Table of Content

30 August 2012, Volume 30 Issue 4

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Effect of Toxoplasma gondii Infection on the Embryonic Neural Stem Cells in Rats

SUN Xiu-Ning, LIU Zhi-Jun, GUAN Zhi-Yu, LIANG Rui-Wen, ZHANG Hao-Yun, WU Xiao-Yan, YU Li, GUAN Ying-Jun

2012, 30(4):  1-253-257. 

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Objective   To investigate the effect of Toxoplasma gondii infection on the proliferation， differentiation and migration of the embryonic neural stem cells （NSCs） in early pregnancy of rat.  Methods  Twelve pregnant Sprague-Dawley rats were randomly divided into control and infection groups. Rats in the infection group were each inoculated intraperitioneally with 1×10⁵ T. gondii RH strain tachyzoites at day 1 （E1 day）. Same amount of physiological saline was intraperitioneally injected for rats in control group. At E5 day， blood samples were taken from caudal vein and Giemsa staining of blood cells was performed to find T. gondii. At E9， E10 and E11 day， two rats in each group per time point were sacrificed and reverse transcription PCR （RT-PCR） was performed to detect B1 gene expression of T. gondii in amniotic fluid to confirm T. gondii infection. NSCs were cultured in vitro. The proliferation level was detected by methyl thiazolyl tetrazolium （MTT） assay. After differentiation culture of NSCs， the immunofluorescence assay was conducted to detect the expression of nestin， microtubule-associated protein 2 （MAP2） and glial fibrillary acidic protein （GFAP） to calculate the ratio of NSCs which differentiated to neurons and astrocytes. The embryonic nerve tissues at E9， E10 and E11 day in each group were taken to make frozen sections. The immunofluorescence assay was carried out to detect the expression of neuronal cell adhesion molecule （NCAM） in the nerve tissues at different developmental stages.  Results  Both the results of blood smears and RT-PCR confirmed that the pregnant rats and embryos were all infected by T. gondii in infection group. The morphology of the cultured NSCs under microscope was consistent with the characteristics of the normal NSCs. In addition， the NSC biomarker nestin protein was stained positive. The MTT assay showed that the proliferation level was lower in infection group than that of the control， and statistical differences were found between the two groups at day 3 and 4 after passages （P<0.05）. The immunofluorescence staining of MAP2 and GFAP showed that the percentage of neuron differentiation was 15.15%（55/363） in control group and 8.73%（31/355） in infection group， respectively, with a statistical difference （P<0.05）, and the percentage of astrocyte differentiation was 53.35%（199/374） and 67.48% （249/369）， respectively （P>0.05）. In both groups， NCAM protein was found expressed at E9， E10 and E11 day in embryo nerve tissues. The fluorescence became stronger with time. The expression level in control group was significantly higher than that in infection group （P<0.01）.  Conclusion  T. gondii infection at early gestation may inhibit the proliferation， differentiation and migration of neural stem cells in rats.

Immune Response of Th17 Cells in Mesenteric Lymph Node of Mice Infected by Schistosoma japonicum

LUO Xue-ping, CHEN Dian-hui, XIE Hong-yan, GAO Zhi-yan, FANG Hui-long, HUANG Jun

2012, 30(4):  2-258-261,267. 

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Objective  To observe the immune response of Th17 cells in mesenteric lymph node （MLN） of C57BL/6 mice infected by Schistosoma japonicum.  Methods  Twenty C57BL/6 mice were randomly divided into infected group and control group each with ten mice. The mice in infected group were infected each with 40±5 S. japonicum cercariae. Five to six weeks later, MLN lymphocytes were separated and stimulated for 4 h by anti-CD3 (1 μg/ml） and anti-CD28 (1 μg/ml） before examination of IL-17 and retinoic acid receptor-related orphan receptor γt(ROR-γt) mRNA by reverse transcription PCR. The level of IL-17 and IFN-γ was detected by ELISA after culturing with supernatant for 72 h. MLN lymphocytes were stimulated for 5 h by 10 ng/ml phorbol myristoyl acetate(PMA) and 1 μg/ml ionomycine. The intracellular cytokines were stained and the content of Th17 and other cytokines was examined by flow cytometry.  Results  The level of IFN-γ ［(214.3±62.6) pg/ml］ and IL-17 [（176.8±62.1) pg/ml] in the supernatant of culured MLN cells from the infected mice was significantly higher than that of normal mice [（46.7±13.9) and 0 pg/ml]（P<0.05）. The expression level of IL-17 and ROR-γt mRNA was also considerably higher than that of normal mice. IL-17+ IL-4+, IL-17+IFN-γ+,  IL-17+ IL-5+ and IL-17+ IL-9 cells accounted for 0.06%, 0.02%, 0.02%， and 0.01% of the mesenteric lymph node CD4+ T cells of the infected mice, respectively. However, IL-17+ IL-10+ and IL-17+ Foxp3+ cells were undetected.  Conclusion  The MLN of S. japonicum-infected C57BL/6 mice can induce the production of Th17 cells, and these cells can secrete IL-4, less IFN-γ, IL-5 and IL-9, but not IL-10, and can not express Foxp3 in the infected mice.

