Primary pelvic Echinococcus granulosus infection:A case report

BACKGROUND Primary pelvic Echinococcus granulosus infection is clinically rare.The reported cases of pelvic Echinococcus granulosus infection are considered to be secondary to cystic echinococcosis in other organs.Single Echinococcus granulosus infection is very rare.CASE SUMMARY In this report,we presented a case of primary pelvic Echinococcus granulosus infection admitted to the First Affiliated Hospital of Xinjiang Medical University.We described the key diagnostic points and surgical treatment of this case.We also summarized the epidemiological characteristics and pathogenesis of the disease.CONCLUSION Our case may provide clinical data for the diagnosis and treatment of primary pelvic Echinococcus granulosus infection.

Echinococcus granulosus钙结合蛋白基因的克隆和序列分析

目的 获得细粒棘球蚴Calcium bindingprotein(CaBPs)基因序列资料 ,进行序列分析 ,为包虫病免疫预防研究奠定基础。方法 从包囊内获取细粒棘球蚴原头蚴 (protoscolices) ,提取RNA ,逆转录成cDNA ,用特异引物扩增CaBPs基因 ;并将其克隆到T载体后进行序列测定和分析。然后登录Gene/Bank数据库。结果 以cDNA为模板获得 3个核苷酸长度不同的基因 ,分别是CaBP1为 914bp ,CaBP2为 10 95bp ,CaBP3为 10 39bp。同源性比较结果表明 :来自新疆羊源CaBPs基因序列和巴西 (Brazil)羊源基因CaBPs同源性为 90 %以上 ;从cDNA获得的 3个CaBPs基因序列的比较分析说明钙结合蛋白可能存在一个家族 ,有不同的成员组成。结论 CaBPs在E .granulosus虫体发育过程中具有十分重要的功能 ,到目前为止其机理尚不完全明了 ,CaBPs基因的获得为进一步研究其结构和功能 ,以及对包虫病的防治具有重要意义。

细粒棘球蚴(Echinococcusgranulosus)CO1与ND1基因序列分析

目的为了明确内蒙古锡林郭勒盟西乌旗羊株和锡林浩特市人株细粒棘球蚴之间的基因型及其遗传变异情况。方法利用分子生物学技术对采自两地区细粒棘球蚴的线粒体CO1基因与ND1基因进行了克隆和测序,并运用DNAStar5.0中的MegAlign工具对已测出的序列进行了分析。结果西乌旗羊株细粒棘球蚴与锡林浩特市人株细粒棘球蚴CO1基因片段和ND1基因片段大小分别均为936bp和895bp。西乌旗羊株细粒棘球蚴CO1基因序列与新疆羊株细粒棘球蚴的CO1基因序列同源性为99.3%;锡林浩特市人株细粒棘球蚴CO1基因序列与新疆人株细粒棘球蚴的CO1基因序列同源性为98.6%;且西乌旗羊株和锡林浩特市人株细粒棘球蚴ND1基因与国内外已报道的G1型ND1基因序列完全相同。结论表明内蒙古上述两地区细粒棘球蚴的同源性很高,且基因型均为G1型,这为内蒙古某些地区细粒棘球蚴流行虫株的确定提供了重要依据,同时也对当地细粒棘球蚴病的预防和控制具有重要意义。

E.granulosus皮下感染小鼠肝切除术后Myd88/IL-23表达和肝再生

目的利用细粒棘球绦虫(Echinococcus granulosus,E.granulosus)感染小鼠模型,结合70%肝切除手术,探索E.granulosus感染对肝脏组织再生的影响,初步探讨MyD88/IL-23在E.granulosus感染小鼠术后肝再生中的表达及意义。方法建立了E.granulosus皮下感染后的小鼠70%肝切除模型。选择18只E.granulosus感染的小鼠,以及18只作为对照的健康小鼠。在手术后的不同时间点,比较2组小鼠的肝脏再生量和再生率。ELISA法监测不同时间点小鼠血清中的MyD88和IL-23水平及肝功能指标AST和ALT的变化。结果与对照组小鼠比较,E.granulosus感染小鼠术后肝再生量和再生率降低,在术后的12 h、1 d和2 d表现出显著性差异。与对照组小鼠比较,E.granulosus感染小鼠外周血中AST和ALT水平在术后1 d明显升高;MyD88和IL-23水平在术后2 h明显升高。结论E.granulosus感染显著减缓了小鼠肝脏切除术后的再生过程,与此同时,感染小鼠的MyD88和IL-23水平显著增加,暗示它们可能在此过程中起作用。

