Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PDAO

Heath, Sekou; Pak, Son-Il; Marshall, S.; Prager, Ellen M.; Orrego, Cristián

doi:10.3354/dao041019

Cited by 22 publications

(12 citation statements)

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“…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).…”

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confidence: 50%

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

Reid

Griffen

Birkbeck

2004

Appl Environ Microbiol

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Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.Salmonid rickettsial septicemia (SRS), or piscirickettsiosis, is a systemic disease of cultured salmonids initially recognized in coho salmon in Chile (1), where it causes significant losses in aquaculture (4). Subsequently, disease has been recorded in Canada (3), Norway (19), Ireland (21), and Scotland (A. N. Grant, A. G. Brown, D. I. Cox, T. H. Birkbeck, and A. A. Griffen, Letter, Vet. Rec. 423:138, 1996), but the impact in the Northern Hemisphere (0.6 to 18% mortalities) has not yet reached the levels experienced in Chile (30 to 90% mortalities) (1). Coho salmon appear to be more susceptible to SRS than Atlantic salmon, which has altered the balance of species cultured in Chile (18). Rickettsia-like organisms have been observed globally in a wide range of marine organisms (7), but difficulty of culture means their gram-negative classification and usual intracellular location within host cells are often all that is known of them. The etiologic agent of SRS was isolated (8) and later named Piscirickettsiosis salmonis as a new genus and species (9). The 16S ribosomal DNA (rDNA) sequence of the P. salmonis type strain, LF-89, indicated taxonomic placement within the ␥ subdivision of the Proteobacter group, rather than alongside true rickettsiae in the ␣ subdivision (9). Ensuing 16S rDNA analysis of isolates from Chile, Canada, and Norway by PCR and restriction fragment length polymorphism revealed a homogeneous group with the presence of one outlier, Chilean strain EM-90 (16), which was substantiated from the 16S rDNA sequence (17). More subtle differences between strains are illuminated by analyzing the 16S-to-23S intergenic transcribed spacer (ITS), which possesses a higher level of sequence diversity (17), meaning that ITS sequence data can be used to infer evolutionary and phylogenetic relationships not evident from 16S rDNA. Both 16S rDNA and ITS data can be complicated by the presence of multiple rDNA operons, documented in P. salmonis strains LF-89 and EM-90 (4). However, the P. salmonis-specific ITS primers described by Marshall et al. (14) are reported to amplify only one ITS region, allowing strain-to-strain comparisons (4). Of the isolates studied by Mauel et al. (17)-four Chilean isolates, one Canadian isolate, and one Norwegian isolate-ITS data analysis indicates phylogenetic diversity between isolates from different geographic regions. Here we apply 16S and ITS sequence data analysis to the widest range of SRS isolates yet to be studied, representing the first report of sequences from Irish and Scottish isolates.Infected fish samples. Samples wer...

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

Reid

Griffen

Birkbeck

2004

Appl Environ Microbiol

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“…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, ).…”

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confidence: 89%

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

Isla

Saldarriaga‐Córdoba

Fuentes

et al. 2019

Journal of Fish Diseases

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Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic‐resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty‐two Chilean P. salmonisisolates, as well as the type strain LF‐89T, were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF‐89T and EM‐90 strains, as well as a seven‐isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.

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“…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. …”

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

Na¹,

Calvo²,

Reece³

et al. 2002

Dis. Aquat. Org.

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Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffinembedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species. KEY WORDS: Hard clams · Disease · QPX · Molecular techniques · PCR · ProbeResale or republication not permitted without written consent of the publisher Dis Aquat Org 52: [233][234][235][236][237][238][239][240][241][242][243][244][245][246][247] 2002 observed in the late 1950s in New Brunswick, Canada, where it caused high mortalities of wild hard clams (Drinnan & Henderson 1963), it did not become a recognized threat to hard clam aquaculture until the late 1980s and early 1990s. Morphological and molecular evidence suggests that the organism is a member of the phylum Labyrinthulomycota (Whyte et al. 1994, Maas et al. 1...

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Monitoring Piscirickettsia salmonis by denaturant gel electrophoresis and competitive PDAO

Cited by 22 publications

References 20 publications

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

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

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

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

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