Echinococcus Granulosus Hydatid Cyst Fluid Research Articles

Effect of Echinococcus granulosus hydatid cyst fluid protein on allergic rhinitis induced by ovalbumin in mice

To investigate the protective effect of Echinococcus granulosus hydatid cyst fluid protein (HCFP) on ovalbumin (OVA)-induced allergic rhinitis (AR) in mice. Twenty-four BALB/c mice at ages of 8 to 10 weeks, each weighing approximately 20 g, were randomly divided into four groups, including groups A (blank control group), B (blank intervention group), C (AR model group) and D (AR+HCFP intervention group), with 6 mice in each group. On days 0, 2, 4, 6, 8, 10 and 12, mice in groups A, B, C and D were injected with 200 μL sterile phosphate buffered saline (PBS), 200 μL sterile PBS containing 20 μg HCFP, 200 μL sterile PBS containing 50 μg OVA and 5 mg Al(OH)3 gel, and 200 μL sterile PBS containing 50 μg OVA, 5 mg Al(OH)3 gel and 20 μg HCFP, respectively. On days 14 to 20, mice in groups A, B, C and D were administered with 40 μL sterile PBS, 40 μL sterile PBS containing 20 μg HCFP, 40 μL sterile PBS containing 2 mg OVA and 40 μL sterile PBS containing 2 mg OVA and 20 μL HCFP by nasal drop, respectively. Mouse behavioral changes were observed and behavioral scores were estimated. The serum levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-5, IL-10, transforming growth factor-β (TGF-β) and OVA-specific IgE antibody (OVA-sIgE) were measured using enzyme-linked immunosorbent assay (ELISA), and the pathological changes of mouse nasal mucosa were observed by hematoxylin and eosin (HE) staining. The mean behavioral score was significantly greater in Group C (6.83 ± 0.50) than in groups A (1.17 ± 0.52) and B (1.33 ± 0.52) (P < 0.05), while a lower mean behavioral score was estimated in Group D (3.50 ± 0.50) than in Group C (P < 0.05). There were significant differences among the groups in terms of serum IFN-γ (F = 4.08, P < 0.05), IL-4 (F = 275.90, P < 0.05), IL-5 (F = 96.82, P < 0.05), IL-10 (F = 77.67, P < 0.05), TGF-β (F = 9.98, P < 0.05) and OVA-sIgE levels (F = 44.69, P < 0.05). The serum IFN-γ level was significantly lower in Group C than in groups A, B and C (P < 0.05), and the serum levels of IL-4, IL-5 and OVA-sIgE were significantly higher in Group C than in groups A, B and C (P < 0.05), while the serum IL-10 and TGF-β levels were significantly greater in Group D than in Group C (P < 0.05). Microscopy showed apparent loss of nasal mucosa cilia, increased number and enlargement of goblet cells, interstitial edema and submucous vascular dilation in Group C, while the pathological changes of nasal mucosa were alleviated in Group D relative to Group C. E. granulosus HCFP has a protective activity against OVA-induced allergic rhinitis in mice.

Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation, characterization and evaluation of immunomodulatory functions on T cells

Cystic echinococcosis is a chronic and complex zoonotic disease. The mechanisms underlying the parasite’s establishment, growth and persistence are not completely understood, and are thought be modulated by a crosstalk through extracellular vesicles. Here, EVs were isolated from the hydatid cyst fluid of patients with cystic echinococcosis and protoscolex culture supernatant. Proteomic analysis of these EVs revealed several parasite- and human-derived proteins. Very few studies have performed proteomic analysis of EVs isolated from HCF and PCS. Our proteomic analysis of the EVs derived from HCF and PCS facilitated identification of 1175 proteins, wherein 1026 and 38 proteins were exclusively identified in the EVs derived from HCF (HCF-EVs) and PCS (PCS-EVs), respectively, and 111 proteins were shared in both. The results of co-culture of PCS-EVs with murine peripheral blood mononuclear cells showed that PCS-EVs significantly regulated T lymphocyte functions in a dose-dependent manner. Collectively, our results provide valuable information on parasite survival strategies and new insights into the role of these EVs in the establishment and persistence of hydatid cysts.

Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis.

BackgroundClinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples.Methodology/Principal FindingsA total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number.Conclusions/SignificanceThe two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits.

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

BackgroundCurrently, 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.Methodology/Principal FindingsHere, 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.Conclusions/SignificanceThe 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.

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

BackgroundProduction of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.Methodology/Principal FindingsMaking use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.Conclusions/SignificanceThis study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

An extensive ultrasound and serologic study to investigate the prevalence of human cystic echinococcosis in northern Libya.

