RESUMEN
Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.
Asunto(s)
Toxocara canis , Toxocariasis , Animales , Antígenos Helmínticos , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Humanos , Immunoblotting/veterinaria , Inmunoglobulina G , Pruebas Inmunológicas/veterinaria , Proteómica , Proteínas Recombinantes , Toxocara , Toxocariasis/diagnósticoRESUMEN
Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis.
Asunto(s)
Antiasmáticos/farmacología , Caspasa 3/inmunología , Caballos/inmunología , Inmunidad Innata/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Femenino , Citometría de Flujo/veterinaria , Immunoblotting/veterinaria , Masculino , Membranas Mitocondriales/fisiología , Neutrófilos/inmunologíaRESUMEN
Bovine cysticercosis remains as one of the most important cause of carcasses and viscera condemnation in Brazilian slaughterhouses. However, the efficiency of post-mortem inspection for the diagnosis of this zoonotic disease is relatively low, and few available studies were performed through serological exams. This study evaluated the frequency of bovine cysticercosis in cattle herds located in different farms of the state of Rondônia, Brazil. Among the 987 animals slaughtered from 33 farms, 21 animals (Frequency: 2.13%; 95C.I. 1.23-3.03) were considered as positive through indirect ELISA and confirmed by Immunoblot tests and the cysticercosis was detected in 12 farms (36.36% - C.I. 95% 19.95-52.78). The disease was detected in the municipalities Vale do Paraíso (12.50%), Theobroma (8.11%), Guaporé (7.27%), Rolim de Moura (5.71%), Presidente Médici (5.0%), Ouro Preto do Oeste (4.69%), Nova União (1.77%), Nova Brasilândia d'Oeste (1.14%) and Ministro Andreazza (1.01%). Therefore, prophylactic measures should be taken to improve beef production, control bovine cysticercosis and reduce costs to public health in this Brazilian state.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Cisticercosis/veterinaria , Animales , Brasil/epidemiología , Bovinos , Enfermedades de los Bovinos/parasitología , Cisticercosis/epidemiología , Cisticercosis/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Immunoblotting/veterinaria , Masculino , PrevalenciaRESUMEN
BACKGROUND: Taenia solium is an important zoonotic parasite that infects humans as definitive host (taeniasis) and pigs as intermediate host (cysticercosis). Serological diagnosis of porcine cysticercosis is limited to antigen detection using ELISA, which is known to cross-react with other Taenia species, and antibody detection using the lentil-lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB), which has not been adequately evaluated for cross-reactivity to other parasites. Field studies suggest that the GP50 diagnostic band of the LLGP EITB may cross-react to Taenia hydatigena, a common non-zoonotic parasitic infection of pigs. The objective of this study was to evaluate the specificity of the LLGP EITB assay in pigs infected experimentally with T. hydatigena and Echinococcus granulosus. RESULTS: Twelve three-month-old seronegative were divided into two groups; six were each given an oral challenge with a single gravid proglottid of T. hydatigena and the other six were each given an oral challenge with 50 gravid proglottids of E. granulosus. Serum samples were collected biweekly until 14 weeks when all pigs underwent a detailed necropsy. Taenia hydatigena cysticerci were found in two of six pigs from the first group. Four T. hydatigena-exposed pigs were seropositive at the GP50-band only on EITB LLGP; two of these had cysts at necropsy while no seronegative pigs had cysts. One E. granulosus-exposed pig was positive to EITB LLGP, again with reactivity only to GP50; all six pigs had hepatic echinococcosis on necropsy. CONCLUSION: These results provide definitive evidence that the GP50 diagnostic band in pigs cross-reacts with T. hydatigena. Evidence of cross-reaction with E. granulosus was not conclusive.
