated mice that served as control.Results Typical ultrastructural alterations included swelling, lysis and vacuolization of the tegumental matrix, and disappearance of basal membrane. In sensory organelles and tubercles, there was extensive or local lysis of internal structure. In the musculature, parenchymal tissues, syncytium and gut epithelial cells, focal or extensive lysis, decrease in granular endoplasmic reticulum, vacuolization and degeneration of mitochondria were observed. These alterations became apparent both in male and female worms 24 hours post-treatment. In female worms, severe damage to the vitelline cells was also observed, resulting in the emergence of vacuoles, a decrease in granular endoplasmic reticulum,fusion of vitelline balls or even collapse of damaged vitelline cells. The most extensive tegumental alterations were observed 3-7 days post-treatment. Whilst 14 days post-treatment ultrastructural damage was still apparent, the tegument of some worms showed similar features to those recovered from untreated control mice. Conclusion Administration of artemether to mice infected with adult S. haematobium results in extensive damage to the ultrastructure in the tegument and subtegument tissues of the worms, confirming previous results with other schistosome species.

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Study on the Transmission of Toxoplasma gondii by Semen in Rabbits

Shiguo LIU ; Chuan QIN ; Zhijun YAO ; Dong WANG

Chinese Journal of Parasitology and Parasitic Diseases.2006;24(3):166-170.

Objective To confirm the transmission of Toxoplasma gondii by semen and to investigate the impact of vaginal status on the transmission of T. gondii in female rabbits. Methods Sixteen male rabbits were infected with T. gondii by intraperitoneal injection each with 1 ×105 RH tachyzoites. Eight rabbits died in 8-14 d after infection.Artificial vagina was used to collect semen from male rabbits weekly before and after infection for 8 weeks. If more than 2 portions of semen from 8 survived male rabbits were collected after infection, the collected semen was mixed weekly for later use. Twenty-seven female rabbits were divided into 4 groups: group 1 with normal vagina (7 rabbits), group 2with wounded vagina (7), group 3 with trichomonas vaginitis (7) and group 4 with colpomycosis infection (6). Tachyzoites were found in mixed semen digested by trypsinase, and were used for endovaginal artificial insemination to female rabbits by uterine cavity tube once a week for 8 consecutive weeks. 2-3 d after every insemination, 2 ml blood was collected from helix vein of each rabbit, and stored at -40 ℃ for use. Anti-T. gondii antibody was examined by ELISA and the B1 gene of T. gondii was detected by PCR. Results Anti-T. gondii antibody was detected in some rabbits (2, 3, 1, and 1 rabbits from each of the groups respectively) on the 16th day after the first insemination. The positive rate of ELISA was 25.9%. The amplification of B1 gene (200 bp) by PCR appeared positive from the blood samples on the 3rd day after the first insemination and the last positive one was proved on the 51th day after the first insemination.Number of positive samples was 2, 1, 3 and 1 in the 4 groups respectively, with an overall PCR positive rate of 18.5%.Only 3 of the 27 rabbits were positive by both ELISA and PCR. Conclusions T. gondii can be transmitted by semen and the health status of vagina shows no impact on it.

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Study on Protective Immunity against Infection of Plasmodium yoelii 17XL in DBA/2 Mice

Wei ZHENG ; Jun LIU ; Dongya MENG ; Xiaofang HU ; Yaming CAO

Chinese Journal of Parasitology and Parasitic Diseases.2006;24(1):14-18.

