The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB ( ), healthy animals ( ), and animals experimentally infected with M. bovis ( ). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB ( ), healthy animals ( ), and animals experimentally infected with M. bovis ( ). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB. 1. Introduction PTB is a prevalent and economically important disease that affects cattle and thus impacts on the cattle industry. It is caused by MAP. Clinical PTB is characterized by chronic granulomatous enteritis with clinical signs of diarrhea, weight loss, decreased milk production, and mortality. However, most infected cattle show no clinical signs during the prolonged incubation stage of infection [1]. On the other hand, a number of theories have proposed that the principal infective agent of Crohn’s disease, a chronic enteropathy in humans, is MAP [2–4]. The economic impact and possible link to Crohn's disease highlights the importance of the development of control programs at the herd level. To this end, it is necessary to improve the diagnostic methods of PTB. Cattle are most often infected as young calves, before 6 month of age, but some studies have shown that infection may also occur in adult cattle. Fecal shedding of MAP generally starts after 2 years, and clinical symptoms appear after an incubation period of 2–10 years. In addition, the elimination of the agent through the stool is very variable [5]. Cell-mediated immune response wanes with progression of the disease and when this occurs, a humoral immune response becomes measurable. It has been shown that cattle are more likely to have a combined antibody and
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