Asunto(s)
Antígenos Helmínticos/inmunología , Equinococosis/veterinaria , Echinococcus granulosus/inmunología , Immunoblotting/veterinaria , Enfermedades de los Porcinos/diagnóstico , Taenia/inmunología , Teniasis/veterinaria , Animales , Reacciones Cruzadas , Equinococosis/diagnóstico , Equinococosis/inmunología , Epítopos , Proteínas del Helminto/inmunología , Immunoblotting/métodos , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/parasitología , Teniasis/diagnóstico , Teniasis/inmunologíaRESUMEN
This study was conducted to evaluate caprine arthritis encephalitis virus (CAEV) transmission among sheep using 15 lambs that were distributed in 2 experimental groups. The exposed group consisted of 10 lambs that remained with their mothers, who were experimentally infected with CAEV. The non-exposed group was characterized as the control group and was comprised of 5 lambs that remained with their CAEV-negative mothers. Blood samples were collected monthly from birth until 1 year of life. To evaluate the transmission, an agar gel immunodiffusion test (AGID), enzyme immunoassay (ELISA), immunoblotting (IB), and nested polymerase chain reaction (nPCR) techniques were used. The non-exposed group was negative in all of the tests throughout the whole experiment. In the exposed group, 2 individuals had positive nPCR results. Positive nPCR samples were sequenced for comparison with the original goat strains and were shown to be similar to the CAEV-Cork strain. Seroconversion was not detected, and clinical manifestations were not observed. Thus, after 1 year of observation, it was verified that CAEV transmission among sheep is possible; however, with discreet frequency. This was an initial study, and other experiments are needed to analyze the adaptive capacity of the CAEV to remain in an infected sheep flock and cause the disease.(AU)
O estudo foi conduzido para avaliar a transmissão do vírus da artrite encefalite caprina (CAEV) entre ovinos, utilizando 15 cordeiros, distribuídos em dois grupos experimentais. O grupo exposto foi constituído por 10 cordeiros, mantidos com suas mães, que foram infectadas, experimentalmente, com CAEV. O grupo não exposto caracterizou-se como grupo controle e foi formado por cinco cordeiros, mantidos com suas matrizes, negativas para CAEV. Foram colhidas amostras de sangue mensalmente, do periodo que compreende o nascimento até um ano de vida. Para avaliar a transmissão, foram utilizadas as técnicas de imunodifusão em gel de agarose (IDGA), ensaio imunoenzimático (ELISA), immunoblotting (IB) e reação em cadeia da polimerase do tipo nested (nPCR). O grupo não exposto se manteve negativo aos testes durante todo o experimento. Já no grupo exposto, dois indivíduos apresentaram resultados positivos na nPCR. As amostras positivas na nPCR foram sequenciadas para serem comparadas com as cepas originais de caprinos, comprovando se tratar de lentivírus semelhante à cepa CAEV-Cork. A soroconversão não foi detectada e a manifestação clínica não foi observada. Sendo assim, após um ano de observação, verificou-se que a transmissão do CAEV entre ovinos é possível, entretanto, com discreta frequência. Este foi um estudo inicial, e outros experimentos são necessários para analisar a capacidade adaptativa do CAEV de permanecer em rebanho ovino infectado e, com isso, causar doença.(AU)
Asunto(s)
Animales , Virus de la Artritis-Encefalitis Caprina , Infecciones por Lentivirus/transmisión , Infecciones por Lentivirus/veterinaria , Ovinos , Virus Visna-Maedi , Ensayo de Inmunoadsorción Enzimática/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Immunoblotting/veterinariaRESUMEN
Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)
In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)
Asunto(s)
Animales , Bovinos , Cisticercosis/diagnóstico , Teniasis/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Estudios Epidemiológicos , Immunoblotting/veterinariaRESUMEN
Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)
In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)
Asunto(s)
Animales , Bovinos , Cisticercosis/diagnóstico , Cisticercosis/etiología , Teniasis/diagnóstico , Factores de Riesgo , Ensayo de Inmunoadsorción Enzimática/veterinaria , Immunoblotting/veterinaria , Estudios EpidemiológicosRESUMEN
The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.
O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.
Asunto(s)
Animales , Anticuerpos Antiprotozoarios/análisis , Caballos/parasitología , Diagnóstico Prenatal/veterinaria , Sarcocystis/patogenicidad , Encefalomielitis/veterinaria , Immunoblotting , Immunoblotting/veterinaria , Intercambio Materno-Fetal , Estudios SeroepidemiológicosRESUMEN
The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.(AU)
O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.(AU)
Asunto(s)
Animales , Caballos/parasitología , Sarcocystis/patogenicidad , Diagnóstico Prenatal/veterinaria , Anticuerpos Antiprotozoarios/análisis , Encefalomielitis/veterinaria , Intercambio Materno-Fetal , Estudios Seroepidemiológicos , Immunoblotting , Immunoblotting/veterinariaRESUMEN
Given the limited knowledge about the diagnosis of bovine cysticercosis by immunoblot, the aim of this study was to assess the applicability of this test, identifying key peptides with diagnostic value. Immunoblot assays were performed using total larval antigen of Taenia crassiceps and 60 sera of positive bovines for cysticercosis (30 naturally and 30 experimentally infected with T. saginata eggs), 30 sera of negative bovines for cysticercosis and 30 sera of bovines with other diseases (fascioliasis, hydatidosis and tuberculosis). The peptides of greater diagnostic importance, in descending order of accuracy (%), were as follows: 6-8kDa (90.8%), 129-143kDa (74.2%), 99-105kDa (71.7%) and 14-19kDa (71.1%). Cross-reactions, due to fascioliasis and hydatidosis, were observed in the four intervals of peptides highlighted. The results demonstrate that the total antigen of T. crassiceps has peptides with a high diagnostic potential; therefore, the immunoblot is useful in the diagnosis of bovine cysticercosis.
