2D-PAGE analysis of Taenia solium metacestode 10-30 kDa antigens for the serodiagnosis of neurocysticercosis in children

Neurocysticercosis (NCC) caused by T. solium metacestode is an increasingly important health issue in Indian children. The sensitivity and specificity of available serological techniques were low in case of single cysticercus granuloma cases which is a more common feature in Indian patients who are children. Serum samples were collected from 13 clinically and radiologically suggestive NCC children and seropositive by ELISA, 25 clinically and radiologically suggestive NCC children and seronegative by ELISA and 25 control subjects. The 10–30 kDa antigens of T. solium metacestode were subjected to 2-dimensional gel electrophoresis (2D-PAGE) followed by enzyme-linked immunoelectrotransfer blot (EITB) assay to detect antibody in serum. Analysis of 10–30 kDa antigenic fraction 2D-PAGE map showed 31 proteins between 10 and ≤28 kDa and innumerable proteins between >28 and 30 kDa with the Isoelectric point of 3–10. All the 13 (100%) NCC seropositive and 15 (60%) out of 25 NCC seronegative samples were reactive with 2D fraction antigens. In the control group, none of the serum was reactive except 2 hydatid samples (92% specificity). The sensitivity and specificity of 2D-PAGE EITB assay were significantly higher than the ELISA which is the routine diagnostic method used in the endemic countries for the serodiagnosis of neurocysticercosis.     

猪带绦虫虫卵感染家猪致肝纤维化相关病理分析

目的观察猪带绦虫虫卵实验感染中间宿主家猪后囊尾蚴致肝纤维化作用,并对病变程度进行定量分析。方法实验猪分成3组,将亚洲猪带绦虫虫卵以高剂量(60 000个/ml)、中剂量(40 000个/ml)和低剂量(10 000个/ml)分别灌服感染家猪,每种剂量感染2头。健康家猪2头为对照组。30 d后剖杀,取肝脏,制作病理切片,HE和Cason染色,对实验猪肝脏病变组织进行病理变化观察及胶原纤维含量分析。结果与健康对照组比较,实验组家猪肝组织发生不同程度的变性、坏死、炎细胞侵润,肝、脾指数增加(P<0.05或P<0.01);肝纤维化改变明显,胶原纤维总面积和总吸光度值增加,胶原纤维含量显著增加(P<0.05或P<0.01)。结论猪带绦虫虫卵感染家猪后以肝脏囊尾蚴寄生居多,同时发生以肉芽肿和纤维化改变为主的病理损害且较为严重。     

Estimation of the cost of Taenia solium cysticercosis in Eastern Cape Province, South Africa

OBJECTIVES: To provide a comprehensive estimate of the societal costs of Taenia solium cysticercosis for the Eastern Cape Province (ECP), South Africa, as an objective measure of its impact in this endemic area. METHODS: Epidemiological data on the prevalence of epilepsy, proportion of epilepsy cases due to neurocysticercosis (NCC) and consequences of cysticercosis were gathered from published and unpublished sources. Economical data were mostly obtained from governmental sources. Three methods were used for estimating productivity losses. Monte Carlo sampling was used to represent the uncertainty of the estimates with 95% Credible Intervals (95% CI). The estimation is for 1 year using a societal approach. All costs are reported in 2004 US Dollars. RESULTS: Overall, there were an estimated 34 662 (95% CI: 17 167-54 068) NCC-associated cases of epilepsy in ECP in 2004. The overall monetary burden (in million of US Dollars) was estimated to vary from US Dollars 18.6 (95% CI: US Dollars 9.0-32.9) to US Dollars 34.2 (95% CI: US Dollars 12.8-70.0) depending on the method used to estimate productivity losses. The agricultural sector contributed an average of Dollars 5.0 million. The prevalence of epilepsy, proportion of productivity reduction and the proportion of epilepsy cases attributable to NCC had the largest impact on the overall estimates. CONCLUSION: This preliminary estimate suggests that T. solium cysticercosis results in considerable monetary costs to a region that is already economically constrained. Because this infection is preventable, these results could guide stakeholders in deciding where to invest scarce health and agricultural resources in their countries.     

