Echinococcus Granulosus Antigen Research Articles

Human and camel cystic echinococcosis – a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22 kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22 kDa. The purified IgG PsAb had been resolved at two bands at 52 kDa and at 32 kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks’ infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels’ sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Echinococcus granulosus as a Promising Therapeutic Agent against Triplenegative Breast Cancer

Abstract:Breast cancer is a major cause of cancer deaths in women, with approximately 1.2 million new cases per year. Current treatment options for breast cancer include surgery, radiation, hormone therapy, and chemotherapy. However, the non-selective cytotoxicity of chemotherapeutic agents often leads to severe side effects, while drug resistance can worsen patient outcomes. Therefore, the development of more effective and less toxic anticancer drugs is a critical need. This study aimed to review the literature on Echinococcus granulosus antigens with anticancer potential against triple-negative breast cancer. Recent studies have suggested that certain parasite antigens may have potential anticancer effects. Specifically, research has shown that echinococcosis, a disease caused by the parasitic cestode Echinococcus granulosus, may have a protective effect against cancer. These findings offer new insights into the potential use of E. granulosus antigens in the development of novel cancer therapies and tumor cell vaccines. The findings of recent studies suggested that E. granulosus antigens may have the potential to be used in effective and less toxic cancer treatments. However, further research is needed to fully understand the mechanisms behind the anticancer effects of these antigens and develop new cancer therapies and vaccines

Impact of Echinococcus granulosus Antigens on Monocyte Development and Dendritic Cell Differentiation.

Different subtypes of dendritic cells (DCs) can induce different types of immune responses. Our previous study found that Echinococcus granulosus (E. granulosus) antigens (Eg.ferritin, Eg.mMDH and Eg.10) stimulated DC differentiation to different subtypes and produced different immune responses. To further understand whether Eg.ferritin, Eg.mMDH and Eg.10 affect the DC-mediated immune response by promoting the differentiation of monocytes to DCs. Bone marrow-derived monocytes were exposed to three antigens of E. granulosus on days 0, 3, 5, and 7. The percentage of monocyte-derived DCs (moDCs), DCs subsets, and the expression of surface molecules of DCs at different time points in different groups were assessed by flow cytometry. The levels of cytokines of IL-1β, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, IL-12p70, IL-18, IL-23, and IL-27 in the cell culture supernatant were detected by multi-factorial detection technology. The percentage of moDCs revealed that none of the three antigens blocked monocyte differentiation to DCs. The monocytes of 7-day-old cultures showed increased sensitivity to these antigens. The Eg.ferritin induced more mature DCs, which expressed high levels of MHC II and costimulatory molecules, and secreted Th1 cytokines. Eg10 and Eg.mMDH induced lower degrees of DC maturation, however differentiated DCs were in a semi-mature state due to low expression of MHC II and costimulatory molecules and secretion of higher Th2 and lower Th1 cytokines. Eg.ferritin promotes full maturation of DCs and induces Th1 immune response, whereas Eg.10 and Eg.mMDH induce semi-mature DCs producing higher levels of Th2 cytokines.

A Novel Designed Sandwich ELISA for the Detection of Echinococcus granulosus Antigen in Camels for Diagnosis of Cystic Echinococcosis.

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels' organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks' infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels' echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment.

The mouse model of chronic asthma: Airway remodelling and disease exacerbation by somatic antigen of Echinococcus granulosus.

