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Identification of Mycobacterium avium subspecies paratuberculosis by PCR techniques and establishment of control programs for bovine paratuberculosis in dairy herds Identificación de Mycobacterium avium subspecies paratuberculosis mediante pruebas de PCR y definición de programa de control de la paratuberculosis bovina en hatos lecheros Identifica o de Mycobacterium avium subespécie paratuberculosis por PCR e defini o do programa de controle da paratuberculose em rebanhos bovinos leiteiros

Keywords: bovinos de leche , caquexia en vacas , cultivo bacteriológico , enfermedad de Johne , paratuberculosis bovina , PCR en tiempo real , a doen a de Johne , caquexia em vacas , cultura bacteriológica , gado leiteiro , paratuberculose bovina , real-time PCR , bacterial culture , paratuberculosis , caquexia in cows , dairy cows , Johne's disease , real–time PCR

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Abstract:

The aim of this study was to establish the protocol of conventional and real time PCR for amplification of Mycobacterium avium subsp. paratuberculosis (MAP) genome from bovine fecal samples, as a way to define strategies for establishing a prevention and control program in a dairy herd at the Universidad de Antioquia (Medellín, Colombia). Fecal samples were individually taken of clinical healthy cows or cows with diarrhea bred in a herd enzootic for Johne's disease, were processed them for culture in liquid Middlebrook 7H9 media supplemented with mycobactin under two different protocols: with or without inhibitors. Fecal samples from clinically healthy cows were used as negative control. Conventional and real time PCR were performed with MAP DNA obtained of fecal or cultured samples. The MAP- specific IS900 segment was amplified by using the respective forward and reverse primers. DNA isolated from a reference MAP strain was used as positive control of PCR. Data were analyzed by descriptive statistics and simple regression analysis between PCR and culture results were performed. All samples cultured in media with or without mycobactin gave a positive result compatible with MAP growth. However, only 13,3% of samples were positive by real time PCR. There was no relationship neither between PCR and culture results, nor between clinical condition of the cow and MAP positivity. These results support the need combine culture of feces with PCR diagnosis for identification of MAP-excreting cows in a dairy herd. Finally, a strategy of prevention and control of bovine paratuberculosis is proposed for this enzootic herd and for dairy herds in Colombia. El objetivo de este trabajo fue establecer un sistema de detección y amplificación del Mycobacterium avium paratuberculosis (MAP) a partir de muestras de materia fecal bovina, mediante el uso de la técnica de PCR en tiempo real, como estrategia de apoyo para el establecimiento de un programa de prevención detección y control de la paratuberculosis bovina en el hato lechero de la Universidad de Antioquia. Muestras de materia fecal fueron tomadas de bovinos provenientes de un hato enzoótico para la enfermedad de Johne, fueron cultivadas en medio de cultivo líquido Middlebrook 7H9 suplementado con micobactina, bajo dos protocolos de aislamiento: con o sin inhibidores. Como control negativo fue utilizada muestras de materia fecal de bovinos clínicamente sanos. Adicionalmente, con el DNA de las muestras aisladas en cultivo y de las muestras de materia fecal, fueron hechas pruebas de PCR convencional y en tiempo real, para

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