Research Project: Reducing Impacts of Disease on Rainbow Trout Aquaculture Production

Location: Office of The Director

2023 Annual Report

Objectives
Rainbow trout are an important recreational and food fish species in the U.S., and it is thus important to improve disease resistance and improve methods for combatting outbreaks of disease to increase production and profitability of U.S. aquaculture. This research plan will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Identify virulence factors in pathogenic flavobacterium species and develop strategies to control disease. • Sub-Objective 1.A: Isolate and characterize F. columnare mutants and identify virulence factors associated with disease in rainbow trout. • Sub-Objective 1.B: Develop vaccines to control columnaris disease. • Sub-Objective 1.C: Develop improved genetic manipulation techniques for F. psychrophilum, which causes bacterial cold-water disease. Objective 2: Characterize salmonid antimicrobial peptides and evaluate their biocidal effects against pathogens. • Sub-Objective 2.A: Identify new antimicrobial peptides by mining the rainbow trout big data sets and characterize their actions and regulation. • Sub-Objective 2.B: Characterize the immunomodulatory actions of new trout AMPs in vitro. • Sub-Objective 2.C: Assess effects of new rainbow trout AMPs on flavobacterial biofilms. Objective 3: Identify rhabdoviral virulence factors and develop strategies to reduce pathogenesis in salmonids. • Sub-Objective 3.A: Identify and characterize potential viral targets and strategies for vaccine development. • Sub-Objective 3.B: Characterize the involvement of stress granule formation and function in pathogen response and the establishment of protective immunity in fish cell-lines.

Approach
Objective 1: For this objective, our scientific aim is to develop genetic techniques to characterize F. psychrophilum and F. columnare virulence mechanisms. Using these genetic techniques, genes encoding specific secreted proteins and components of flavobacterial secretion systems will be mutated and the effects of these modifications on bacterial pathogenesis will be evaluated using in vitro and in vivo systems. Data from these approaches will improve understanding of host-pathogen interactions and generate attenuated bacterial strains for vaccine development. Objective 2: For this objective, our scientific aim is to identify and characterize new antimicrobial peptides (AMPs) in rainbow trout, understand their actions against flavobacterial and rhabdoviral pathogens, and ascertain their physiological control to improve health of rainbow trout. Using established in vitro systems with trout cell-lines, biological actions of AMPs will be assessed and ability of AMPs to kill important flavobacterial and rhabdoviral pathogens, and flavobacterial biofilms, will be characterized. Objective 3: For this objective, our scientific aim is to understand the innate immune response and components of virulence in two important rainbow trout rhabdoviruses (IHNV and VHSV). Work will involve sequential characterization of the effects of critical rhabdoviral proteins, and modifications to these proteins, on the stress response and host immunity using trout cell-lines, in vitro. The goal is to characterize components of rhabodoviral virulence and how these components (viral proteins) influence host response as a means to identify possible viral targets for new vaccine candidates. Developing new vaccine candidates based on improved understanding of rainbow trout antiviral immune functions should advance our abilities to combat these pathogens in rainbow trout.

