The objective of this study was to determine the association of ante mortem test results with presence of disseminated MAP infection at slaughter. Twenty adult lactating Holstein cows were obtained for slaughter from October 2003 to February 2004 from 4 Colorado dairies with a known history of clinical JD. Cows that were purchased had a history of at least one positive serum ELISA result. Prior to euthanasia, serum was collected for ELISA and feces for conventional and BACTEC culture and liver biopsy was performed for histologic exam. At necropsy 16 specimens, including feces, lymph nodes, and muscle were submitted for conventional culture. Cows with at least one positive tissue culture beyond the intestine and its associated LN were considered to have disseminated infection (DI). Sixteen of 20 cows had confirmed MAP infection via culture. Twelve of these had disseminated infection (DI). Of the 12 cows with DI, 5 had no evidence of clinical JD, 3 had weight loss without diarrhea, and 4 had obvious clinical JD. The most common tissue found culture positive for MAP in DI cows was the hepatic LN (11 of 12 DI cows). Fecal culture (FC) with BACTEC and conventional FC detected fecal shedding in 7 and 11 of 12 DI cows, respectively. Of 19 ante mortem liver biopsies collected, 4 were affected with multifocal granulomatous hepatitis, but no acid-fast bacteria were identified. Of the four cows that had negative ELISA results at euthanasia, 3 were infected and 2 had DI, despite previous positive serum ELISA results. If the perceived human health risk leads to mandatory exclusion of cows with disseminated MAP infection from the food supply, serum ELISA may be an option for detecting a large proportion of DI cattle ante mortem.