Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PCR

Reported strains of Piscirickettsia salmonis, a pathogen of salmonid fishes , were analyzed by amplifying part of the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) operon followed by denaturing gradient gel ele ctrophoresis (DGGE) of the amplicons. All amplified fragments differing in sequence were distinguished by migration during DGGE. A simpler format, con stant denaturant gel electrophoresis (CDGE), allowed the same diagnostic di stinctions among strains. Sampling during 1997 and 1998 of salmonids from 5 different sites on and near Chiloe Island in southern Chile displaying pis cirickettsiosis revealed only P. salmonis resembling LF-89, the type strain first isolated in 1989. These observations are encouraging for control str ategies, which might otherwise be compromised by unpredictable shifts of P. salmonis types in salmon farms. A competitive PCR assay offered insight ab out the power of PCR for quantification and about specific tissue invasiven ess by this intracellular pathogen. This approach revealed that the PCR cou ld amplify approximately 1 to 10 P. salmonis genome equivalents against a b ackground of > 99.9 % salmonid DNA. Ii: also raised the possibility that th e salmonid brain is an important site for P. salmonis survival, with its ba cterial load in 1 individual having been about 100 times the loads observed in liver and kidney. Pathogen detection by competitive PCR in a surface se awater sample from a netpen in use indicated a density of about 3000 to 400 0 P. salmonis cells (or their DNA remnants) 1(-1). Such quantitative estima tes should aid decisions about disease prevention and management as, for ex ample, choice of netpen sites following fallow periods and certification of ova, which are known conduits of infection.