Purified listeriolysin O (LLO) was evaluated as a specific antigen to detect both humoral and cell mediated immune responses of sheep infected with Listeria monocytogenes. Six sheep (two in each group) were orally inoculated with 10(10) organisms of L. monocytogenes, L. ivanovii, or L. innocua. Only the L. monocytogenes inoculated sheep had an elevated temperature (> 42 degrees C) and after 15 days had anti-LLO antibodies as assessed by an ELISA. In a blastogenesis assay, only peripheral blood mononuclear cells (PBMC) from L. monocytogenes-infected sheep responded to LLO, while PBMC from all the sheep responded somewhat to heat-killed L. monocytogenes bacteria. In a skin test, only L. monocytogenes-infected sheep exhibited a positive reaction to injected LLO, while all the Listeria-infected sheep reacted to heat-killed bacteria. On day 120 postinfection, all of the sheep were orally inoculated with L. monocytogenes. Only the four that had not been previously given L. monocytogenes exhibited an elevated temperature (> 42 degrees C). 80 days later, sera from all of the animals were positive for anti-LLO antibodies. Thus, prior exposure to L. ivanovii or L. innocua does not protect against a L. monocytogenes challenge. These results suggest LLO is an excellent antigen for use in detecting Listeria infection in sheep. However, whether LLO will be useful in differentiating chronically infected animals from animals that have recovered, has yet to be investigated.