Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

Mitochondrial ultrastructural and atpase changes during the life cycle of Ascaris Suum

Ultrastructural morphology and ATPase specific activities of mitochondria isolated from 1-celled fertilized egg, 10-day embryo, 21-day infective larvae and adult body wall muscle of Ascaris suum and rat liver were determined and compared. Although cristae of both muscle and egg mitochondria contained numerous elementary particles with head pieces of conventional diameter (85 A), each muscle mitochondrion contained relatively few, short cristae with a diminished frequency of elementary particles and associated ATPase activity. These morphological relationships are related to the previous conclusion that the transition from an aerobic to an essentially anaerobic metabolism is intimately associated with the mitochondrion and is a normal and mandatory feature of development.

Molecular paleoparasitological diagnosis of Ascaris sp. from coprolites: new scenery of ascariasis in pre-Colombian South America times

Paleoparasitological studies using microscopy showed that Ascarisand Trichuris trichiura are the human intestinal parasites most found in archaeological sites. However, in pre-Columbian South American archaeological sites, Ascaris is rare. In this work we standardized a molecular methodology for Ascaris diagnosis directly from ancient DNA retrieved from coprolites. Using cythochrome b gene (142 bp) target, ancient DNA sequences were retrieved from South American samples, negative by microscopy. Moreover, the methodology applied was sensitive enough to detect ancient DNA extracted from 30 Ascaris eggs from an European coprolite. These results revealed a new scenery for the paleodistribution of Ascaris in South America.

Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA) report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-a levels (mean ± SD; 2 days: control = 0, BCG = 169 ± 13, BCG + Asc = 202 ± 37, and Asc = 0; 7 days: control = 0, BCG = 545 ± 15.5, BCG + Asc = 2206 ± 160.6, and Asc = 126 ± 26; 14 days: control = 10 ± 1.45, BCG = 9 ± 1.15, BCG + Asc = 126 ± 18, and Asc = 880 ± 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean ± SD; 2 days: control = 0, BCG = 3.7 ± 1.59, BCG + Asc = 0.82 ± 0.005, Asc = 0.48 ± 0.33; 7 days: control = 0, BCG = 2.78 ± 1.54, BCG + Asc = 3.07 ± 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 ± 0.53, BCG + Asc = 9.61 ± 0.81, Asc = 10.5 ± 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean ± SD; 2 days: BCG = 1.13 ± 0.07 and BCG + Asc = 0.798 ± 0.305; 7 days: BCG = 1.375 ± 0.194 and BCG + Asc = 0.548 ± 0.0226; 14 days: BCG = 0.473 ± 0.184 and BCG + Asc = 0.675 ± 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.

Extracts of Ascaris suum egg and adult worm share similar immunosuppressive properties

Adult Ascaris suum body extract (Asc) prepared from male and female worms (with stored eggs) down-regulates the specific immune response of DBA/2 mice to ovalbumin (OA) and preferentially stimulates a Th2 response to its own components, which is responsible for the suppression of the OA-specific Th1 response. Here, we investigated the participation of soluble extracts prepared from male or female worms or from eggs (E-Asc) in these immunological events. Extracts from either sex (1 mg/animal) or E-Asc (0.35 or 1 mg protein/animal) suppressed the delayed-type hypersensitivity (DTH) reaction (60-85%), proliferative response (50-70%), IL-2 and IFN-gamma secretion (below detection threshold) and IgG1 antibody production (70-90%) of DBA/2 mice to OA. A dose of 0.1 mg E-Asc/animal did not change DTH or proliferation, but was as effective as 0.35 mg in suppressing IL-2 and IFN-gamma, and OA-specific IgG1 antibodies. Lymph node cells from DBA/2 mice injected with Asc (1 mg/animal) or a high dose of E-Asc (1 mg protein/animal) secreted IL-4 upon in vitro stimulation with concanavalin A. As previously demonstrated for Asc, the cytokine profile obtained with the E-Asc was dose dependent and changed towards Th1 when a low dose (0.1 mg protein/animal) was used. Taken together, these results suggest that adult worms of either sex and eggs induce the same type of T cell response and share similar immunosuppressive properties.

