The Acute Inflammatory Potential of Particles From the Echinococcus granulosus Laminated Layer Is Moderated by Its Calcium Inositol Hexakisphosphate Component.

Cystic echinococcosis is caused by the tissue-dwelling larva (hydatid) of Echinococcus granulosus sensu lato. A salient feature is that this larva is protected by the acellular laminated layer (LL). As the parasite grows, the LL sheds abundant particles that can accumulate in the parasite's vicinity. The potential of LL particles to induce inflammation in vivo has not been specifically analysed. It is not known how each of its two major components, namely highly glycosylated mucins and calcium inositol hexakisphosphate (InsP6) deposits, impacts inflammation induced by the LL as a whole. In this work, we show that LL particles injected intraperitoneally cause infiltration of eosinophils, neutrophils and monocytes/macrophages as well as the disappearance of resident (large peritoneal) macrophages. Strikingly, the absence of calcium InsP6 enhanced the recruitment of all the inflammatory cell types analysed. In contrast, oxidation of the mucin carbohydrates caused decreased recruitment of neutrophils. The carbohydrate-oxidised particles caused cell influx nonetheless, which may be explained by possible receptor-independent effects of LL particles on innate immune cells, as suggested by previous works from our group. In summary, LL particles can induce acute inflammatory cell recruitment partly dependent on its mucin glycans, and this recruitment is attenuated by the calcium InsP6 component.

Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses.

Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of beclin-1, atg16l1 and atg12, indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of il-6, il-10, il-12, tnf-α, ifn-γ and tgf-ß genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.

Comparative analysis of host immune responses to Hydatid cyst in human and ovine hepatic cystic Echinococcosis.

BACKGROUND: Hydatid disease is caused by the larval stages of the canine tapeworm Echinococcus granulosus. It is one of the most critical helminthic diseases, representing worldwide public health and socio-economic concern. AIM: This study aimed to investigate the expression of apoptosis and immune response within hepatic tissues of humans and sheep infected with the Hydatid cyst. METHODS: Paraffin-embedded tissue was prepared from each tissue sample and used for histopathological examination by Haematoxylin- Eosin. Also, toluidine blue staining was used for mast cell detection, while an immunohistochemical study was performed to assess CD3 T lymphocytes, CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, CD20 memory B lymphocytes, CD68 macrophage, and caspase-3 antibodies. RESULTS: The histological examination revealed significant changes, including the infiltration of inflammatory cells, predominantly lymphocytes with scattered giant cells, necrotic hepatic tissue, and fibrosis. Toluidine blue stain revealed a higher number of mast cells (5 cells/field) in humans compared to sheep (3.6 cells/field). The immunohistochemical analysis confirmed that the CD3 were the most predominant inflammatory cell in the hepatic tissue of humans (intensive 70%), and sheep (moderate 38.47%). Caspase-3 was observed in all samples in different grades and mostly in human liver tissue. CONCLUSION: This data could aid in recognizing immunological markers for differentiating disease progression, as well as enhance the understanding of local immune responses to cystic Echinococcosis (CE). The findings could provide preliminary data for future studies on immune responses associated with Hydatid cysts.

Immunization with a Mu-class glutathione transferase from Echinococcus granulosus induces efficient antibody responses and confers long-term protection against secondary cystic echinococcosis.

Cystic echinococcosis, a zoonosis caused by cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) genetic complex, affects humans and diverse livestock species. Although a veterinary vaccine exhibiting high levels of antibody-mediated protection has successfully reached the market, the large genetic diversity among parasite isolates and their particular host preferences, makes still necessary the search for novel vaccine candidates. Glutathione transferases (GSTs) constitute attractive targets for immunoprophylaxis due to their outstanding relevance in helminth detoxification processes, against both exogenous and endogenous stressors. Among the six GSTs known to be expressed in E. granulosus s.l., EgGST1 (Mu-class), EgGST2 (Sigma-class), and EgGST3 (a still non-classifiable isoenzyme), show the highest proteomic expression. Therefore, their recombinant forms -rEgGST1, rEgGST2 and rEgGST3- were herein analyzed regarding their potential to induce long-term antiparasite protection in mice. Only immunization with rEgGST1 induced long-lasting protection; and accordingly, rEgGST1-specific antibodies enhanced the parasite killing through both the classical activation of the host complement system and the antibody-dependent cellular cytotoxicity by macrophages. These results support further testing of rEgGST1 as a vaccine candidate in diverse hosts due to the broad expression of EgGST1 in different parasite stages and tissues.