There is now sufficient evidence to support an inverse association between helminth infection and secreted products with allergic/autoimmune disorders. Accordingly, several experimental studies have shown that Echinococcus granulosus infection and hydatid cyst compounds are able to suppress immune responses in allergic airway inflammation. This is the first study on effects of somatic antigens of E. granulosus on chronic allergic airway inflammation in BALB/c mice. Mice in OVA group were intraperitoneally (IP) sensitized with OVA/Alum. Subsequently, were challenged by nebulizing of OVA 1%. The treatment groups received somatic antigens of protoscoleces on the specified days. Mice in PBS group were received PBS in both sensitization and challenge. The effects of somatic products on development of chronic allergic airway inflammation were evaluated by examining histopathological changes, the recruitment of inflammatory cells in the bronchoalveolar lavage, cytokines production in the homogenized lung tissue, and total antioxidant capacity in serum. Our findings show that the co-administration of somatic antigens of protoscoleces simultaneously with the development of asthma intensifies allergic airway inflammation. The identification of effective components involved in exacerbation of allergic airway inflammation manifestations will be a crucial approach to understanding the mechanism of these interactions.

Design of highly sensitive nano-biosensor for diagnosis of hydatid cyst based on gold nanoparticles

Cystic echinococcosis, a zoonotic parasitic infection, is a major public health and economic concern, with worldwide distribution. The development of sensitive diagnostic methods for hydatid disease is important. We designed a highly sensitive nano-biosensor for the diagnosis of hydatid cyst based on gold nanoparticles (AuNPs). AuNPs were synthesized. Echinococcus granulosus antigen was coated on the ELISA microwells. Then, the E. granulosus IgG antibody was added to the microwells. After incubation and washing, the Ag-Ab complex was incubated with a human IgG HRP​-conjugated antibody. Then, the synthesized AuNPs and tetramethylbenzidine (TMB), as a chromogenic substrate of HRP, were added to the reaction. Finally, the absorption rate was measured by spectrophotometry. The results showed that the enzyme peroxide and TMB change the color of the reaction from red to yellow by oxidizing AuNPs. The sensitivity and specificity of the designed method were investigated. The linear equation and regeneration of nanobiosensor designed for red color Y = 0.0312X + 0.649, R2 9962 and for yellow color Y = 0.013X + 0.398, R2 9851 were determined. The limit of detection of the designed nanobiosensor was 0.001 μg mL−1. The results confirmed that the designed nanobiosensor was completely specific for the detection of E. granulosus antibody.

Exacerbation of allergic asthma by somatic antigen of Echinococcus granulosus in allergic airway inflammation in BALB/c mice

BackgroundThere is ample evidence demonstrating a reverse relationship between helminth infection and immune-mediated diseases. Accordingly, several studies have shown that Echinococcus granulosus infection and hydatid cyst compounds are able to suppress immune responses in allergic airway inflammation. Previous studies have documented the ability of hydatid cysts to suppress aberrant Th2 immune response in a mouse model of allergic asthma. However, there is a paucity of research on the effects of protoscoleces on allergic asthma. Thus, this study was designed to evaluate the effects of somatic antigens of protoscoleces in a murine model of allergic airway inflammation.MethodsOvalbumin (OVA)/aluminum hydroxide (alum) was injected intraperitoneally to sensitize BALB/c mice over a period of 0 to 7 days, followed by challenge with 1% OVA. The treatment group received somatic antigens of protoscoleces emulsified with PBS on these days in each sensitization before being challenged with 1% OVA on days 14, 15, and 16. The effects of somatic antigens of protoscoleces on allergic airway inflammation were evaluated by examining histopathological changes, the recruitment of inflammatory cells in the bronchoalveolar lavage, cytokine production in the homogenized lung tissue (IL-4, IL-5, IL-10, IL-17, and IFN-γ), and total antioxidant capacity in serum.ResultsOverall, administration of somatic antigens of protoscoleces exacerbated allergic airway inflammation via increased Th2 cytokine levels in the lung homogenate, recruitment of eosinophils into bronchoalveolar lavage fluid, and pathological changes. In addition, total antioxidant capacity and IFN-γ levels declined following the administration of somatic antigens.ConclusionsThe results revealed that the co-administration of somatic products of protoscoleces with OVA/alum contributed to the exacerbation of allergic airway inflammation in BALB/c mice. Currently, the main cause of allergic-type inflammation exacerbation is unknown, and further research is needed to understand the mechanism of these interactions.Graphical