Tendo em vista o conhecimento limitado sobre o diagnóstico da cisticercose bovina pelo immunoblot, o objetivo deste trabalho foi avaliar a aplicabilidade desse teste, identificando os principais peptídeos com valor diagnóstico. Foram realizados ensaios de immunoblot, utilizando antígeno total de larva de Taenia crassiceps e 60 soros de bovinos positivos para a cisticercose (30 naturalmente e 30 experimentalmente infectados com ovos de T. saginata), 30 soros de bovinos negativos para a cisticercose e 30 soros de bovinos com outras patologias (fasciolose, hidatidose e tuberculose). Os peptídeos de maior importância diagnóstica, em ordem decrescente de acurácia (%), foram os seguintes: 6-8 kDa (90,8%), 129-143 kDa (74,2%), 99-105 kDa (71,7%) e 14-19 kDa (71,1%). Reações cruzadas, em decorrência da fasciolose e hidatidose, foram observadas nos quatro intervalos de peptídeos ressaltados. Os resultados demonstram que o antígeno total de T. crassiceps possui peptídeos com alto potencial diagnóstico, sendo, portanto, o immunoblot útil no diagnóstico da cisticercose bovina.
Asunto(s)
Animales , Bovinos/parasitología , Cisticercosis/veterinaria , Immunoblotting/veterinaria , Taenia saginata/parasitología , Antígenos , Cisticercosis , PéptidosRESUMEN
Given the limited knowledge about the diagnosis of bovine cysticercosis by immunoblot, the aim of this study was to assess the applicability of this test, identifying key peptides with diagnostic value. Immunoblot assays were performed using total larval antigen of Taenia crassiceps and 60 sera of positive bovines for cysticercosis (30 naturally and 30 experimentally infected with T. saginata eggs), 30 sera of negative bovines for cysticercosis and 30 sera of bovines with other diseases (fascioliasis, hydatidosis and tuberculosis). The peptides of greater diagnostic importance, in descending order of accuracy (%), were as follows: 6-8kDa (90.8%), 129-143kDa (74.2%), 99-105kDa (71.7%) and 14-19kDa (71.1%). Cross-reactions, due to fascioliasis and hydatidosis, were observed in the four intervals of peptides highlighted. The results demonstrate that the total antigen of T. crassiceps has peptides with a high diagnostic potential; therefore, the immunoblot is useful in the diagnosis of bovine cysticercosis(AU)
Tendo em vista o conhecimento limitado sobre o diagnóstico da cisticercose bovina pelo immunoblot, o objetivo deste trabalho foi avaliar a aplicabilidade desse teste, identificando os principais peptídeos com valor diagnóstico. Foram realizados ensaios de immunoblot, utilizando antígeno total de larva de Taenia crassiceps e 60 soros de bovinos positivos para a cisticercose (30 naturalmente e 30 experimentalmente infectados com ovos de T. saginata), 30 soros de bovinos negativos para a cisticercose e 30 soros de bovinos com outras patologias (fasciolose, hidatidose e tuberculose). Os peptídeos de maior importância diagnóstica, em ordem decrescente de acurácia (%), foram os seguintes: 6-8 kDa (90,8%), 129-143 kDa (74,2%), 99-105 kDa (71,7%) e 14-19 kDa (71,1%). Reações cruzadas, em decorrência da fasciolose e hidatidose, foram observadas nos quatro intervalos de peptídeos ressaltados. Os resultados demonstram que o antígeno total de T. crassiceps possui peptídeos com alto potencial diagnóstico, sendo, portanto, o immunoblot útil no diagnóstico da cisticercose bovina(AU)
Asunto(s)
Animales , Immunoblotting/veterinaria , Cisticercosis/veterinaria , Bovinos/parasitología , Taenia saginata/parasitología , Cisticercosis/diagnóstico , Péptidos , AntígenosRESUMEN