Untargeted metabolomics to discriminate liver and lung hydatid cysts: Importance of metabolites involved in the immune response.

The Echinococcus granulosus sensu lato species complex is responsible for the neglected zoonotic disease known as cystic echinococcosis (CE). Humans and livestock are infected via fecal-oral transmission. CE remains prevalent in Western China, Central Asia, South America, Eastern Africa, and the Mediterranean. Approximately one million individuals worldwide are affected, influencing veterinary and public health, as well as social and economic matters. The infection causes slow-growing cysts, predominantly in the liver and lungs, but can also develop in other organs. The exact progression of these cysts is uncertain. This study aimed to understand the survival mechanisms of liver and lung CE cysts from cattle by determining their metabolite profiles through metabolomics and multivariate statistical analyses. Non-targeted metabolomic approaches were conducted using quadrupole-time-of-flight liquid chromatography/mass spectrometry (LC-QTOF-MS) to distinguish between liver and lung CE cysts. Data processing to extract the peaks on complex chromatograms was performed using XCMS. PCA and OPLS-DA plots obtained through multiple statistical analyses showed interactions of metabolites within and between groups. Metabolites such as glutathione, prostaglandin, folic acid, and cortisol that cause different immunological reactions have been identified both in liver and lung hydatid cysts, but in different ratios. Considering the differences in the metabolomic profiles of the liver and lung cysts determined in the present study will contribute research to enlighten the nature of the cyst and develop specific therapeutic strategies.

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.

Effects of annexin B18 from Echinococcus granulosus sensu lato on mouse macrophages.

Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ß，IL-6，IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.

Comparison of Methods in the Serologic Diagnosis of Cystic Echinococcosis.

PURPOSE: Cystic echinococcosis (CE) is caused by the larval form of Echinococcus granulosus. Clinical, radiologic, pathologic, and serologic findings should be evaluated together for the diagnosis of CE. The sensitivity and specificity oalf serologic tests may vary depending on the method used. In this study, we aimed to detect IgG antibodies specific to E. granulosus using indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodies (IFA) and western blot (WB) tests. METHODS: In our study, the serum samples of 74 patients sent to our laboratory with suspicion of CE were studied using two different commercial IHA tests, ELISA, IFA and WB test. The test results were evaluated along with radiological findings and histopathological examinations, the latter being the gold standard. RESULTS: Of all the patients, 51 (69%) were female and 23 (31%) were male. There was a statistically significant difference between males and females (χ2 = 9.7, p = 0.002). Out of 74 patients, positivity rates for Siemens IHA, Fumouze IHA, ELISA, IFA and WB test were positive as 33 (44.6%), 35 (47.3%), 43 (58.1%), 42 (56.7%) and 38 (51.3%), respectively. The sensitivity and specificity of the tests were as follows: 66.67 and 2.31% for Siemens IHA; 70.83% and 96.15% for Fumouze IHA; 85.42%, and 88.46% for ELISA; 83.33% and 88.46% for IFA; 72.92% and 88.46% for WB test. CONCLUSION: There were statistically significant differences in between all five methods (p < 0,001). While the tests with the highest specificity was Fumouze IHA, the test with the highest sensitivity was the ELISA test. It was concluded that IHA and ELISA tests were more practical in practice because of their greater applicability.

Comparison of the Diagnostic Performance of Antigen B Purified from Sheep Hydatid Cyst Fluid (HCF) with Commercial ELISA Kit.