Modulation of the mTOR pathway plays a central role in dendritic cell functions after Echinococcus granulosus antigen recognition

Immune evasion is a hallmark of persistent echinococcal infection, comprising modulation of innate immune cells and antigen-specific T cell responses. However, recognition of Echinococcus granulosus by dendritic cells (DCs) is a key determinant of the host's response to this parasite. Given that mTOR signaling pathway has been described as a regulator linking metabolism and immune function in DCs, we reported for the first time in these cells, global translation levels, antigen uptake, phenotype, cytokine transcriptional levels, and splenocyte priming activity upon recognition of the hydatid fluid (HF) and the highly glycosylated laminar layer (LL). We found that LL induced a slight up-regulation of CD86 and MHC II in DCs and also stimulated the production of IL-6 and TNF-α. By contrast, HF did not increase the expression of any co-stimulatory molecules, but also down-modulated CD40 and stimulated the expression of the anti-inflammatory cytokine IL-10. Both parasitic antigens promoted protein synthesis through mTOR activation. The use of rapamycin decreased the expression of the cytokines tested, empowered the down-modulation of CD40 and also reduced splenocyte proliferation. Finally, we showed that E. granulosus antigens increase the amounts of LC3-positive structures in DCs which play critical roles in the presentation of these antigens to T cells.

LncRNA028466 regulates Th1/Th2 cytokine expression and associates with Echinococcus granulosus antigen P29 immunity

BackgroundCystic echinococcosis (CE) is a parasitic disease that is caused by Echinococcus granulosus (Eg). The recombinant Echinococcus granulosus antigen P29 (rEg.P29) was shown to confer effective immunity to sheep and mice during E. granulosus secondary infection in our previous study. In this study, we sought to investigate the ability of long noncoding RNA 028466 (lncRNA028466) as a regulator for the protective immunity mediated by rEg.P29 vaccination and to study the effects of lncRNA028466 on CD4+T cell differentiation in mice spleen.MethodsFemale BALB/c mice were divided into two groups and were vaccinated subcutaneously with rEg.P29 antigen and PBS as a control (12 mice each group). Following prime-boost vaccination, CD4+T, CD8+T, and B cells from the spleen were isolated by flow cytometry. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression of lncRNA028466 in these three kinds of cells. Then, lncRNA028466 was overexpressed and knocked down in naive CD4+T cells, and Th1 and Th2 cytokine expression was detected. qRT-PCR, western blot, and ELISA were performed to evaluate the production of IFN-γ, IL-2, IL-4, and IL-10, and flow cytometry was performed to detect the differentiation of Th1 and Th2 subgroups.ResultslncRNA028466 was significantly decreased after the second week of immunization with rEg.P29 antigen. The proportion of CD4+ T cells was increased after rEg.P29 immunization. Overexpression of lncRNA028466 facilitated the production of IL-4, IL-10 and suppressed the production of IFN-γ, IL-2. Furthermore, after transfection with siRNA028466, IL-2 production was facilitated and IL-10 production was suppressed in naive CD4+ T cells.ConclusionsImmunization with rEg.P29 downregulated the expression of lncRNA028466, which was related to a higher Th1 immune response and a lower Th2 immune response. Our results suggest that lncRNA028466 may be involved in rEg.P29-mediated immune response by regulating cytokine expression of Th1 and Th2.

Applicability of Brugia malayi immune antibody library for the isolation of a human recombinant monoclonal antibody to Echinococcus granulosus antigen B

Echinococcus granulosus is a worldwide zoonotic infection that causes human cystic echinococcosis (CE) or hydatid disease. The present study describes the isolation and production of a monoclonal antibody against recombinant AgB protein using the developed Human AntibodY Disease ENhanced (HAYDEN)-Filariasis library. The DNA sequences of the isolated clones were analyzed, followed by gene analysis and binding assays. Clone E1 showed a full-length sequence and represents the IgHV5-LV3 antibody gene family. The antibody protein yield was satisfactory, and it reacted specifically against rAgB. The novel E1 protein is potentially useful for the development of an antigen detection assay for CE. The ability of the Brugia malayi immune antibody library to isolate antibodies against Echinococcus granulosus antigens highlights the broad coverage of immune antibody libraries.