O estudo de conidiobolomicose ovina tem sido realizado nos seus aspectos clínicos, epidemiológicos, patológicos e moleculares. Informações, entretanto, sobre a resposta imune do hospedeiro na infecção por Conidiobolus lamprauges são inexistentes. Este estudo teve por objetivo a identificação de proteínas imunorreativas que possam desempenhar papel importante na resposta imune de ovinos naturalmente infectados por C. lamprauges. Para a caracterização protéica e imunológica foi utilizada a cepa de C. lamprauges (FIOCRUZ-INCQS 40316) isolada de ovino com sinais clínicos de conidiobolomicose no Estado do MT e cinco amostras de soro de ovinos infectados naturalmente pelo fungo. A presença de anticorpos IgG foi observada em todos os animais doentes com títulos reagentes em diluições de até 1:1.600. Na técnica do immunoblot, o perfil antigênico frente aos soros ovinos com a doença apresentou doze bandas reativas, com massas moleculares variando de 35 a 198 kDa. Dentre estas, a proteína de 198 kDa foi reativa em 3 soros de ovinos e a de 53 kDa apresentou a maior intensidade comparativamente com outras bandas, sendo provavelmente imunodominante. Amostras de soro de animais sadios não apresentaram reatividade demostrando a especificidade da técnica. A presença de proteínas antigênicas de C. lamprauges e IgG específicos em soros de ovinos observados no presente trabalho poderá auxiliar no desenvolvimento de métodos de diagnóstico precoces e na utilização de proteínas candidatas a vacinas para o controle e prevenção da infecção em animais e humanos.(AU)
The study of sheep conidiobolomycosis has been carried out in its clinical, epidemiological, pathological and molecular aspects. Information, however, about the host immune response in infection Conidiobolus lamprauges is absent. This study aimed to identify immunoreactive proteins that may play an important role in the immune response of sheep naturally infected by C. lamprauges. For protein and immunological characterization, C. lamprauges (strain FIOCRUZ-INCQS 40316) isolated from a sheep with clinical signs of conidiobolomycosis in the MT state and five sera samples of naturally infected sheep were used. The presence of IgG antibody was observed in all patients with reagent titers in dilutions up to 1:1600. In immunoblot technique, the antigenic profile against infected sheep sera showed twelve reactive bands with molecular weights ranging from 35 to 198 kDa. Among them, the 198 kDa protein was reactive against sera from three sheep and the 53 kDa showed increased intensity compared to other bands probably being immunodominant. Healthy animal serum samples showed no reactivity demonstrating the specificity of the technique. The presence of antigenic proteins of C. lamprauges and specific IgG in sheep sera observed in this study may assist in the development of early diagnostic methods and the use of protein as candidate vaccines for the control and prevention of infection in animals and human.(AU)
Asunto(s)
Animales , Ovinos , Conidiobolus , Cigomicosis/veterinaria , Immunoblotting/veterinaria , Reacciones Antígeno-AnticuerpoRESUMEN
Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.
Asunto(s)
Cisticercosis/diagnóstico , Electroforesis en Gel Bidimensional/métodos , Immunoblotting/métodos , Enfermedades de los Porcinos/diagnóstico , Taenia solium/aislamiento & purificación , Animales , Antígenos de Protozoos/sangre , Cisticercosis/parasitología , Electroforesis en Gel Bidimensional/veterinaria , Immunoblotting/veterinaria , Proteómica/métodos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Porcinos , Enfermedades de los Porcinos/parasitología , Taenia solium/inmunologíaRESUMEN
Thirty-two water buffalo (Bubalus bubalis) calves aged 610 months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose-response study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I-III received the recommended vaccine dose (RD) twice 4 weeks apart, RD twice 18 weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16 weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson's correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P > 0.05).