Cloning, Expression and Activity Analysis of Full-length Gene Encoding Thioredoxin Peroxidase from Oncomelania hupensis

MA Xian-liang，LIU Qin，ZHANG Yi

2012, 30(4):  3-262-267. 

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Objective   To clone and express full-length thioredoxin peroxidase (TPx) gene of Oncomelania hupensis and study on the peroxidase activity of the recombinant protein.  Methods  Total RNA was obtained from the cultivated O. hupensis and a cDNA sequence of the TPx gene was cloned by RT-PCR. The TPx cDNA ends were amplified by the SMARTer RACE cDNA Amplification Kit. After sequencing, blasting and matching, the full-length cDNA of the TPx gene was obtained. The TPx cDNA was ligated with the pGEM-Teasy and transformed into E. coli DH5α. After sequencing and blasting, the characteristics of biological information of the TPx gene was analyzed. The positive recombinants with pGEM-Teasy/TPx and expression vector pET-28a were digested by the double restriction enzymes, ligated each other, transformed into E. coli BL21（DE3）, and induced by IPTG for expression. The recombinant TPx was expressed as a histidine fusion protein and was purified with Ni chromatography and NTA cation exchange chromatography. The expressed and purified TPx was analyzed by SDS-PAGE. The different concentrations of TPx recombinant protein （10, 20, 30, 40， and 50 μg/ml）were added into hydrogen peroxide （H₂O₂） reduction test in vitro to calculate the clearance rate of H₂O₂， each concentration with parallel control of dithiothreitol sugar alcohol （DTT）. In the protection test of super-coiled DNA, the TPx protein was added with a concentration of 2.5, 5.0, and 10 μg/ml, respectively, to observe the protective effect of super-coiled DNA in metal-catalyzed oxidation （MCO）.  Results  The complate cDNA encoding TPx was 992 bp. ORF was 747 bp with GenBank accession number of JN831437. The ORF encoded 249 amino acids, and the relative molecular weight （M_r） of predicted protein was 27 000. The recombinant plasmid pET28a/TPx was built, and the soluble recombinant protein was obtained by induction and purification. The results of SDS-PAGE showed that the M_r was 27 000. H₂O₂ reduction test in vitro showed that the H₂O₂ clearance rate of reaction system containing DTT was significantly higher than the clearance rate without DTT （P<0.05）, the difference among various concentrations was not statistically significant （P>0.05）. The protection of super-coiled DNA showed that the protective effect of 5.0 μg/ml group was better than 2.5 μg/ml group, but there was no difference between the protective effect of 5.0 μg/ml group and 10 μg/ml group.  Conclusion  The full-length cDNA of the TPx gene of O. hupensis is obtained, and the recombinant TPx protein shows a certain antioxidant activity.

Study on the Genetic Differences among Oncomelania hupensis Population in Middle and Lower Reaches of Yangtze River using Microsatellite DNA Markers

LI Shi-zhu，ZHANG Li，LIU Qin，LV Shan，WANG Qiang，QIAN Ying-jun，YANG Kun，ZHOU Xiao-nong

2012, 30(4):  4-268-273. 