Comparison of different monoclonal antibodies against immunosuppressive proteins of Ascaris suum

The extract of Ascaris suum suppresses the humoral and cellular immune responses to unrelated antigens in the mouse. In order to further characterize the suppressive components of A. suum, we produced specific monoclonal antibodies which can provide an important tool for the identification of these proteins. The A. suum immunosuppressive fractions isolated by gel filtration from an extract of adult worms were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells and the cloned hybrid cells obtained were screened to determine the specificity of secreted antibodies. Three monoclonal antibodies named MAIP-1, MAIP-2 and MAIP-3 were selected and were shown to react with different epitopes of high molecular weight proteins from the A. suum extract. All antibody molecules have kappa-type light chains but differ in heavy chain isotype. MAIP-1 is a mouse IgM, MAIP-2 is an IgA immunoglobulin and MAIP-3 is an IgG1 immunoglobulin and they recognize the antigen with affinity constants of 1.3 x 10(10) M-1, 7.1 x 10(9) M-1 and 3.8 x 10(7) M-1, respectively. The proteins recognized by these monoclonal antibodies (PAS-1, PAS-2 and PAS-3) were purified from the crude extract by affinity chromatography and injected with ovalbumin in BALB/c mice in order to determine their suppressive activity on heterologous antibody production. It was demonstrated that these three proteins are able to significantly suppress anti-ovalbumin antibody secretion, with PAS-1 being more efficient than the others.

Prevalência de enteroparasitas na população urbana do 2.° subdistrito de Botucatu, SP (Brasil)

Procurou-se conhecer a prevalência de enteroparasitoses na população urbana do 2.° subdistrito de Botucatu, SP (Brasil) através de exames coprológicos realizados pelos métodos de FAUST, HOFFMAN e processo de tamização. A prevalência de enteroparasitoses foi relacionada com atributos da população, tais como sexo, idade, cor e com fatores ligados ao meio ambiente. O processo de amostragem empregado foi o casual simples em duplo estágio, sendo o quarteirão a unidade primária do primeiro estágio e o domicílio a unidade do segundo estágio. Os resultados mostraram que 53,76% das 895 pessoas amostradas apresentavam-se infestadas por uma ou mais espécies de parasitas intestinais. As prevalências foram as seguintes: Ancylostomidae, 17,54%; T. trichiurus, 13,63%; A. lumbricoides, 10,69%; S. stercoralis, 6,03%; E. vermicularis, 3,69%; H. nana, 1,79%; Taenia sp, 1,22%; S. mansoni, 0,22%; E. coli, 15,53%; G. lamblia, 14,07%; E. nana, 2,35%; I. bütschlii, 1,01% e E. histolytica, 0,22%.

Estudo comparativo dos métodos coprológicos de Lutz, Kato-Katz e Faust modificado

Foram estudados, comparativamente, em 500 pacientes, os métodos de exames de fezes de Lutz, Faust modificado e Kato-Katz para o diagnóstico parasitológico de fezes. O método de Kato-Katz proporcionou maiores índices de positividade do que as outras duas técnicas no diagnóstico de ancilostomideos, T. trichiurus e S. mansoni. Nenhuma diferença foi observada quanto ao diagnóstico de A. lumbricoides. Para o diagnóstico das protozooses não houve diferença significativa entre os métodos de Faust modificado e de Lutz. Baseado nos dados obtidos, eficiência, simplicidade e rapidez de execução recomenda-se a associação dos métodos de Kato-Katz e Faust modificado na rotina de diagnóstico parasitológico de fezes.

Estudo da ocorrência de enteroparasitas em hortaliças comercializadas na região metropolitana de São Paulo, SP, Brasil: I - Pesquisa de helmintos

Hortaliças in natura, comercializadas na região metropolitana de São Paulo, SP (Brasil), foram analisadas através de metodologia própria, visando à pesquisa e à identificação de formas de transmissão de helmintos intestinais de interesse médico. As hortaliças examinadas, constituídas de 50 amostras de cada variedade, foram de: alface (Lactuca sativa), variedades lisa e crespa, escarola (Chichorium sp) e agrião (Nasturtium officinale). Os resultados evidenciaram elevados percentuais de contaminação em todas as variedades analisadas, porém, as freqüências de helmintos foram maiores no agrião. A escarola apresentou valores médios, geralmente situados entre as alfaces e o agrião. Os números médios de ovos e larvas por 100 gramas de amostra, embora elevados, não apresentaram diferenças estatisticamente significantes entre as quatro variedades de hortaliças estudadas. Uma grande variedade de helmintos, de ocorrência freqüente na população residente na Região Metropolitana de São Paulo, foi observada nas amostras. Os mais freqüentes, no entanto, foram: ancilostomídeos e Ascaris sp. Recuperaram-se também ovos de Toxocara sp, Fasciola sp e de tricostrongilídeos, comprovando a ocorrência de contaminação das hortaliças por fezes de animais domésticos. Considerando-se os resultados obtidos, ressalta-se a importância das hortaliças na transmissão de helmintíases intestinais, bem como a necessidade de medidas que propiciem uma melhoria na qualidade higiênico-sanitária destes alimentos.

