Improving Aquaculture: the Impact of Bacterial Disease Treatments on Salmonid Immune Performance

Aquaculture is a global multibillion-dollar industry that is continuously threatened by infectious diseases, including those of bacterial origin. Currently, the only method to combat bacterial disease outbreaks as they occur is the use of antibiotics. Though effective, this method increases the prevalence of antibiotic resistant bacteria. Any prophylactic treatments available provide slightly increased protection but require improvements. To facilitate vaccine design and/or treatments, a deeper understanding of fish immunity is essential. When considering Canadian finfish aquaculture, the industry is dominated by salmonids including freshwater rainbow trout and saltwater Chinook salmon. The contents of this thesis examine bacterial diseases that are relevant to the intensive culture of these two salmonid species. More specifically, both in vitro and in vivo analyses were used to explore the immune performance of salmonids when assessing breeding strategies, bacterial pathogenesis and alternative treatment options. The freshwater rainbow trout model analyzed infection with Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD). Initially, forty families were created and assessed for resistance/susceptibility to F. psychrophilum following infection. Based on survival, the immune performance of the highest and lowest performing crosses was evaluated through serum IgM production, MHC II β1 genotype and respiratory burst activity (RBA). Despite not finding significant differences between the resistance and susceptible families, RBA of leukocytes was observed to significantly decrease when fish were presenting disease symptoms. This observation led to the use of the rainbow trout immune cell line, RTS11, as a model to learn more about the pathogenesis of the organism and to test the efficacy of a promising antimicrobial peptide (AMP) called pituitary adenylate cyclase-activating polypeptide (PACAP). The saltwater Chinook salmon model used Vibrio anguillarum, the causative agent of vibriosis, for immune studies. In this system, outbreeding was used to determine if the immune performance of an inbred aquaculture stock could be enhanced via hybrid vigor. The inbred stock was crossed with seven wild populations and, following infection with V. anguillarum, survival, serum IgM production, MH II β1 genotype and immune transcript expression were assessed. This revealed that higher performing crosses had reduced inflammatory transcript expression as well as heterozygosity of the MH II β1 gene. To explore infection at the cellular level, a stromal cell line was created and characterized from Chinook salmon spleen so that immune transcript levels to heat-killed V. anguillarum could be explored. The results obtained from this thesis were able to provide novel and functional information regarding the immune response of salmonids and the pathogenesis of industrially relevant bacteria. Additionally, a greater understanding of fish immunity in response to bacterial infection was obtained and could help enhance future disease treatments for aquaculture. A steady and increasing supply of fish protein from responsible aquaculture will reduce pressures on wild stocks, some of which are extensively exploited and in danger of collapse

Long-term implantation of acoustic transmitters induces chronic inflammatory cytokine expression in adult rainbow trout (Oncorhynchus mykiss)

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.vetimm.2018.10.003 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Telemetry transmitters are frequently used in studies of wild fish migration and behavior. Although the effects of surgically implanted transmitters on survival, tag retention, healing and growth have been studied, there has been little research regarding the potential immune response induced by these transmitters. In the current study, mature rainbow trout received either surgical implantation of an acoustic transmitter or a sham surgical procedure. These fish were then sampled over a 10-week period so that pro-inflammatory cytokine expression in the spleen, peritoneal cavity lymphocytes and muscle at the surgical site could be analyzed. There were no significant differences in transcript expression for the spleen and muscle tissue between fish that had a transmitter and those that received the surgical procedure alone. However, transmitter presence significantly increased the expression of IL-6, IL-1β and TNFα in the peritoneal cells at 10 weeks indicating that tagged fish may be coping with chronic inflammation. Furthermore, tagged male fish had a higher inflammatory response in 10-week peritoneal lavage samples when compared to their tagged mature female counterparts, providing some evidence that mature female rainbow trout may have suppressed immune function when sexually mature. Externally, fish appeared to heal at similar rates regardless of the presence or absence of the transmitter, but the tag itself was prone to encapsulation and adhesion to the body wall and/or surgical site. This suggests that fish tagged with large intraperitoneal implants may not behave similarly to their wild counterparts. This research could aid in the development of improved telemetry tags that are more biocompatible, economical and better able to track fish behavior/movement.Canada Research Council Research ChairNSERC Discovery Grant [217529

Long-term implantation of acoustic transmitters induces chronic inflammatory cytokine expression in adult rainbow trout (Oncorhynchus mykiss)