Objective To investigate the development and dynamic changes of host immune response in DBA/2 mice infected with Plasmodium yoelii 17XL. Methods Female DBA/2 mice were infected by intraperitoneal ( i. p. ) injection of 106 P. yoelii 17XL parasitized erythrocytes ( PRBC). Levels of IL-12, IFN-γ, IL-4, IL-10 and P. yoelii 17XL-specific antibody in sera were measured by ELISA. Concentrations of NO in cell supernatants were measured by the Griess reaction. Parasitemia,percentage of mononuclear-macrophages of individual mice were monitored daily, and phagocytosis of mononuclear macrophages was also observed. Results Primary parasitemia in vein blood was developed on day 3 postinfection, which peaked with a level of 46. 9% on day 9. Most mice cleared the infection and survived by day 20 postinfection. From day 6 to day 16, the phagocytosis of PRBC by rodent macrophages was observed on the blood smear. Infected mice had a continuously increased level of IL-12 in serum from day 1 postinfection. Accordingly, high level of IFN-γ was also detected in sera from day 1 postinfection,which peaked on day 6. Infected mice produced higher level of IL-4 and IL-10 in serum on day 6 postinfection, which peaked on day 9 and day 15 postinfection respectively. In addition, splenocytes from infected mice produced significantly higher level of NO on day 6 and 20 postinfection. Level of P. yoelii 17XL-specific IgG was determined in the sera of infected mice with a steadily increased trend after infection, which peaked on day 70 postinfection. Conclusions Effective polarizing of Thl cells is significant in inhibition of parasitemia and eventual clearance of the Plasmodium parasites. Activated mononuclear-macrophages play a key role in inhibiting parasitemia in the early phase of infection with P. yoelii 17XL.

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Comparison of the role of dendritic cells and macrophages in inducing protective immunity against Schistosoma japonicum

Dingwen SHEN ; Yonglong LI ; Wenqi LIU ; Xiaochun LONG ; Juan LIU ; Ruppel ANDREAS

Chinese Journal of Parasitology and Parasitic Diseases.2006;24(1):19-22.

Objective To compare a potential role of dendritic cells (DCs) and macrophages in inducing protective immunity against infection with Schistosoma japonicum. Methods DCs and macrophages were pulsed in vitro with soluble egg antigen (SEA) of S. japonicum. BALB/c mice were injected three times with DCs or macrophages, either antigen-pulsed or not,and challenged with 40 ± 2 cercariae of S. japonicum per mouse. Worms were collected 42 days later by portal perfusion of the mice and egg number of liver was calculated. To evaluate whether protective immunity had been induced by preparations of DCs or macrophages, the worm burden and fertility ( eggs per female per mouse liver) were compared between the groups of mice. The antibody level against SEA was detected by ELISA. Results With respect to mice injected with untreated cells, numbers of worms and eggs per female worms were significantly reduced in the groups of mice having received pulsed DCs (26. 3% and 37.9%, respectively), or pulsed macrophages (22. 0% and 30.7%). Untreated DCs and macrophages induced no significant effects. The antibody level against SEA rose in sera of all groups of mice up to 42 days after the challenge, but most pronounced in those immunized with pulsed DCs, although this was not significantly different from other groups. Conclusion The results suggest that the protective immunity against S. japonicum might be induced by DCs to a higher extent than by macrophages after in vitro pulsing with egg antigen.

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Reduction of Total Antioxidant Capacity in Artemether-treated Female Schistosoma japonicum

Zili ZHAI ; Jingyan MEI ; Peiying JIAO ; Shuhua XIAO

Chinese Journal of Parasitology and Parasitic Diseases.2002;20(6):354-357.

Objective To study the effect of artemether (Art) on total antioxidant capacity (T-AOC) in adult Schistosoma japonicum. Methods In vitro, the T-AOC was determined in five-week old worms incubated without or with Art and/or hemin for 24 h, and the worms were continuously incubated for 96 h, then worm survival was assessed. In vivo, T-AOC was determined in worms freshly recovered from mice 6 - 24 h after treatment with Art 300 mg/kg. Results Throughout 96 h incubation no worms were killed by 50 μmol/L Art or 50 μmol/L hemin alone, but approximatdy 80% of them were killed by Art plus hemin. Addition of reduced glutathione and vitamin E could significantly block the cidal action of the combined treatment. No effect on T-AOC was seen in the worms exposed to Art or heroin alone for 24 h, but the combined treatment led to a pronounced T-AOC reduction in female worms in vitro. Such a drug effect on female worms was demonstrated in vivo. After female worms were exposed to Art for 6 - 24 h in vivo, their T-AOC was significantly reduced by 40% - 64%. However, no drug effect on male worms' T-AOC was observed in vivo and in vitro exposed to Art plus hemin. Conclusion Art-induced T-AOC reduction in female worms may sensitize them to lethal damages of endogenous and exogenous oxygen radicals.