In vitro evaluations on canine monocyte-derived dendritic cells of a nanoparticles delivery system for vaccine antigen against Echinococcus granulosus.

Since dogs play a central role in the contamination of humans and livestock with Echinococcus granulosus, the development of an effective vaccine for dogs is essential to control the disease caused by this parasite. For this purpose, a formulation based on biodegradable polymeric nanoparticles (NPs) as delivery system of recombinant Echinococcus granulosus antigen (tropomyosin EgTrp) adjuved with monophosphoryl lipid A (MPLA) has been developed. The obtained nanoparticles had a size of approximately 200 nm in diameter into which the antigen was correctly preserved and encapsulated. The efficiency of this system to deliver the antigen was evaluated in vitro on canine monocyte-derived dendritic cells (cMoDCs) generated from peripheral blood monocytes. After 48 h of contact between the formulations and cMoDCs, we observed no toxic effect on the cells but a strong internalization of the NPs, probably through different pathways depending on the presence or not of MPLA. An evaluation of cMoDCs activation by flow cytometry showed a stronger expression of CD80, CD86, CD40 and MHCII by cells treated with any of the tested formulations or with LPS (positive control) in comparison to cells treated with PBS (negative control). A higher activation was observed for cells challenged with EgTrp-NPs-MPLA compared to EgTrp alone. Formulations with MPLA, even at low ratio of MPLA, give better results than formulations without MPLA, proving the importance of the adjuvant in the nanoparticles structure. Moreover, autologous T CD4+ cell proliferation observed in presence of cMoDCs challenged with EgTrp-NPs-MPLA was higher than those observed after challenged with EgTrp alone (p<0.05). These first results suggest that our formulation could be used as an antigen delivery system to targeting canine dendritic cells in the course of Echinococcus granulosus vaccine development.

Camel hydatidosis diagnostic kit: optimization of turnip and horseradish peroxidase conjugates using glutaraldehyde method.

Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130kDa and one minor band at 55kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.

An improved system for constructing marker-free recombinant goatpox viruses to express foreign proteins

Goatpox virus (GTPV), belonging to the genus Capripoxvirus in the family Poxviridae, causes a contagious disease affecting goats and sheep. Homologous recombination as a conventional method is commonly used to construct recombinant GTPVs but generally with genetic markers, such as enhanced green fluorescent protein (eGFP) and guanine phosphoribosyl transferase (gpt). We have previously constructed a recombinant GTPV, which can efficiently express the EG95 antigen of Echinococcus granulosus, but contains eGFP and gpt markers in viral genome. In this study, our previous GTPV-generating system was modified by reconstruction of a Loxp-containing transfer plasmid for deleting markers using the Cre/Loxp system. Meanwhile, the previous method was significantly improved by introduction of an immortalized goat testis cell line as a substitute for primary cells. Based on the latest system, a marker-free recombinant GTPV was reconstructed for expressing the EG95 antigen, and showed neither a significant difference in replication kinetics from its parental virus nor mutations in the foreign sequence during serial 10 passages in vitro.

Survey of echinococcoses in southeastern Qinghai Province, China, and serodiagnostic insights of recombinant Echinococcus granulosus antigen B isoforms

