Trachea and cloaca swabs were stored in cold tryptose phosphate broth supplemented with antibiotics. The medium was clarified by low-speed centrifugation and the supernatant was harvested, aliquoted and stored at 270uC. Upon thawing, trachea and cloaca swabs sampled from the same bird on the same day were pooled and the viral RNA was extracted from 200 ml using a MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche). Subsequently, cDNA was synthesized using reverse primer 59-CACTGGGCACGGTGAGC-39 and part of the M1 gene was amplified by running 45 cycles of Light Cycler PCR using primer 59-CTTCTAACCGAGGTCGAAACGTA-39 as the reverse primer in the presence of the TaqMan fluorescent probe 59-6FAM-TCAGGCCCCCTCAAAGCCGA-X-ph. Negative and positive control samples were tested in parallel. The lower limit of detection was determined to be approximately 500 TCID 50 . Some samples gave inconclusive results, meaning that they gave only a very weak signal (fluorescence,0.07) after .31 cycles.