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Not AvailableCell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne’s Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies againstMAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.ICA