Renibacterium salmoninarum and Aeromonas salmonicida pathogenesis and virulence in lumpfish (Cyclopterus lumpus)

Gnanagobal, Hajarooba (2023) Renibacterium salmoninarum and Aeromonas salmonicida pathogenesis and virulence in lumpfish (Cyclopterus lumpus). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Renibacterium salmoninarum, the etiological agent of Bacterial Kidney Disease (BKD), and Aeromonas salmonicida, which causes furunculosis, are economically important pathogens of marine fish. The marine teleost lumpfish (Cyclopterus lumpus) is an eco-friendly cleaner fish in Atlantic salmon (Salmo salar) farming. As the lumpfish demand in salmonid aquaculture continues to rise, understanding how lumpfish interact with well-known Gram-positive and Gram-negative fish pathogens is certainly required. Therefore, in my Ph.D. thesis, I studied the interactions between lumpfish host and Grampositive R. salmoninarum or Gram-negative A. salmonicida with a particular focus on the fundamental aspects of bacterial pathogenicity and virulence. First, I evaluated the lumpfish susceptibility and immune response to R. salmoninarum infection. Lumpfish showed typical BKD clinical signs and 35 % mortality when infected with a high dose of R. salmoninarum (1×109 cells dose-1). High bacterial loads were observed in tissues (i.e., spleen, liver, and head kidney) at 28 days post-infection (dpi), and R. salmoninarum continued to persist in tissues until 98 dpi. Further, gene expression analysis using qPCR in the fish head kidney found that R. salmoninarum causes immune suppression at 28 dpi and lumpfish induce a cell-mediated immune response at 98 dpi. Second, I profiled the lumpfish head kidney transcriptome response to R. salmoninarum at early (28 dpi) and chronic (98 dpi) infection using RNA sequencing. Compared to 98 dpi, lumpfish induced many molecular pathways and genes at 28 dpi. For instance, R. salmoninarum-induced genes at 28 dpi were linked to innate and adaptive immunity, while R. salmoninarum-suppressed genes were involved in amino acid metabolism, cellular and developmental processes. In contrast, the transcriptome response of the lumpfish head kidney to this pathogen was minimal at 98 dpi, with R. salmoninarumdependent dysregulation of genes primarily connected to cell-mediated adaptive immunity. Third, I described the riboflavin supply pathways of A. salmonicida. Using in silico tools and RT-PCR, I found that A. salmonicida has a riboflavin biosynthesis pathway (RBP) and a riboflavin transporter. Moreover, I constructed the deletion mutants of riboflavin biosynthesis genes, their duplicated copies, and the transporter (ribN) of A. salmonicida and studied their role in virulence and potential as live-attenuated vaccine candidates using the lumpfish infection model. The results showed that riboflavin biosynthesis is crucial for A. salmonicida virulence. Overall, the thesis provided fundamental insights into the pathogenicity and virulence of R. salmoninarum and A. salmonicida and lumpfish response. The findings presented here are valuable for developing immunoprophylactic measures for lumpfish against BKD and furunculosis.

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