Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis

Yáñez, Alejandro J.; Valenzuela, Karla; Silva, H.; Retamales, J.; Romero, Alex; Enríquez, Ricardo; Figueroa, Jaime; Claude, Alejandro; Gonzalez, Jose Emanuele; Avendaño‐Herrera, Rubén; Cárcamo, Juan G.

doi:10.3354/dao02403

Cited by 83 publications

(66 citation statements)

References 27 publications

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“…In some cases, the PCR products of 16S and ITS-1 were acquired through 2 rounds of amplification in order to facilitate cloning and sequencing. Additionally, the PCR products obtained from native fish corresponded in size to products obtained from the DNA of the P. salmonis PPT005 strain, which was grown in cell culture (Yañez et al 2012). Moreover, despite the identification of P. salmonis DNA, autopsies of fish from which the samples were taken did not present pathognomonic signs that could be linked to those known for this causative agent in salmonids.…”

Section: Piscirickettsia Salmonis 16s Rna and Its-1 Gene Amplificatiomentioning

confidence: 95%

“…Phylogenetic analysis through 16S rRNA places this bacterium within a new family in the Proteobacteria, the gamma subdivision Gammaproteobacteria (Fryer et al 1992, Mauel et al 1999, Rojas et al 2008. In relation to this, several research groups have been able to cultivate this bacterium in cell-free, artificial media, leading to the postulation that P. salmonis should rather be classified as a facultative intracellular bacterium (Mauel et al 2008, Yañez et al 2012, 2013.…”

Section: Introductionmentioning

confidence: 99%

“…Bacteria can also be cultivated in cell lines (Fryer et al 1990, Lannan & Fryer 1991) and bacteriological media (Mauel et al 2008, Yañez et al 2012, 2013. Likewise, techniques have also been applied that are based on fluorescent antibodies and enzymatic immunoassays (ELISA) (Lannan & Fryer 1991), PCR analysis, using primers designed to amplify specific sequences of the 16S rRNA gene (Mauel et al 1996, Karatas et al 2008, and the intergenic internal transcribed spacer (ITS-1) sequence, located between the 16S and 23S rRNA (Marshall et al 1998).…”

Section: Introductionmentioning

confidence: 99%

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Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile

Contreras-Lynch¹,

Olmos²,

Vargas³

et al. 2015

Dis. Aquat. Org.

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Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, a severe disease causing high mortalities in salmonids. This bacterium has been previously identified and isolated in all cultivated salmonids in Chile and worldwide, including Salmo salar, Oncorhynchus kisutch, and O. mykiss, in addition to being found in non-salmonid species such as Dicentrarchus labrax and Atractoscion nobilis. In this study, the 16S rRNA gene and intergenic spacer ITS-1 of P. salmonis were amplified by PCR from DNA samples extracted from the native Chilean fish species Eleginops maclovinus, Odontesthes regia, Sebastes capensis, and Salilota australis. Analysis of the 16S rRNA sequences from O. regia demonstrated a close phylogenetic relationship with the 16S rRNA gene in the Chilean EM-90 strain. The 16S rRNA sequences from E. maclovinus, S. capensis, and S. australis were related to the Chilean LF-89 sequence and Scottish strains. To confirm these findings, analysis of P. salmonis ITS-1 sequences obtained from the 4 sampled native species demonstrated a high degree of identity and a close phylogenetic relationship with Chilean P. salmonis sequences, including LF-89 and EM-90. These results suggest a strong relationship between the nucleotide sequences from the 16S rRNA and ITS-1 genes amplified from native fish with those sequences described in the first P. salmonis strains to be identified and isolated in Chile.KEY WORDS: 16S gene · ITS-1 · rDNA sequences · Piscirickettsiosis · Native fish · SalmonidsResale or republication not permitted without written consent of the publisher

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Section: Piscirickettsia Salmonis 16s Rna and Its-1 Gene Amplificatiomentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile

Contreras-Lynch¹,

Olmos²,

Vargas³

et al. 2015

Dis. Aquat. Org.