Progress Report
This is the final report for this project which terminated in October 2022. See reports for replacement projects 5090-31000-028-000D (Developing Strategies to Improve Dairy Cow Performance and Nutrient Use Efficiency with Nutrition, Genetics, and Microbiology), 5090-12630-006-000D (Managing Nutrients and Assessing Pathogen Emission Risks for Sustainable Dairy Production Systems), and 5082-21000-001-000D (Sustainable Production and Pest Management Practices for Nursery, Greenhouse, and Protected Culture Crops) for additional information. In support of Objective 1, ARS collaborators in Milwaukee, Wisconsin, focused on the use of genetic techniques to characterize mechanisms of Flavobacterium (F.) columnare and F. psychrophilum virulence and identify potential strategies to control bacterial disease in rainbow trout aquaculture. Sub-objective 1A: Proteomic analyses revealed over 50 secreted F. columnare proteins as potential virulence factors. A virulent F. columnare strain (MS-FC-4) was identified as more amenable to genetic manipulation than other strains. Using this strain, more than 35 targeted F. columnare mutants were constructed with single or multiple deletions of genes predicted to encode virulence factors, including genes that encode proteases, chondroitinases, cytolysins, adhesins, and proteins involved in motility and iron uptake. Some mutants had as many as ten genes deleted from different locations on the chromosome, demonstrating the efficiency of these genetic techniques. Mutants were examined for virulence in zebrafish and in rainbow trout (in collaboration with ARS scientists in Leetown, West Virginia). Deletion of some genes encoding motility proteins (GldJ, SprB), cytolysins (CylA), proteases, and iron uptake proteins resulted in reduced virulence. Mutants defective for motility but competent for secretion exhibited reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Sub-objective 1.B: Results described above identified virulence factors associated with disease that may lead to vaccines or other control measures for columnaris disease. Mutants with attenuated virulence were evaluated as protective vaccines. Naïve rainbow trout fry exposed to the mutants by immersion were maintained for 28 days and then challenged with wild-type (WT) F. columnare. Exposure to several of the mutants resulted in increased resistance to later exposure to the WT. These potential vaccine strains included mutants lacking multiple genes involved in iron utilization, a mutant lacking a secreted protease, and several mutants deficient in motility, suggesting potential paths toward development of protective vaccines. Research demonstrated that development of a biosynthetically crippled aroA deletion mutant was not feasible in F. columnare. Sub-objective 1.C: Novel genetic techniques for F. psychrophilum strain THCO2-90 were developed to construct mutants lacking the type IX protein secretion system (T9SS) and demonstrate that F. psychrophilum T9SS is required for virulence in rainbow trout. Tools to allow genetic manipulation of F. psychrophilum strain CSF-259-93 – a preferred model strain for studies of disease in rainbow trout that previously resisted all attempts at genetic manipulation - were developed in collaboration with a scientist in Mankato, Minnesota, by identifying F. psychrophilum restriction enzymes that digest foreign DNA and developing methods to protect DNA from these enzymes. To overcome the restriction enzyme barrier to gene transfer, four methyltransferase genes were expressed to modify plasmid DNA in Escherichia coli. This protected the DNA and greatly increased the efficiency of gene transfer into F. psychrophilum. This high-risk approach resulted in a major breakthrough that now allows genetic manipulation of important F. psychrophilum strains to identify virulence factors and construct attenuated mutants as potential vaccines. These improved genetic techniques were used to construct targeted gene deletion mutants of F. psychrophilum strain CSF-259-93. ARS researchers in Leetown, West Virginia, discovered several of these mutants were avirulent against rainbow trout, demonstrating the value of this approach in identifying F. psychrophilum genes required for bacterial cold water disease. In support of Objective 2, ARS researchers in Milwaukee, Wisconsin, characterized salmonid antimicrobial peptides (AMPs) and evaluated biocidal effects against pathogens and immunomodulatory effects in rainbow trout. Approximately 200 putative AMPs were identified, including a family of AMPs called Nk-lysins. In vitro culture of rainbow trout gill (RTgill) cells was optimized and this system was used to evaluate effects of AMPs (cecropin B and cecropin P1). After treatments of AMPs (cecropin B and cecropin P1 independently) and co-stimulators (lipopolysaccharide and polyinosinic polycytidylic acid), time-course sampling identified twenty genes of interest that were further analyzed by reverse transcription-quantitative polymerase chain reaction, and eight of them were shown to have immunomodulatory effects on RTgill cells. In vitro culture of primary lymphocytes and leukocytes from rainbow trout head kidney was optimized and used to address modulation of immune responses from energy metabolism hormones, RT-Ghrelin and RT-desGhrelin (truncated ghrelin analog). Both ghrelin isoforms exerted immunomodulatory effects in multiple immune pathways, but they play divergent roles in trout immune system. The AMP cecropin was not deleterious to F. columnare at concentrations up to 300 micromolar. In support of Objective 3, ARS collaborators in Toledo, Ohio, focused on host-pathogen interactions and mechanisms of rhabdoviral virulence in rainbow trout. Sub-objective 3A: Infectious hematopoietic necrosis virus (IHNV) matrix protein (IHNV-M) is implicated in downregulating host cell transcription through unknown mechanisms, which allows IHNV to evade host immune response. Host cell transcription was measured by transfecting cells with IHNV-M to observe the effects of the WT gene compared to control vector and IHNV-M deletion mutants. By comparing luciferase reporter levels in transfected cells, deletions were identified that attenuated inhibitory effects of IHNV-M on host cell transcription. This finding shows a crucial role of a particular amino acid sequence on IHNV-M’s inhibitory effect on host cell transcription. A series of deletion mutants in the IHNV non-virion (NV) gene were produced to understand the role of NV in regulation of host immune responses. All NV deletion mutants showed attenuation of WT NV modulatory activities. In some cases, these mutants suppressed, rather than augmented, luciferase expression as compared to the empty vector control. Ten IHNV matrix-protein (M) mutant constructs were developed to assess the ability to impact host transcription, six of which were significantly suppressed in their anti-host functions, and two mutants were targeted for insertion into the IHNV genome for attenuation studies. A proposed full length, modified IHNV genome was developed to allow swapping of mutant NV and M genes. The WT version, containing introduced restriction sites to allow the aforementioned gene swaps, was synthesized and tested in cell culture replication studies in Epithelioma Papulosum Cyprini (EPC) cells. Whereas transfection of the full-length genome alone or the full-length genome along with IHNV N, P and L plasmids gave modest cytopathic effects, co-transfection with IHNV NV plasmid gave rise to significant cytopathic effects, indicating a critical role for NV in viral production. These studies provide new information on IHVN replication and virulence that will inform novel vaccine candidates. Sub-objective 3B: Concurrent work showed that the viral hemorrhagic septicemia virus (VHSV) strain IVb NV protein is involved in viral pathogenesis by mediating a crucial immunological signaling pathway to enable viral protein synthesis and inhibit interferon signaling to evade the host cell immune and stress responses. Formation of canonical stress granule (SG) was found to be conserved in multiple fish cell types, and the role of NV in SG induction was determined using WT and recombinant viruses lacking the entire NV coding sequences or with a mutation in the ATG codons preventing NV protein production while not impacting RNA synthesis. Results implicate VHSH strain IVb NV in stress granule formation during infection. Removal of functional NV during VHSV IVb infection resulted in a significant increase in infected cells forming SG-like structures compared to recombinant WT VHSV infection, suggesting an important role of NV in regulating the formation of SGs. Stable cells of EPC, rainbow trout gonad (RTG2) and RTgill expressing green fluorescent protein-Ras GTPase-activating protein-binding protein 1 (G3BP1) were developed to monitor SG formation in real time following virus infection and vaccination, as well as G3BP1-knockout RTG2 and RTgill cells using CRISPR/Cas9 technology. Research demonstrated that cells respond to heat shock, oxidative stress, and VHSV infection by forming SG that localized a key SG marker, G3BP1, and that PKR-like endoplasmic reticulum kinase (PERK), but not (dsRNA)-dependent protein kinase (PKR), is required for VHSV-induced SG formation. Furthermore, in VHSV strain Ia infected cells, PERK activity is required for interferon production, antiviral signaling and viral replication. SG formation required active virus replication as individual VHSV Ia proteins or inactive virus did not induce SG. Cells lacking G3BP1 produced increased interferon, antiviral genes and viral mRNA, but viral protein synthesis and viral titers were reduced. This research shows a critical role of activation of the integrated stress response pathway and SG formation highlighting a novel role of G3BP1 in regulating VHSV protein translation and replication.

