This study describes the response of cattle to use of a dot-ELISA test when using serum absorbed with Mycobacterium phlei. In addition, results obtained when using visual observation are compared with those obtained when using a densitometer. Infection status of cattle was determined by fecal culture. Cattle of different level of exposure and disease manifestation were examined. A significant increase of dot-ELISA response was found in animals with heavy Mycobacterium paratuberculosis shedding using both unadsorbed and adsorbed serum, as compared to animals that were fecal culture negative or shedding M. paratuberculosis at lower levels (P < 0.05). Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture positive clinical suspects, and in 85 of 93 (91.4%) of the fecal culture negative cattle when using unadsorbed serum. On adsorbing the serum the sensitivity of the visual determination decreased to 34.1% (15/44) and the specificity increased to 97.9% (91/93). About 75% of the dot-ELISA positive cattle were heavy bacterial shedders at the time of serologic testing with < 1500 colonies/g of feces tested in the fecal culture test. Effects of sensitivity and specificity at various cut-off points were determined by use of video densitometric measurements because sera have not discretely segregated into distinct groups of positive and negative results. Comparing the dot-ELISA results determined by visual vs the objective densitometric measurement showed compatible results for test specificity. Test sensitivity using visual evaluation was 66% and 87.5% using the objective densitometric evaluation for unadsorbed sera at an ELISA cut-off value of 0.2 optical density. This difference was even more pronounced when adsorbed sera were used.