Self Cite

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“…In vitro can be cultured in cell lines as CHSE-214 (Chinook salmon embryo), macrophage/monocyte (RTS-11) or Sf21 (Spodoptera frugiperda; ECACC 89070101) cell culture, where P . salmonis has high titers) [4]; and also in cell-free culture media, as CHAB agar, AUSTRAL-SRS broth or MC medium [5–7]. …”

Section: Introductionmentioning

confidence: 99%

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism

Machuca

Martı́nez

2016

PLoS ONE

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The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.

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“…This may be due to the technically challenging process of developing polyclonal antisera using cells propagated by cell culture, although methods to purify the bacterium from cell culture debris have been optimised (Kuzyk et al 1996). The establishment of agar (Mauel et al 2008, Mikalsen et al 2008) and liquid (Vera et al Yañez et al 2012, Henríquez et al 2013) media for P. salmonis may also assist in the production of highly specific antisera.As yet, the antigenic basis for the distinction between the 3 (or possibly 4) serotypes of TRLO is unknown. Serotypic diversity among many groups of Gram-negative bacteria is driven by variability in the heat-stable lipopolysaccharide O-antigens.…”

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confidence: 99%

Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

Morrison¹,

Young²,

Knowles³

et al. 2016

Dis. Aquat. Org.

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Atlantic salmon Salmo salar L. farmed in south-east Tasmania, Australia, are susceptible to infection by the Tasmanian Rickettsia-like organism (TRLO), a Gram-negative bacterium. Here, we report the first isolation of TRLO from south-east Tasmania in pure culture and show that the bacterium is culturable on both specialised enriched agar and in cell culture using the CHSE-214 cell line. In vitro cultured TRLO was used to reproducibly elicit disease in Atlantic salmon parr held in fresh water. In inoculated fish, TRLO was observed intracytoplasmically in peripheral blood leucocytes, suggesting that these cells are responsible for haematogenous dispersal of the bacterium within the host. Fish with experimentally induced disease presented with gross and histopathological changes similar to TRLO-infected fish at commercial marine farms. TRLO was also isolated in culture from farmed Atlantic salmon in the Tamar River and Macquarie Harbour production areas in Tasmania, both of which have no history of TRLO-associated disease. These TRLO isolates appear to be serologically distinct from each other as well as from isolates obtained from south-east Tasmania, linking each serotype to a specific geographical location within Tasmania. Despite the lack of clinical evidence of TRLO-linked disease in fish grown in the Tamar River and Macquarie Harbour, experimental infection trials demonstrably showed the pathogenic potential of these TRLO serovars. Together, these data provide evidence that TRLO is a fastidious, facultative intracellular bacterium and confirm TRLO as a pathogen of Atlantic salmon, causing a disease designated Tasmanian salmonid rickettsiosis.KEY WORDS: Rickettsia-like organism · TRLO · Atlantic salmon · Salmo salar · Culture · Pathogenicity · Tasmania Resale or republication not permitted without written consent of the publisherDis Aquat Org 122: [85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] 2016 RLO (Khoo et al. 1995, Athanassopoulou et al. 2004, Corbeil et al. 2005, Wu et al. 2005 or P. salmonis-like organism (Antonio et al. 2000, Chen et al. 2000a,b, Mauel et al. 2003 until their taxonomic status can be formally resolved. This includes the Tasmanian RLO (TRLO), a bacte rium detected in Atlantic salmon Salmo salar L. farmed on the south-east coast of Tasmania, Austra lia (Corbeil et al. 2005).TRLO is an intracellular, Gram-negative coccoid bacterium that emerged in the summer of 2001 (Department of Primary Industries, Parks, Water and Environment [DPIPWE] unpublished data). Since then, TRLO has been implicated in minor sporadic outbreaks of disease which have typically coincided with the annual peak in water temperature. The disease is characterised by a high morbidity rate which results in significant production loss through reduced feed intake but relatively low mortality, less than 10% of affected stock. During outbreaks, affected fish are commonly co-infected with the Tasmanian Atlantic salmon reovirus (TSRV; Zainathan et al. 2015), and it is not known wh...

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Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis

Cited by 83 publications

References 27 publications

Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile

Identification and genetic characterization of Piscirickettsia salmonis in native fish from southern Chile

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism

Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

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