Analysis on the reactivity of five subunits of antigen B family in serodiagnosis of echinococcosis

In this study, the reactivity and differences of five subunits of echinococcus antigen B (AgB) family, recognizing specific antibodies in echinococcosis patient serum, were analyzed. Eight recombinant subunit antigens from Echinococcus granulosus (EgAgB1-EgAgB4) and Echinococcus multilocularis (EmAgB1-EmAgB3 and EmAgB5) were tested by ELISA using a panel of 243 serum samples collected from cystic echinococcosis (CE), alveolar echinococcosis (AE), cysticercosis (CC) patients and clinically normal individuals (NH). The results showed that the diagnostic sensitivity of the subunits for CE sera were 83.06%, 62.90%, 29.03%, 75.81% and 41.13%, and the specificities were 73.95%, 72.27%, 76.47%, 73.11% and 85.71%, respectively. The reactivity of three paralogous subunits, EgAgB1, EgAgB2 and EgAgB3 from E. granulosus and EmAgB1, EmAgB2 and EmAgB3 from E. multilocularis were compared by serological assay. All of the orthologous subunits showed no statistical difference (P>0.05) in detecting CE and AE sera; it revealed that the reactive epitopes may be similar between the orthologous subunits. In a total of 124 CE sera, the positive recognition rate by EgAgB1 was the highest (103/124), yet cocktail subunit antigens may detect even more positives from 100/124 to 112/124 using different subunit combinations. IgG4 subclass was the predominant antibody in reacting with subunit antigens. To conclude, the epitopes of orthologous AgB subunits from E. granulosus and E. multilocularis that recognize specific antibodies may be similar. The paralogous subunits EgAgB1, EgAgB2 and EgAgB4 were the main reactive subunit in sera detection and may have utility as echinococcosis diagnostics, with EgAgB1 possessing the greatest potential. Cocktail subunits may improve the positive detection rate.     

Molecular characterization of a novel gene encoding an 8-kDa-subunit of antigen B from Echinococcus granulosus genotypes 1 and 6

Antigen B in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa which are encoded by a multigene family. Four genes encoding 8-kDa-subunit monomers of the antigen B have been identified from E. granulosus. Recently, we have isolated another novel gene from Echinococcus multilocularis encoding a fifth 8-kDa-subunit of AgB (named EmAgB8/5), predominantly transcribed in the adult worm, but not in vesicles of metacestodes. In this study, we cloned and characterized two EmAgB8/5 homologue genes from E. granulosus genotypes 1 and 6 by PCR, and named as EgG1AgB8/5 and EgG6AgB8/5, respectively. The phylogenetic relationship of these genes with other genes encoding the antigen B 8-kDa-subunit monomers was also discussed.     

Molecular cross-talk in host-parasite relationships: the intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis

Cystic echinococcosis (CE), a zoonosis caused by the development of Echinococcus granulosus tapeworm larvae in the internal organs of ungulates and humans, continues to pose a major public health burden in underdeveloped and industrialised areas worldwide. Research designed to improve parasitic disease control and find out more about parasite biology has already identified a number of E. granulosus antigenic molecules. The major E. granulosus immunomodulant antigen isolated from hydatid fluid is antigen B, a 120kDa polymeric lipoprotein consisting of various 8kDa subunits. By inhibiting elastase activity and neutrophil chemotaxis and eliciting a non-protective Th2 cell response, antigen B helps the parasite evade the human response. In this review, we briefly discuss current information on the molecular characteristics and immunomodulatory properties of E. granulosus antigen B. Besides focusing on findings that provide intriguing insights into the complex interplay between host and parasite, we suggest how this information could extend the current therapeutic options in inflammatory diseases.     

Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2)

A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.     

The Echinococcus granulosus antigen B shows a high degree of genetic variability

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.     

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers

Echinococcus granulosus antigen B is an oligomeric protein of 120-160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed approximately 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.     

Characterisation of the native lipid moiety of Echinococcus granulosus antigen B

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.     