Dípteros muscóides como vetores mecânicos de ovos de helmintos em jardim zoológico, Brasil

OBJETIVO: Verificar as espécies de dípteros muscóides capazes de veicular ovos e larvas de helmintos e avaliar o potencial de contaminação dos dípteros capturados. MÉTODOS: A pesquisa foi realizada em dois pontos distintos do Jardim Zoológico da cidade do Rio de Janeiro, RJ, no período de maio de 1996 a abril de 1998. As capturas dos dípteros foram realizadas semanalmente com armadilhas contendo peixe em putrefação, que permaneceram expostas durante uma hora nos dois pontos: local 1- próximo à lixeira do zoológico e o local 2- perto do recinto do hipopótamo e das aves de rapina. Foram capturadas 41.080 moscas, sendo a espécie Chrysomya megacephala mais representativa com 69,34%, seguida de Chrysomya albiceps 11,22%, Musca domestica 7,15%, Chrysomya putoria 4,52%, Fannia sp. 3,12%, Ophyra sp. 2,53% e Atherigona orientalis 2,08%. As moscas capturadas tiveram a superfície dos corpos lavadas com água destilada e os tubos digestivos dissecados. RESULTADOS: Das espécies estudadas, C. megacephala e M. domestica apresentaram maior quantidade de ovos de helmintos na superfície do corpo e no conteúdo intestinal. Ovos de Ascaridoidea e Trichinelloidea prevaleceram no conteúdo intestinal de C. megacephala. Dos ovos de helmintos encontrados na superfície do corpo e no conteúdo intestinal foram identificados: Ascaris sp., Toxascaris sp., Toxocara sp., Trichuris sp., Capillaria sp., Oxiurídeos, Triconstrogilídeos e Acantocephala. Também foram encontradas larvas de helmintos na superfície do corpo dos dípteros. Houve diferenças significativas (nível de 5%, pelo teste F) entre os diferentes pontos de capturas em relação ao número de ovos de helmintos encontrados nos dípteros. CONCLUSÕES: As fezes dos animais do jardim zoológico, encontradas freqüentemente nos abrigos e lixeiras, contribuíram para a proliferação dos dípteros muscóides, que assumem importante papel na veiculação de ovos de helmintos, principalmente pelo contato direto do corpo do díptero com o alimento dos animais.

Glucogeno sintasa en helmintos parasitos: inhibicion por benzimidazoles

Se ha determinado el effecto inhibidor sobre la actividad Glucogeno sintetasa (E.C.2.41.11) por parte de cuatro antihelminticos: Albendazol, Mebendazol, Parbendazol y Tiabendazol. Observandose que en todos los casos, es el Parbendazol quien ha demostrado un mayor poder inhibidor sobre la glucógeno sintetasa de Ascaris suum, Fasciola hepatica y Moniezia expansa. El Tiabendazol es el anti-helmintico que menor efecto inhibidor ha presentado sobre la enzima en los tres parasitos objeto de nuestro estudio. Con el presente trabajo y otros previstos en la misma linea, se pretende aportar nuevos datos acerca del aun desconocido locus de acción de estos antihelminticos.

Metodo para montaje permanente de huevos de helmintos enteroparasitos

Se comunican resultados obtenidos empleando Medio de Hoyer para el montaje de huevos de helmintos enteroparásitos, destinado a preparaciones para colecciones docentes y/o de investigación. La utilización de esta técnica en muestras fecales conteniendo huevos de A. lumbricoides, T. trichiura, Uncinaria sp., Taenia sp., Diphyllobothrium sp., H. nana, H. diminuta y F. hepática, permitió la correcta observación de ellos en lecturas iniciadas a las 24 horas y mantenidas hasta 180 días después.

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.