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Objective    To identify the genetic structure of Oncomelania hupensis in the middle and lower reaches of Yangtze River by using microsatellite DNA molecular markers.  Methods  O. hupensis snails were collected from the provinces of Anhui， Hunan， Hubei， Jiangxi， and Jiangsu， of which 6 polymorphic microsatellite DNA loci （P84, T5-13, T5-11, T4-22, T6-27 and P82） were amplified with fluorescence labeled universal primer. 20-50 snail samples were collected at each spot， adding up to 165 samples. The number of alleles （N_a）， inbreeding coefficient （F_IS）， heterozygosity （H）， fixation index （F_ST） of each group snails, genetic distance between groups， and the polymorphic information content （PIC） were calculated. Cluster analysis was then carried out based on genetic distance， and hierarchical AMOVA calculation was done.  Results  The number of alleles in each population ranged from 3 to 33， and the inbreeding coefficient ranged from 0.143 to 0.539. The average expected heterozygosity and observed heterozygosity ranged from 0.600 to 0.883 and 0.308 to 0.759, respectively， being the highest in Hubei population and the lowest in Jiangsu population. The range of F_ST value between paired populations was from 0.0006 to 0.0531. The small F_ST value suggested that genetic differentiation did not occur among the populations. The average polymorphic information content in the populations ranged from 0.511 to 0.850， showing a high polymorphism except the Jiangsu population. Hierarchical AMOVA calculations showed that inter-individual variation of the snails occupied 95.2% of the total variations. Cluster analysis revealed that Anhui group clustered first to Jiangsu， followed with Hunan， Jiangxi and Hubei.  Conclusion  There is a rich diversity in O. hupensis. Cluster analysis shows a consistency with the geographical distribution pattern. The genetic differences among the 5 snail populations are trivial with microsatellite DNA variation mostly present in individuals.

Prokaryotic Expression of Chimeric Gene Derived from the Group 1 Allergens of Dust Mites and Bioactivity Identification

GUO Wei，JIANG Yu-xin，LI Chao-pin

2012, 30(4):  5-274-278. 

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Objective   To express a chimeric gene R8 derived from the group 1 allergens of dust mites using prokaryotic expression system and detect their bioactivities.  Methods  PCR amplification was performed using specific primers of Derf1 gene and the pUCm-T recombinant plasmid containing the R8 chimeric gene as a template. The PCR products were inserted into the pET28a(+) empty vector after double digestion using restriction endonuclease BamHⅠ and XhoⅠ, respectively. The recombinant plasmid was transferred into E. coli line BL21 and induced by 1 mmol/L isopropyl-β-D-1-thiogalactopyranoside (IPTG）. The expressed product was detected by SDS-PAGE and the target protein was purified. IgE binding assay of the purified protein R8 was detected by ELISA using dust mite allergic patient sera. For determining immunogenicity of R8 protein， 75 BALB/c mice were randomly divided into 5 groups， namely PBS (negative control）， rDer f 1 group and rDer p 1 group (positive groups）， R8 group and asthma group. The mice were treated with dust mite extract at 0, 7, 14 day by intraperitoneal injection of allergens (100 μl， 0.1 μg/μl) and inhaled challenge as aerosol （0.5 μg/ml, 30 min/d） on day 21 for 7 days. Before inhalation in immunotherapy groups at 25~27 day， specific allergen immunotherapy was performed using rDer f 1， rDer p 1 and R8 allergens respectively. Mice in negative control group were treated with PBS all the time. Twenty-four hours after the last challenge， mice in every group were sacrificed. The bronchoalveolar lavage fluid （BALF） was collected. ELISA was used to detect the level of interferon-γ （IFN-γ） and interleukin 4 （IL-4） in BALF.  Results   SDS-PAGE analysis revealed that chimeric gene R8 was expressed with a band of approximately M_r 35 000. Compared with groups of rDer f 1 and rDer p 1 ［（80.44±15.50） and （90.79±10.38） μg/ml， respectively］， IgE binding capacity of the protein R8 （37.03±12.46） μg/ml was statistically lower （P<0.001）. The level of IFN-γ in sera of R8 group [（343.43±38.79） pg/ml] was higher than that of the PBS and asthma groups ［（393.93±50.68） and （208.44±46.11） pg/ml， respectively］ （P<0.01）， but no statistical difference to that of the rDer f 1 and rDer p 1 groups （P>0.05）. IL-4 level in R8 group was lower markedly than the others （P<0.05 or P<0.01）. Conclusion   Chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.

Epitope Structure Prediction of Antigen B of Echinococcus granulosus and Construction of Multi-epitope Recombinant Antigen

JIANG Li， ZHANG Yao-guang， JIANG Shou-fu， FENG Zheng

2012, 30(4):  6-279-283. 

