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An easy and efficient method for native and immunoreactive Echinococcus granulosus antigen 5 enrichment from hydatid cyst fluid.

ABSTRACT: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis.Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results.The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.

SUBMITTER: Pagnozzi D

PROVIDER: S-EPMC4132071 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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An easy and efficient method for native and immunoreactive Echinococcus granulosus antigen 5 enrichment from hydatid cyst fluid.

Pagnozzi Daniela D Biosa Grazia G Addis Maria Filippa MF Mastrandrea Scilla S Masala Giovanna G Uzzau Sergio S

PloS one 20140813 8

<h4>Background</h4>Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis.<h4>Methodology/principal findings</h4>Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure i ...[more]

PMID: 25119821

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Project description:The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.

Project description:Cystic echinococcosis is a worldwide chronic zoonotic disease that threatens human health and animal husbandry. Exosome-like vesicles (ELVs) have emerged recently as mediators in the parasite-parasite intercommunication and parasite-host interactions. Exosome-like vesicles from parasites can transfer non-coding RNAs (ncRNAs) into host cells to regulate their gene expression; however, the ncRNAs profiles of the ELVs from Echinococcus granulosus remain unknown. Here, we isolated protoscolece (PSC)-ELVs and hydatid fluid (HF)-ELVs from the culture medium for E. granulosus PSCs in vitro and the HF of fertile sheep cysts, respectively. The microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) profiles of the two types of ELVs were analyzed using high-throughput sequencing, and their functions were predicted using Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis. In PSC-ELVs and HF-ELVs, 118 and 58 miRNAs were identified, respectively, among which 53 miRNAs were present in both ELVs, whereas 65 and 5 miRNAs were unique to PSC-ELVs and HF-ELVs, respectively; 2,361 and 1,254 lncRNAs were identified in PSC-ELVs and HF-ELVs, respectively, among which 1,004 lncRNAs were present in both ELVs, whereas 1,357 and 250 lncRNAs were unique to PSC-ELVs and HF-ELVs, respectively. Intriguingly, the spilled PSCs from cysts excrete ELVs with higher numbers of and higher expression levels of miRNAs and circRNAs than HF-ELVs. The miRNA sequencing data were validated by quantitative reverse transcription-polymerase chain reaction. Furthermore, the target lncRNAs and mRNAs regulated by the 20 most abundant miRNAs were screened, and a ceRNA regulatory network containing 5 miRNAs, 41 lncRNAs, and 23 mRNAs was constructed, which provided new ideas and the molecular basis for further clarification of the function and mechanism of E. granulosus ELVs ncRNAs in the parasite-host interactions. Egr-miR-125-5p and egr-miR-10a-5p, sharing identical seed sites with host miRNAs, were predicted to mediate inflammatory response, collagen catabolic process, and mitogen-activated protein kinase cascade during parasite infections. In conclusion, for the first time, we identified the ncRNAs profiles in PSC-ELVs and HF-ELVs that might be involved in host immunity and pathogenesis, and enriched the ncRNAs data of E. granulosus. These results provided valuable resources for further analysis of the regulatory potential of ncRNAs, especially miRNAs, in both types of ELVs at the parasite-host interface.

Project description:Cystic echinococcosis (CE), caused by Echinococcus granulosus metacestode, invokes a serious public health concern. Early diagnosis has great impacts on reduction of disability-adjusted life years. Several antigen B-related molecules (EgAgB; EgAgB1-5) are known to be immunopotent, but detection of EgAgB is variable in many patients and may not allow reliable interpretation of its immunological relevance. More importantly, the immunoproteome profile of hydatid fluid (HF) has not been addressed.We conducted a proteome analysis of the HF of a single fertile cyst of CE1 and CE2 stages through two-dimensional electrophoresis (2-DE). Each protein spot was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We subsequently determined the immunoproteome profile employing patient sera of entire disease spectrum from CE1 to CE5 stages.We identified 40 parasite proteins, of which EgAgB (28 spots) and antigen 5 (EgAg5; 5 molecules) were abundant. EgAgB proteoforms constituted the majority, mostly EgAgB1 (24 spots), followed by EgAgB2 and EgAgB4 (2 spots each). EgAgB3 was detected only by liquid chromatography-MS/MS. EgAgB5 was not recognized. We also detected 38 host proteins, which were largely composed of serum components, antioxidant/xenobiotic enzymes, and enzymes involved in carbohydrate metabolism. CE1 and CE2 HF exhibited comparable spotting patterns, but CE2 HF harbored greater amounts of EgAgB and EgAg5 complexes. CE sera demonstrated complicated immune recognition patterns according to the disease progression; CE2 and CE3 stages exhibited strong antibody responses against diverse EgAgB and EgAg5 proteoforms, while CE1, CE4, and CE5 stages mainly reacted to EgAg5 and cathepsin B. Patient sera of alveolar echinococcosis (AE) cross-reacted with diverse EgAgB isoforms (36%). EgAg5 and cathepsin B also demonstrated cross-reactions with sera from neurocysticercosis and sparganosis.Our results demonstrated that detection of a single defined molecule may not properly diagnose CE, since specific immunodominant epitopes changed as the disease progresses. Immunoproteome analysis combined with imaging studies may be practical in the differential diagnosis of CE from AE and other cystic lesions, as well as for staging CE, which are pertinent to establish appropriate patient management.

Dataset Information

An easy and efficient method for native and immunoreactive Echinococcus granulosus antigen 5 enrichment from hydatid cyst fluid.

Publications

An easy and efficient method for native and immunoreactive Echinococcus granulosus antigen 5 enrichment from hydatid cyst fluid.

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