ITS-1 ribosomal DNA sequence variants are maintained in different species and strains of Echinococcus

This study investigated sequence heterogeneity in the first internal transcribed spacer (ITS-1) of ribosomal DNA within and among species and strains of Echinococcus. Different ITS-1 sequence variants exist in Echinococcus granulosus and Echinococcus multilocularis, which represent at least four evolutionary lineages: (1) a sheep strain-lineage of E. granulosus, (2) a sister lineage of a cervid and camel E. granulosus ITS-1 variants, (3) a lineage including the ITS-1 variants representing horse, bovine and camel strains of E. granulosus, as well as variants from E. multilocularis, Echinococcus oligarthrus and Echinococcus vogeli and (4) a distinct lineage of ITS-1 variants including E. granulosus strains from sheep and cervid, and E. multilocularis. At least two of the species (E. granulosus and E. multilocularis) were paraphyletic for ITS-1. Divergent ITS-1 variants from these two species shared distinct evolutionary lineages. The sequence data provided evidence that at least two turnover mechanisms, namely slippage and unequal crossing over/transposition, have led to the divergence and maintenance of sequence variants in Echinococcus species and strains.     

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Canine echinococcosis in Kyrgyzstan: using prevalence data adjusted for measurement error to develop transmission dynamics models

Echinococcosis is a major emerging zoonosis in central Asia. A cross-sectional study of dogs in four villages in rural Kyrgyzstan was undertaken to investigate the epidemiology and transmission of Echinococcus spp. A total of 466 dogs were examined by arecoline purgation for the presence of Echinococcus granulosus and E. multilocularis. In addition, a faecal sample from each dog was examined for taeniid eggs. Any taeniid eggs found were investigated using PCR techniques (multiplex and single target PCR) to improve the diagnostic sensitivity by confirming the presence of Echinococcus spp. and to identify E. granulosus strains. A total of 83 (18%) dogs had either E. granulosus adults in purge material and/or E. granulosus eggs in their faeces as confirmed by PCR. Three genotypes of E. granulosus: G1, G4 and the G6/7 complex were shown to be present in these dogs through subsequent sequence analysis. Purge analysis combined with PCR identified 50 dogs that were infected with adult E. multilocularis and/or had E. multilocularis eggs in their faeces (11%). Bayesian techniques were employed to estimate the true prevalence, the diagnostic sensitivity and specificity of the procedures used and the transmission parameters. The sensitivity of arecoline purgation for the detection of echinococcosis in dogs was rather low, with a value of 38% (credible intervals (CIs) 27-50%) for E. granulosus and 21% (CIs 11-34%) for E. multilocularis. The specificity of arecoline purgation was assumed to be 100%. The sensitivity of coproscopy followed by PCR of the isolated eggs was calculated as 78% (CIs 57-87%) for E. granulosus and 50% (CIs 29-72%) for E. multilocularis with specificity of 93% (CIs 88-96%) and 100% (CIs 97-100%), respectively. The 93% specificity of the coprological-PCR for E. granulosus could suggest coprophagia rather than true infections. After adjusting for the sensitivity of the diagnostic procedures, the estimated true prevalence of infection of E. granulosus was 19% (CIs 15-25%) and the infection pressure in the dog population was estimated to be 0.29 infections per year (CIs 0.014-0.75). Logistic regression analysis failed to identify any significant risk factors for infections for E. granulosus. After adjusting for the sensitivity of the test procedures, the estimated true prevalence for E. multilocularis was 18% (CIs 12-30%). Dogs that were restrained had a significantly lower prevalence of E. multilocularis of 11% (CIs 6-29%) compared with 26% in free-roaming dogs (CIs 17-44%) and independently within these groups hunting dogs were more likely to be infected than non-hunting dogs.     

细粒棘球蚴AgB8/2基因的原核表达及免疫学鉴定

旨在克隆细粒棘球蚴 AgB8/2基因,优化原核表达体系,鉴定纯化蛋白并初步确定其诊断价值.采用 RT-PCR扩增 AgB8/2基因 cDNA,构建原核表达载体pET-AgB8/2并在大肠杆菌 BL21(DE3)中表达,优化表达条件.纯化的AgB8/2融合蛋白经 Western blot鉴定正确后,采用斑点免疫金渗滤法初步验证其血清学诊断价值.结果显示,克隆的 AgB8/2基因包含了 273 bp的完整ORF,序列分析表明与其它已报道的AgB8/2 cDNA序列的同源性在 99%～100%之间.优化的表达条件为,当菌液 OD600值为0.6时,加入终浓度为0.05 mmol/L的 IPTG,37℃,振荡培养5 h.纯化的蛋白经 Western blot鉴定为目的蛋白.克隆的 AgB8/2基因高度保守,原核表达载体pET-AgB8/2构建正确并高效表达可溶性融合蛋白,可作为特异性抗原应用于斑点免疫金渗滤法检测血清抗体.     