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella abortus/inmunología , Brucelosis/veterinaria , Búfalos/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Vacuna contra la Brucelosis/administración & dosificación , Brucelosis/inmunología , Brucelosis/prevención & control , Búfalos/microbiología , Pruebas de Fijación del Complemento/veterinaria , Femenino , Inmunización Secundaria/veterinaria , Immunoblotting/veterinaria , Ganglios Linfáticos/microbiología , Trinidad y TobagoRESUMEN
Human pathogens have evolved to infect vertebrate hosts other than human beings without causing symptoms of the disease, thus permitting them to complete their life cycle and to develop into infectious forms. The identification and management of infected animals are alternatives to control dissemination of the disease and to prevent human illness. In the current study, the potential use of staphylococcal A or streptococcal G proteins was evaluated with enzyme-linked immunosorbent assays (ELISAs) for seroepidemiological studies. Sera were collected from animals that were representative of 23 different Brazilian wild mammals. A high protein A binding rate was observed in all animals, except for the orders Didelphimorphia, Artiodactyla, and Rodentia, in which affinity was medium or low. Affinity for streptococcal G protein was higher in animals of the order Artiodactyla, whereas no streptococcal G protein binding was observed in samples obtained from felines (order Carnivora). Bacterial protein binding to mammalian immunoglobulins was confirmed by immunoblotting. The results suggest that secondary detection systems should be better investigated in ELISA protocols before their implementation in seroepidemiological studies involving wild mammals.
Asunto(s)
Animales Salvajes/microbiología , Inmunoglobulinas/inmunología , Staphylococcus/inmunología , Streptococcus/inmunología , Animales , Animales Salvajes/inmunología , Artiodáctilos/inmunología , Artiodáctilos/microbiología , Proteínas Bacterianas/inmunología , Brasil , Carnívoros/inmunología , Carnívoros/microbiología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Immunoblotting/veterinaria , Roedores/inmunología , Roedores/microbiologíaRESUMEN
Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Considering the scarce information on the occurrence of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlândia, Minas Gerais State, Brazil, this study aimed to investigate the frequency of antibodies against these parasites in sheep sera from this region by using different serological methods. A total of 155 sheep serum samples were analyzed by the indirect fluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for the detection of IgG against T. gondii and N. caninum. Seroreactivity by IFAT showed 80% of samples with titers between 512 and 2048 for T. gondii (cutoff ≥ 64) and 78% presenting titers between 50 and 200 for N. caninum (cutoff ≥ 50). Seroreactivity by ELISA showed 75% of samples with ELISA index (EI) between 2.0 and 3.0 for T. gondii (cutoff ≥ 1.3) and 54% presenting EI between 1.3 and 2.0 for N. caninum (cut off ≥ 1.3). Discordant results by both tests were analyzed by immunoblot, resulting in a total seropositivity of 61% for T. gondii and 23% for N. caninum, with 41% to T. gondii only, 3% to N. caninum only, and 20% to both parasites. There was a significant positive association between seropositivity to T. gondii and age over one year (P<0.001), but such association was not found for N. caninum infection. In conclusion, as T. gondii and N. caninum infections are simultaneously present in sheep flocks of this region, it should be emphasized the importance to carry out a regular monitoring of Toxoplasma infection due to its high prevalence, its zoonotic potential and induction of reproductive disorders leading to economic losses. For neosporosis, sheep farmers should be instructed about the presence of the parasite in the flock, its risk factors and potential abortifacient role in sheep. Differential flock management could be valuable tool to establish the association of serological positivity and reproductive disease induced by N. caninum in sheep.