A prevalence study of abdominal cystic echinococcosis (CE) was undertaken in the northwest, north-central, and northeast regions of Libya. A total of 36 villages along the coast were included, in which 20,220 people were screened by portable ultrasound. Three hundred thirty-nine (1.7%) were diagnosed with CE. There was no significant difference between CE prevalence rates in the three regions of Libya (mean = 1.6%); however, intervillage rates were variable, ranging from 0% to 4.5%. The prevalence of CE increased significantly with age (P < 0.0001) and females were significantly more affected (2%) than males (1.3%) (P < 0.0001). Cases of CE were distributed among 3.2% of housewives, 2.6% of farmers, 2.3% of male civil servants, 1.3% of female students, and 1.1% of male students. Housewives (P < 0.0001) and students (P < 0.0001) were significantly more at risk for CE. A statistically significant proportion (62% [210 of 339]) of CE cases kept dogs (P < 0.0001). Of the ultrasound-positive CE cases 69% (233 of 339) were antibody seropositive by ELISA using Echinococcus granulosus hydatid cyst fluid antigen B. Blood samples (n = 10,096) collected onto filter papers were taken randomly from ultrasound-negative people who entered the study, of which 11.2% were found to be seropositive. Seropositivity increased with age (P < 0.0001), with females exhibiting higher seroprevalence than males for all age groups (P < 0.0001). Seroprevalence was distributed occupationally among 17.3% of female students, 11.2% of male students, 8.3% of housewives, 7.3% of farmers, and 6.4% of male civil servants. Housewives and students were more likely to be seropositive (P < 0.0001). Forty-seven percent (526) of the seroreactors kept dogs. All liver hydatid cysts detected by ultrasound during community screening were classified according to morphology and size into six types. Type I (17.5% of all cases) were small univesicular cysts less than 50 mm in diameter with no laminations or daughter cysts. Type II (34%) were univesicular cysts with only laminations. Type IIIa (8.5%) were univesicular cysts with the appearance of laminations and daughter cysts. Type IIIb (13%) were univesicular with laminations and less prominent daughter cysts. Type IV (5.6%) presented as a solid mass. Type V were degenerated calcified or partially calcified cysts (13.2%). Type VI presented as multiple cysts (8% of all cases). The CE cases that exhibited Types II, IIIa, IIIb, IV or VI cysts showed the highest seropositivity (86%, 96%, 95%, 100%, and 96%, respectively), while Types I and V were the least seroreactive (38% and 22%, respectively). Cases of CE occurred in 311 families, with 93% having only one member as a CE case while 7% of the families had two or more cases. However, 25% of the ultrasound-negative persons belonging to families with an index CE case were seropositive for antibodies to Echinococcus. These results confirm the importance of human CE in Libya. They also confirm the usefulness of ultrasound combined with serology as a mass screening approach for CE in north African communities.

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid.

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF-I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF-I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF-I activity respectively was destroyed by periodate oxidation. Furthermore, partial deglycosylation with Peptide N-Glycosidase F of SHCF-I which had been digested with Pronase E, released an active fraction (MW < 14 KDa) which bound to Soybean agglutinin, suggesting that N-linked oligosaccharides containing alpha- or beta-linked N-acetyl galactosamine play a role in C activation by SHCF.

Partial purification and comparison of the kinetic properties of ovine liver Echinococcus granulosus hydatid cyst fluid malate dehydrogenase and the cytoplasmic enzyme from the host liver

Ovine liver Echinococcus granulosus hydatid cyst fluid and cytoplasmic healthy ovine liver malate dehydrogenases were purified 24- and 30-fold by Sephadex G-200 and DEAE-Sephadex chromatography. Both enzymes were eluted with the same elution volume and the same salt concentration from the respective columns. The pH optimum of the enzymes from both sources was 8.4 in either Tris-HCI or barbital buffer. The K m values for oxaloacetate were 0.211 and 0.200 mM for hydatid cyst fluid and healthy ovine liver enzymes, respectively. The m values for NADH were 0.220 and 0.213 mM hydatid cyst fluid and healthy ovine liver enzymes, respectively. Enzyme from both sources demonstrated similar heat denaturation patterns. Both enzyme preparations were inhibited at high concentrations of either substrate. Neither enzyme was inhibited by para-hydroxymercuribenzoate or fumarate, and both enzyme preparations were specific for NADH as a cofactor. The results are discussed in terms of the possible infiltration of the host enzyme into the cyst fluid.

Isolation and partial characterization of species-specific and cross-reactive antigens of Echinococcus granulosus cyst fluid

Two parasite antigens have been isolated from Echinococcus granulosus hydatid cyst fluid using hydrophobic interaction chromatography, anion exchange chromatography and gel filtration chromatography. Initial characterization of the antigens indicates that both are glycoproteins, of approximately 20 and 48 kDa (Eg20 and Eg48). When the two antigens were tested with a battery of antisera from patients with heterologous parasitic infections, only Eg20 was found to be specific for E. granulosus. The Eg48 antigen cross-reacted with the sera of 33% of E. multilocularis patients. In both antigens, some of the epitopes recognized by antibodies in the sera of hydatid patients were periodate-sensitive. This suggests the involvement of carbohydrates in at least some of the antigenic determinants. Due to the abundance of the Eg48 antigen in the hydatid cyst fluid, it would be the more practically useful antigen for disease diagnosis, especially in countries where only E. granulosus is endemic.

Specific and cross-reactive antigens of Echinococcus granulosus hydatid cyst fluid

The parasite antigens of Echinococcus granulosus hydatid cyst fluid have been characterised using the techniques of radioiodination, immunoprecipitation, SDS-PAGE, and immunoblotting. Five major subunit antigens of the parasite have been identified of relative molecular mass ( M r) 12 000, 16 000, two at 20 000, and 38 000 when reduced. The 12 and 16 kDa molecules are specific to E. granulosus and are excreted/secreted by both U.K. strains of the parasite and all human isolates examined, although not all hydatid disease patients produce antibodies to them. These molecules may be suitable for detection in specific immunodiagnosis. The 38 kDa molecule associates, via a disulphide linkage, with one of the 20 kDa molecules to form a single molecule of 60 kDa. This antigen is cross-reactive with human antibody to other cestode, trematode, and nematode parasites. Part of this cross-reactivity is associated with the presence of phosphorylcholine bound to the 38 kDa subunit.