Thus, it is important to better understand the potential for cross-reactivity among these mycoplasmas and determine their ability to impact assay specificity as has been previously described (Ameri et al.,2006; Freeman et al., 1984). There are few sets of definedstatus serum samples from pigs infected with only a single species of mycoplasma and no similar pen-base oral fluid samples to investigate these potential cross-reactions. Therefore, the goals of this study were to generate paired serum and oral fluid samples from swine infected with a single-species of mycoplasma, estimate the extent of cross-reactivity between these mycoplasmas using both commercial ELISAs and in-house Tween 20 detergent-base ELISAs, and evaluate the use of pen-based oral fluids for antibody detection