Accomplishments
1. Genetic studies reveal virulence factors involved in columnaris disease of freshwater fish. Columnaris disease, caused by Flavobacterium columnare, is a major problem for freshwater aquaculture. An understanding of how this bacterium causes disease is lacking, and control measures are inadequate. ARS collaborators in Milwaukee, Wisconsin, developed efficient genetic techniques to make targeted mutations in Flavobacterium columnare. Mutation of iron utilization systems in Flavobacterium columnare resulted in reduced ability of the pathogen to cause disease in rainbow trout and zebrafish. Exposure of rainbow trout and zebrafish to some mutants that had attenuated virulence, particularly those deficient in multiple steps of iron uptake, resulted in partial protection against later exposure to the wild-type pathogen. This research identified critical components involved in columnaris disease and will inform development of effective attenuated vaccines.

Review Publications
Han, Y., Leaman, D.W., Shepherd, B.S. 2023. Ghrelin modulates differential expression of genes relevant to immune activities and antimicrobial peptides in primary head kidney cells of rainbow trout (Oncorhynchus mykiss). Animals. 13(10). https://doi.org/10.3390/ani13101683.
Thunes, N.C., Mohammed, H.H., Evenhuis, J., Lipscomb, R.S., Perez-Pascual, D., Stevick, R.J., Birkett, C., Conrad, R.A., Ghigo, J., Mcbride, M.J. 2023. Secreted peptidases contribute to virulence of fish pathogen Flavobacterium columnare. Frontiers in Cellular and Infection Microbiology. 13. Article:1093393. https://doi.org/10.3389/fcimb.2023.1093393.
Conrad, R.A., Evenhuis, J., Lipscomb, R.S., Perez-Pascual, D., Stevick, R.J., Birkett, C., Ghigo, J., Mcbride, M.J. 2022. Flavobacterium columnare ferric iron uptake systems are required for virulence. Frontiers in Microbiology. Volume 12. https://doi.org/10.3389/fcimb.2022.1029833.