Echinococcus Granulosus 14-3-3 Protein:A Potential Vaccine Candidate Against Challenge with Echinococcus Granulosus in Mice

Objective To investigate the protective immunity against Echinococcus granulosus in mice immunized with rEg14-3-3. Methods ICR mice were subcutaneously immunized three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally and then sacrificed after six months of post-challenge to detect the proliferation of splenocytes by MTT assay, and to measure the secretion of IL-2, IL-4, IL-20, and IFN -y by ELISA. The rate of reduced hydatid cyst and the levels of IgE, igG and IgG subclasses in sera were examined. Results Mice vaccinated with rEg14-3-3 and challenged with protoscoleces revealed significant protective immunity of 84.47%. ELISA analysis indicated that the immunized mice generated specific high levels of IgG and the prevailing isotypes of IgG were IgG1 and IgG2a. Splenocytes from mice immunized with rEg14-3-3 showed a significant proliferation response. The secretion of IFN-V and IL-2 increased significantly in the vaccinated mice whereas there was no significant difference in IL-4 and IL-20 levels between vaccinated and control mice. Conclusion The results indicate that the rEg24-3-3 vaccine could induce a high level of protective immunity as a promising vaccine candidate to prevent cystic echinococcosis.

Analysis of Protoscoleces-specific Antigens from Echinococcus Granulosus with Proteomics Combined with Western Blot

Objective To establish and optimize the proteomic analysis of protoscoleces-specific antigens from Echinococcus granulosus. To provide a foundation for identifying specific antigens in the soluble proteins of E. granulosus protoscoleces for further research. Methods Brood capsules were collected aseptically from fertile E. granulosus cysts from the livers of an infected patient. The fertile E. granulosus cysts were fractured, and protoscoleces were collected by centrifugation. The soluble proteins of protoscoleces were acquired using the 2D Quant kit according to the manufacturer＇s instructions. We employed two-dimensional electrophoresis （2-DE） combined with immunoblot assay （Western blot） to analyze the soluble components of E. granulosus protoscoleces antigens. The 2-DE and immunoblot maps obtained were analyzed with PDQuest 8.0 image analysis software. Results About 233 soluble protein spots were identified with Coomassie-stained gels. Most of the proteins had a molecular weight of 16 000 Da to 117 000 Da, and an isoelectric point value of 3.0 to 10.0. 2-DE immunoblot was conducted and 57 specific antigen spots were observed, among which 23 spots were identified. Conclusion 2-DE combined with Western blot is the key to successful proteomic analysis and presents a new possibility for searching the specific E. granulosus protoscoleces antigens.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus:Implications for EG95 vaccine application

Objective:To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus(E. granulosus). Methods:We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1(DNASTAR Inc.,Madison,WI),and the phylogenetic analysis was performed by using MEGA5.1(CEMI,Tempe,AZ,USA). In addition,linear and conformational epitopes were analysed,including secondary structure,NXT/S glycosylation,fibronectin type ecoⅢ(Fnndary Ⅲ) domain and glycosylphosphatidylinositol anchor signal(GPIanchor). The s structure was predicted by PSIPRED method. Results:Our results indicated that most isolates overall shared 72.6-100% identity in EG95 gene sequence with the published standard EG95 sequence,X90928. However,EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates,respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine,X90928,and they belonged to Subgroup 1. However,in comparison to X90928,several amino acid mutations occurred in most isolates besides oncosphere,which potentially altered the immunodominant linear epitopes,glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions:This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates,and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Echinococcus Granulosus: Suitable in vitro Protoscolices Culture Density

The present study is to determine the suitable protoscolices （PSCs） density for long-time culturing in vitro. The PSCs were divided into eight groups with different densities and the viability tests were carried out with 0.1% methylene blue staining. Then the infection ability of cultured PSCs was assessed by the mean cyst weight of mice inoculated intraperitoneally with PSCs after 8 months post-infection.