亚洲带绦虫烯醇化酶基因的原核表达、鉴定与组织定位

目的 表达亚洲带绦虫（Taenia asiatica,Ta）成虫烯醇化酶（enolase,ENO）基因,并对其进行组织定位和免疫反应性分析。 方法 通过大规模测序从亚洲带绦虫cDNA文库中确定ENO基因,PCR扩增目的片段,将其克隆至表达质粒pET-30a（+）,在大肠埃希菌BL-21/DE3中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）观察重组蛋白的表达情况,用镍离子金属螯合剂亲和层析柱纯化重组蛋白,蛋白质印迹（Western blotting）分析该重组蛋白的免疫反应性。将重组蛋白免疫SD大鼠制备免疫血清,ELISA检测抗体水平,间接免疫荧光检测确定ENO在亚洲带绦虫成虫组织中的定位。 结果 PCR、双酶切和DNA测序结果均表明,重组质粒pET-30a（+）-TaENO构建成功。SDS-PAGE结果显示,重组蛋白TaENO的相对分子质量（Mr）为47 000。经亲和层析获高纯度的蛋白,蛋白浓度为0.37 mg/ml。重组蛋白TaENO能被SD大鼠抗血清、感染亚洲带绦虫的猪血清和患者血清识别。间接免疫荧光检测结果显示,TaENO主要定位于成虫的表膜。 结论 纯化后的亚洲带绦虫成虫烯醇化酶重组蛋白具有较强的免疫反应性,烯醇化酶主要定位于成虫表膜。     

中国西部地区牛带绦虫的分子鉴定和生物行为研究进展

本文对近年来涉及中国西部7省(区)10县（市）的牛带绦虫病的流行病学调查和驱虫治疗、成虫标本的形态学观察、11个地理虫株的分子生物学鉴定、牛带绦虫和亚洲带绦虫感染中间宿主猪和牛后囊尾蚴的发育和生物行为观察,以及中间宿主组织病理学变化的比较等进行综述,认为将亚洲带绦虫定为新物种比作为牛带绦虫的亚种更合适。     

Distribution of the cestode Taenia parva (Taeniidae) along the digestive tract of the common genet (Genetta genetta)

Summary  The small intestine of the common genet is the habitat of Taenia parva (Taeniidae), a common parasite whose intermediate host is Apodemus spp. (Rodentia). Here we divided the small intestine of 51 common genets into sections and analysed them separately. Cestodes were classified into four categories: larval, pre-adult, adult and gravid specimens. A total of 4,443 individuals were recovered and classified: 543 larval stages, 2,326 pre-adults, 872 adults and 702 gravid specimens. A double analysis was performed by checking the distribution of the parasite along the digestive tract and comparing this distribution with reproductive fitness. This is the first study to test the border effect on the distribution of T. parva in its habitat. We also tested and demonstrated the Ideal Free Distribution Model (IFM model). A non-random distribution was found, with the lowest parasite frequencies in the top and the bottom portions of the small intestine. Maturity classes of cestodes vs. intestine portion were tested and no differences between portions were detected. We also analysed seasonal changes in parasite frequency. Log-linear analysis showed that the mean frequency of cestode occurrence per individual host was higher during winter, followed by spring, autumn and summer.     

Evaluation of an IgG-ELISA strategy using Taenia solium metacestode somatic and excretory-secretory antigens for diagnosis of neurocysticercosis revealing biological stage of the larvae

Diagnosis of neurocysticercosis (NCC) is complicated because of the variability in clinical presentations and course of the disease where viability of parasite is a major determinant. The present study describes evaluation of ELISAs using Taenia solium metacestode somatic and excretory-secretory (ES) antigens for detection of anti-T. solium metacestode IgG antibodies in serum and cerebrospinal fluid (CSF). And results of the ELISAs in cases with a definitive diagnosis of NCC are correlated with the biological stages of the parasite such as live vesicular or degenerated stage. The sensitivity of the IgG-ELISA using ES antigen is observed to be much higher in serum (88.2%) than in CSF (64.28%) although it is only marginally higher in serum (76.4%) than in CSF (75%) when somatic antigen is used in the ELISA. Whereas, the specificities of the ELISA using either somatic or ES antigen for detection of IgG antibodies in serum (97.97%; 96.96%) and CSF (96.42%; 97.61%) are comparable. A strong association is observed between live stage of the parasite and detection of antibodies in sera and CSF from more number of NCC patients by ELISA using ES antigens. Similarly, detection of antibodies by ELISA using somatic antigens could be associated with the dead or degenerated stage of the parasite in brain. The IgG-ELISA strategy developed in the present study opens up an avenue for diagnosis of NCC in hospitals or in population prevalence studies. The use of crude extracts of ES proteins might improve the serodiagnosis of the cases of NCC carrying live vesicular stage of the parasite larvae.     

Genetic variation of the 8-kDa glycoprotein family from Echinococcus granulosus, Taenia multiceps and Taenia hydatigena

Background  Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.

Methods  The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.

Results  Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.

Conclusion  We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.

     

猪带绦虫AgB疫苗的研制进展及展望

猪囊尾蚴病是一种严重危害人类健康的人兽共患寄生虫病,主要流行于中非、南非、拉丁美洲、东亚和南亚等地区.采用疫苗防治该病是当前研究的热点.AgB的DNA疫苗种类有pV93-AgB、pCD-AgB、pCMV-SynVW2-1和pBU-CD58/IFN-γ-AgB等,AgB的蛋白疫苗种类包括VW7-3多肽、VW2-1多肽、V...