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Objective   To improve the reactivity of Echinococcus granulosus antigen B （EgAgB） by constructing multi-epitope antigens using the gene fragment from 3 subunits， EgAgB1， EgAgB2， and EgAgB4.  Methods   Bioedit， Discovery Studio Visualizer software and I-TASSER on-line server were used to predict protein structure and analyze the gene sequence of the 3 subunits and their combinations in different way. The epitope or subunit combination which had a higher prediction scores was selected for gene recombination. The target sequence was amplified with specific overlap primers and by using overlap extension PCR technology. The target sequence was then cloned to pET32a(+) vector for constructing expression plasmid and expressing recombinant proteins. The expressed products were served as multi-epitope recombinant antigens after purification. The immuno-response of the recombinant multi-epitope antigens were explored by Western blotting analysis.  Results   Structure prediction showed that all the three subunits EgAgB1， EgAgB2 and EgAgB4 are in a “Z” word structure. The epitope region is located in the central part of the sequence. For combinations from the three subunits and four reactive epitopes （KK36，RK30，B4-2，and B4-3）， 57 different combinations were tried for structure prediction. Six of them were selected for recombination and expression. Western blotting analysis on the six multi-epitope antigens （MEA-8，MEA-20，MEA-26，MEA-36，MEA-49, and MEA-52）suggested that the band reactivity of multi-epitope antigen was much stronger than AgB subunit antigens when the positive serum of cystic echinococcosis were used.  Conclusion  By using protein tertiary structural prediction and screening the higher prediction score of combinations， six multi-epitope recombinant antigens were constructed. Western blotting shows that the band reactivity of multi-epitope antigen is much stronger than that of AgB subunit antigens.

Effect of Anti-osteopontin Antibody on Expression of IL-2 and IL-5 in Hepatic Alveolar Hydatid Tissue of Gerbil

GAO Liang-liang，ZHANG Shi-jie，WU Xiang-wei，ZHANG Yong-guo，ZHANG Long，PENG Xin-yu，CAO Yu-wen，SUN Hong

2012, 30(4):  7-286-289. 

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Objective  To observe the effect of anti-osteopontin antibody on the level of IL-2 and IL-5 in the liver of gerbil infected with Echinococcus multilocularis(Em).  Methods  180 gerbils were infected with echinococcus protoscoleces (approximately 400 for each gerbil) by abdominal opening inoculation in liver. The gerbils were randomly divided into three groups with 60 each: experiment group (group A, with anti-osteopontin antibody interference)， control group (group B, with normal rabbit serum injection) and model group (group C, with no interference). Ten gerbils from each group were sacrificed at 20， 60， 100, 140, 180, and 220 days post-infection respectively. The liver tissue with hydatid cysts was collected and the expression of IL-2 and IL-5 was observed after immunohistochemistry staining (SP method).  Results  E. multilocularis hydatid tissue spreaded over the liver and abdominal cavity. The positive expression rate of IL-2 in the tissue showed no statistical difference among the three groups (P＞0.05). On the days 140 and 180, however, the positive expression rate of IL-5 in group A was 40% and 20% respectively， considerably lower than that in group B (100% and 90%) and group C (90% and 80% respectively).  Conclusion  The anti-osteopontin antibody can reduce Th2 type cytokine response in the Em-infected gerbils, which may strengthen the immunity of the host.

Efficacy of Tribendimidine Against Three Isolates of Trichinella spiralis in Mice

GAO Yun，YANG Xiao-dong，WANG Li-na，CHEN Xiao-ning，LI Li-na，ZHAO Xiang-ju，LIU Dan

2012, 30(4):  8-290-293. 

