Identification of IgE and IgG1 specific antigens in Echinococcus granulosus cyst fluid

Cystic echinococcosis (CE) is an anthropozoonotic disease with worldwide distribution and is caused by the cestode Echinococcus granulosus. Anaphylactic shock induced by CE rupture is a serious complication especially in patients with hydatid infections, as the resulting leakage of fluid contains highly toxic endogenous antigen. We aimed to isolate and identify the antigens of specific IgE and IgG1 (sIgE and sIgG1) in E. granulosus cyst fluid (EgCF). Crude antigen for EgCF was prepared from E. granulosus-infected sheep liver. Antigens were separated and identified by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and immunoblotting. Results of 1D SDS-PAGE and immunoblotting showed that 40.5 kDa protein was the major antigen of sIgE, and 35.5 kDa protein was the major antigen of sIgG1 in EgCF. Results of 2-DE and immunoblotting showed that main antigens of sIgE in EgCF were four proteins with pI values ranging from 6.5 to 9.0 and a molecular weight of 40.5 kDa. Main antigens of sIgG1 in EgCF were five proteins with pI values ranging from 6.5 to 9.0 and a molecular weight of 35.5 kDa. The antigens identified for sIgE and sIgG1 can provide critical insights into cellular and molecular mechanisms underlying anaphylactic shock induced by CE.

Humoral response and evolution of Echinococcus infection in experimentally infected sheep / Resposta humoral e evolução à infecção por Echinococcus em ovinos experimentalmente infectados

There is little information on the humoral response of sheep experimentally infected with Echinococcus granulosus. Thus, the objective of this study was to evaluate this response and measure its evolution. Doses of 10, 100, 1000 and 10000 E. granulosus eggs were prepared and inoculated via intraruminal puncture. Blood samples were obtained before inoculation and every 48 h after inoculation, until they became seropositive. Thereafter, they were taken monthly for the first year and then every three months until 1700 days of observation had been completed. An ELISA test, with total hydatid fluid antigen, was used for immunodiagnosis. The average optical density of the 12 inoculated sheep was found to be above the mean cutoff value 10 days after inoculation, went on increasing until 180 days after inoculation and remained above the cutoff level until the end of the observation period. This confirms that the antibody response of sheep to E. granulosus infection occurs before production of hydatid fluid and that activation, mobilization and establishment of oncospheres in the tissues generates a persistent response from the host's immune system.

Existe pouca informação sobre a resposta imune humoral de ovinos experimentalmente infectados por Echinococcus granulosus. O objetivo deste estudo é avaliar a resposta imune por anticorpos em ovinos infectados. Os ovinos receberam doses de 10, 100, 1.000 e 10.000 ovos de E. granulosus por via intrarruminal. Amostras de sangue foram colhidas antes e após infecção, a cada 48 horas, até a detecção de anticorpos anti-E. granulosus e após, colheram-se amostras mensal e trimestralmente, no primeiro ano até 1.700 dias de infecção. No imunodiagnóstico, utilizou-se o ensaio imunoenzimático indireto (ELISA-teste) e como antígeno total, líquido hidático. Na detecção de anticorpos anti-E. granulosus no soro das 12 ovelhas, a densidade ótica esteve acima do ponto de corte, após 10 dias de infecção, aumentando até 180 dias pós-infecção, e permanecendo acima desses dias até o final do experimento. Isso confirma que a resposta por anticorpos em ovinos infectados por E. granulosus antecede a produção de líquido hidático, e que a ativação, mobilidade e permanência das oncosferas nos tecidos possibilita a resposta imune dos hospedeiros.

Detección de coproantígenos para el diagnóstico de echinococosis canina en la zona fronteriza de La Quiaca-Villazón / Coproantigens detection for the diagnosis of canine Echinococcosis in the border area of La Quiaca-Villazón