Identification and bioinformatics analysis of lactate dehydrogenase genes from Echinococcus granulosus

Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adult Echinococcus granulosus(E.granulosus) and to predict the structure and function of its encoding protein using bioinformatics methods.Methods:With the help of NCBI,EMBI, Expasy and other online sites,the open reading frame(ORF),conserved domain,physical and chemical parameters,signal peptide,epitope,topological structures of the protein sequences were predicted and a homology tertiary structure model was created:Vector NT1 software was used for sequence alignment,phylogenetic tree construction and tertiary structure prediction. Results:The target sequence was 1 233 bp length with a 996 bp biggest ORF encoding 331 amino acids protein with typical L-LDH conserved domain.It was confirmed as full length cDNA of LDH from E.granulosus and named as EgLDH(GenBank accession number:HM748917).The predicted molecular weight and isoelectric point of the deduced protein were 3 5516.2Da and 6.32 respectively.Compared with LDHs from Taenia solium,Taenia saginata asiatica,Spirometra erinaceieuropaei.Schistosoma japonicum,Clonorchis sinensis and human,it showed similarity of 86% ,85% ,55% ,58% ,58% and 53% ,respectively.EgLDH contained 3 putative transmembrane regions and 4 major epitopes(54aa-59aa.81aa-87aa,97aa-102aa,307aa-313aa),the latter were significant different from the corresponding regions of human LDH.In addition,some NAD and substrate binding sites located on epitopes 54aa-59aa and 97aa-102aa,respectively.Tertiary structure prediction showed that 3 key catalytic residues 105R,165D and 192H forming a catalytic center near the epitope 97aa-102aa,most NAD and substrate binding sites located around the center.Conclusions:The full length cDNA sequences of EgLDH were identified.It encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.

In-vitro scolicidal activity of Mallotus philippinensis (Lam.) Muell Arg. fruit glandular hair extract against hydatid cyst Echinococcus granulosus

Objective: To investigate new scolicidal agent from natural resources to cope with the side effects associated with synthetic drugs in Echinococcosis. Methods: The scolicidal potential of methanolic fruit powder extract (10 and 20 mg/mL) of Mallotus philippinensis ( M. philippinensis ) was investigated. Viability of protoscoleces was confirmed by trypan blue exclusion method, where mortality was observed at concentration of 10 and 20 mg/mL in 60 min treatment against Echinococcus granulosus ( E. granulosus ), under in-vitro conditions with reference to the known standard drug Praziquantel . Results: At concentration 10 and 20 mg/mL, the mortality rate was observed 97% and 99% respectively for 60 min treatment; while up to 93% mortality was observed with 20 mg/mL for only 10 min treatment. The concentration above 20 mg/mL for above 2 h showed 100% mortality, irrespective of further incubation. Conclusions: As compared with the standard anti-parasitic drug Praziquantel our extract has significant scolicidal activity with almost no associated side effects.

Morphometric differentiation between camel and sheep strains of Echinococcus granulosus using computer image analysis system(CIAS)

Objective:To find importance of morphometrie criterion of larval rostellar hook of Echinococcus granulosus(E.granulosus) and the easy and reliable method for distinguish sheep and camel strains in epidemiologic studies.Methods:Larval rostellar hooks(n=1860) of 31 camel and sheep isolates in Iran,which aheady had been characterized by PCR.were carefully processed by computerized imagime analysis system(CIAS) and acquired data about rostellar books were analyzed using software SPSS.Results:Measurement analysis of rostellar hooks[mean Iength (24.23±3.12lμm]indicated that length of the large hook was a remarkable parameter for strain differentiation.Data analysis demonstrated that CIAS could be used as a reliable tool to distinguish camel from sheep strains with high sensitivity(95.2%) and specificity(91.5%). Conclusions:CIAS as a specific,sensitive,economic,fast,and reliable means might be used for differentiation of E.granulosus strains.Although perimeter and area were measured by digital technology,they were not shown as discriminative criterion as total hook length did.

Study of Infection of Echinococcus granulosus in Yak in Spring and Its Potential Role in Transmission of Cystic Echinococcosis in Rangtang County of Sichuan,China

Dear Editor, Cystic echinococcosis （CE）, caused by Echinococcus granulosus in larval stage, is considered as one of the most dangerous parasitic zoonosis in the world. The obligate 2-host parasitic cycle of Echinococcus granulosus is predominantly synanthropic. Dogs are the usual definitive hosts, and lots of mammalian species can be intermediate hosts, including domestic livestock and human[I2]. In the Tibetan plateau, China, the population is mainly Tibetans primarily engaged in livestock husbandry and CE is therefore a health problem for both people and animal in Tibetan communities. The reported infection rate of Echinococcus gronulosus in slaughtered yak in slaughterhouses is usually very high, being about 50% or higher as reported, and the liver and lungs are the main affected organs[34].