Asunto(s)
Coccidiosis/veterinaria , Neospora/inmunología , Enfermedades de las Ovejas/epidemiología , Toxoplasma/inmunología , Toxoplasmosis Animal/epidemiología , Factores de Edad , Animales , Anticuerpos Antiprotozoarios/sangre , Brasil/epidemiología , Coccidiosis/epidemiología , Coccidiosis/inmunología , Coccidiosis/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Immunoblotting/veterinaria , Inmunoglobulina G/sangre , Masculino , Ratones , Neospora/patogenicidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Ovinos , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/parasitología , Oveja Doméstica , Toxoplasma/patogenicidad , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/parasitologíaRESUMEN
During a seroepidemiological survey 2004-2006 from areas in Brazil endemic for American cutaneous leishmaniasis (ACL), serum samples from 10 dogs with ulcerated cutaneous lesions (S-ACL) and 52 asymptomatic dogs (AS-ACL) of unknown age and breed living in areas endemic for ACL were monitored for 1 year for ulcerated cutaneous lesions and immunoblotting using peroxidase-conjugated secondary anti-IgG, anti-IgG1 and anti-IgG2 dog antibodies. We reported that antibodies against Leishmania (Viannia) braziliensis in the sera of 22/52 dogs with asymptomatic disease showed intense reactivity to peptides larger than 66 kDa. We believe that dogs harboring subclinical amastigotes show an immunoblotting profile similar to that of symptomatic animals because a dog with self-healing presented antigens greater than 66 kDa. Such patterns can be exploited for diagnostic and epidemiological research for leishmaniasis.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Immunoblotting/veterinaria , Inmunoglobulina G/clasificación , Leishmania braziliensis/aislamiento & purificación , Leishmaniasis Cutánea/veterinaria , Animales , Enfermedades de los Perros/sangre , Enfermedades de los Perros/parasitología , Perros , Leishmaniasis Cutánea/sangre , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/inmunologíaRESUMEN
A dificuldade de se localizar cisticercos em bovinos com infecção discreta torna o diagnóstico tradicional da cisticercose bovina em matadouros um recurso de baixa sensibilidade, o que gera uma demanda de diagnóstico por métodos alternativos como o immunoblot. Para tanto, neste trabalho, foram realizados ensaios de immunoblot a fim de analisar os peptídeos responsáveis pela reação do antígeno vesicular de Taenia crassiceps. Foram utilizadas 28 amostras de soro de bovinos comprovadamente negativos e 28 amostras coletadas de bovinos infectados experimentalmente com ovos de Taenia saginata. Os resultados dos ensaios de immunoblot com as amostras de soros-controle mencionadas indicaram que os peptídeos de 4-6, 14 e 18kDa destacaram-se entre os demais, mostrando altas taxas de desempenho no diagnóstico da cisticercose bovina e uma aparência diferenciada, com área e largura maiores, ao contrário dos peptídeos de média e alta massa molecular, que se apresentaram sob a forma de linha e com reações inespecíficas. O immunoblot mostrou ter um potencial como uma alternativa de diagnóstico da cisticercose bovina, incluindo sua aplicação em animais vivos, como método auxiliar de diagnóstico em estudos epidemiológicos da doença.
The diagnosis of cysticercosis in bovines that presents discreet infection is committed by the conventional methodology of detection employed in slaughterhouses. This deficiency leads to the development of alternative methods, such as immunoblot, in order to improve the detection of this disease. An immunoblot assay was developed to detect peptides from Taenia crassiceps antigenand it was used to test 56 samples of bovine serum (28 confirmed as negative and 28 from experimentally infected animals with Taenia saginata eggs). The peptides 4-6, 14 and 18 kDa showed better performance in bovine cysticercosis diagnosis due to an enhanced appearance, once they showed higher area and wide, contrasting with medium and high molecular weight peptides, that showed unspecific reactions and discreet appearance. The obtained results indicated that immunoblot can represent a potential alternative in bovine cysticercosis diagnosis, including its use in live animals, improving the detection of this disease even in epidemiological studies.
Asunto(s)
Animales , Bovinos , Enfermedades de los Bovinos , Cisticercosis/diagnóstico , Cisticercosis/veterinaria , Immunoblotting/veterinaria , PéptidosRESUMEN
The Taenia solium larva causes cysticercosis in pigs, as well as economic losses for farmers and taeniosis in humans, constituting a public health problem. Infested pigs must therefore be identified before they enter the food chain. To this end, a dot blot assay was developed for the immunodiagnosis of porcine cysticercosis. A study was made of 44 pigs from different areas of Colombia that had all tested positive to cysticercosis, both by necropsy and the Western blot technique. Another group was formed comprising 44 pigs that had all tested negative to Western blot and necropsy. After analysing these 88 samples to validate the diagnostic assay, the result was a sensitivity of 86.4% and a specificity of 93.2%. The dot blot assay proved useful in diagnosing porcine cysticercosis. As the assay is easy to use in laboratories in endemic areas, as well as under field conditions, it is also appropriate for epidemiological studies.
Asunto(s)
Anticuerpos Antihelmínticos/análisis , Cisticercosis/veterinaria , Cysticercus/inmunología , Immunoblotting/veterinaria , Enfermedades de los Porcinos/diagnóstico , Animales , Cisticercosis/diagnóstico , Cisticercosis/epidemiología , Prevalencia , Sensibilidad y Especificidad , PorcinosRESUMEN
The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n=10) and G2 (uninfected group, n=8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r=0.62, P=0.05), MAT(s) x MAT (ah) (r=0.97, P<0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r= 0.14, P=0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis.