Marine Microbial Pathogenesis and Immunity of Marine Cold-Water Fishes

Javier Santander1, Sathees Duglas1, Ignacio Vasquez1, Rebecca Kwabiah1

Marine Microbial Pathogenesis and Vaccinology Laboratory, Department of Ocean Sciences Memorial University of Newfoundland, NL, Canada

 The research at the Marine Microbial Pathogenesis and Vaccinology Laboratory is focus on host immunity and microbial pathogenesis, including design, construction, and evaluation of vaccines in the context of marine finfish aquaculture in cold environments. We use cellular and organism models of marine infectious diseases to understand bacterial and host interaction in marine environment under climate change conditions. We also contribute to studies related to marine biotechnology including, aquafeeds and functional feed ingredients, fish response to gases and hydrostatic pressure, synthetic fish meat production, and biomedicine. We closely collaborate with the aquaculture, pharma, oil & gas industries, international research institutions, indigenous communities and government agencies to promote sustainable seafood production. The presentations of this section represent part of our research related to bacterial pathogenesis and fish host response.  Macrophages are among the first immune cells to respond to infectious diseases. We have developed several infectious models using primary macrophages from different fish species and bacterial pathogens. Vibrio anguillarum is a frequent marine pathogen of fish and invertebrates. Several V. anguillarum serovars have been described, while O1, O2 and O3 have been associated to outbreaks. Serovar O1 and O2 are the most common in salmonid infections. Using RNA-seq studies here we described the response of Atlantic salmon (Salmo salar) primary macrophages to V. anguillarum O1 and O2 infection. We found that V. anguillarum extracellularly infected salmon macrophages. Serovar O1 triggered a fast and acute immune response in contrast to O2 that triggered a slow and modest immune response. Similarly, Aeromonas salmonicida subspecies salmonicida is common pathogen in salmonids. We have characterized a highly virulent intracellular strain of A. salmonicida. Using dual RNA-Seq we studied Atlantic salmon macrophages response to the A. salmonicida infection and the bacterial gene response to the macrophage intracellular conditions. We found that A. salmonicida induce partial apoptosis inhibition and impaired lysosome formation. A. salmonicida upregulate several virulence factors encoding genes related to the effects observed in the macrophages.

In collaboration with the Division of Biomedical Sciences at the Faculty of Medicine of Memorial University we had explored the immune response of eye tissues in several marine teleost fish. In this study the expression of CD14, a common macrophage marker in mammals, in lumpfish (Cyclopterus lumpus) and spotted wolffish (Anarhichas minor) during V. anguillarium infection and temperature variations was reported. CD14 epitope expression increased in the ocular choroid rete and head kidney upon bacterial infection in lumpfish and a six-week hypothermia challenge in spotted wolffish. The tissue spatial regulation of CD14 epitope in lumpfish and spotted wolffish we observed herein might serve as a tool toward a better understanding of the mechanisms involved in tissue damage responses.

Comparative Immune Response of Atlantic Salmon Primary Macrophages to Vibrio anguillarum Serovars O1 & O2

Sathees Duglas1*, Ignacio Vasquez1, Ahmed Hossain1, Vimbai Irene Machimbirike1, Jennifer Hall2, Trung Cao1 and Javier Santander1

1Marine Microbial Pathogenesis and Vaccinology Laboratory, Department of Ocean Sciences, Memorial University of Newfoundland, St. John's, NL, Canada.; 2 The Aquatic Research Cluster, Department of Ocean Sciences, Memorial University of Newfoundland, St. John's, NL, Canada.

 Vibrio anguillarum, a Gram-negative marine pathogen, causes vibriosis is several fish and invertebrate species worldwide. Among the 23 identified serovars, O1, O2, and O3 are associated with outbreaks. V. anguillarum O1 strains have higher virulence than O2 strains. While genomic analyses revealed differences in virulence genes, infection mechanisms and host immune responses across serovars remain unclear. This study aimed to explore the immune responses of primary Atlantic salmon macrophages to V. anguillarum O1 and O2 infection. Gentamycin exclusion assay was conducted to assess cell viability and V. anguillarum infection at 1, 2, 3, and 4 hours post-infection (hpi). Both serovars progressively reduce cell viability over time, with O1 causing slightly greater mortality. Despite higher infection rates at 1 hpi, both serovars showed low recovered CFU at later time points, suggesting an extracellular infection mechanism, as confirmed Transmission Electron Microscopy, which showed no intracellular bacteria. RNA-seq at 1 and 2 hpi revealed distinct immune response profiles. Macrophages exhibit a rapid and robust immunological response to V. anguillarum O1, characterized by strong activation of TLR4 signaling, cytokine-cytokine receptors, and NF-κB-mediated transcription of pro-inflammatory cytokines. In contrast, infection with O2 triggered a more modest response, including TLR signaling and cytokine activity, associated to stress regulation, immunomodulation via negative control of T-cell proliferation, and engagement of G-protein-coupled receptor pathways. These findings suggest that V. anguillarum O1 is more immunogenic and virulent in Atlantic salmon primary macrophages, aligning with previous animal assays, while O2 may delay or modulate host response to evade immune detection. 