Molecular differentiation of cryptic stage of Echinococcus granulosus and Taenia species from faecal and environmental samples

Objective:To differentiate cryptic stage of Echinococcus granulosus(E.granulosus) and Taenia by PCR-RFLP and sequence information of amplicon.Methods:DNA were isolated from metacestodes stage of Taenia and E.granulosus using DNA isolation kit(Q-BIOgene kit,USA), the amplified and purified DNA product was then cloned and sent for sequencing.The generating sequence information was used for amplicons identification.Results:Out of 112 faecal and environmental samples,16 exhibited positive result.The product size of amplicon positive for E.granulosus was 310 bp;whereas,for Taenia spp.sizes varied from 379 to 388 bp.Restriction pr of ile of actinⅡwith Csp61 also differed Taenia spp.and E.granulosus.Conclusions:The result of the study indicated that,the primers were useful to differentiate cryptic stage of the two genera which is yet to be reported earlier.

Molecular characterization of Echinococcus granulosus in paraffin-embedded human tissues from Southwest Iran

Objective:To investigate Echinococcus(E.)granulosus genotypes as the causative agents of hydatidosis in humans in the southwest of Iran(Khuzestan province).Methods:In this study,isolates of 80 archived human paraffin embedded hydatid cysts were collected from pathology laboratories in Ahvaz city,Khuzestan province.DNA was extracted and examined by nested-PCR of ribosomal DNA(rDNA)internal transcribed spacer 1(ITS1),and PCR-RFLP.In addition,the sequences of fragments of genes coding for Cox space1 and NADH dehydrogenase 1(ND1)were also examined.Results:Of the 80 paraffin samples,44(55.0%)were from the liver,27(33.8%)from the lung,and the rest from other organs.The amplified hydatid genomic DNA showed that the cysts were E.granulosus strains.The results of PCR-RFLP and sequencing analysis revealed the presence of G1 genotype(sheep strain)in all human isolates.Furthermore,no camel strain(G6)was detected among all samples in the regions studied.Conclusions:The molecular findings indicate that the predominant genotype involved in E.granulosus transmission in southwest of Iran is the common sheep strain(G1),which occurs in human populations.These results may have important implications for hydatid disease control in the studied areas.

In vitro treatments of Echinococcus granulosus with fungal chitosan,as a novel biomolecule

Objective:To determined the antiparasitic activity of the isolated chitosan from Penicillium viridicatum,Penicillium aurantiogriseum and commercial chitosan against protoscolicidal of hydatid cysts were determined.Methods:After isolating chitosan from fungal cell walls,four concentrations(50,100,200,400μg/mL)of each type of prepared chitosan and commercial chitosan were used for 10,30,60,and 180min,respectively.Results:Among different type of chitosan,commercial chitosan with the highest degree of deacetylation showed high scolicidal activity in vitro.Fungal chitosan could be recommended,as good as commercial chitosan,for hydatic cysts control.Conclusions:It seems to be a good alternative to synthetic and chemical scolicidal.

Molecular Characterization of <i>Echinococcus granulosus</i>in South of Iran

The focus of present study was to determine the epidemiological and molecular aspects of different strains of cystic echinococcosis in Fars province, Iran. Liver and lung samples from 410 sheep, 206 goats and 315 cattle were collected. In cattle, the infestation rate was 18.1% (57/315), with 11.1% hepatic cysts and 7.0% pulmonary cysts. Out of all identified cysts, 31.4% of the hepatic and 31.8% of the pulmonary cysts were found fertile. Incidence rate of hydatid cyst infection in sheep was 15.5% (64/410) with 11.9% hepatic cysts and 3.6% pulmonary cysts, of which 24.5% and 20% of hepatic and pulmonary cysts were respectively identified as fertile. The infestation rate was 16.0% (33/206) in goat, in which 10.2% and 5.8% cysts were collected from liver and lung, correspondingly. The prevalence of fertile hepatic and pulmonary cysts was recorded as 23.8% and 16.7%, respectively. Genotyping the cystic materials using PCR showed that the most prominent strains responsible for cystic echinococcosis in the Fars province are G1 and G6/7, while no evidence of E. multilocularis was recorded. This information may give us some clues to find out more about strains distribution in different regions in Iran, which may finally use to find tools in the eradication program of the disease, here and elsewhere.

