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An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV.

ABSTRACT: BACKGROUND: Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting. FINDINGS: The expression stability of five commonly used housekeeping genes [beta-actin (ACTB), elongation factor 1-alpha (EF1A), ubiquitin (UBQ), glyceraldehyd-3-phosphate dehydrogenase (GAPDH) and tubulin alpha (TUBA)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium Piscirickettsia salmonis and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, UBQ and EF1A appeared as the most stable, although EF1A was slightly upregulated at late stages of P. salmonis infection in RTS11. ACTB instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of GAPDH and TUBA was also demonstrated. CONCLUSION: Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of UBQ, ACTB and EF1A as reference genes in qRT-PCR assays for studying the effect of P. salmonis and IPNV on the host immune response.

SUBMITTER: Pena AA

PROVIDER: S-EPMC2873344 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV.

Peña Andrea A AA Bols Niels C NC Marshall Sergio H SH

BMC research notes 20100414

<h4>Background</h4>Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.<h4>Findings ...[more]

PMID: 20398263

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Project 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, ymt, 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 high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.

Project description:Piscirickettsia salmonis is the etiologic agent of salmon rickettsial syndrome (SRS) and is responsible for considerable economic losses in salmon aquaculture. The bacterium affects coho salmon (CS; Oncorhynchus kisutch), Atlantic salmon (AS; Salmo salar), and rainbow trout (RT; Oncorhynchus mykiss) in several countries, including Norway, Canada, Scotland, Ireland, and Chile. We used Bayesian genome-wide association study analyses to investigate the genetic architecture of resistance to P. salmonis in farmed populations of these species. Resistance to SRS was defined as the number of days to death and as binary survival (BS). A total of 828 CS, 2130 RT, and 2601 AS individuals were phenotyped and then genotyped using double-digest restriction site-associated DNA sequencing and 57K and 50K Affymetrix® Axiom® single nucleotide polymorphism (SNP) panels, respectively. Both traits of SRS resistance in CS and RT appeared to be under oligogenic control. In AS, there was evidence of polygenic control of SRS resistance. To identify candidate genes associated with resistance, we applied a comparative genomics approach in which we systematically explored the complete set of genes adjacent to SNPs, which explained more than 1% of the genetic variance of resistance in each salmonid species (533 genes in total). Thus, genes were classified based on the following criteria: i) shared function of their protein domains among species, ii) shared orthology among species, iii) proximity to the SNP explaining the highest proportion of the genetic variance, and iv) presence in more than one genomic region explaining more than 1% of the genetic variance within species. Our results allowed us to identify 120 candidate genes belonging to at least one of the four criteria described above. Of these, 21 of them were part of at least two of the criteria defined above and are suggested to be strong functional candidates influencing P. salmonis resistance. These genes are related to diverse biological processes, such as kinase activity, GTP hydrolysis, helicase activity, lipid metabolism, cytoskeletal dynamics, inflammation, and innate immune response, which seem essential in the host response against P. salmonis infection. These results provide fundamental knowledge on the potential functional genes underpinning resistance against P. salmonis in three salmonid species.

Project description:BackgroundVertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis.ResultsEffects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells.ConclusionThe absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

Dataset Information

An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV.

Publications

An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV.

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