Temperature impacts Atlantic salmon's (Salmo salar) immunological response to infectious salmon anemia virus (ISAv).

Ocean temperatures continue to rise annually due to the ever-growing consequences of global climate change. These temperature changes can have an impact on the immunological robustness of cultured fish, especially cold-water species such as Atlantic salmon. The salmon farming industry already loses hundreds of millions of dollars each year to infectious and non-infectious diseases. One particularly important and WOAH reportable disease is infectious salmon anemia caused by the orthomyxovirus ISAv. Considering the changing environment, it is necessary to find ways to mitigate the effect of diseases on the industry. For this study, 20 Atlantic salmon families were housed in each of 38 different tanks at the AVC, with half of the fish being kept at 10 °C and half being kept at 20 °C. Donor Atlantic salmon IP- injected with a highly virulent ISAv isolate (HPR4; TCID50 of 1 × 105/mL) were added to each tank as the source of co-habitation infection. Both temperatures were sampled at onset of mortality in co-habited fish and at resolution of mortality. Family background and temperature significantly impacted ISAv load, as assessed by qPCR, time to mortality and overall mortality. Mortality was more acute at 20 °C, but overall mortality was higher at 10 °C. Based on percent mortality calculated over the course of the study, different families demonstrated different levels of survival. The three families that demonstrated the highest percent mortality, and the three families with the lowest percent mortality were then assessed for their antiviral responses using relative gene expression. Genes significantly upregulated between the unexposed fish and ISAv exposed fish included mx1, il4/13a, il12rb2, and trim25, and these were further impacted by temperature. Understanding how ISAv resistance is impacted by temperature can help identify seasonal risks of ISAv outbreaks as well as ideal responses to be targeted through immunopotentiation.

Pathologic features of mycobacteriosis in naturally infected Syngnathidae and novel transcriptome assembly in association with disease.

Syngnathidae (seahorses, seadragons and pipefish) suffer significant losses from non-tuberculous mycobacteria. However, they produce markedly different lesions in response to the disease compared to other teleost species, notably infrequent granuloma formation. This study evaluated 270 syngnathid fish, from which 92 were diagnosed with mycobacteriosis by histopathology, culture or both. Microscopic lesions variably consisted of random foci of coagulative necrosis in multiple organs, containing high numbers of free bacteria and large aggregates or sheets of macrophages with cytoplasm laden with acid-fast bacilli. Mycobacterial associated granulomas were identified in only six seahorses. Five fish had positive cultures with no observed microscopic changes. RNA-seq of the head kidney was performed to investigate the transcriptome of two infected and six non-infected lined seahorses Hippocampus erectus. Assembled and annotated putative transcripts serve to enrich the database for this species, as well as provide baseline data for understanding the pathogenesis of mycobacteriosis in seahorses. Putative components of the innate immune system (IL-1ß, IL-6, TNF, NOS, Toll-like receptor 1, MHC Class I, NF-κß, transforming growth factor beta, MyD88) were identified in the RNA-seq data set. However, a homolog for a key component in the TH1 adaptive immune response, interferon-gamma, was not identified and may underlie the unique pathologic presentation.

Stress, nutrition and parental care in a teleost fish: exploring mechanisms with supplemental feeding and cortisol manipulation.

Parental care is an essential life-history component of reproduction for many animal species, and it entails a suite of behavioural and physiological investments to enhance offspring survival. These investments can incur costs to the parent, reducing their energetic and physiological condition, future reproductive capabilities and survival. In fishes, relatively few studies have focused on how these physiological costs are mediated. Male smallmouth bass provide parental care for developing offspring until the brood reaches independence. During this energetically demanding life stage, males cease active foraging as they vigorously defend their offspring. Experimental manipulation of cortisol levels (via implantation) and food (via supplemental feeding) in parental males was used to investigate the fitness consequences of parental care. Improving the nutritional condition of nest-guarding males increased their reproductive success by reducing premature nest abandonment. However, supplemental feeding and cortisol treatment had no effect on parental care behaviours. Cortisol treatment reduced plasma lymphocyte numbers, but increased neutrophil and monocyte concentrations, indicating a shift in immune function. Supplemental feeding improved the physiological condition of parental fish by reducing the accumulation of oxidative injury. Specifically, supplemental feeding reduced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) on DNA nucleotides. Increasing the nutritional condition of parental fish can reduce the physiological cost associated with intensive parental activity and improve overall reproductive success, illustrating the importance of nutritional condition as a key modulator of parental fitness.