Las técnicas de inmunodiagnóstico basadas en la identificación de antígenos parasitarios en las heces secas de los perros han sido desarrolladas para la vigilancia de la echinococosis canina. En la región fronteriza de La Quiaca-Villazón, se encontraron las condiciones ambientales que favorecerían el ciclo del parásito, dada la presencia del hospedador definitivo (perro) y de hospedadores intermediarios (ovejas y cabras). La actividad más importante de la Puna es la cría de ovinos y camélidos; la faena se realiza en el campo y a manos del dueño de los ovinos, y no se aplican medidas preventivas de sanidad. El objetivo de este trabajo fue estimar la presencia de caninos parasitados por Echinococcus granulosus en esta región. Durante el año 2006 se recolectaron 168 muestras de materia fecal. En La Quiaca se tomaron muestras de las siguientes localidades: Barrios (área semirrural), Santa Catalina, Yavi Chico, El Portillo, Pumahuasi y Cara Cara (zonas rurales) y La Quiaca (área urbana). En Villazón se seleccionó el área urbana y el área semirrural de Ojo de Agua, Lampaya y Matancillas. Las muestras se analizaron por la prueba de copro-ELISA y copro-Western blot. Las localidades de San Francisco y Barrios tuvieron una prevalencia de 14,3 % y 6,7 %, respectivamente. En Villazón se encontró un 3,4 % de prevalencia en el área urbana. En Lampaya se encontró un 30 % de prevalencia. Estos resultados sugieren la necesidad de implementar estrategias para el control de la hidatidosis, tanto a nivel urbano como rural, para evitar el aumento y la dispersión de la echinococosis en la región.

Cystic Echinococcosis is a major public health issue. Immunodiagnostic techniques based on the identification of parasite antigens in dog dry faeces have been developed as alternatives for the surveillance of canine Echinococcosis. The environmental conditions favouring the parasite cycle were met in the border of La Quiaca-Villazón, given the presence of definitive (dog) and intermediate hosts (sheep and goats). The most important activity in La Puna is sheep and goat rearing; slaughtering is carried out almost exclusively in the field by sheep's owners, and preventive health measures do not apply. The aim of this study was to determine the presence of dogs parasitized by Echinococcus granulosus in this border region. A hundred and sixty eight (168) stool specimens were collected during 2006. La Quiaca samples were taken from the following selected areas: the semi-rural area of Barrios, the rural areas of Santa Catalina, Yavi Chico, El Portillo, Pumahuasi and Cara Cara and the urban area of La Quiaca; selected urban areas in Villazón and the semi-rural area of Ojo de Agua, Lampaya and Matancillas. The samples were analyzed by copro-ELISA -Western blot test. The cities of San Francisco and Barrios had a prevalence of 14.3% and 6.7%, respectively. A prevalence of 3.4% was observed in the urban area of Villazón, which indicates that dogs become infected in the rural areas and bring the risk into the city. Lampaya showed a prevalence of 30%. These findings suggest the need to implement strategies for the control of hydatidosis, both in urban and rural areas to avoid the increase and spread of Echinococcosis in the region.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Serological evaluation of EgAgB16 kDa, a recombinant antigen from echinococcus granulosus for diagnosis of human hydatidosis

Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits [EgAgB16 kDa] from Echinococcus granulosus [Iranian G1 strain] and its evaluation by ELISA test. DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus [Iranian G1 strain] protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individuals [n=72], healthy individual [n=48], toxoplasmosis [n=4], strongyloidosis [n=4], kala-azar [n=5] and tuberculosis [n=5] were examined using this recombinant antigen. Recombinant protein was purified by affinity chromatography using His-Tag column. After purification, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. While the produced recombinant AgB16 kDa showed promising results in diagnosing human hydatidosis, but more investigations should be implemented to reach an accurate gold standard

Serum antibody & Th2 cytokine profiles in patients with cystic echinococcosis.

Background & objectives: Human cystic echinococcosis (CE), caused by Echinococcus granulosus, is one of the most important and widespread parasitic zoonoses. T helper cell-2 (Th2) dominated immunity in CE is associated with increased susceptibility to the disease, while T helper cell-1 (Th1) cell activation is assumed to induce protective immunity. Hence, in order to investigate in vivo Th2 cell activation and serum complement levels, the present study was aimed to detect serum levels of specific IgG, IgE, interleukin (IL)-4, IL-10, C3c and C4 in confirmed CE patients. Methods: Specific IgG levels in serum was measured by enzyme linked immunosorbent assay using recombinant E. granulosus antigen-B/2 (RecEg-AgB/2) and serum IgE, IL-4, IL-10, C3c and C4 were quantified by nephelometry in 45 surgically confirmed patients with CE, and 10 healthy controls. Results: Specific IgG (P<0.0001), IgE (P<0.05), IL-4 (P=0.0197) and IL-10 (P<0.01) levels were significantly elevated in CE cases compared to healthy controls. IL-4 could be detected in 34 patients (75.55%) and six controls (60%) in a low concentration. The IgE concentration was elevated (>120 U/ml) in 36 (80%) cases of CE and in one healthy control. Interpretation & conclusion: Our results showed higher C3c and C4 levels in CE patients than healthy controls. No significant association was found between IgE concentrations and cytokine levels. The results of this study point to a cytokine profile suggestive of Th2 cell dominance in vivo in CE.