INTRODUCTION: Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus. CE is a health problem in Middle Eastern countries, such as Iran. The purpose of this study was to purify subunit 8 KDa antigen B from crude sheep hydatid cyst fluid (HCF) and compare its sensitivity and specificity with a commercial human ELISA kit (PT-Hydatid-96). METHODS: 28 sera samples were collected from hydatid cyst patients who had surgery for a hydatid cyst and had their disease confirmed by pathology after the surgery. Furthermore, 35 samples of healthy individuals with no history of hydatid cysts were collected, as were nine serum samples from parasite-infected non-CE patients. HCF was obtained from sheep fertile cysts at a Sari slaughterhouse and used as an antigen. In an indirect ELISA test, the B antigen was employed, and the results were compared to those from a commercial ELISA kit. RESULTS: The results of this study were analyzed using the Kappa test. The commercial ELISA kit showed 17 cases (23.6%) positive, 44 cases (61.1%) negative, and 11 cases (15.3%) borderline. B antigen showed that 18 (25%), 43 (59.7 %), and 11 (15.3%) were positive, negative, and borderline, respectively. One sample (1.4% of 72 total samples) of 35 serum samples from healthy individuals was positive using B antigen-based ELISA. In addition, all nine serum samples from parasite-infected non-CE patients were negative for both tests. The sensitivity and specificity of the commercial ELISA kit have been evaluated at 60.7% and 100%, respectively. For B antigenbased ELISA, these values are 64.3 and 97.7%, respectively. CONCLUSION: Antigen B produced from hydatid cyst fluid is a promising option for serological identification of hydatid cysts in both infected and healthy individuals. In an indirect ELISA test, hydatid fluid antigen could be used as a precise source of detection.

Equinococosis quística músculo-esquelética primaria de evolución crónica / Primary musculo-skeletal echinococcosis of chronic evolution

Resumen La equinococosis quística es una zoonosis parasitaria crónica de alta prevalencia en Chile. Se presenta el caso clínico de un varón de 66 años, proveniente de la Región del Maule, con una equinococosis quística músculo-esquelética. Consultó por dolor, aumento de volumen y una fístula en muslo izquierdo, con salida de líquido cristalino. En el estudio imagenológico se identificaron múltiples lesiones quísticas en el ala sacra, hueso ilíaco y tejidos blandos de zona inguinal y muslo izquierdo. La serología Elisa IgG para Equinococcus granulosus fue positiva. Se realizó la resección quirúrgica de las lesiones musculares y se inició terapia antiparasitaria combinada con albendazol y praziquantel, con buena respuesta clínica; sin embargo, al suspender la terapia, por iniciativa del paciente, se reiniciaron los síntomas.

Cystic echinococcosis is a chronic parasitic zoonosis of high prevalence in Chile. We report a clinical case of a 66-year-old man, domiciled in an urban area of the Maule Region, who presents skeletal muscle cystic echinococcosis. Consultation for pain, volume increase and left thigh fistula that gives out crystalline fluid. In the study with imaging techniques, multiple cystic lesions are identified in the sacral wing, iliac bone, soft tissues of the groin and left thigh. No cysts were evident in other organs. Serology Elisa IgG was positive Echinococcus granulosus. Surgical resection of soft tissue injuries. Combined antiparasitic therapy with albendazole and praziquantel was started, with good clinical response. Upon discontinuation of antiparasitic therapy at the initiative of the patient, symptoms are reinitiated.

Detección de coproantígenos de Echinococcus granulosus en canes de trabajadores de camales y comercializadores de vísceras en Lima metropolitana / Detection of stool antigens of Echinococcus granulosus in dogs belonging to slaughterhouse workers and offal merchants in Metropolitan Lima

RESUMEN Objetivo Demostrar la presencia de Echinoccocus granulosus en el hospedero definitivo en la ciudad de Lima, Perú, mediante la detección de antígenos del parásito en heces de canes pertenecientes a trabajadores y comercializadores de vísceras de centros de beneficio autorizados en Lima metropolitana. Métodos Se recolectaron muestras de heces de 58 canes, que fueron evaluadas utilizando la técnica coproELISA para detectar antígenos secretorio/excretorio de E. granulosus. Mediante una encuesta se obtuvo información sobre las prácticas de alimentación y el manejo de las mascotas. Resultados El 13,8% (8/58) de canes fue positivo a E. granulosus. En 27,8% (5/18) de los hogares se encontró al menos un animal positivo y se estimó que en las familias que tenían más de cuatro canes las posibilidades de encontrar al menos uno positivo eran mayores. En todos los hogares con al menos un can positivo sus mascotas se alimentaban con vísceras. El 94,4% (17) de los participantes no tenía conocimiento de las formas de contagio de la equinococosis. Conclusiones Los resultados muestran la presencia de hospederos definitivos en la zona urbana de Lima y subrayan la necesidad de aumentar la difusión de las prácticas para evitar la transmisión del parasito.