Spatial patterns in markers of contaminant exposure, glucose and glycogen metabolism, and immunological response in juvenile winter flounder (Pseudoplueronectes americanus).

Inshore winter flounder (Pseudoplueronectes americanus) populations in NY, USA have reached record low numbers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gradient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253 transcripts of >100bp were differentially expressed, with 60% showing strong homology to mostly teleost sequences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in individual flounder from each site. Statistically significant site-specific differences were observed in expression of all but one gene, although patterns in expression were complex with only one (vitellogenin), demonstrating a west to east gradient consistent with known loadings of municipal sewage effluent. Principal components analysis (PCA) identified relationships among the genes evaluated. Our data indicate that juvenile winter flounder are responding to estrogenic chemicals in more urbanized coastal bays, and suggests potential mechanistic links between immune response, contaminant exposure and energy metabolism.

Measures of immune system status in young-of-the-year winter flounder Pseudopleuronectes americanus.

The immune status of young-of-the-year (YOY) winter flounder Pseudopleuronectes americanus was evaluated in fish collected from six areas around Long Island, NY, U.S.A. representing more urban areas with high population density in the west, to less densely populated more rural areas in to the east. Gene expression markers for innate immunity (pleurocidin) and contaminant exposure (cytochrome P4501A; cyp1a) were measured in liver and fin of fish collected at each site. Expression of pleurocidin was significantly higher in fin than liver, but was highly variable among individuals. Some statistically significant differences in pleurocidin expression among sites were observed, although elevated levels were not associated with degree of urbanization. Expression was related in part to fish size: a positive correlation between expression and total length (LT ) of fish was observed with the largest LT class (>125 mm) exhibiting significantly elevated pleurocidin expression as compared with fish in the smaller LT class. This indicates that immune competency may increase with age. No site-specific differences in cyp1a expression were observed. These data suggest that exposure to aromatic hydrocarbon contaminants is fairly widespread throughout the study area and that any differences in pleurocidin expression in YOY P. americanus are probably due to other factors. Antimicrobial activity was also measured as a functional indicator of immune response. Activity was highly variable, showing no significant site-specific differences, and no significant correlation to pleurocidin expression. The lack of correlation between pleurocidin expression and antimicrobial activity indicates that other antimicrobial peptides may be active against the bacteria tested or that other factors are influencing antimicrobial activity. This is the first quantitative evaluation of pleurocidin expression in YOY P. americanus from an urban area. Further work is needed to characterize factors controlling pleurocidin expression, as well as other indicators of immune response in young fish.

Immunostimulation of Salmo salar L., and its effect on Lepeophtheirus salmonis (Krøyer) P-glycoprotein mRNA expression following subsequent emamectin benzoate exposure.

Control of sea lice, Lepeophtheirus salmonis, on farmed Atlantic salmon, Salmo salar, relies heavily on chemotherapeutants. However, reduced efficacy of many treatments and need for integrated sea lice management plans require innovative strategies. Resistance to emamectin benzoate (EMB), a major sea lice parasiticide, has been linked with P-glycoprotein (P-gp) expression. We hypothesized that host immunostimulation would complement EMB treatment outcome. Lepeophtheirus salmonis-infected Atlantic salmon were fed immunostimulatory or control feeds. Sea lice were collected for 24-h EMB bioassays 1 and 2 weeks prior to commencement of EMB treatment of the fish. Two weeks after cessation of immunostimulant-treated feed, EMB was administered at 150 µg kg(-1) fish biomass for 7 days. The bioassay revealed stage, gender and immunostimulant-related differences in EMB EC(50) . Sea lice attached to salmon with a history of immunostimulation exhibited significantly greater survival than those on control feeds, despite similar levels of EMB in host tissues. Lepeophtheirus salmonis from salmon with a history of immunostimulation also exhibited higher P-gp mRNA expression as well as greater survivability compared to controls. Administration of immunostimulants prior to EMB treatment caused increased expression of P-gp mRNA which could have consequently caused decreased efficacy of the parasiticide.