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.vetimm.2018.10.003 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Telemetry transmitters are frequently used in studies of wild fish migration and behavior. Although the effects of surgically implanted transmitters on survival, tag retention, healing and growth have been studied, there has been little research regarding the potential immune response induced by these transmitters. In the current study, mature rainbow trout received either surgical implantation of an acoustic transmitter or a sham surgical procedure. These fish were then sampled over a 10-week period so that pro-inflammatory cytokine expression in the spleen, peritoneal cavity lymphocytes and muscle at the surgical site could be analyzed. There were no significant differences in transcript expression for the spleen and muscle tissue between fish that had a transmitter and those that received the surgical procedure alone. However, transmitter presence significantly increased the expression of IL-6, IL-1β and TNFα in the peritoneal cells at 10 weeks indicating that tagged fish may be coping with chronic inflammation. Furthermore, tagged male fish had a higher inflammatory response in 10-week peritoneal lavage samples when compared to their tagged mature female counterparts, providing some evidence that mature female rainbow trout may have suppressed immune function when sexually mature. Externally, fish appeared to heal at similar rates regardless of the presence or absence of the transmitter, but the tag itself was prone to encapsulation and adhesion to the body wall and/or surgical site. This suggests that fish tagged with large intraperitoneal implants may not behave similarly to their wild counterparts. This research could aid in the development of improved telemetry tags that are more biocompatible, economical and better able to track fish behavior/movement.Canada Research Council Research ChairNSERC Discovery Grant [217529

Extracellular dsRNA induces a type I interferon response mediated via class A scavenger receptors in a novel Chinook salmon derived spleen cell line

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.dci.2018.08.010 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Despite increased global interest in Chinook salmon aquaculture, little is known of their viral immune defenses. This study describes the establishment and characterization of a continuous cell line derived from Chinook salmon spleen, CHSS, and its use in innate immune studies. Optimal growth was seen at 14–18 °C when grown in Leibovitz's L-15 media with 20% fetal bovine serum. DNA analyses confirmed that CHSS was Chinook salmon and genetically different from the only other available Chinook salmon cell line, CHSE-214. Unlike CHSE-214, CHSS could bind extracellular dsRNA, resulting in the rapid and robust expression of antiviral genes. Receptor/ligand blocking assays confirmed that class A scavenger receptors (SR-A) facilitated dsRNA binding and subsequent gene expression. Although both cell lines expressed three SR-A genes: SCARA3, SCARA4, and SCARA5, only CHSS appeared to have functional cell-surface SR-As for dsRNA. Collectively, CHSS is an excellent cell model to study dsRNA-mediated innate immunity in Chinook salmon.Natural Sciences and Engineering Research Council of CanadaCanada Research Counci

CK-2 of rainbow trout (Oncorhynchus mykiss) has two differentially regulated alleles that encode a functional chemokine

The final publication is available at Elsevier via https://doi.org/10.1016/j.vetimm.2018.02.003. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Rainbow trout chemokine 2 (CK-2) is currently the only known CC chemokine to have a mucin stalk. Further analysis of the mucin stalk region revealed a second, related CC chemokine sequence, denoted here as CK-2.1. This second sequence was determined to be an allele of CK-2 following genomic PCR analysis on several outbred individuals. Furthermore, in both in vivo and in vitro trials, CK-2 and CK-2.1 were both present, but appeared to have differential tissue expression in both control and PHA stimulated samples. Upon the development of a polyclonal antibody to rCK-2, CK-2 was only observed in the brain, liver and head kidney of PHA stimulated rainbow trout tissues. In comparison, when using the rainbow trout monocyte/macrophage-like cell line, RTS-11, CK-2 protein was observed in both control and PHA stimulated conditions. When studying the function of CK-2, a chemotaxis assay revealed that both peripheral blood leukocytes and RTS-11 cells migrated towards rCK-2 significantly at all concentrations studied when compared to truncated β2m. Interestingly, this migration was lowest at both the highest concentration and the lowest concentrations of CK-2. Thus, teleostean chemokine receptors may become desensitized when overstimulated as has been observed in mammalian models. The observed chemotactic function was indeed due to rCK-2 as cell migration was inhibited through pre-treatment of both the cells and the polyclonal antibody with rCK-2. As has been observed thus far with all other chemokines, CK-2 does appear to function through binding to a G-coupled protein receptor as chemotaxis could be inhibited through pre-treatment with pertussis toxin. Overall, the results of this study indicate that CK-2 is a functional chemokine that is encoded by two differentially expressed alleles in rainbow trout, CK-2 and CK-2.1.Natural Sciences and Engineering Research Council || 21752

PACAP Is Lethal to Flavobacterium psychrophilum Through Either Direct Membrane Permeabilization or Indirectly, by Priming the Immune Response in Rainbow Trout Macrophages

