Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

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.

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.

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.

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.

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.

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].

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.

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.

A COMPREHENSIVE ECHINOCOCCUS PREVENTION LAUNCHED IN THE TIBET AUTONOMOUS REGION

Echinococcus has been shared by both humans and animals for quite a long time now.The Tibet Autonomous Region（TAR）is one of the areas with severe epidemic of echinococcus,with patients and their families encountering devastating health problems and heavy economic burdens in relation to the disease,not to mention a dramatic loss of agriculture and animal husbandry production.In March 2017,

A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto,Echinococcus multilocularis and Echinococcus canadensis in China

Background:Echinococcosis caused byEchinococcus is one of the most major infectious diseases in north-west highland of China.E.granulosus sensu strict,E.multilocularis,andE.canadensis are known to be the only three species related to human health transmitting in the areas.To achieve targeted treatment and control of echinococcosis,the accurate identification and discrimination of the species are important.However,currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.Methods:In the study,a set of primers were designed to aim at the three human-pathogenicEchinococcus species in China.The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity.A total of 73parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.Results:The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products.The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately.When DNA mixtures of the targeted species containing the same concentration were used as templates,the lowest amount of DNA which can be detected was 50 pg/μl,10 pg/μl and 5 pg/μl forE.granulosus s.s.,E.multilocularis,andE.canadensis respectively.No cross-reactivity was observed when DNA from eight genetically close species was used as control templates.The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected withE.shiquicus,which showed negative signals in the developed assay.Of all the tested stool materials,16 were previously found positive forEchinococcus by visual and microscopic examination.Among these 16 samples,13 were confirmed by the multiplex PCR,and the other three tested negative.Additionally,the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.Conclusions:The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation,which proven to be a promising tool utilized in clinic and surveillance system.

Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library

Objective To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 μg) was submitted to reverse transcription using random hexamers ［pd(N6)］. The double-strand blunt-ended cDNAs were ligated with an EcoRI/NotI adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector λgt11 EcoRI arms. After being packaged in vitro, λgt11 was put to an infectious bacteria Echinococcus coli (E.coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments.Results The recombinant ratio was nearly 100% and approximately 1×106 clones could be derived from this λgt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. Conclusions A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

Impact of overgrazing on the transmission of Echinococcus multilocularis in Tibetan pastoral communities of Sichuan Province,China

Background Overgrazing was assumed to increase the population density of small mammals that are the intermediate hosts of Echinococcus multilocularis, the pathogen of alveolar echinococcosis in the Qinghai Tibet Plateau. This research tested the hypothesis that overgrazing might promote Echinococcus multilocularis transmission through increasing populations of small mammal, intermediate hosts in Tibetan pastoral communities. Methods Grazing practices, small mammal indices and dog Echinococcus multilocularis infection data were collected to analyze the relation between overgrazing and Echinococcus multilocularis transmission using nonparametric tests and multiple stepwise logistic regression. Results In the investigated area, raising livestock was a key industry. The communal pastures existed and the available forage was deficient for grazing. Open （common） pastures were overgrazed and had higher burrow density of small mammals compared with neighboring fenced （private） pastures; this high overgrazing pressure on the open pastures measured by neighboring fenced area led to higher burrow density of small mammals in open pastures. The median burrow density of small mammals in open pastures was independently associated with nearby canine Echinococcus multilocularis infection （P=-0.003, OR=1.048）. Conclusion Overgrazing may promote the transmission of Echinococcus multilocularis through increasing the population density of small mammals.

Seasonal pattern of Echinococcus re-infection in owned dogs in Tibetan communities of Sichuan, China and its implications for control

Background:Human cystic echinococcosis(CE)and alveolar echinococcosis(AE)are highly endemic in Tibetan communities of Sichuan Province.Previous research in the region indicated that domestic dog was the major source of human infection,and observations indicated that domestic dog could have more access to intermediate hosts of Echinococcus spp.:both domestic livestock(CE)viscera and small mammals(AE),in early winter and again in spring.We hypothesized that there would therefore be a significant increase in the risk of canine infection with Echinococcus spp.in these two seasons and conducted a reinfection study to investigate this further.Methods:Faecal samples were collected from owned dogs in seven townships in Ganze Tibetan Autonomous Prefecture(Sichuan Province,China),and Echinococcus spp.infection status was determined using copro-antigen ELISA.Dogs were sampled in April(spring),July(early summer),September/October(autumn/early winter)and December(winter)in 2009;and in April(spring)2010.Dogs were treated with praziquantel following each of the five sample collections to eliminate any tapeworms.Information on dog sex,age and body weight was also collected.The t-test,Fisher’s exact test,Poisson regression and logistic regression were used to compare means and prevalences,and to identify factors associated with infection status.Results:The proportion of female dogs was significantly lower than that of male dogs;female dogs had significantly higher(22.78%)baseline copro-ELISA prevalence than males(11.88%).Dog body weight,sex,age,county and previous infection status at any sampling point had no influence on the re-infection prevalence in general.Poisson regression did not found a significant influence on the re-infection prevalence due to different deworming/sampling time spans.Dogs exhibited significantly higher re-infection prevalences in spring and early summer of 2009 and in early winter between September/October and December of 2009,suggesting a higher infection pressure in these seasons comparing with other seasons.Conclusion:Following praziquantel treatment,dog body weight,sex,age,county,deworming time span and previous infection status at any sampling point had no influence on the re-infection prevalence in the region in general.The differences between re-infection prevalences were probably due to the seasonality in Echinoccocus spp.infection pressure in the region.Early winter,spring and early summer should be important seasons for optimal dog deworming intervention in these Tibetan communities.