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Objective   To evaluate the efficacy of tribendimidine （TBD) against 3 geographical isolates of Trichinella spiralis in mice.  Methods  Isolates of T. spiralis from Henan (hereinafter referred to as HnT.s), Yunnan (referred to as YnT.s) and Heilongjiang (referred to as HljT.s) were used in the study. 144 Kunming strain mice were divided into 2 groups: 72 mice in group A (adult stage, treatment at 5 d after infection), and 72 mice in group B (encapsulated larva stage, treatment at 53 d after infection). Group A was further divided equally into 12 sub-groups. Mice in every 3 sub-groups were each infected orally with 200 T. spiralis larvae of the 3 isolates respectively, and the remained 3 sub-groups served as untreated control. Mice in the 3 sub-groups infected with one isolate were orally treated with TBD at a single dose of 10, 20， and 30 mg/kg, respectively. Group B was treated as group A but with a course of TBD once daily at a dose of 100, 200， and 300 mg/(kg·d) for 7 d, respectively. Mice in group A were sacrificed 2 d post-treatment and adult worms were recovered from the small intestine and counted. Those in group B were sacrificed 10 d after completion of 7 d treatment. The intact diaphragm was removed and digested for collecting larvae. Worm burden and worm reduction of each treated sub-group were calculated and statistically compared with the respective control. Results  In group A, the mean worm burden in the treated sub-groups infected with HnT.s and YnT.s were all significantly lower than that of the controls (P＜0.01), with a mean worm reduction rate of 39.0%, 57.9%， and 86.0% in HnT.s sub-groups, and of 34.9%, 69.3%， and 92.2% in YnT.s sub-groups, respectively, showing an increase with the dosage, 2 mice in each of the 30 mg/kg sub-groups were cured. The worm burden in the 10 mg/kg of HljT.s subgroup was similar to that of the control (P>0.05), but was significantly lower in the other 2 sub-groups than that of the controls (P＜0.01). The worm reduction rate in the 3 sub-groups was 27.9%, 57.4%, and 60.7%, respectively. In all treated sub-groups of group B, the mean worm burden was significantly lower than that of the controls (P＜0.05), with a mean worm reduction rate of 57.8%, 75.4%, and 87.5% in HnT.s sub-groups, of 74.5%, 92.4%, and 99.1% in YnT.s sub-groups, and of 50.5%, 53.3%, and 61.6% in HljT.s sub-groups, respectively, with the 3 dosages.  Conclusion  Tribendimidine shows adequate efficacy on Trichinella spiralis adults and on encapsulated larvae of the 3 geographical isolates in mice, with better effect on Yunnan isolate.

Cloning and Expression of D-like Aspartic Protease of Anisakis simplex

NI Fang，XU Shi-san，ZHANG Shao-lei，XU Chang-mao，LUO Da-min

2012, 30(4):  9-294-298. 

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Objective   To clone and express the full length of D-like aspartic protease gene (AsAP) of the third stage larvae of Anisakis simplex.  Methods  According to the partial information of D-like aspartic protease encoding gene of A. simplex from GenBank, specific primers were designed to amplify 3′end and 5′ end of AsAP gene using rapid amplification of cDNA ends (RACE), and the full length of the D-like aspartic protease gene was obtained. Using total RNA of the third-stage larvae of A. simplex, coding sequence of the AsAP gene was amplified by reverse transcription-PCR (RT-PCR). The PCR product was digested by EcoRⅠ and SalⅠ, and cloned into pET32 vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into E. coli BL21 (DE3). Expression of the protein induced by IPTG under gradient concentration and different time was conducted.  Result  A 1 753 bp full length of AsAP was obtained, which contained 30 bp 5′UTR, 361 bp 3′UTR and a 1 362 bp open reading frame (ORF) encoding 453 amino acids with a predicted molecular mass of M_r 50 726.  It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum. The predicted amino acid sequence contains two conserved catalytic motif, an active site flap, an S2 subsite and an S3 subsite. A 20 amino acids signal peptide was found in the N-terminus, with significant hydrophobic property. Different concentration of the IPTG (0.2~1.6 mmol/L) showed little effect on the expression, and the production of the protein was up to maximum after 2 hours induction. Conclusion  The AsAP gene has been cloned and expressed.

Detection of Immunity and Antioxidant Indexes in Dairy Cows Infected with Cryptosporidium

ZHOU Qing-xin，LI Jin-chun，XU Qian-ming，ZHAO Chang-cheng，WANG Xi-chun，WANG Ju-hua，LIU Wei，LI Pei-ying

2012, 30(4):  10-301-304. 

