Total concentrations of immunoglobulin subsets were measured by ELISA as previously described [38] . Plasma samples were diluted 1:6000, 1:4000 and 1:60,000 to detect IgM, IgA and IgG, respectively, in Tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (Bethyl laboratories Inc., Interchim, France). The different subsets were detected with the appropriate peroxidase anti-pig IgM, IgA or IgG (Bethyl laboratories Inc.) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. Anti-M. hyopneumoniae IgG titers were also measured by ELISA using a commercial kit (ELISA ID ScreenH M. hyopneumoniae Indirect, IDVET, France). Absorbance was read at 450 nm using an ELISA plate reader (Spectra thermo, Tecan, NC, USA) and the Biolise 2.0 data management software.