To determine the level of viral shedding of the different recombinants, 10 birds per group were inoculated with 200 μL allantoic fluid containing 105 EID50 of the rH120, rIBYZ, or rH120-S1/YZ strains, respectively. 200 μL PBS per chick was administered to the control group via eye drop and the intranasal route. . Oral and cloacal swabs were collected from each bird into 1 mL PBS at 7, 14, 18, 22, 26, and 30 dpi and stored at −80 °C. After freezing and thawing three times and centrifuging at 8,000 × g for 5 min at 4 °C, relative amount of virus present in 200 μL of supernatants of each sample was quantified by RNA extraction, reverse transcription, and real-time PCR as described previously.