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Objective  To detect the immune status and antioxidant system indexes of cows infected with Cryptosporidium.  Methods  Fecal samples of 325 dairy cows were collected at a farm in Anhui and examined by floating saturated solution. 7 positive cows and 7 negative cows from the farm were selected as infection group and non-infection group， respectively. Blood samples were taken from cow′s jugular vein before feeding in the morning. 19 indexes of total protein (TP)， albumin (ALB)， IgG， IgM， IgA， phagocytic rate of white blood cells， T lymphocyte transformation rate， IL-2， superoxide dismutase (SOD)， glutathione peroxidase (GSH-Px)， malondialdehyde (MDA)， NO， alanine aminotransferase (ALT)， aspartate aminotransferase (AST)， alkaline phosphatase (ALP)， glucose (GLU)， triglyceride (TG)， Cl^－， and Ca²⁺ were tested， respectively.  Results  The infection rate of 325 cows was 31.7%（103/325）. The Cryptosporidium was identified as C. andersoni according to the morphology and size of oocysts. Compared with the non-infection group， there was no significant difference in the concentration of TP， ALB， IgM， IgA， GSH-Px， ALT， AST， ALP and Cl^－ （P>0.05）. The concentration of MDA and NO in the infection group increased by 59.9% and 28.1% （P<0.05 or 0.01）， and that of IgG， SOD， GLU， TG， Ca²⁺， IL-2 and the activities of T lymphocyte transformation rate， phagocytic rate of white blood cells decreased by 32.9%， 11.1%， 18.6%， 78.9%， 14.5%， 7.0%， 22.0%， and 20.2%, respectively （P<0.05）.  Conclusion  The change of antioxidant and immune indexes shows that the capability of eliminating free radicals and the immune function have decreased in the Cryptosporidium andersoni-infected cows.

A Cross Analysis on the Capability of Examining Helminths： National Technique Competition for Parasitic Disease Diagnosis in 2011

ZHANG Li，LI Shi-zhu，LI Yu，WANG Qiang，FU Qing，LIU Wei，ZHU Hong-qing，XU Jing，CHEN Ying-dan，CHEN Shao-hong，CHEN Jia-xu，CHEN Zhao，WANG Li-ying， ZHOU Xiao-nong

2012, 30(4):  11-305-308. 

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Objective  To understand the comprehensive capability of helminth detection among professionals at different level of parasitic disease control institutions and promote the overall strength of diagnosis.  Methods  Four professionals from each parasitic diseases control institutions were selected as contestant (age<45 and at least two contestant from county-level institution). The content of contest included making stool slides with Kato-Katz method (five slides in thirty minutes, a total score of 15 and 9 as passing score) and identification of eleven common helminth eggs with microscopy (ten slides, five minutes per slide, a total score of 60, 36 as passing score).  Results  The average score of making slides in 119 contestants from 30 provinces was 11.4, and 119 contestants passed accouted for 93.3%. The average score of film-reading was 22.0, and 20 contestants passed accouted for 16.8%. There were no statistically significant differences between the results in different gender, age (≤30, 31-40, >40), job title (the junior, intermediate, and senior), institution level (provincial, municipal, and county level)(P>0.05). By Kato-Katz slide-making and film-reading, the scores in contestants from provinces with schistosomiasis control task (12.1±1.7，32.1±11.5, respectively) were better than contestants from other provinces (11.1±1.8，18.1±10.5, respectively). The scores in contestants from western (18.4±11.4) were lower than those from eastern (25.2±12.4) and central (24.1±13.1) for film-reading.  Conclusion  The overall capability of parasitic disease examination is unbalanced among regions, and evidently there is a need to strengthen the capacity of phthogen detection in the disease control programs.

Investigation on the Sensitivity of Anopheles sinensis to Insecticide

LIU Ying，CHEN Jian-she，ZHOU Rui-min，QIAN Dan, CHEN Qing-wei，XU Bian-li，ZHANG Hong-wei

2012, 30(4):  12-309-311. 