A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa

We describe the development of a specific and sensitive PCR/semi-nested PCR system for the rapid diagnosis of Echinococcus granulosus genotype G1, E. granulosus genotype G6/7, and Echinococcus ortleppi (G5). Diagnosis of G1 and the group G5/6/7 is performed by a simple PCR, while discrimination between E. ortleppi (G5) and G6/7 involves a subsequent semi-nested PCR step. The target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 species of cestodes, including Echinococcus multilocularis, Echinococcus equinus, E. ortleppi and three strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25pg of DNA. This new approach was compared with published protocols of restriction fragment length polymorphism-PCR and sequencing of mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes were developed, one hybridising only with G1, the other with G5, G6 and G7 amplification products. Preliminary epidemiological results obtained with this PCR approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man

An enzyme-linked immunosorbent assay (ELISA) was adapted for the serological differential diagnosis of cystic or alveolar echinococcosis in man caused by Echinococcus granulosus or E. multilocularis respectively. By affinity chromatography using rabbit anti hydatid fluid IgG coupled covalently to CNBr-Sepharose 4B a protein fraction (Em 1) containing shared antigens of both parasites could be isolated from an extract of E. multilocularis metacestode tissue. From the same source another antigen fraction (Em 2) with a high degree of specificity for E. multilocularis was prepared by immunosorption. Antigen Em 1 was equally sensitive for the detection of antibodies against E. granulosus and E. multilocularis, whereas antigen fraction Em 2 appeared to be more specific for E. multilocularis. A correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human cystic or alveolar echinococcosis by the simultaneous use of both antigen fractions in the ELISA and by comparison of their reactivities.     

Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites

Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.     

Identification of critical residues of an immunodominant region of Echinococcus granulosus antigen B

Immune evasion strategies often shape the immunogenicity of parasite components. We recently found that the N-terminal extension of the major subunit of Echinococcus granulosus antigen B (AgB), the causative agent of hydatid disease, concentrates the immunoreactive B cell epitopes of the native molecule. The nature of this immunodominance was analyzed using four monoclonal antibodies (mAbs) defining overlapping epitopes in this region of the AgB molecule. The minimal epitope requirements of these mAbs were determined using phage display peptide libraries. The consensus sequences isolated with the mAbs, and alanine replacement analysis with synthetic peptides mapped the relevant molecular contacts within a short stretch corresponding to residues 17-24 of the AgB major subunit. Substitution of two critical residues within this stretch produced a dramatic loss of antigenicity, as determined by using patient sera. The circular dichroism spectra of the antigen, together with the distribution of the contact residues, suggest that this region adopts an amphipathic alpha-helix structure that clusters the contact residues on its polar side. To provide further insight in the interpretation of the structure activity relationships for this immunoreactive region of E. granulosus AgB, we developed a model for the N-terminal extension of the AgB major subunit, which helps to rationalize our data.     

Modeling the transmission of Echinococcus granulosus and Echinococcus multilocularis in dogs for a high endemic region of the Tibetan plateau

Echinococcus granulosus and Echinococcus multilocularis abundance and prevalence data, for domestic dogs of Shiqu County, Sichuan Province, People's Republic of China, were fitted to mathematical models to evaluate transmission parameters. Abundance models, assuming the presence and absence of immunity, were fit for both E. granulosus and E. multilocularis using Bayesian priors, maximum likelihood, and Monte Carlo sampling techniques. When the models were compared, using the likelihood ratio test for nested models, the model assuming the presence of immunity was the best fit for E. granulosus infection, with a purgation based prevalence of 8% (true prevalence interval of 8-19% based on the sensitivity of purgation) and a mean abundance of 80 parasites per dog, with an average infection pressure of 560 parasites per year. In contrast, the model assuming the absence of immunity was the best fit for E. multilocularis infection, with a purgation based prevalence of 12% (true prevalence interval of 13-33% based on the sensitivity of purgation) and a mean abundance of 131 parasites per dog, with an average infection pressure of 334 or 533 parasites per year assuming a 5 or 3 month parasite life expectancy, respectively. The prevalence data for both parasites was then fit to a set of differential equations modeling the transition between infection states in order to determine number of infectious insults per year. Infection pressure was 0.21, with a 95% credibility interval of 0.12 to 0.41, infections per year for E. granulosus and 0.52, with a 95% credibility interval of 0.29-0.77, infections per year for E. multilocularis assuming a 5 month parasite lifespan or 0.85, with a 95% credibility interval of 0.47-1.25 infections per year, assuming a 3 month E. multilocularis lifespan in dogs.