Fisiopatología y respuesta inmune de ovinos experimentalmente infectados con Echinococcus granulosus / Pathophysiology and immune response in sheep experimentally infected with Echinococcus granulosus

La respuesta inmune a la infección por Echinococcus granulosus en el ovino ha sido poco estudiada. El objetivo del presente trabajo fue aportar información sobre la fisiopatología y la respuesta inmune a la infección experimental con E. granulosus en ovinos. Se inocularon experimentalmente ovinos con tres dosis distintas de huevos de E. granulosus, evaluándose la repuesta inmune por seguimiento mediante enzimo inmuno ensayo con tres preparaciones antigénicas (líquido hidatídico total, fracción purificada de líquido hidatídico total y fracción lipoproteica purificada) durante 500 días. Se sacrificaron animales en forma escalonada para observar macroscópica y microscópicamente el desarrollo del parásito. La respuesta inmune se detectó a partir de los 10 días y se mantuvo durante el período de observación, resultando inicialmente proporcional a la carga de huevos inoculados, y disminuyendo las diferencias con el tiempo. Se identificaron quistes fértiles a los 10 meses post inoculación y oncósferas vivas 500 días post inoculación. La respuesta de anticuerpos en el ovino a la infección por E. granulosus fue anterior a la formación de líquido hidatídico y resultó generada por la movilidad de la oncósfera. La temprana fertilidad identificada histológicamente indica que la alimentación de canes con vísceras de ovinos jóvenes puede producir ciclos de infección. La presencia de oncósferas vivas en el hígado, por su parte, aporta información sobre la patogenia de la enfermedad y permite expresar hipótesis sobre las causas de nuevas operaciones en el hombre luego de la extirpación de un quiste hidatídico lo que podría liberar el freno inmunitario sobre dichas oncósferas.

The immune response to Echinococcus granulosus in sheep has not been extensively investigated. The objective of this study was to increase the information on the physiopathology of E. granulosus and the immune response elicited in sheep. Animals were experimentally inoculated with three different doses of E. granulosus eggs and the immune response was evaluated over 500 days using enzyme immunoassay with three antigenic preparations: total hydatid fluid, purified fraction of hydatid fluid and purified lipoprotein fraction. Sheep were slaughtered at different intervals to observe the macroscopic and microscopic development of the parasite. Immune response was detected at 10 days and was maintained throughout the observation period, being initially proportional to the load of inoculated eggs and then decreasing over time. Fertile cysts were identified 10 months after inoculation and live onchosphere 500 days after inoculation. Antibody response to E. granulosus in sheep preceded hydatid fluid formation and was generated by the mobility of the onchosphere. Early histological identification of fertile cysts indicates that feeding dogs with viscera of young sheep can produce cycles of infection. Furthermore, the presence of live onchosphere in the liver here found contributes to a better knowledge of the pathogenesis of this disease it could be hypothetically considered as a cause for the repeated surgeries necessary in man after the extirpation of a hydatid cyst.

Immunosuppressive effect of purified pyocyanine pigment on T-lymphocytes viability against experimental infection with hydatid cyst protoscolices

The effect of pyocyanine pigment, which was isolated and purified from Pseudomonas aeruginosa, on specific lymphocytes viability inside the body of white male Balb/c mice against the experimental secondary hydatidosis and the infectivity of protoscolices was studied in comparison with negative control mice groups, phosphate buffered saline [PBS] and positive control group [immunoferon]. Four groups of male Balb/c mice were intraperitoneally [IP] inoculated with four purified concentrations of pyocyanine [25, 50, 75, 100 micro m/ml]. Seven days later, they were given the same concentrations as a booster dose of the pigment, then 7 days later they were intraperitoneally infected with 2000 protoscoleces/ mL [PBS] as a challenge dose. The fifth group was intraperitoneally inoculated with 1ml of sterile PBS and used as a negative control group, while the sixth group was intraperitoneally inoculated with 100 micro mg/ml immunoferon and received the challenge dose of 2000 protoscoleces/ ml PBS and served as the positive control group. The concentrations of 50, 75 and 100 micro m/ml of this pigment had suppressive effect on these specific immune response cells. This effect was statistically significant [p<0.01] after six weeks from the challenge dose with intraperitoneal protoscolices infection. This effect revealed that the protoscolices infectivity increased due to suppression viability of T lymphocytes, while the immunoferon showed a significant stimulation of these specific cellular cells, which decrease the protoscolices infectivity in comparison with higher pigment concentrations. Pyocyanine is a toxic pigment causing suppression of T-cells activity, especially at higher concentrations which allow protoscolices development and growth

