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In Experiment 1, birds were injected intracerebroventricularly (i.c.v).
After a period of two weeks for acclimatisation, birds were injected in the left patagium with 20μl of 5 1 PHA-P (L8754 Sigma-Aldrich) in PBS, following Smits et al. [4].
Birds were injected subcutaneously with 0.1 mL of a bacterial suspension containing 1×107 CFU/ml of bacteria or with 0.1 ml of sterile PBS in the back of the neck.
A sample of six birds from the same species were used as controls; these birds were injected with just 20μl of PBS (thus do not eliciting an immune response; 2) and manipulated in the same way to obtain blood samples at the same time intervals.
Control birds were injected with PBS.
Seven weeks after onset of the experiment, birds were injected subcutaneously in the lower-neck region with 50 μl of 10 mg/ml KLH.
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The right wing-web of each bird was injected with 0.1 ml of a 5 mg/ml PHA-P solution (Sigma L8754, St Louis, MO, USA) diluted in phosphate-buffered saline.
To test disease susceptibility, at 2 days of age, each bird was injected intra-abdominally with 2000 pfu MDV (JM strain) and housed for up to 8 weeks in Horsfall-Bauer isolators.
Homogenates of bird organs (turkey spleen and chicken kidneys) were injected into the allantoic cavity of 10-day-old embryonated chicken eggs.
They were injected (i.p).
Mice were injected (i.t).
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