Nonspecific reactions were blocked by use of normal swine serum for 20 min.
Following fixing, cells were washed in TBS (50 mM Tris-HCl, 150 mM NaCl pH 7.4) and blocked using 5% normal swine serum diluted in TBS.
Sections were blocked using 5% normal swine serum diluted in PBS with 5% BSA.
The infected cells were fixed with methanol 48 hours after infection, and an indirect immunofluorescence assay was conducted by using anti-SIV swine serum (primary antibody) and a secondary fluorescein isothiocyanate conjugated anti-swine antiserum (Sigma).
A serologic survey was conducted of 214 fattening pigs on June 30 and every week thereafter by using ELISA (HerdChek Swine Influenza H1N1 Antibody Kit; IDEXX Laboratories, Westbrook, ME, USA).
For anti-Müllerian hormone (AMH) and α-smooth muscle actin (SMA) immunostaining, normal rabbit serum was used; for all other antibodies, normal swine serum was used (Scottish Antibody Production Unit, Carluke, Scotland, UK).
After the nonspecific staining had been blocked by normal swine serum, sections were incubated with the primary antibodies for between 50 and 60 min.
Isolated influenza virus was subtyped by RT-PCR analysis by using primers specific for canine, swine, and avian hemagglutinin 3 (H3) genes.
Methane production from swine wastewater was evaluated by using three inoculums: rumen (I1), stabilized swine wastewater (I2) and sewage sludge (I3).
Inasmuch as we identified a swine lineage virus in waterfowl, we first investigated laboratory contamination by using trace back and history of swine virus isolations during the time the surveillance samples were processed.
