Abstract
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.
Highlights
To expand upon the existing toolset available to the P. salmonis research community, we developed a high-throughput screening method to study P. salmonis infection in different hosts using a cell viability reagent, including salmon-derived cell lines and primary cell culture systems of an alternative animal model, the zebrafish
In order to establish zebrafish as a suitable model to study P. salmonis infection, we sought to compare the infection generated by the bacterium in the primary cell cultures and the well-studied S. salar cell lines, SHK-1 and Anterior Salmon Kidney (ASK)
Since the zebrafish kidney primary cell cultures (ZKPCC) is composed of different cell types, we aimed to identify the effect of P. salmonis infection in immune-related cells present in the primary cell culture [35,38]
Summary
That causes salmonid rickettsial septicemia (SRS) or piscirickettsiosis, an infectious disease that affects diverse fish species worldwide and causes significant economic losses in Microorganisms 2021, 9, 2516. First described as an epizootic disease, causing high mortality in the southern region of Chile, P. salmonis was responsible for a cumulative mortality of up to. SRS disease has been reported in every species of farmed salmonids (chinook salmon, Oncorhynchus tshawytscha; masu salmon, Oncorhynchus masou; rainbow trout, Oncorhynchus mykiss; Atlantic salmon, Salmo salar) [7,8] and other non-salmonid hosts, such as the European sea bass Dicentrarchus labrax [9], the white seabass Atractoscion nobilis [10], the Hawaiian tilapia [11], and a variety of native wild fish in southern Chile [12], among others. The limited effectiveness of current management highlights the need to develop novel approaches to prevent this disease and combat this intracellular pathogen
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