BackgroundEchinococcoses, caused by metacestodes of Echinococcus granulosus (cystic echinococcosis; CE) and E. multilocularis (alveolar echinococcosis; AE), represent major emerging parasitic diseases. These enzootic helminthiases invoke significant public health concerns and social burdens in endemic areas. The diseases are prevalent in the Qinghai-Tibetan Plateau, China, while community-based epidemiological studies have been scarcely reported. We surveyed echinococcosis patients in the southeastern Qinghai Province, China, to better understand the concurrent epidemiological situation in this area.MethodsDuring July and August of 2013 and 2014, we screened echinococcosis patients at Yushu and Golog Prefectures, Qinghai Province, China, in a diagnostic campaign. A total of 2856 people (male:female ratio, 1:1.12; mean age, 34.6 years; age range, 6–88 years) were ultrasonographically examined for the presence of hepatic echinococcal cysts. We also collected serum samples from patients and analyzed antibody reactivity against recombinant forms of diverse E. granulosus antigen Bs (rEgAgB1-5) by enzyme-linked immunosorbent assay.ResultsWe detected 134 patients whose imaging scans were compatible with CE (115 cases) and AE (20 patients). One patient might have been infected with both CE and AE. The overall incidence was 4.7% (CE, 4.0%; AE, 0.7%). A large proportion (67.5%) of CE patients was diagnosed at active and transitional CE1-CE3 stages in their late 30s. The AE cases were generally detected at advanced stage in patients at early 20s (60%). Analysis of the receiver operating characteristic curve and Youden’s index indicated that rEgAgB2 was the most promising biomarker, followed by rEgAgB3 and rEgAgB1. Overall, sensitivity and specificity of rEgAgB1-3 were 84.5–92.7% and 91.9–94.6%, respectively. rEgAgB4 and 5 showed low sensitivity with high cross-reactivity.ConclusionsOur results strongly suggest that disability-adjusted life years related to echinococcoses in Qinghai-Tibetan areas might be more serious than previously considered. Control and prevention strategy against CE and AE are highly required in these areas. In addition to ultrasonography, serological tests might provide supportive data. However, serological data should be carefully interpreted for differential diagnosis, especially in areas where both CE and AE are co-endemic.

PUC18-CpG stimulates RAW 264.7 via TBK1-mediated pathway and presents adjuvanticity in mice.

Phosphorothioate CpG oligodeoxynucleotides (ODN) are reported to be recognized by the membrane-bound TLR9 and trigger the MyD88-dependent up-regulation of Type I interferons and pro-inflammatory cytokines. Whether plasmids containing multiple CpG motifs stimulate the same signaling pathway is yet to be determined. The present results show that the CpG motifs enrich plasmid pUC18-CpG stimulates RAW 264.7 in vitro, mainly through the TBK1-mediated signaling pathway, causing the up-regulation of IFN-β, and pro-inflammatory cytokines TNF-α and IL-6. When pUC18-CpG is co-administered with the recombinant Echinococcus granulosus antigen, the antigen-specific antibody titers are markedly increased compared to the Quil-A adju- vanted group. Antigen specific cytokine quantification shows that cytokine profiles from the pUC18-CpG adjuvanted-group are switched to a Th1-biased immune response.

Expansion of a highly sensitive and specific ELISA test for diagnosis of hydatidosis using recombinant EgB8/2 protein.

Objective(s):Hydatidosis is a zoonotic infection and endemic in Iran. Due to the serological cross-reactivity (of sera) with other parasitic infection, diagnosis of hydatid cyst is considered to be problematic. In this regard, application of recombinant antigens improves serological diagnosis for human hydatidosis. Here, we present an ELISA test based on B8/2 recombinant antigen of Echinococcus granulosus with particular regard to its capability to diagnose human hydatidosis.Materials and Methods:The synthesized E. granulosus B8/2 (EgB8/2) gene was sub-cloned into pET28b (+) plasmid. Nde1 and Hind3 restriction enzymes were used to confirm the recombinant plasmid extraction. Cloning was verified by colony PCR, digestion enzymes, and sequence determination methods. To express rtEgB8/2, strains of Escherichia coli BL21 (DE3) pLysS and Rosetta (DE3) were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). A Ni-NTA column was used for purification, and the expressed protein was analyzed by SDS-PAGE as well as western blotting. ELISA test was used to identify the antigenicity of produced protein.Results:The presence of EgB8/2 gene fragment in the recombinant plasmid was confirmed. SDS-PAGE showed that the BL21 (DE3) pLysS strain had the highest level of expression and a protein band of 11 kDa was observed in induced bacteria. Western blotting approved the purity of rtEgB8/2 protein, and ELISA test measured sensitivity and specificity as 95% and 97.5%, respectively.Conclusion: E. granulosus metacestode contains a high amount of antigen B protein. These results confirm the reproducibility of high-quality rtEgB8/2 recombinant antigen as a reliable candidate in serological test.