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Objective  To investigate the sensitivity of Anopheles sinensis to DDT, cyfluthrin, malathion and deltamethrin.  Methods  In June 2009, 417, 421 and 433 mosquitoes were respectively captured from the fields of Tongbai county, Huaibin county, Yongcheng city, and tested by the adult Anopheles filter contact method recommended by WHO. The KT₅₀ and the mortality rate of 24 h-post-exposure to 4% DDT （1.428 g/m²）, 0.15% cyfluthrin （0.0 534 g/m²）, 5% malathion （1.78 g/m²）, 0.05% deltamethrin （0.0 178 g/m²） were calculated. The resistance level was graded as sensitive group （S） with a mortality rate of 98%-100%, preliminary resistance group （M） with mortality rate of 80%-97%, and resistance group （R） with mortality rate lower than 80%.   Results  The KT₅₀ of An. sinensis to DDT in Tongbai, Huaibin and Yongcheng were 206.13, 877.04 and 826.81 min, respectively; the KT₅₀ to cyfluthrin was 206.43, 85.39, and 427.60 min, respectively; the KT₅₀ to malathion was 19.98, 48.05 and 97.79 min, respectively; the KT₅₀ to deltamethrin was 1122.50, 89.65, and 960 min, respectively. The mortality rate of An. sinensis in 24h-post-exposure to 4% DDT was 82.52%, 57.41%, and 65.69%, and the resistance degree was judged as M, R and R, respectively.  That to 0.15% cyfluthrin was 91.89%, 85.00%, and 72.73%, with a resistance degree of M, M, and R, respectively. That to 5% malathion was 95.10%, 95.37%, and 93.16%, all with a resistance degree of M. That to 0.05% deltamethrin was 92.08%， 77.14%, and 63.46%， with a resistance degree of M， R and R， respectively.  Conclusion  An.sinensis in Henan Province has developed high degree of resistance to DDT, cyfluthrin and deltamethrin， and certain degree of resistance to malathion.

Research Progress on Peptidoglycan Recognition Proteins of Medical Shells and Molluscs

ZHANG Zong-lu，GUO Yun-hai，LUO Tai-chang，Zhang Yi

2012, 30(4):  13-312-316. 

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Peptidoglycan recognition proteins （PGRPs） are highly conserved pattern recognition receptors in evolution, and they can recognize peptidoglycan（PGN） and bacteria that contain PGN in their cell wall component in early immune process of host, then provide signal transduction and activate a series of immune proteins. PGRPs are extensively present in insects, molluscs, echinoderms and vertebrates. Research progress and frontiers on PGRPs gene, type, structure, express localization, function, and evolution in medical molluscs and other snails were briefly reviewed in this article.

Research Progress on the Mechanisms of Antigenic Variation in Giardia lamblia

Feng Xian-min，Wang Yue-hua，Ju Xiao-hong

2012, 30(4):  14-317-320. 

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The intestinal protozoan parasite Giardia lamblia is one of the most common causes of diarrhoea and undergoes antigenic variation. In pathogenic microorganisms, antigenic variation is often described as a mechanism to evade the host immune system, resulting in chronic and/or recurrent infections. In the recent years, significant advances in the knowledge of the antigen switching process have been achieved. Here we review the principal knowledge on the mechanisms that regulate this process, including genomic organization, post-tanscriptional gene silencing, expressional modifications, and processing and turnover of VSPs.

Application of PCR and PCR-derived Technologies in the Detection of Angiostrongylus cantontensis

CUI Li-yun，ZHANG Xiao-xiao，LI Kun-hua，YANG Yi-mei

2012, 30(4):  15-321-324. 

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Angiostrongyliasis is a zoonotic disease and has become one of the potentially threatening food-borne parasitic infections in China. This article reviews the advances in the application of PCR and PCR-derived techniques in detection of Angiostrongylus cantontensis.

Research Progress on the Biology and Epidemiology of  Cyclospora cayetanensis

ZHANG Xiao-li，ZHANG Wei-zhe，SHEN Yu-juan，LIU Ai-qin

2012, 30(4):  16-325-328. 

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Cyclosporiasis is one of the emerging parasitic diseases. Cyclospora cayetanensis is so far the only species infecting humans in the Cyclospora genus. This paper reviews mainly the biological characteristics of C. cayetanensis and the current epidemiology status of human infection.

Facial Demodex Infestation among Urban and Rural Residents in Shangqiu City of Henan Province

CUI Jin-huan，WANG Chen

2012, 30(4):  17-283-285. 

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A survey with improved transparent tape method indicated that the prevalence of Demodex infestation among 565 urban and rural residents in Shangqiu was 21.2%(120/565). Farmers (32.3%, 53/164) and service employees (33.7%, 29/86) showed higher prevalence than other occuptions （P<0.05）． Among age groups, lowest prevalence was found in people under 20 years old （4.8%， 5/105）, while the highest in people over the age of 50 (44.4%， 40/90). Prevalence among females, the rural residents and those sharing public toiletries and people with oily skin and acne or other facial sickness was statistically higher than others (P＜0.05). Among the couples with demodex infestation, 79.6% of the couples only had one side infested while both sides got infested in 20.4% of the couples.

