Serological patterns of Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Pasteurella multocida and Streptococcus suis in pig herds affected by pleuritis

Wallgren, Per; Nörregård, Erik; Molander, Benedicta; Persson, Maria; Ehlorsson, Carl-Johan

Detection of antibodies to S. suis was made by an indirect ELISA designed for that purpose. Each well in a microtiter plate (Greiner Bio-one, Sigma-Aldrich) was coated with 100 µL of the sonicated antigen diluted 1/10,000 in PBS-T in room temperature for 18 h. Thereafter the microtiter plate was washed three times with PBS-T, and 100 µL serum diluted 1/100 in PBS was added to duplicate wells and the plates were incubated at 37 °C for 1 h. The plates were again washed three times with PBS-T and 100 µL of the conjugate (Protein A-horseradish peroxidase conjugate, Bio-Rad, Richmond, USA) diluted 1/5000 with PBS-T was added to each well and the microtiter plates were stored for 1 h in 37 °C. Then the plates were again washed three times with PBS-T and 100 µL of the substrate with tetra methylbenzidine (TMB, SVANOVA Biotech, Uppsala, Sweden) was added to each well. The reaction was stopped with 100 µL H 2 SO 4 after 10 min and the absorbance was read at 450 nm by a spectrophotometer (Multiscan MCC/340 ® MK type II, Labsystem OY, Helsinki, Finland). The results obtained were adjusted to A 450 = 1.0 for a positive standard serum and absorbance values exceeding 0.5 were regarded as positive reactions, based on the mean absorbance value +2 standard deviations of samples from 72 pigs without clinical signs of S. suis infection (A 450 = 0.26 ± 0.12).