We investigated the antibody response to the Salmonella O4 antigen using the microplate agglutination test (MAT) and MAT combined with dithiothreitol (DTT) treatment using serum samples sequentially obtained from horses with equine paratyphoid, apparently healthy horses kept in the same stable, horses kept on farms near the farm that raised Salmonella enterica subsp. enterica serovar Abortusequi-infected horses, and horses kept in a non-endemic area of equine paratyphoid. DTT-MAT titers of samples collected from all symptomatic horses were tentatively positive (1:20 or more) and were maintained for a long time although those in MAT were negative (less than 1:320). In DTT-MAT, antibodies of the O4 antigen were detected in 8 of 15 (42.1%) serum samples collected from apparently healthy horses kept in the same stable. The antibody-positive rate of DTT-MAT in horses kept on farms near the farm where S. Abortusequi-infected horses were raised and horses kept in a non-endemic area of equine paratyphoid were 4.5% and 0.1%, respectively. The presence of IgG or IgA antibody against S. Abortusequi LPS in ELISA were confirmed in almost all DTT-MAT-positive sera. DTT-MAT would be useful in the serological diagnosis of equine paratyphoid because of its prolonged ability to detect a specific antibody against the somatic antigen produced by an S. Abortusequi infection.