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ODNSIRUCTION AND ANALYSIS OF cDNA LIBRARY OF NECATOR AMERICANUS THIRD STAGE LARVAE

Bin ZHAN ; Hawdon JOHN ; Qiang SHAN ; Hainan REN ; Huiqing QIANG ; Shuhua XIAO

Chinese Journal of Parasitology and Parasitic Diseases.2000;18(1):26-29.

[Objective] To obtain the genetic information on Necator americanus and to search for the purpose genes.[Methods] Mrna was isolated from the third stage larvae of Necator americanus maintained in hamsters.Double strand Cdna was synthesized and ligated to ΛzapⅡ vector to construct the Cdna library.Expresed se-quence tages (ESTs) were obtained by single pass sequencing of randomly isolated Cdna clones from the es-tablished library.[Results] A Cdna librazy of N.americanus was successfully constructed with high recombi-nant efficiency.The titer of unamplified library was 1×107.The insert size was about 750～3000bp.Of 11 ESTs obtained from the library,7 have a significant homology with certain functional genes.[Conclunsion]A high quality and high representative Cdna library of N.americanus was constructed at the first time and ome functional genes were identified from the library by ESTs.

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ALLOZYME-BASED GENETIC VARIATION WITHIN AN UNSTABLE"POPULATION" OF CHINESE ONCOMELANIA HUPENSIS(GASTROPODA: RISSOACEA: POMATIOPSIDAE)

Yi ZHANG ; Hexiang LIU ; Ting FENG

Chinese Journal of Parasitology and Parasitic Diseases.2000;18(1):21-23.

[Objective] To answer the following questions:① For Oncomelania snails collected two years apart from the same locality,has there been genetic divergence?②How much experimental error has there been in studying subsets of these populations? ③As this is an unstable population,what has the net effect been on Hardy-Weinberg equilibrium(Hwe)?[Methods] Allozymes were studied using horizontal starch gel electrophoresis.Data collected from numbers of experiments were conapiled.Data from each collection were divided into two equal subsets based on chronology of the experiments.Thirty-four loci were studied using 72 to 180 snails per subset.[Results] The mean number of alleles per locus ranged frcra 1.5～1.9.With each consecutive subset,the 96 polymorphic loci dropped from 38.2 to 17.6.The mean heterozygosity was very low:0.033 to 0.049 and not significantly different from Hardy-Weinberg expectations.Ten loci and 11 alleles exclusive to the first group were eliminated from the overall study reducing the number of polymorphic loci from 19 to 10.There were significant departures from Hwe at five loci having a substantial number of individuals for each allele.Nei's and Wright's D were 0.003±0.001 and 0.054±0.006 respectively.[Conclusion] ①There were significant errors seen primarily in the results scored' in the earliest experiments.②These earlier errors involving scoring difficult to resolve loci,and interpretation of rare alleles that were not found in later experiment had no significant effect on overall genetic distance.③The use of Wright's D for closely related populations is explained.Results with Nei's D indicated no significant difference among the four subunits; Wright's D yielded significant difference between the collections made two years apart,attributed to the annual flooding of the Yangtze River mixing snails from different localities.④ Major polymorphic loci were not in Hwe as predicted using the unstable population model.⑤One must study 25 or more individuals to find relatively rate alleles and study population genetics.

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