Mechanisms underlying immune tolerance caused by recombinant Echinococcus granulosus antigens Eg mMDH and Eg10 in dendritic cells.

Mice immunized with recombinant Echinococcus granulosus antigens Eg10 and Eg mMDH do not show elevated resistance to E. granulosus infection but show aggravated infection instead. To gain a deeper insight in the immune tolerance mechanisms in mice immunized with Eg10 and Eg mMDH, this study simulated the immune tolerance process in vitro by culturing bone marrow-derived dendritic cells (BMDCs) in the presence of Eg10 or Eg mMDH. Scanning electron microscopy revealed that Eg10- and Eg mMDH-treated DCs exhibited immature cell morphology, while addition of LPS to the cells induced changes in cell morphology and an increase in the number of cell-surface protrusions. This observation was consistent with the increased expression of the cell-surface molecules MHCII and CD80 in Eg10- and Eg mMDH-treated DCs pretreated with LPS. DCs exposed to the two antigens had a very weak ability to induce T-cell proliferation, but could promote the formation of Treg cells. Introduction of the indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyl tryptopha (1-MT) enhanced the ability of the antigens to induce T cells and inhibited the induction of Treg cells. Eg mMDH-treated DCs showed a strong response to 1-MT: the DCs had high mRNA levels of IDO, IL-6, and IL-10, while 1-MT decreased the expression. In contrast, DCs treated with Eg10 did not show significant changes after 1-MT treatment. Eg mMDH inhibited DC maturation and promoted IDO expression, which, on the one hand, decreased the ability of DCs to induce T-cell proliferation, resulting in T-cell anergy, and on the other hand, induced the formation of Tregs, resulting in an immunosuppressive effect. In contrast, the escape mechanisms induced by Eg10 did not primarily depend on the IDO pathway and might involve other mechanisms that need to be further explored.

The diagnostic value of the recombinant antigen B1 for the serological diagnosis of hepatic cystic hydatid

Objective: Get echinococcus granulosus antigen B1 recombinant protein (rEgAgB1) by genetic engineering, discuss its' serology diagnosis value for HCE, and analysis the relationship between its' expression and clinical test. Methods: Construct rEgAgB1, then ELISA and Immunoblotting method of serum samples were taken by recombinant protein and HCF for 25 patients with HCE and 25 normal people respectively, retrospectively analyzed the clinical data of 25 patients with hepatic hydatid cyst, study the relationship between age, sex, symptom,subtype,other organs affected or not, quantity of the cyst, size of the cyst and the expression level of rEgAgB1 in serum. Results: Use rEgAgB1 as antigen, detect the IgG1 specific antibody in the serum of 25 patients with HCE, 19 of which were positive, serum positive rate is 76%. Use rEgAgB1 as antigen, detect the IgG4 specific antibody in the serum of 25 patients with HCE, 9 of which were positive, serum positive rate is 36%. Analysis the clinical data of 25 patients with HCE, the result showed there is no correlation between age, ethnic, symptom, size of cyst and expression level of rEgAgB1 in serum. Conclusion: Serological diagnosis by rEgAgB1 has high sensitiviy and specificity, The recombinant AgB indicates the advantage of no cross-reaction with other parasite diseases or nonparasite hepatic diseases. Recombinant antigen B can improve the specificity but decrease the sensitivity.

Impairment of Macrophage Presenting Ability and Viability by Echinococcus granulosus Antigens.

