Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767 type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 μm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 μm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 μm), it did facilitate growth at lower iron concentrations (10 μm and 1 μm). An exception was the H-2 isolate, which only grew with 100 μm of iron sulphide. In turn, 100 μm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209 genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron‐acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT‐qPCR to assess the effects of normal and iron‐limited conditions on iron‐uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H‐2 and the type strain DSM20767T. Further evaluated was the in vitro immune‐related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron‐limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H‐2 than DSM20767T. These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF‐α, IL‐1β, TLR1 and INF‐γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron‐limited conditions, especially H‐2. These observations indicate that iron‐acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron‐uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.