Animal experiments:
To generate clinical samples for validation of the triplex assay, a total of 19 Landrace x large white crossbred pigs (11 piglets at three weeks of age and eight weaner pigs at seven weeks of age) were purchased from a commercial swine farm in Southern Manitoba and transported to the National Centre for Foreign Animal Disease (NCFAD) animal facility. After seven days of acclimatization, pigs were randomly assigned to three groups: Group 1 (eight 7-weeks old pigs for PRV-JS-2012, Pig# 5-12), Group 2 (eight 3 weeks-old piglets for PRV-JS-2012, pig # 13-20) and Group 3 (three 3 weeks old piglets for PRV-Bristol, Pig # 24-26). Each group was housed in an individual pen. Each pig in Group 1 and 2 received 2 ml of PRV Strain JS-2012 (105 TCID50/ml, 1 ml in each nostril) and each pig in Group 3 received 2 ml of PRV Bristol strain (105 TCID50/ml, 1 ml in each nostril). After the challenge, all animals were monitored twice daily for clinical signs (fever, neurological, respiratory etc.); and oral fluid, nasal and oropharyngeal swabs were collected every other day starting at 2 days post infection (dpi) until 14 dpi. Pigs that reached humane endpoint were euthanized and tissue samples (tonsils, spleen, lung, brain, trigeminal ganglia, kidney, and lymph nodes) were collected. The remaining animals were euthanized at 14 days post infection and similar samples as above were collected. All the samples were stored at -80ÂșC until tested. The animal experiment was approved by the Animal Care Committee at the Canadian Science Centre for Human and Animal Health. The guidelines from the Canadian Council for Animal Care were observed during all procedures with animals.