ABSTRACT
La fasciolosis es una parasitosis cosmopolita de importancia médico-veterinaria ocasionada por Fasciola hepatica, que afecta al ganado ovino, caprino y vacuno; y accidentalmente al hombre ocasionando una infección endemo-epidémica de difícil diagnóstico. El objetivo fue desarrollar un ELISA sándwich indirecto, empleando 3 anticuerpos, para identificar antígenos de secreción-excreción de Fasciola hepatica (ESFh). Para el ELISA se emplearon anticuerpos policlonales de ratón anti ESFh como anticuerpos de captura, anticuerpos policlonales de conejo anti ESFh como anticuerpos de detección, a las concentraciones de 10 y 5 μg/mL, respectivamente. Como conjugado se emplearon anticuerpos monoclonales de ratón anti-inmunoglobulinas totales de conejo ligado a peroxidasa 1/1000). Se analizaron 31 muestras de heces de ganado ovino y los resultados se compararon con los obtenidos por el examen coproparasitológico directo (CD) y contrainmunoelectroforesis (CIEF). El límite de detección obtenido para ELISA sándwich indirecto fue 100 ng/mL. La prueba presentó una sensibilidad de 100%, especificidad de 96.6% y valores predictivos positivos y negativos de 50% y 96.6% respectivamente; con relación al examen CD. Al comparar ELISA tipo sándwich indirecto con CIEF se obtuvo una especificidad de 93.5% y un valor predictivo negativo del 100%. Se concluye que la prueba de ELISA sándwich indirecto diseñada es capaz de detectar antígenos metabólicos en muestras de heces de ovino y se puede utilizar para el diagnóstico Fasciola hepatica.
Fasciolosis is a cosmopolitan parasitosis medical-veterinary importance caused by Fasciola hepatica, which affects sheep, goats and cattle; and it affects man accidentally causing an epidemic-endemic infection difficult to diagnose. The aim was to develop an indirect sandwich ELISA with 3 antibodies for detecting excretory-secretory antigens of Fasciola hepatica (ESFh). For the development of indirect sandwich ELISA were used, as capture antibody, mouse polyclonal antibodies anti ESFh and polyclonal antibodies rabbit anti-ESFh as detection antibody, at the concentrations of 10 and 5 μg/mL respectively. The conjugate used was mouse monoclonal anti- total immunoglobulins rabbit linked to peroxidase (1/1000). Were analized 31 sheep fecal samples, and the results were compared with those obtained by direct coproparasitological examination (DC) and counterimmunoelectrophoresis (CIEP). The detection limit obtained for indirect sandwich ELISA was 100 ng/mL. The test had a 100% sensitivity, 96.6% specificity, positive and negative predictive values of 50% and 96.6% respectively, in relation to DC test. Comparing with CIEP the specificity obtained for indirect sandwich ELISA was 93.5% and a negative predictive value of 100%. We concluded that indirect sandwich ELISA designed is able to detect metabolic antigens in ovine feces samples and can be used for Fasciola hepatica diagnosis.
ABSTRACT
Animal trypanosomiasis is a disease caused by parasites of the genus Trypanosome. This malady is widely distributed in many countries, located in tropical and subtropical areas of the world where blood-sucking flies are present. Water buffaloes are important domestic animals used for meat and milk production, and draught power. Buffalo herds are raised in areas where trypanosomiasis is endemic. In Venezuela, the buffalo industry is becoming a very important and common livestock. However, animals imported from non-endemic areas may suffer severe infections. The development of methods which ensure an efficient epidemiological surveillance against this disease is of great relevance. The immunological tests are of great importance for this purpose, because of the low sensitivity of the current parasitological methods, due to the low parasite burden that occur in subclinical and chronic infections caused by trypanosomes. To estimate the serological prevalence of trypanosome in water buffaloes, an enzyme-linked immunosorbent assay (ELISA) was used in buffalo samples of healthy animals from the municipalities of Rómulo Gallegos, Ricaurte and Girardot, in the State of Cojedes, Venezuela. Additionally, samples were also assessed with the indirect fluorescence antibody test (IFAT) and the microhematocrit test (MHCT). A total of 180 blood samples, none of which had an active parasitemia by TMC, were assessed. The prevalence determined by ELISA was 45.56%, which was significantly (P<0.05) higher than that obtained by IFAT (28.89%). The results of the experiments showed a moderate Kappa index of concordance of 0.45 (95% CI: 0.31-0.58); whereas the concordance value for both tests was 73.33%. Both the sensitivity and specificity of ELISA, compared to the IFAT, was 82.69% and 69.53%, respectively. The predictive positive and negative values were 52.44% and 90.82%, respectively. The findings suggest an endemic condition, with moderate infection values caused by Trypanosoma spp. in buffaloes from these regions of Venezuela and show, for the first time, the usefulness of ELISA for epidemiological studies of trypanosomiasis.
ABSTRACT
Resumen Se estandarizó una prueba de ELISA para la detección de anticuerpos anti-F. hepatica en leche de bovinos. Se emplearon 80 muestras, de las cuales 44 se usaron como control positivo y 36 como control negativo. Como control de reactividad cruzada se utilizaron muestras de bovinos que presentaron en las heces huevos de un paramphistomido. La sensibilidad y especificidad diagnóstica de la prueba fueron del 80% y del 72,2% respectivamente, con un intervalo de confianza del 95%. Los resultados obtenidos son comparables con los reportados en estudios realizados en otros países. Se propone la prueba estandarizada como herramienta diagnóstica alternativa y de tamizaje en estudios epidemiológicos de fasciolosis.
Abstract We standardized an ELISA test for the detection of anti-F. hepatica antibodies in bovine milk. A total of 80 samples were used, of which 44 were used as positive controls and 36 as negative controls. Milk samples from bovines presenting paramphistomidae eggs in feces were used as cross-reactivity controls. Sensitivity and specificity of the diagnostic test were 80 and 72,2%, respectively, with 95% confidence interval. These results are comparable with those reported in studies conducted in other countries. We propose this standardized test as an alternative diagnostic and screening tool in epidemiological studies of fasciolosis.
Resumo Estandardizou-se um teste de ELISA para a detecção de anticorpos anti Fasciola hepatica no leite de bovinos. Empregaram-se 80 amostras, das quais 44 usaram-se como controle positivo e 36 como controle negativo. Como controle da reatividade cruzada utilizaram-se amostras de bovinos que apresentaram ovos de um paramphistomido nas fezes. A sensibilidade e especificidade diagnosticam do teste foi de 80 e 72% respectivamente, com um intervalo de confiança de 95%. Os resultados obtidos são comparáveis com os reportados em estudos realizados em outros países. Propõe-se o teste estandardizado como ferramenta diagnostica alternativa e de tamisado em estudos epidemiológicos de fasciolíase.