Summary auto-generated
Researchers developed a fish cell culture system using Epithelioma papillosum cyprini (EPC) cells to study Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon. The pathogenic bacterium multiplied intracellularly within EPC cells over seven weeks. Immunohistochemical analysis detected production of the p57 antigen, haemolysin, and cytolysin within infected cultures. Transmission electron microscopy confirmed the intracellular nature of infection, with bacteria residing in membrane-bound vesicles. Importantly, bacteria cultured within EPC cells demonstrated significantly greater virulence in challenge experiments with juvenile rainbow trout compared to bacteria cultured on agar plates. Fish challenged with EPC-grown bacteria showed 100% mortality at the highest dose within five weeks, while plate-cultured bacteria required six weeks to produce initial deaths. This cell culture model provides a more physiologically relevant system for studying R. salmoninarum virulence mechanisms than traditional plate culture techniques, allowing investigation of pathogenic mechanisms under conditions more closely resembling the natural in vivo infection environment.
Key findings
- R. salmoninarum successfully infected and multiplied intracellularly within EPC fish cells for up to seven weeks, with bacteria localized in membrane-bound vesicles and large cystic cavities
- Infected EPC cultures produced putative virulence factors including p57 antigen, haemolysin, and cytolysin, with p57 being produced in large quantities both as cell-associated and secreted forms
- Bacteria cultured in EPC cells exhibited markedly increased virulence in challenge experiments with rainbow trout compared to bacteria cultured on nutrient agar plates
- The EPC cell culture system provided a reproducible, physiologically relevant model for studying R. salmoninarum pathogenesis that more closely mimics in vivo conditions than conventional plate culture methods
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Abstract
A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques.