Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1β, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Construction of 3D Model of Protein Drug Targets for Renibacterium Salmoninarum - A Bacterial Pathogen Causing Bacterial Kidney Disease in Young Salmonid Fish

The aim of this study is to construct 3D models of potential drug targets for the Bacterial Kidney Disease (BKD) causing pathogen Renibacterium salmoninarum. The bacterial pathogen Renibacterium salmoninarum was selected for homology modeling studies since there were no known protein structures of the organism reported in the NCBI database. The reported protein sequences were run through DrugBank to pick out drug-targets. Online databases and web tools such as PMDB, UniProt, Drug Bank, and SwissModel were employed in this analysis. An aggregate of 412 protein sequences were identified as potential drug targets and were retrieved from the UniProt. Homology models of the protein sequences were constructed using the SwissModel database for all 412 proteins. These were then refined through a protein blast and Ramachandran plot analysis. Out of the 412 constructed models, 143 models were of reliable quality. These were then submitted to the PMDB database for further reference. To demonstrate the application of these constructed models, protein-ligand docking analysis using Auto Dock Vina was performed. Among the antibiotics that were tested against their known drug targets, trimethoprim demonstrated significant potential for the inhibition of R. salmoninarum’s dihydrofolate reductase protein, with a binding energy of -9.06 Kcal/mol and with the formation of 3 hydrogen bonds. Therefore through protein-ligand docking studies and the construction of 3D models of protein drug targets, Trimethoprim is proposed as a solution to the Bacterial Kidney Disease (BKD) problem in salmonid fishes. Further in-vitro evidences are in demand to prove this hypothesis.

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence

The bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD has proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.

Evidence for the Use of Mucus Swabs to Detect Renibacterium salmoninarum in Brook Trout

Efforts to advance fish health diagnostics have been highlighted in many studies to improve the detection of pathogens in aquaculture facilities and wild fish populations. Typically, the detection of a pathogen has required sacrificing fish; however, many hatcheries have valuable and sometimes irreplaceable broodstocks, and lethal sampling is undesirable. Therefore, the development of non-lethal detection methods is a high priority. The goal of our study was to compare non-lethal sampling methods with standardized lethal kidney tissue sampling that is used to detect Renibacterium salmoninarum infections in salmonids. We collected anal, buccal, and mucus swabs (non-lethal qPCR) and kidney tissue samples (lethal DFAT) from 72 adult brook trout (Salvelinus fontinalis) reared at the Colorado Parks and Wildlife Pitkin Brood Unit and tested each sample to assess R. salmoninarum infections. Standard kidney tissue detected R. salmoninarum 1.59 times more often than mucus swabs, compared to 10.43 and 13.16 times more often than buccal or anal swabs, respectively, indicating mucus swabs were the most effective and may be a useful non-lethal method. Our study highlights the potential of non-lethal mucus swabs to sample for R. salmoninarum and suggests future studies are needed to refine this technique for use in aquaculture facilities and wild populations of inland salmonids.

Molecular detection of Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) from Poland

Renibacterium salmoninarum causes bacterial kidney disease mainly in salmonid fish. Oligonucleotide primers incorporating R. salmoninarum unique sequences were designed to amplify a 501 bp region of the gene encoding a 57 kDa soluble extra-cellular protein. The primers did not amplify other wide varieties of aquatic or piscine bacteria Aeromonas salmonicida or Yersinia ruckeri. This assay provides a molecular description and definitive identification of R. salmoninarum in Poland.