In the BAL fluid, IgG,
IgM and IgA antibodies against M. hyopneumoniae were
detected by using peroxidase-labeled goat anti-porcine
IgG, IgM and IgA polyclonal antibodies (Bethyl Laboratories),
as previously reported by Marchioro et al. (2013).
The BAL fluid was tested undiluted. The Optical Density
(OD) value was measured at 450 nm and the mean OD
value of the serum or BAL fluid from the non-vaccinated
animals plus two folds the SD was used as cut-off value to
determine the number of positive samples in the
vaccinated groups for each immunoglobulin. Values equal
to or higher than the cut-off were considered as positive.