Detection of Clonorchis sinensis Eggs in the Ground Gallbladder Stones by Microscopy

MA Rui-hong，QIAO Tie，LUO Xiao-bing

2012, 30(4):  18-298-300. 

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Sera, feces, bile and gallbladder stones were collected from 179 patients who accepted gallbladder-preserving cholelithotomy during the period of January to June 2010 at the general surgery department in the Second People′s Hospital of Panyu District in Guangzhou. Rapid colloidal gold immunochromatography was used to detect IgG against Clonorchis sinensis. C. sinensis eggs were examined by fecal direct smear, and in bile sediments and ground gallbladder stones. The results showed that the positive rate of rapid colloidal gold immunochromatographic assay for IgG was 51.4%, and the egg positive rate in feces, bile sediments and gallbladder stones was 30.7%, 44.7% and 69.8%, respectively. The detection rate of fecal direct smear was the lowest, while that of the gallbladder stone examination was the highest (P<0.05). Those patients with egg-positive feces and/or bile sediments were all with egg-positive gallbladder stones.

Treatment of 42 Child Cases of Pulmonary Echinococcosis granulosus by Excision of Internal Cyst through Video-assisted Thoracoscopic Surgery

GUO Rui，MA Jin-shan，NU Er-Lan

2012, 30(4):  19-329-330. 

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Among 42 child cases of pulmonary echinococcosis granulosus treated by excision of internal cyst through  video-assisted thoracoscopic surgery (VATS), the cysts were completely removed in 28 cases (66.7%), cysts in 14 cases (33.3%) were removed by puncture, and cyst ruptured accidentally during operation in 1 cases (2.4%). The mean operation time was (96.70±10.90) min, mean volume of blood loss during operation was (8.60±1.31) ml, and the average admission in hospital was (10.20±1.10) d, with post-operation complications in 2 cases (4.8%). Three years' follow-up revealed no recurrence. The VATS is a way for treating pulmonary echinococcosis granulosus in children with light trauma, less bleeding and fewer complications.

Cloning and Expression of Triosephosphate Isomerase Gene of Boophilus microplus

LIANG Yi-lin，GAO Pan，KE Ze-kai，LUO Xin-ping*，LIU Zhi-gang

2012, 30(4):  20-331-332. 

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Total RNA was extracted from adult Boophilus microplus. RT-PCR was used to amplify the gene and the fragment was subcloned into the expression vector pET-28a. The cloned gene was expressed in E. coli Rosseta（DE3）, induced by IPTG， and identified by SDS-PAGE. The results showed that the triosephosphate isomerase （tim） gene of B. microplus has 750 bp and encodes 249 amino acids （GenBank No. JX112888）. The cloned tim gene shares 99% homology with that in tick embryos. The relative molecular weight （M_r） of the expressed recombinant protein is about 27 000.

Detection of Cryptosporidium parvum in Human Stool Using TaqMan Real-time Polymerase Chain Reaction

SHAO Jing-dong, WU Lin, WU Fu-ping, FU Chun-ling, WANG Yi-qian, FAN Li-li

2012, 30(4):  21-333-335. 

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The special DnaJ-like protein gene of Cryptosporidium parvum was amplified through designing special primers and TaqMan probes within the conserved and specific regions for this gene. In this way, a rapid and stable method of real-time PCR assay for the detection of C. parvum was established. The specificity and sensitivity of PCR were also analyzed. By adding standard culture fluid in blank fecal sample, the sensitivity of the method was evaluated. The results showed that the detection limit of pure culture with real-time PCR assay was 26 oocysts/ml. The detection limit for C. parvum in artificially contaminated fecal sample was 2 600 oocysts/ml. The specificity of the method was verified with no amplification on DNA from other enteric parasites and bacteria. These results indicated that the real-time PCR method for C. parvum detection in fecal sample is simple, rapid, with high specificity and sensitivity.

A death case of imported falciparum malaria in Beijing

2012, 30(4):  22-324,328. 

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First case of imported acute schistosomiasis patient after interruption of schistosomiasis transmission in Cixi City

2012, 30(4):  23-封二. 

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