Experimental design

Two experiments were conducted in the present study.

All people who participated in the animal experiments were either veterinarians with over 10 years of experience in conducting animal experiments with birds or specifically trained by attending a FELASA C course, or were animal care takers, which were specialized in managing poultry under experimental conditions. All participants were specifically approved by the Lower Saxony State Office for Customer Protection and Food Safety (LAVES) to contribute to these animal studies.

Forty-eight one-day-old commercial broiler chickens were raised at the Clinic for Poultry and were randomly divided into two groups (vvIBDV-inoculated group and virus-free control). At seven and 14 days post hatch (dph), sera were collected for maternally derived antibody (MDA) detection. Twenty-four chickens were inoculated with vvIBDV at the age of 15 dph with a dosage of 10³ egg-infectious dose (EID)₅₀/bird via eye drop. Twenty-four chickens were kept as virus-free controls which received PBS. Clinical signs were monitored throughout the whole experiment. Six birds of each group were randomly selected and sacrificed at three, seven, 14 and 21 days post inoculation (dpi), when the experiment was terminated. Serum samples were collected for the detection of vvIBDV specific IgG antibodies by ELISA. BF was weighted to calculate the organ to body weight ratio. Pathological lesions were determined. Samples of BF, CT, as well as the middle of the caecum were formalin-fixed and sectioned for the detection of histopathological lesions. Samples of BF, CT and the middle region of the caecum were collected for immunohistochemical detection of vvIBDV-antigen, immune cells, mast and goblet cells. In addition, caecum content was collected at necropsy for gut microbiota composition analysis.

Experiment 2 was partially a repeat of Experiment 1 with a total of thirty-six broiler chickens. Eighteen chickens were inoculated with vvIBDV at a dose of 10³ EID₅₀/bird at 14 dph. The remaining eighteen chickens were kept as virus-free controls. Six broilers of each group were randomly selected and different to Experiment 1 necropsied at 10, 14 and 21 dpi, when the experiment was terminated. As in Experiment 1, serum samples were collected for MDA detection and at necropsy for the detection of vvIBDV specific IgG antibodies by ELISA. Parameters, which were investigated at necropsy in Experiment 2 as a repeat of Experiment 1 include: bursa/body weight ratio, histological lesions, viral-antigen detection, immune cell populations including T and B lymphocytes, mast cell, and goblet cells in the BF, CT, and caecum. Different to Experiment 1, gut microbiota composition of caecum content was only determined at 14 dpi.

In all experiments birds were randomly selected for necropsy (n = 6/group and time point in each experiment). They were stunned using blunt trauma, which was placed on the fronto-parietal region, and subsequently immediately killed by exsanguination, which was approved by the animal welfare committee of the Lower Saxony State Office for Customer Protection and Food Safety.