Therapeutic effects of Sophora moorcroftiana alkaloids in combination with albendazole in mice experimentally infected with protoscolices of Echinococcus granulosus

The objective of the present study was to determine if the combination of alkaloids from Sophora moorcroftiana seeds and albendazole might be effective in the treatment of experimental echinococcosisin female NIH mice (6 weeks old and weighing 18-20 g, N = 8 in each group) infected withprotoscolices of Echinococcus granulosus. Viable protoscolices (N = 6 x 103) were cultured in vitro in 1640 medium and mortality was calculated daily. To determine the in vivo efficacy, mice were inoculated intraperitoneally with viable protoscolices and then treated once daily by gavage for three months with the alkaloids (50 mg kg-1 day-1) and albendazole (50 mg kg-1 day-1), separately and in combination (both alkaloids at 25 mg kg-1 day-1 and albendazole at 25 mg kg-1 day-1). Next, the hydatid cysts collected from the peritoneal cavity of the animals were weighed and serum IL-4, IL-2, and IgE levels were analyzed. Administration of alkaloids to cultured protoscolices showed significant dose- and time-dependent killing effects. The weight of hydatid cysts was significantly decreased upon treatment with each drug (P < 0.01), but the decrease was more prominent and the rate of hydatid cyst growth inhibition was much higher (76.1 percent) in the group receiving the combined treatments (18.3 ± 4.6 mg). IL-4 and total IgE were decreased (939 ± 447 pg/mL and 2.03 ± 0.42 IU/mL, respectively) in serum from mice treated with alkaloids and albendazole compared with the untreated control (1481 ± 619 pg/mL and 3.31 ± 0.37 IU/mL; P < 0.01). These results indicate that S. moorcroftiana alkaloids have protoscolicidal effects and the combination of alkaloids and albendazole has significant additive effects.

Immunization of lambs against infection with echinucoccus granulosus using protoscolices and hydatid fluid antigens

In the present study, protoscolices and hydatid fluid were prepared from livers or lungs of sheep with hydatid cyst in sterile conditions. The protein concentration of samples was then measured by Bradford method. 12 lambs 4 - 6 months of age of mixed sex were randomly allocated to 3 groups of 4 lambs. Each lamb in groups 1 and 2 was immunized subcutaneously in the neck with a 2 - ml dose of vaccine [1 mg of protoscolex and hydatid fluid proteins dissolved in 1 ml of PBS] and emulsified with an equal volume of Freund's complete adjuvant [FCA] respectively. Control lambs were immunized with adjuvant in PBS. Lambs were boosted at day 28 with the same preparation except that FCA was replaced by Freund's incomplete adjuvant [FIA]. Three weeks after the second immunization, each lamb received a challenge infection with 2000 protoscolices intraperitoneally and 10 adult E. granulosus. All lambs were euthanized after 7 months and examined for hydatid cysts. Results showed that, the number of cysts in immunized lambs with protoscolices were lower than in control group and also the number of cysts in immunized lambs with hydatid fluid antigen was significant [p < 0.05]. This result indicated that the protective immunity in lambs with protoscolices and hydatid fluid was 54.5% and 75.75% respectively

Seroprevalencia de hidatidosis humana en la Región de Coquimbo. Chile / Human hydatidic seroprevalence in Coquimbo Region, Chile

