Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PDAO

Abstract

Reported strains of Piscirickettsia salmonis, a pathogen of salmonid fishes, were analyzed by amplifying part of the internal transcribed spacer (ITS) of the nbosomal RNA (rRNA) operon followed by denaturing gradient gel electrophoresis (DGGE) of the amphcons. AU 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 Chilok 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. salmonjs 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-' 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.

Isolates of Piscirickettsia salmonis from Scotland and Ireland Show Evidence of Clonal Diversity

“…Their connection could result from the geographic proximity of the sampling locations in Chile or may indicate a specificity of these isolates for non-Atlantic salmonids with which they may have coevolved. Similar focusing of isolates around a geographic locus has been reported by Heath et al (12) in an analysis of ITS fragments by denaturing gradient gel electrophoresis. By this method, samples collected from five sites around one location in Chile were indistinguishable from the type strain of P. salmonis (LF-89).…”

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure

“…Several molecular typing methods have been used to characterize P. salmonis isolates, including comparisons of 23S rRNA, 16S rRNA and the internal transcribed spacer (ITS) regions of several geographically distinct isolates. These comparative analyses show distinct P. salmonis isolates (i.e., from Chile, Norway and Canada) clustering closely into a monophyletic genogroup, excepting one outlier – the Chilean EM‐90 isolate (Heath, Pak, Marshall, Prager, & Orrego, ; Mauel, Giovannoni, & Fryer, ).…”

Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria

“…The conserved nature of SSU rDNA renders it inappropriate for identification of strain differences; however, sequences not under structural constraint, such as the internal transcribed spacer (ITS) regions, can be very useful for such discrimination. Molecular diagnostics targeting the ITS regions recently have been developed to distinguish among species and/or strains of several disease agents, such as Perkinsus (Reece et al 2001) and Piscirickettsia salmonis (Heath et al 2000) and these regions also may be useful for QPX discrimination. …”