Development, validation and field evaluation of an indirect ELISA for the detection of antibodies against Brucella abortus in bulk and individual milk samples in dairy cattle.

Brucellosis is an abortigenic and zoonotic disease. In cattle, it is mainly caused by Brucella abortus. The disease is endemic in low- and middle-income countries, being considered a neglected zoonotic disease. In these countries, it is of high importance to develop and validate sensitive, specific and low-cost diagnostic assays for brucellosis. The aim of the present study was the development of an indirect enzyme-linked immune assay (iELISA) to detect anti-B. abortus antibodies in milk samples. We purified the lipopolysaccharide antigen from B. abortus and produced an anti-bovine IgG monoclonal antibody to develop an iELISA (iELISAINTA). The iELISAINTA was validated using 1730 bulk milk samples and 1734 individual milk samples. The sampled dairy herds had at least 3 years of consistency at their positive or negative official brucellosis status. Individual milk samples were taken in parallel with serum samples from the cows. The status of the cows was defined by the result of the complement fixation test (CFT) performed with their serum sample. The reproducibility of the assay was evaluated in two laboratories. In addition, we evaluated the performance of the assay in the field, using 4385 bulk milk samples and 968 individual milk samples. The results of the iELISAINTA were compared with those obtained using the officially accepted brucellosis techniques: iELISA from Canada (iELISACFIA) in milk samples, and the buffered plate antigen (BPA) and the CFT in serum samples. At validation, the sensitivity (Se) of the iELISAINTA in bulk milk samples was 98.61 %, and the specificity (Sp) 98.79 % with a ≥ 10 % of positivity (PP) cutoff. In individual milk samples, the Se was 98.04 %, and the Sp 98.56 % with a ≥ 16 PP cutoff. The chance-corrected agreement kappa value (κ) between the results obtained in the different laboratories was κ = 0.87. In the field evaluation, in bulk milk samples the κ value between the iELISAINTA and the iELISACFIA was κ = 0.86. On individual milk samples, the κ values were: between the iELISAINTA and the iELISACFIA κ = 0.79, between the iELISAINTA and BPA was κ = 0.85, and between the iELISAINTA and CFT κ = 0.82. The developed iELISAINTA showed a very good performance and it could be used as a screening assay for anti-B. abortus antibodies detection in individual milk samples and for epidemiologic surveillance in bulk milk samples.

Utilización de microoganismos marcadores para la evaluación de las condiciones higiénico-sanitarias en la producción primaria de leche / Use of indicator microorganisms for the hygienic-sanitary conditions evaluation in the milk primary production

Los microorganismos marcadores presentes en los alimentos advierten sobre la manipulación inadecuada de la materia prima o el alimento, la presencia de un peligro para el consumidor o una deficiencia de los procesos destinados a su saneamiento. Estos marcadores son herramientas importantes para poder desarrollar registros microbiológicos históricos, a partir de los cuales se pueden establecer valores propios de referencia alcanzables con la metodología de trabajo desarrollada en cada empresa. Dichos valores permitirán, además, implementar los sistemas que garantizan la inocuidad de los alimentos, como es el caso del Análisis de Peligros y Control de Puntos Críticos (HACCP, por su sigla en inglés). El objetivo del trabajo fue estudiar los principales microorganismos marcadores de higiene en leche cruda como herramienta para evaluar las condiciones higiénico-sanitarias en la producción primaria, de tal forma que permitan elaborar un diagnóstico de la situación inicial como paso previo a la implementación de un sistema HACCP. Para el estudio, y a manera de experiencia piloto, se utilizó leche cruda proveniente de un establecimiento productor de la provincia de Santa Fe (Argentina). Las muestras se recolectaron directamente de la ubre, luego del prefrío (intercambiador a placas con agua a temperatura ambiente), tanque de enfriamiento, manos del ordeñador, pezoneras y agua empleada en el establecimiento. Se realizó 1 muestreo por semana hasta completar 44 muestreos distribuidos a lo largo del año. Los marcadores estudiados fueron: recuento total de microorganismos psicrotrofos, mesófilos, termodúricos, mohos y levaduras, enterobacterias y Staphylococcus aureus. Se determinó, además, la presencia de clostridios sulfito reductores y Salmonella spp. El análisis de correlación entre los microorganismos marcadores no permitió establecer predicciones sobre el comportamiento de un grupo microbiano con base en el proceder de otro...

The indicator microorganisms present in the food notify on the inadequate manipulation of the commodity or the food, the presence of a hazard for the consumer or a processes deficiency. These indicator microorganisms are important tools to develop historic microbiologic records, from which own values of compassable reference with the methodology of work developed in each business can be established. These values will permit, besides, to implement the systems that guarantee the food safety, as is the case of the Hazard Analysis of Critical Control Points (HACCP). The objective of this work was to study the main hygiene indicator microorganisms in raw milk as tool to evaluate the sanitary conditions in the primary production, in such a way that they permit to devise a diagnosis of the initial situation like prior step to the implementation of a HACCP system. For the study, and to way of pilot experience, raw milk originating from a producing establishment of the Santa Fe Province, Argentina, was utilized. The samples were collected directly from the udder, after the first cooling, bulk tank, milker hands, teat cup and water employed in the dairy farm. One sampling was carried out for week until completing 44 samplings distributed along the year. The indicator microorganisms studied were: psychrotrophic count, mesophilic count, thermoduric, molds and yeasts, coliforms and Staphylococcus aureus. It was determined, besides, the presence of sulphite-reducting clostridia and Salmonella spp. The correlation analysis among the indicator microorganisms did not permit to establish predictions on the behavior of a microbe group based on the behavior of another. Nevertheless, the information that each one of the indicator microorganisms turned out to be adequate to evaluate the sanitary conditions in the milk primary production and to generate records that will be useful for the HACCP system implementation.