Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis , appropriate molecular and cellular studies in multiple cell lines with different origins need...
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ftdoajarticles:oai:doaj.org/article:68122db149da4bd986ae20127e90d48a 2023-05-15T15:33:03+02:00 Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis Javiera Ortiz-Severín Julia I. Tandberg Hanne C. Winther-Larsen Francisco P. Chávez Verónica Cambiazo 2021-12-01T00:00:00Z https://doi.org/10.3390/microorganisms9122516 https://doaj.org/article/68122db149da4bd986ae20127e90d48a EN eng MDPI AG https://www.mdpi.com/2076-2607/9/12/2516 https://doaj.org/toc/2076-2607 doi:10.3390/microorganisms9122516 2076-2607 https://doaj.org/article/68122db149da4bd986ae20127e90d48a Microorganisms, Vol 9, Iss 2516, p 2516 (2021) P. salmonis virulence cell culture viability host–pathogen interaction zebrafish kidney primary cell culture salmon cell lines Biology (General) QH301-705.5 article 2021 ftdoajarticles https://doi.org/10.3390/microorganisms9122516 2022-12-31T11:02:19Z Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis , appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3 , ym t, pipB2 and pepO , were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for ... Article in Journal/Newspaper Atlantic salmon Directory of Open Access Journals: DOAJ Articles Microorganisms 9 12 2516 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
P. salmonis virulence cell culture viability host–pathogen interaction zebrafish kidney primary cell culture salmon cell lines Biology (General) QH301-705.5 |
spellingShingle |
P. salmonis virulence cell culture viability host–pathogen interaction zebrafish kidney primary cell culture salmon cell lines Biology (General) QH301-705.5 Javiera Ortiz-Severín Julia I. Tandberg Hanne C. Winther-Larsen Francisco P. Chávez Verónica Cambiazo Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
topic_facet |
P. salmonis virulence cell culture viability host–pathogen interaction zebrafish kidney primary cell culture salmon cell lines Biology (General) QH301-705.5 |
description |
Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis , appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3 , ym t, pipB2 and pepO , were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for ... |
format |
Article in Journal/Newspaper |
author |
Javiera Ortiz-Severín Julia I. Tandberg Hanne C. Winther-Larsen Francisco P. Chávez Verónica Cambiazo |
author_facet |
Javiera Ortiz-Severín Julia I. Tandberg Hanne C. Winther-Larsen Francisco P. Chávez Verónica Cambiazo |
author_sort |
Javiera Ortiz-Severín |
title |
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
title_short |
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
title_full |
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
title_fullStr |
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
title_full_unstemmed |
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis |
title_sort |
comparative analysis of salmon cell lines and zebrafish primary cell cultures infection with the fish pathogen piscirickettsia salmonis |
publisher |
MDPI AG |
publishDate |
2021 |
url |
https://doi.org/10.3390/microorganisms9122516 https://doaj.org/article/68122db149da4bd986ae20127e90d48a |
genre |
Atlantic salmon |
genre_facet |
Atlantic salmon |
op_source |
Microorganisms, Vol 9, Iss 2516, p 2516 (2021) |
op_relation |
https://www.mdpi.com/2076-2607/9/12/2516 https://doaj.org/toc/2076-2607 doi:10.3390/microorganisms9122516 2076-2607 https://doaj.org/article/68122db149da4bd986ae20127e90d48a |
op_doi |
https://doi.org/10.3390/microorganisms9122516 |
container_title |
Microorganisms |
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9 |
container_issue |
12 |
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2516 |
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1766363523172007936 |