Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Abstract

Piscinckettsia salrnonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease To assess the genetic variabihty the 16s ribosomal DNA, the internal transcribed spacer (ITS) and the 23s ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16s rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7 % similarity). Two-thirds of the 23s rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16s rDNA, ITS and 23s rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (:,99.4 % 16s rDNA s d a r i t y , 99.1 to 99.7 % ITS and 99.3 to 99.8 % 23s rDNA slrmlarities). The sequence of one Chilean isolate, EM-90, was unique, with 16s rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23s rDNA from 97.6 to 98.5%.

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

“…A highly sensitive and specific diagnostic DNAbased probe (Kellner-Cousin et al 1993) could serve as a practical tool for detection and quantification of Dreissena's prokaryotes. Efforts to clarify the taxonomic position of Dreissena's prokaryotes could specifically make use of techniques such as antigenic structure determination (Renault & Cochennec 1995), protein electrophoresis (Le Gall & Mialhe 1992), and analysis of genomic content (e.g., 16S, internal transcribed spacer, and 23S ribosomal DNA sequencing, Mauel et al 1999).…”

Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PDAO

“…DNA samples from P. salmonis strains SLGO-94 (rainbow trout) and Cl-95 (coho salmon), both from Chile, as well as from ATL-4-91 (British Columbia, Canada, netpen-raised Atlantic salmon) and NOR-92 (Norway, netpen-raised Atlantic salmon) were donated by M. J. Mauel. These strains are listed by Mauel et al (1996Mauel et al ( , 1999 and House et al (1999) along with additional information on their biological and institutional origins.…”

“…unsequenced sites. GB1 to the nght of the strain designation indicates GenBank 1995-1996 accessions of sequences (LF-89, U36943.1; SLGO-94, U62104 1; Cl-95, U62103.1; ATL-4-91, U36945.1; NOR-92, U36946.1, EM-90, U36944.1), M denotes sequences shown in Mauel et al (1999) and GB2 indicates sequences submitted to GenBank on January 19, 2000 (i.e., LF-89, U36943.2; SLGO-94, U62104.2, and so on). In the numbenng of U36943 1, we sequenced, with no ambiguities, positions 224-458 (the entire segment between primers RTSl and RTS4) for LF-89 and EM-90, 294-451 for SLGO-94, 252-457 for Cl-95, 242-447 for ATL-4-91 and 245-455 for by incubation at 65°C for 1 h. The eluates (1 p1) served as templates for amplification (in a reaction volume of 50 p1) with the corresponding Piscirickettsia salmonisspecific primer pairs, i.e., RTSl/RTS4 and RTSl /RTS2, to reach concentrations suitable for subsequent competitive PCR experiments.…”

“…Symptoms of the disease initially reported in coho salmon Oncorhynchus kisutch were evident after fish underwent transfer to salt water (Bravo & Campos 1989). A microbial agent was isolated (Fryer et al 1990, Cvitanich et al 1991 and further characterized as a novel intracellular organism, Piscirickettsia salmonis (type strain LF-89 = ATCC V R 1361), belonging to the gamma subdivision of the proteobacteria (Fryer et al 1992, Fryer & Mauel 1997, Mauel et al 1999. The agent survived in salt water and less well in fresh water (Lannan & Fryer 'Corresponding author.…”

“…The microbial etiology of these outbreaks has been confirmed by determination of close phylogenetic affiliations to Piscirickettsia salmonis LF-89 (Mauel et al 1996(Mauel et al , 1999, serological criteria (Olsen et al 1997) and comparative biochemistry of the microbial antigens (Jones et al 1998). However, severe epizootics of P. salmonis occur only in the fish farms of southern Chile, while outbreaks of the organism in other parts of the world have been less pronounced and sporadic (for reviews, see Fryer & Lannan 1996, House et al 1999.…”