Echinococcus granulosus:A novel parenchymal sparing surgical treatment

Human echinococcosis is a zoonosis caused by Echinococcus(E.)tapeworms.There are different species,but E.granulosus and E.multilocularis have clinical relevance causing cystic and alveolar echinococcosis,respectively.Cystic echinococcosis(CE)is defined as presence of one or more hydatid cysts,most frequently in the liver and the lungs,less usual in the kidneys,bones,spleen,pancreas,heart or brain.The location,the number and size of the cysts determine the severity of the disease[1].

秉螈Pingia granulosus的重新发现及新模描述

1933年,张孟闻以1号采自浙江天台山的次成体有尾类标本定名粗皮肥螈Pachytriton granulosus,并于1935年建立新属——秉螈属Pingia以辖之。因模式标本在日本侵华战争期间遗失且多年来一直未再采集到标本,后来学者多认为其是无斑肥螈的同物异名。2008年6月1日,两栖动物爱好者周行在浙江龙王山采集到2号有尾类标本,经本文作者仔细研究后发现,除部分色斑及指(趾)端腹面未覆有角质鞘外,其他特征与秉螈Pingia granulosus完全相符。因此作者建议恢复秉螈属Pingia和秉螈Pingia granulosus的有效性,并指定新模标本予以描述。

我国内蒙古大兴安岭北麓泡状肝包虫种类的研究 III.苏俄棘球绦虫(Echinococcus russicensis sp.nov.)

在我国内蒙古大兴安岭北麓草场流行有与在前苏联广泛分布的泡状棘球蚴其成虫具球形子宫相似的病原,过去称之为苏俄的多房棘球绦虫。我们在比较研究了它与欧洲的多房棘球绦虫的全程生活史的发育规律,及成虫和幼虫期的结构之后,发现两者之间有极大差异,认为不能再用多房棘球绦虫称呼之。最近,我们用内蒙古的苏俄型泡状棘球蚴人工感染4只羊羔,全部阴性不能发育,说明内蒙古的本虫种不同于所谓幼虫期可以在牛羊发育的"哈萨克亚种"。为了纪念前苏联学者最早发现和叙述本种成虫,兹暂定名为苏俄棘球绦虫新种(Echinococcus russicensis sp.nov.)。

我国内蒙古大兴安岭北麓泡状肝包虫种类的研究 II.西伯利亚棘球绦虫(Echinococcus sibiricensis Rauschet Schiller,1954)

目的Rausch and Schiller(1954)在美国阿拉斯加白令海峡St.Lawrence岛从北极狐(Alopex lagopus)和雪撬狗(sledge dogs)发现西伯利亚棘球绦虫新种(Echinococcus sibiricensis sp.nov.),但诸多学者认为E.sibiricensis Rausch et Schiller,1954是欧洲的多房棘球绦虫(Echinococcus multilocularis Leuckart,1863)的地理株(或亚种)(Vogel,1957;Skrjabin and Abuladze,1964;Kumaratilake and Thompson,1982;Meyajaki,1991等)。于1985-2002年间,作者在我国内蒙古大兴安岭北麓草场多年进行泡状棘球蚴(alveolar Echinococcus)病原的野外调查和实验室鼠类的人工感染试验,结果发现该地区同地点存在有北美西伯利亚棘球绦虫、欧洲多房棘球绦虫及苏俄多房棘球绦虫三种不同"地理株",而且三种成虫常混合感染于同一终宿主沙狐(Vulpes corsac)的体内,它们的幼虫期也存在于当地的布氏田鼠(Microtus brandti)。2001年,作者再次检查当地沙狐151只,从中检出19只感染有泡状棘球绦虫成虫。含成熟虫体的13只阳性沙狐中,感染西伯利亚棘球绦虫有11只,其中2只单独感染此虫种,另9只还混合感染了欧洲多房棘球绦虫及苏俄多房棘球绦虫三虫种。在呼伦贝尔草原的泡状棘球蚴病原中,西伯利亚棘球绦虫是优势种,它在终宿主沙狐和中间宿主的感染率都高过其他虫种。本文着重介绍西伯利亚棘球绦虫的成虫子宫结构和它幼虫期在野外布氏田鼠及人工感染实验鼠体内的发育情况。