COMPARISON OF FOUR MOLECULAR TYPING METHODS FOR RIEMERELLA ANATIPESTIFER

Abstract

The pathogen causing septicaemiae and infectious serositis in waterfowl, Riemerella anatipestifer has been categorized as a member of Flavobacteriaceae by 16S rDNA sequence analysis and at least 21 serotypes have been characterized through serum agglutination test. So far, the molecular determinants for R. anatipestifer serotyping are not clear; moreover, serotype analysis is time-consuming and labor-intensive. To find a better molecular typing method for R. anatipestifer, 58 field isolates collected from sick birds in central Taiwan from 2006 to 2008 were investigated by molecular methods to determine serotype-associated genetic variations. Genetic variations were determined by the methods of ITS nucleotide sequence, OmpA amino acid sequence, pulsed-field gel electrophoresis (PFGE), and repetitive-sequence PCR (Rep-PCR). The phylogenetic trees constructed by the ITS region showed nine clusters, while the amino acid sequence of OmpA clustered into seven clusters. OmpA sequence correlated much better with serotyping than ITS, PFGE, and Rep-PCR. Meanwhile, the biofilm formation activity was also tested for serotypes 2 and 8, which were the two largest groups in the study. Serotype 2 isolates showed a higher degree of biofilm formation than serotype 8 isolates. In conclusion, we demonstrated that phylogenetic analysis with OmpA amino acid sequence is an effective tool in epidemiology and may be a candidate method substituting serotyping for R. anatipestifer. Moreover, the biofilm forming activity may play an important role in field transmission of R. anatipestifer.