Estudio previos de seroprevalencia de hidatidosis en Chile, realizados en sectores urbanos y rurales encontraron una tasa de 135 x 105, con una estimación probable de 17.002 personas infectadas. En los últimos años la prevalencia humana y animal ha tendido a disminuir en todas las regiones del país, excepto en la IV, VI y IX. Entre ellas destaca la IV Región por mostrar las mayores alzas, tanto en el ganado ovino como en el caprino. Con el objeto de contribuir al conocimiento actual de la hidatidosis humana, se estudió la prevalencia serológica en comunidades rurales ganaderas de la IV Región de Coquimbo con el fin de que dichos resultados sirvan de base para la aplicación de programas de control en la zona. Se estudiaron 4.632 muestras de habitantes de las provincias de Elqui, Limarí y Choapa. La selección del grupo se efectuó por muestreo aleatorio sistemático representativo de la región, según localidades y equivalentes al 3 por ciento de la población rural total de la IV Región. Las muestras de sangre obtenidas del pulpejo del dedo medio, fueron colectadas en papel filtro, según las normas bioéticas, estando supeditado al consentimiento de las personas. A cada eluído se les efectuó ELISA IgG. En forma paralela en dos laboratorios de rutina de inmunodiagnóstico parasitológico. Como antígeno se usó líquido hidatídico hepático ovino, aislado en forma estéril, centrifugado a 12.000 g en un laboratorio y en el otro el antígeno estuvo constituido por la fracción B o antígeno de Oriol. La seroprevalencia de la hidatidosis encontrada en comunidades rurales ganaderas de la IV Región de Coquimbo, alcanzó a un 2,5 por ciento. La tasa global encontrada en la IV Región equivale a una prevalencia estimada de 2.500 x 105, cifra notablemente superior a la tasa de 1,65 x 105 a nivel nacional y de 6,73 x 105 para la Región de Coquimbo notificadas en el 2004. Las prevalencias encontradas para las provincias de Limarí y Choapa, fueron superiores, en forma significativa, al n.

Detection of hydatid fluid and protoscolices antigens in sheep with hydatidosis

Hydatidosis is a disease caused by infection with the metacestode stage of the dog tapeworm, Echinococcus granulosus. This is recognised as one of the world's most important zoonoses, affecting both humans and their domestic animals. In the current study, 20 sheep cystic livers or lungs were collected. Hydatid fluid and protoscolices were isolated and the soluble protein was prepared. Polypeptide profile of hydatid fluid and protoscolices was analysed by SDS-PAGE with 12.5% acrylamide concentration. Humoral immunity and antigenic pattern were evaluated by Western blotting. In electrophoresis of hydatid fluid, five bands with molecular weight of 84, 68-70, 55, 27-28 and 16-17 kDa were observed; the 68-70 and 55 kDa bands were noticeably dominant. In Western blot of hydatid fluid protein, only 68-70 and 55 kDa bands had conciderable positive reaction. A total of 12 bands were also observed in protoscolices polypeptide profile. The molecular weight of the bands were 120, 88-89, 84, 66, 55-56, 49-50, 43-44, 36, 30-31, 24, 20 and 15- 16 kDa. Seven bands with molecular weight of 66, 55-56, 49-50, 43-44, 36, 30-31 and 24 kDa had positive reaction in Western blot We concluded that the determination of specific antigenic bands for sheep hydatid fluid and protoscolices was successfully achieved in this study

Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

Hydatid cyst fluid (HCF), somatic antigens (S-Ag) and excretory-secretory products (ES-Ag) of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemaglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates

Human cystic echinococcosis: diagnostic value of different antigenic fractions of hydatid cyst fluid with different specific immunoglobulin g subclasses by enzyme linked immunoelectrotransfer blot

The present study aimed to evaluate the usefulness of immunoblot analysis of hydatid cyst fluid [HCF] for diagnostic verification of human cystic echinococcosis [CE]. HCF of camel origin was resolved by SDS-PAGE gel electrophoresis and transblotted on nitrocellulose membrane. Forty-four persons with other parasitic infections and 20 normal controls were analyzed. Total IgG and IgG subclass 1-4 in CE sera preferentially recognized several polypeptide bands in the range of 14-200 kDa. Sera from the normal controls did not recognize any of these polypeptides. These data suggested that detection of any of these polypeptides bands could be used for the confirmation of human cystic echinococcosis in Egypt

Identification of immunodeominant antigens by immunoelectrotranfer in hydatid fluid

A través de electroforésis en geles de poliacrilamina (SDS-PAGE) e inmunoelectrotransferencia (Western Blot), fueron identificados y caracterizados los antígenos immunodominantes del líquido hidatídico. Para ello, se estudiaron 23 pacientes con hidatidosis confirmadas, 12 con sospecha clínica de infección y serología positiva con los métodos convencionales (doble difusión con detección del arco 5 y ELISA), 28 individuos sanos y 23 con otras infecciones parasitarias y no parasitarias. Los resultados demostraron 7 bandas antigénicas localizadas entre los 8 y los 120 kDa. Dos bandas immunodominantes fueron reconocidas en los pacientes con hidatidosis comprobada y con serología positiva, sus pesos moleculares (PM) fueron de 8 y 12 kDa. Dos bandas inespecíficas fueron detectadas con los sueros de individuos sanos y con cisticercosis. Se concluye que las bandas de PM de 8 y 12 kDa son las de mayor valor diagnóstico a diferencia de las de 32 y 60 kDa que son inespecíficas