Dual-Seq Transcriptomics of Aeromonas salmonicida Infection in Atlantic salmon (Salmo salar) Primary Macrophages Reveals Lysosome and Apoptosis Impairments

Ignacio Vasquez1*, Manuel Soto-Davila1, Ahmed Hossain1, Hajarooba Gnanagobal1, Jennifer R. Hall2, Javier Santander1*

1Marine Microbial Pathogenesis and Vaccinology Laboratory, Department of Ocean Sciences Memorial University of Newfoundland, NL, Canada; 2Aquatic Research Cluster, CREAIT Network, Ocean Sciences Centre, Memorial University of Newfoundland, 0 Marine Lab Road, St. John’s, NL, A1C 5S7, Canada.

A. salmonicida subsp. salmonicida is one of the oldest-known marine pathogens causing furunculosis in freshwater to marine fish species, particularly salmonids, including emergent aquaculture fish species such as lumpfish, Atlantic cod, and sablefish. A. salmonicida causes septicemia and fish death due to a systemic shock. Early stages of A. salmonicida infection, including intracellular macrophage infection, immune escape, and dissemination to lymphoid organs, are not fully comprehended. Here, we conducted a dual RNA-seq study and functional analyses in Atlantic salmon primary macrophages infected with A. salmonicida to identify relevant genes for fish cellular immunity and A. salmonicida pathogenesis. RNA samples were taken from non-infected primary macrophages, 1- and 2-hour post-infection (hpi) with A. salmonicida. A total of 871 and 1,683 differential expressed genes (DEGs) were identified in at 1 and 2 hpi, respectively. At 1 hpi, A. salmonicida modulated the expression of host genes associated with inflammation, fatty acids synthesis, and apoptosis. While at 2 hpi A. salmonicida hijacked pathways related to myeloid cell differentiation, cytoskeleton and actin filament organization, lysosome maturation, and apoptosis. In contrast, A. salmonicida shown an upregulation of genes encoding for hemolysin, aerolysin, type IVa pili, and T3SS effectors. In conclusion, these results suggest that A. salmonicida induces endocytosis, impairs lysosome maturation, and reduces apoptosis.

Spatial Regulation of CD14-like Epitope in Hematopoietic and Ocular Tissues of Lumpfish (Cyclopterus lumpus) and Spotted Wolffish (Anarhichas minor) During Bacterial Infection and Temperature Variations

 1,2Rebecca R. Kwabiah, 1Lauren E. Murphy, 1Hélène Paradis, 1Rand Al-Badoosh, 2Trung Cao, 2Vimbai I. Machimbirike, 2Hajarooba Gnanagobal, 2Ignacio Vasquez, 2Aqsa Maqsood, 1Kenneth Kao, 2Javier Santander, 1Robert L. Gendron

1Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada; 2Marine Microbial Pathogenesis and Vaccinology Lab, Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada

Innate immunity is one of the first lines of defense in response to tissue damage, with macrophages being key players in these processes. The expression of macrophage protein markers in North Atlantic marine teleost fish have not been extensively studied. To that end, here we used an anti-mammalian CD14 antibody to explore CD14 epitope expression in hematopoietic and ocular tissues of lumpfish (Cyclopterus lumpus) and spotted wolffish (Anarhichas minor). Western blotting indicated that proteins reactive with an anti-mammalian-CD14 antibody displayed molecular weights similar to mammalian CD14 isoforms in both lumpfish and spotted wolffish. Immunohistochemistry (IHC) with this anti-mammalian-CD14 antibody was used to assess CD14-like reactive epitope expression in head kidney and ocular choroid rete mirabile in both normal healthy lumpfish and in a Vibrio anguillarum marine bacterial pathogenesis paradigm in lumpfish, and in a six-week hypothermia and hyperthermia challenge paradigm in spotted wolffish. As infection proceeded in lumpfish, CD14-like expression increased in the head kidney and ocular choroid rete mirabile vasculature. In spotted wolffish, the hypothermia challenge led to higher levels of CD14-like expression in the ocular choroid rete mirabile compared to control temperature or hyperthermia challenge. The tissue spatial regulation of the CD14-like epitope in lumpfish and spotted wolffish we observed herein might serve as a tool in emerging novel model organism-based technologies toward a better understanding of the mechanisms involved in responses to tissue damage.