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 electrophoresis (DGGE) of the amplicons. All amplified fragments differing in sequence were distinguished by migration during DGGE. A simpler format, constant denaturant gel electrophoresis (CDGE), allowed the same diagnostic distinctions among strains. Sampling during 1997 and 1998 of salmonids from 5 different sites on and near Chiloé Island in southern Chile displaying piscirickettsiosis revealed only P. salmonis resembling LF-89, the type strain first isolated in 1989. These observations are encouraging for control strategies, which might otherwise be compromised by unpredictable shifts of P. salmonis types in salmon farms. A competitive PCR assay offered insight about the power of PCR for quantification and about specific tissue invasiveness by this intracellular pathogen. This approach revealed that the PCR could amplify approximately 1 to 10 P. salmonis genome equivalents against a background of > 99.9% salmonid DNA. It also raised the possibility that the salmonid brain is an important site for P. salmonis survival, with its bacterial load in 1 individual having been about 100 times the loads observed in liver and kidney. Pathogen detection by competitive PCR in a surface seawater sample from a netpen in use indicated a density of about 3000 to 4000 P. salmonis cells (or their DNA remnants) 1(-1). Such quantitative estimates should aid decisions about disease prevention and management as, for example, choice of netpen sites following fallow periods and certification of ova, which are known conduits of infection.
Heath
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Department of Biology, San Francisco State University, California 94132, USA.
Pak
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Marshall
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Prager
E M
EM
Orrego
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C
D000818:Animals; D017756:Aquaculture; D001424:Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D001483:Base Sequence; D002677:Chile; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D005393:Fish Diseases / Q000382:microbiology*; D020563:Gammaproteobacteria / Q000235:genetics*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D017684:Oncorhynchus kisutch; D017686:Oncorhynchus mykiss; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011110:Polymorphism, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012487:Salmonidae; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012335:RNA, Ribosomal