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

Location: Office of The Director

2022 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
The aim for Objective 1 was to use genetic techniques to characterize mechanisms of Flavobacterium columnare and F. psychrophilum virulence and identify potential strategies to control bacterial disease in rainbow trout aquaculture. Our previous results highlighted the importance of the Flavobacterium type IX secretion system (T9SS) and the many proteins that it secretes for virulence in fish. Proteomic analyses revealed over 50 secreted F. columnare proteins as potential virulence factors and a similar number for F. psychrophilum. We isolated more than 35 F. columnare mutants with single or multiple deletions of genes predicted to encode virulence factors. The mutants were examined for virulence in zebrafish and in rainbow trout (collaboration with ARS researchers in Leetown, West Virginia). We determined that deletion of some of these genes resulted in reduced virulence. Ongoing experiments will identify the secreted proteins that are most important for columnaris disease. This will help us understand the disease process and may lead to vaccines or other control measures. We collaborated with a scientist at Minnesota State University-Mankato to improve gene transfer into F. psychrophilum strain CSF-259-93, a preferred model strain for studies of cold water disease in rainbow trout. Previously, this and most other F. psychrophilum strains had resisted gene transfer attempts. To overcome the restriction enzyme barrier to gene transfer, we expressed four genes to modify plasmid DNA in Escherichia coli. This greatly increased the efficiency of gene transfer into F. psychrophilum. This was a high-risk approach that resulted in a major breakthrough that now allows us to perform genetic experiments on relevant F. psychrophilum strains to identify virulence factors and to construct attenuated mutants as potential vaccines. In the last few months, we used these improved genetic techniques to construct several targeted gene deletion mutants of F. psychrophilum strain CSF-259-93. Two of these mutants were avirulent against rainbow trout as determined by ARS researchers in Leetown, West Virginia, demonstrating the value of this approach in identifying F. psychrophilum genes required for cold water disease. We are currently characterizing these genes and preparing the work for publication. The aim for Objective 2 was to characterize salmonid antimicrobial peptides (AMP) and evaluate their biocidal effects against pathogens. Six Nk-lysin paralogs of rainbow trout were synthesized in 30 mg and water solubility were tested. Pending availability of the BSL3 lab, challenge studies (combination of AMP and bacterial treatment) will be conducted to determine minimal inhibitory and bactericidal concentrations. For the study of immunomodulatory effects of AMPs in rainbow trout, we optimized the culturing system of rtGill- w1 cells and used this system to evaluate the effects of animal AMPs (cecropin B and cecropin P1). After treatments of AMPs (cecropin B and cecropin P1 independently) and co-stimulators (LPS and Poly:IC), time- course samples were collected and differential expression levels of immune relevant genes were profiled. Twenty genes of interest were analyzed by reverse transcription-quantitative polymerase chain reaction, and eight of them were shown, in vitro, to have immunomodulatory effects on rt-Gill cells. According to these preliminary results, we are designing more primers to evaluate differential expression levels of more immune relevant genes in multiple immune pathways. In addition, we also developed an in vitro cell culture system of primary lymphocyte and leukocyte from rainbow trout head kidney. Using these primary head-kidney cells, we addressed the modulation of immune responses from rainbow trout 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. For Objective 3, we have shown previously that infection of rainbow trout cells (RTG2 and RTgill) as well as Epithelioma papulosum cyprini cells with Viral Hemorrhagic Septicemia Virus (VHSV) IVb and Ia strains induced Stress Granule (SG) formation with different kinetics. Stress kinases protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK) have differential roles in inducing SG formation and interferon (IFN) production. Studies using non-virion (NV) protein deleted viruses showed that NV inhibits SG formation during viral infection. To determine which viral proteins when expressed in RTG2 and RTgill cells may induce SG formation, we transfected individual viral proteins with Myc- tagged expression plasmids and showed that none of the proteins when expressed directly induced SG formation. It is likely that SG formation required expression of more than one protein. To test if viral infection, which in turn will produce viral proteins, is required, we infected RTG2 or RTgill cells with ultraviolet (UV)-inactivated virus incapable of replicating in host cells and showed that viral replication is required for SG formation. We generated galectin 3-binding protein-1 (G3BP1) knockout (KO) cells using CRISPR/Cas9 technology in RTG2 and RTgill cells. However, the cells were not stable and required repeated establishment of KO cells prior to experimentation. Cells were compared with wild type (WT) and cells with significantly decreased expression of G3BP1 were used in our studies. Cells with reduced G3BP1 levels showed reduced SG formation with IVb or Ia infection. Role of PKR and PERK were determined in inhibitor studies. While 77% of infected cells formed SG, only 36% of PKR inhibited and 8% of PERK inhibited cells formed SG on VHSV Ia infection. Using antibodies generated against host proteins, we were able to show reduced viral replication and increased IFN induction that correlated with more interferon-stimulated gene (ISG) expression. Both of these effects were reduced in cells treated with PKR and PERK inhibited cells, with greater impact of PERK on both SG formation and viral replication. Similar roles of stress kinases were determined in cells expressing green fluorescent protein (GFP)-G3BP1 with the caveat that in some cells over-expression of G3BP1 previously established was less successful as the cells did not retain GFP expression following passaging in cell culture. However, using Neon transfection we have obtained greater than 70% of cells expressing the transgene and we are in the process of testing. Using our expertise of purifying SG in mammalian cells, we have standardized SG immunoprecipitation along with appropriate conditions and controls and have established the method in infected cells and will use this method to analyze protein and RNA content using mass spectrometry and RNA-sequencing.

Accomplishments
1. Identified critical components for potential vaccine against columaris disease in freshwater fish. Columnaris disease, caused by Flavobacterium columnare, is a prevalent disease affecting freshwater aquaculture that can cause significant mortality. An understanding of how this bacterium causes disease is lacking, and control measures are inadequate. ARS researchers in Milwaukee, Wisconsin, developed efficient techniques to make targeted mutations in F. columnare. Mutation of the type-nine secretion system, or of a combination of secreted proteins, reduced ability to cause disease in rainbow trout and zebrafish. This identifies critical components involved in columnaris disease, and researchers are using these methods to construct attenuated strains for vaccine evaluation.

Review Publications
Lu, X., Deng, D., Huang, F., Casu, F., Kraco, E.K., Newton, R., Zohn, M., Teh, S.J., Watson, A.M., Shepherd, B.S., Ma, Y., Dawood, M.A., Mendoza, L.R. 2022. Chronic exposure to high-density polyethylene microplastic through feeding alters the nutrient metabolism of juvenile yellow perch (Perca flavescens). Animal Nutrition. 9:143-158j. https://doi.org/10.1016/j.aninu.2022.01.007.
Thunes, N.C., Conrad, R.A., Mohammed, H.H., Zhu, Y., Barbier, P., Evenhuis, J., Perez-Pascual, D., Ghigo, J., Lipscomb, R.S., Schneider, J., Li, N., Erbes, D.H., Birkett, C.L., LaFrentz, B.R., Welch, T.J., McBride, M.J. 2021. Type IX secretion system effectors and virulence of the model Flavobacterium columnare strain MS-FC-4. Applied and Environmental Microbiology. 88(3). Article e01705-21. https://doi.org/10.1128/aem.01705-21.
Conrad, R.A., Evenhuis, J., Lipscomb, R.S., Birkett, C., McBride, M.J. 2022. Siderophores produced by the fish pathogen Flavobacterium columnare strain MS-FC-4 are not essential for its virulence. Applied and Environmental Microbiology. 88(17). Article e00948-22. https://doi.org/10.1128/aem.00948-22.