^aG, biotype gravis; M, biotype mitis. Cultures were kept lyophilized at room temperature or were stored in defibrinated sheep blood and held at -70°C until needed. Before use, the strains were inoculated onto blood agar plates (trypticase soy agar with 5% sheep blood; Becton Dickinson, Cockeysville, MD) and were incubated at 37°C overnight.
^bDNA for ribotyping was isolated by the universal isolation procedure (5). Hybridization of restricted DNA fragments was performed using a mixture of five digoxigenin-labeled oligonucleotide probes at 37°C for 4 hours as recently described by Regnault et al. (6). Posthybridization washes were also performed at 37°C in 2X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS) for 2x5 minutes and in 0.1X SSC, 0.1% SDS for 2x10 minutes. Detection was performed by using the DIG Wash and Block Buffer Set (Boehringer Mannheim Biochemicals, Indianapolis, IN), sheep anti-digoxigenin antibody conjugated with alkaline phosphatase, nitroblue tetrazolium chloride (NBT), and 5-bromo-4-chloro-3-indolylphosphate (BCIP).
^cNew = pattern has not been previously observed.