The kinetics of antibody production against listeriolysin O (LLO), a major virulence factor of the intracellular bacterial pathogen Listeria monocytogenes, was studied by dot blot analysis with highly purified LLO during oral infection of sheep. Specific antibodies appeared as soon as day 9 of an oral infection and peaked by day 20 of infection; specific antibody levels then remained almost stable for at least 4 months. A subclinical infecting dose (approximately 10(6) viable bacteria) was capable of eliciting a significant antibody response to LLO, almost at the same level as that observed with a high-dose oral challenge (approximately 10(10)). Antibodies to LLO were mostly constituted by immunoglobulin G (IgG), since an IgA response was not detectable and only a transient and inconstant IgM response was observed between day 9 and day 20 of an oral infection. These results show that antibodies to LLO are constantly produced during oral infection even with a low infecting dose, thus confirming that LLO is highly immunogenic. Detection of antibodies to LLO can therefore be used to detect sheep that have been previously exposed to L. monocytogenes.