Despite advances toward an improved understanding of the evasive mechanisms leading to the establishment of cystic echinococcosis, the discovery of specific immunosuppressive mechanisms and related factors are of great interest in the development of an immunotherapeutic approach. To elucidate immunosuppressive effects of bioactive factors contained in chromatographic fractions from hydatid cystic fluid (HCF) of Echinococcus granulosus. Hydatid cystic fluid was fractionated by reverse phase chromatography. Non-specific Concanavalin A-driven proliferation of spleen cells was used to determine specific inhibitory fractions. Trypan blue exclusion test and flowcytometry analysis were performed to check whether highly inhibitory fractions of HCF have apoptotic effect on peritoneal macrophages. Western blot analysis was used to determine proteolytic effects of parasitic antigens on major histocompatibility complex (MHC) class II (I-a) contained in membrane proteins extract from macrophages. High concentrations of HCF and few of chromatographic fractions suppressed spleen cells proliferation. Fractions 7 and 35 were the highest inhibitory fractions. Specifically fraction 35 and to a lesser extent HCF induced apoptosis in peritoneal naive macrophages. However, HCF and the fraction 7 proteolytically altered the expression of MHC class II molecules on peritoneal macrophages. The proteolytic molecule was identified to be a serine protease. Macrophages taken at the chronic and end phase from cystic echinococcosis-infected mice were able to uptake and process C-Ovalbumine-FITC. These cells expressed a drastically reduced level of (I-a) molecules. Our study present new aspects of immune suppression function of E. granulosus. Further molecular characterization of apoptotic and proteolytic factors might be useful to develop immunotherapeutic procedure to break down their inhibitory effects.

Sero-epidemiology of cystic echinococcosis (Echinococcus granulosus) in the livestock of Oman.

A cross-sectional serological survey of cystic echinocossis was carried out on 2802 randomly collected sera of camels (n=706), cattle (n=687), goats (n=701) and sheep (n=708) from all governorates of Oman. The samples were analysed by in house indirect ELISA (iELISA) using Echinococcus granulosus antigen B (EgAgB) of naturally infected camels. The overall percentage of antibodies against EgAgB was found to be 14.6%. The highest percentage of positive was observed in sera from camels (22.4%) followed by cattle (12.9%), sheep (12.2%) and goats (10.9%). The highest percentage of seropositivity was observed in females (15.4%) as compared to male animals (10.6%). The imported livestock were found more seropositive (15.2%) as compared to local (14.7%) and crossbred livestock (14.1%). The highest exposure was observed in animals with the age group of above 5years (18.3%) followed by the age group of up to 2years (15.1%,) and those between 2 and 5years (12.4%). The univariate analysis has indicated that camels (OR:2.33, CI 1.74, 3.14), cattle (OR:1.21, CI 0.87, 1.67), sheep (OR:1.12, CI 0.81, 1.55) were more likely to test positive than goats. Furthermore, females (OR: 1.53, CI 1.11, 2.11) were more likely to test seropositive. Sera from animals above 5years of age (OR:1.58, CI 1.25, 2.01) and between 2 and 5years old (OR:1.30, CI 0.98, 1.71) were found more likely to test seropositive than those up to 2years of age. The multivariable analysis at individual level indicated that camels (OR: 2.07, CI 1.66, 2.56, p<0.001) and female (OR: 1.43, CI 1.04, 1.98, p=0.030) were more likely to acquire CE. At herd level, the final multivariable model indicated that herds located in Dofar and Musandam (OR: 4.48, CI 2.69, 7.45, p<0.001), in areas receiving seasonal rains (OR: 2.54, CI 1.09, 5.90, p<0.001) and practicing transhumance (OR: 4.22, CI 1.84, 9.65, p<0.001) and sedentary (OR: 2.07, CI 1.38, 3.12, p=0.001) farming system were more likely to acquire CE in Oman. The study documents the serological evidence of CE in livestock of Oman and a carefully planned control program should be devised after further epidemiological and molecular investigations in the intermediate and final hosts.