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed in mammals and is capable of performing roles as a neurotransmitter, neuromodulator, and vasodilator. This polypeptide belongs to the glucagon/secretin superfamily, of which some members have been shown to act as antimicrobial peptides in both mammalian and aquatic organisms. In teleosts, PACAP has been demonstrated to have direct antimicrobial activity against several aquatic pathogens, yet this phenomenon has never been studied throughout a live bacterial challenge. The present study focuses on the influence of synthetic Clarias gariepinus 38 amino acid PACAP on the rainbow trout monocyte/macrophage-like cell line, RTS11, when exposed to the coldwater bacterial pathogen Flavobacterium psychrophilum. PACAP was shown to have direct antimicrobial activity on F. psychrophilum when grown in both cytophaga broth and cell culture media (L-15). Further, the ability of teleostean PACAP to permeabilize the membrane of an aquatic pathogen, F. psychrophilum, was demonstrated for the first time. The viability of RTS11 when exposed to PACAP was also observed using a trypan blue exclusion assay to determine optimal experimental doses of the antimicrobial peptide. This displayed that only concentrations higher than 0.1 μM negatively impacted RTS11 survival. Interestingly, when RTS11 was pre-treated with PACAP for 24 h before experiencing infection with live F. psychrophilum, growth of the pathogen was severely inhibited in a dose-dependent manner when compared to cells receiving no pre-treatment with the polypeptide. Relative expression of pro-inflammatory cytokines (IL-1β, TNFα, and IL-6) and PACAP receptors (VPAC1 and PAC1) was also analyzed in RTS11 following PACAP exposure alone and in conjunction with live F. psychrophilum challenge. These qRT-PCR findings revealed that PACAP may have a synergistic effect on RTS11 immune function. The results of this study provide evidence that PACAP has immunostimulatory activity on rainbow trout immune cells as well as antimicrobial activity against aquatic bacterial pathogens such as F. psychrophilum. As there are numerous pathogens that plague the aquaculture industry, PACAP may stimulate the teleost immune system while also providing an efficacious alternative to antibiotic use

Serum IgM, MH class IIβ genotype and respiratory burst activity do not differ between rainbow trout families displaying resistance or susceptibility to the coldwater pathogen, Flavobacterium psychrophilum

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.aquaculture.2017.10.020 © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) is a significant threat to global aquaculture. True to its name, BCWD tends to occur at temperatures between 8 and 12°C and presents as a systemic disease with characteristic skin ulcerations. Juvenile rainbow trout are particularly susceptible and in these fish the condition is referred to as rainbow trout fry syndrome (RTFS). Resistance to F. psychrophilum is heritable and is not adversely correlated with the growth of fish, thus selective breeding appears to be an achievable approach to its control. The current study explores the connection between resistance to BCWD and several immunological markers. After determining resistance/susceptibility to F. psychrophilum following experimental infection in 40 full-sibling families of rainbow trout, selected families were experimentally infected with F. psychrophilum and differences in antibody production, major histocompatibility (MH) class IIβ genotype and respiratory burst activity (RBA) throughout infection were compared. Serum IgM production increased over time but significant differences between resistant and susceptible families were not observed at either 28days or 120days. Of the six families that were genotyped for MH class IIβ, there did not appear to be specific genotypes that conferred resistance or susceptibility to F. psychrophilum. Further, the RBA of both head kidney leukocytes and whole blood was not significantly different between the resistant and susceptible rainbow trout families. Although the selected immune markers did not differ based on resistance status, the RBA of head kidney leukocytes in all families studied dramatically decreased seven days after infection while total blood RBA remained constant. Day seven was also when severe symptoms and/or mortality due to BCWD was first observed, thus these results may reveal information regarding the pathogenesis of the organism. A better understanding of appropriate immune defenses could provide the basis for breeding programs to effectively combat this costly pathogen, but further study of functional immune markers particularly during the fry stage of development is required.Natural Sciences and Engineering Research Council’s Strategic Project Grant program [STPGP 430654-12]Canada Graduate Scholarship programOntario Graduate Scholarship progra

First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost

8 Pág.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.This work was supported by the Center for Genetic Engineering and Biotechnology, Havana, Cuba; by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-85494-C2-1-R) and by the Comunidad de Madrid (grant 2016-T1/BIO-1672). Mobility between laboratories was supported by the grant from the IUBMB Wood-Whelan Research Fellowships to J. Velázquez. It was also supported through a Canada Research Council Research Chair and Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC DG; Grant # RGPIN-2018-04116) to Brian Dixon, as well as an Ontario Graduate Scholarship in Science and Technology to Shawna L. Semple.Peer reviewe

Salmonid Antibacterial Immunity: An Aquaculture Perspective

The aquaculture industry is continuously threatened by infectious diseases, including those of bacterial origin. Regardless of the disease burden, aquaculture is already the main method for producing fish protein, having displaced capture fisheries. One attractive sector within this industry is the culture of salmonids, which are (a) uniquely under pressure due to overfishing and (b) the most valuable finfish per unit of weight. There are still knowledge gaps in the understanding of fish immunity, leading to vaccines that are not as effective as in terrestrial species, thus a common method to combat bacterial disease outbreaks is the use of antibiotics. Though effective, this method increases both the prevalence and risk of generating antibiotic-resistant bacteria. To facilitate vaccine design and/or alternative treatment efforts, a deeper understanding of the teleost immune system is essential. This review highlights the current state of teleost antibacterial immunity in the context of salmonid aquaculture. Additionally, the success of current techniques/methods used to combat bacterial diseases in salmonid aquaculture will be addressed. Filling the immunology knowledge gaps highlighted here will assist in reducing aquaculture losses in the future