Whole-Blood Infection Assay for Poultry Immunity

The whole-blood infection assay was performed as described [24 (link)]. Six chickens per line (in total 12 animals) were used. Blood samples from two to four animals (one or two from each line) were tested on the same day. Briefly, peripheral blood was drawn by wing venipuncture (Vena ulnaris) into commercial hirudin-coated syringes (S-Monovette®, 2.7 mL Hirudin, Sarstedt, Germany). Hirudin was chosen as anticoagulant as it was previously shown to have no effect on thrombocytes and complement activation [24 (link), 26 (link)]. The number of 10⁶ GFP-expressing microbial cells was added to 1 mL of blood. Samples were incubated at 41 °C, 5% CO₂ under constant rotation (20 rpm, Tube rotator, VWR, Darmstadt, Germany, product number VWRI444-0500) for 240 min. Every 30 min, a volume of blood was taken for flow-cytometric analysis (20 µL), determining the colony forming units of the pathogens (cfu, 10 µL) and for establishing the transcription levels of selected immune-related genes by RT-PCR (50 µL; 0, 30, 90, 150 and 240 min after inoculation of bacteria).