Blood samples were collected from 20 stalking-harvested wild boar. Blood was drawn from the thoracic major veins by exsanguination immediately after death. Two blood collection tubes were filled per animal and stored at 4°C during transport until the laboratory; although for three animals only one tube could be filled. Serum was obtained by centrifugation from one of the tubes and stored at 4°C. The second tube was used to obtain haemolysed serum after freezing the whole blood simulating the treatment that occurs when blood samples are sent frozen. In a subjective haemolysis-scale from 1 (almost nil) to 4 (severe), the haemolysed obtained sera were classified as level 3. After obtaining all fresh and haemolysed sera, the first ELISA test was performed. Sera were stored at -20°C for one week and thawed at 4°C for one night. One hour before performing the ELISA analysis, sera were removed from 4°C storage and brought to room temperature. After analysis, sera were frozen again to -20°C. This process was repeated 5 times.
All animal samples used for this study came from opportunistic sampling during legal hunting. No life animal was handled and no special permits were required.
A commercially available blocking ELISA was used for detection of antibodies to the gpI antigen of Suid Herpesvirus 1 (IDEXX HerdCheck Anti-ADV gpI, IDEXX, Inc., USA). This ELISA technique has been broadly used for testing antibodies to ADV in different wild boar populations [9–11].
The ELISA was performed following the manufacturer's instructions, in an ADV antigen-coated microwell plate, using a 1:2 serum dilution. One hundred microliters of diluted sera were added in each microwell and incubated for 1 hour at room temperature (RT). Samples, positive and negative controls were tested in duplicate in each plate. Subsequent to a wash step, 100 μl of anti-ADVgpI monoclonal antibody conjugate was added and incubated at RT for 20 minutes. If no gpI antibodies were present in the tested serum, the conjugated gpI antibodies were free to react with the gpI antigen. Conversely, if gpI antibodies were present in the tested serum, the enzyme-conjugated monoclonal antibodies were blocked from reacting with the antigen. Following the incubation, the unreacted conjugate was washed out and the reaction was revealed by adding 100 μl of substrate/chromogen solution. In the presence of substrate enzyme, reaction generated blue colour. After 15 minutes of revealing, the reaction was stopped with 50 μl/well of Stop solution and optical density (OD) was measured in a spectrophotometer at 650 nm.
Results were expressed as a percentage of inhibition (%IN) value using the following formula: [%IN = (mean negative control OD - mean sample OD/mean negative control OD) × 100]. The quantity of antibodies to ADV-gpI was inversely proportional to the OD and directly proportional to the %IN. According to the manufacturer's instructions, only samples with %IN values equal to or greater than 40% were considered positive. Samples with a %IN value between 30-40% were considered doubtful and sera with %IN values < 30% were classified as negative.