ABSTRACT Objective To demonstrate the presence of Echinoccocus granulosus in the definitive host in the city of Lima, Perú, by detecting parasite antigens in the stool of dogs belonging to offal handlers and merchants in authorized slaughterhouses in Metropolitan Lima. Methods Stool samples were collected from 58 dogs and examined using the coproELISA technique for the detection of secretory/excretory antigens of E. granulosus. A survey was conducted to obtain information on pet feeding and handling practices. Results Positivity to E. granulosus was detected in 13.8% (8/58) of the dogs. In 27.8% (5/18) of the homes, at least one animal showed positivity, and in families that had more than four dogs the chances of finding positivity in at least one dog were higher (P < 0.05). In all homes where at least one dog tested positive the pets were fed on offal. Of study participants, 94.4% (17) knew nothing about the routes of transmission of hydatid disease. Conclusions Results show the presence of definitive hosts in the urban area of Lima and underscore the need to more widely disseminate practices for the prevention of parasite transmission.

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.

Effectiveness of the Live Births Information System in the Far-Western Brazilian Amazon / Eficácia do Sistema de Informação de Nascidos Vivos na Amazônia Extremo-Ocidental Brasileira

The Live Birth Information System (SINASC) was implemented in 1990 for the purpose of providing information about the live-birth characteristics for the establishment of specific health indicators. This work evaluates the information quality of SINASC in relation to its data completeness and coverage for five municipalities from the State of Acre from 2005 to 2010. Lack of information (not filled out or stated as "unknown") was estimated for each variable. Coverage was estimated comparing the Civil Register office statistics in accordance with the mother's municipality of residence. An increase in incompleteness of the majority of variables was observed, and also a decrease in coverage between 2005 and 2010 in these municipalities. These findings do not tally with results from the majority of studies that use SINASC as a data source. The results of this work highlight the relevance of continuous capacity building and the incentive for accurate and complete data inclusion, as well as awareness of the importance of SINASC for public health policies.

O Sistema de Informação de Nascidos Vivos (SINASC) foi implantado no ano de 1990 com o objetivo de fornecer dados sobre as características de nascidos vivos para o estabelecimento de indicadores de saúde específicos. Objetivo: O presente trabalho avalia a qualidade da informação do SINASC quanto à incompletude dos seus dados e da cobertura para cinco municípios do estado do Acre nos anos de 2005 e 2010. Métodos: Foi calculada a incompletude (definida como dados em branco/ignorado) de cada variável, assim como a cobertura desse sistema através da comparação com as estatísticas do Registro Civil, segundo município de residência da mãe. Resultados: Observou-se um aumento da incompletude da maioria das variáveis e uma diminuição da cobertura de 2005 para 2010 no conjunto dos municípios avaliados, destoando dos resultados obtidos na maioria dos estudos que utilizam o SINASC como fonte de dados. Conclusões: Os resultados deste trabalho apontam para a importância da contínua capacitação e também para o incentivo ao preenchimento dos dados de forma correta e completa, bem como a conscientização da importância do SINASC para as políticas públicas de saúde.

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.

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.

Notes on human cases of cystic echinococcosis in Peru

Cystic echinococcosis (CE) is a high prevalent zoonosis in the central and southern Peruvian Andes. Serum samples (n50)frompatients presenting presumptive clinical and radiological diagnosis of CE (group 1), were tested for antibodies against Echinococcus granulosus metacestode using Arc-5 double diffusion assay (DD5), immunoelectrophoresis (IEF), and immunoelectrotransfer blot (EITB) techniques. Serum samples (n18) from patients presenting other parasite infections (paragonomiasis, cysticercosis, and fascioliasis) or healthy blood donors (n15), were designated as control groups. The overall sensitivity of the tests was of 94 percent (DD5 and IEF tests) or 96 percent (EITB test). Only patients from group 1 were seropositive for CE. Polypeptides of 21, 31, and 48 kDa were considered positive for CE. Based on these results, this study demonstrates that CE also occurs in other coastal departments (Piura, Ancash, Ica, Arequipa, and Tacna) besides Lima.

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.

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.