Combinatorial effects of administration of immunostimulatory compounds in feed and follow-up administration of triple-dose SLICE® (emamectin benzoate) on Atlantic salmon, Salmo salar L., infection with Lepeophtheirus salmonis.

Several immunostimulatory feed additives have shown the ability to induce protective responses in Atlantic salmon to infection with Lepeophtheirus salmonis. However, even the most encouraging results rarely surpass a 50% protective index in the host. That fact coupled with the well-documented limitations of single-therapy strategies in the effective management of parasitic infections generally make it imperative to identify therapies that can be combined in an integrated pest management approach for sea lice. With this in mind, we hypothesized that immunostimulatory feeds could enhance the protection provided by SLICE® emamectin benzoate (EMB). To test this hypothesis, Atlantic salmon were fed one of two different immunostimulatory feeds (CpG ODN or Aquate®) for c. 7 weeks, challenged with L. salmonis copepodids early within that immunostimulatory feed period and then placed on a triple-dose (150 µg kg(-1) ) feed of SLICE® for 1 week following the completion of the immunostimulatory feeding period. CpG ODN (2 mg kg(-1) ) and the commercial yeast extract (Aquate® 0.2%) inclusion in feeds were able to successfully induce inflammatory gene expression (interleukin-1ß) in the head kidneys of infected fish at 13 and 26 days post-exposure (DPE), and 13 DPE, respectively. Lice burdens were lower on fish fed CpG ODN (18%) or Aquate® (19%) diets; however, due to variability, these were not statistically significant over time. Despite no statistically significant reductions in lice numbers, by 33 DPE fish on immunostimulatory feeds had significantly reduced cortisol levels when compared to infected fish on control diet. Cortisol levels in fish receiving an immunostimulatory diet were no different from initial baseline levels prior to infection, whereas the levels in control diet fish were significantly elevated from all other time points. Despite the positive effects on infection of fish fed immunostimulatory feeds, no synergism was observed with follow-up treatment with SLICE® . In fact, highest survival of lice was observed in fish with prior immunostimulation.

CpG inclusion in feed reduces sea lice, Lepeophtheirus salmonis, numbers following re-infection.

Lepeophtheirus salmonis infections in Atlantic salmon, Salmo salar, have been characterized by little to no hyperplastic response and a biphasic immune response that results in chronic inflammation with tissue repair as the infection progresses. We hypothesized that CpG administration with prior lice exposure would enhance epithelial inflammatory mechanisms and boost the Atlantic salmon immune response to L. salmonis, leading to greater protection against infection. We administered multiple exposures of L. salmonis to two groups of Atlantic salmon and compared responses against first-time exposed Atlantic salmon. Following re-exposure, CpG fed fish exhibited increased skin expression of interleukin (IL)-1ß and IL-12 ß compared to control previously exposed (CPE) and control first-time exposed (CFE) animals, respectively. This inflammatory enhancement occurred with significantly lower expression of matrix metalloproteinase-9 (MMP 9), both systemically (spleen) and locally (skin). Reduced MMP 9 expression was a hallmark of the re-infected fish (occurred in both tissues at both times). When significant differences were present in the skin or spleen, the two re-exposed groups showed greater similarity than with the first exposure group. Lice numbers on CpG fed fish were significantly lower than CFE fish at 7 days post-re-infection (dpri), and although they were not significantly different at 17 dpri, the trend of lower lice levels remained. CpG fed fish also showed nearly twofold greater protection than CPE when compared to the CFE group (48.5% vs. 27.0% reductions at 7 dpri and 27.2% vs. 13.1% reductions at 17 dpri, respectively). The enhanced protection of CpG oligodeoxynucleotide administration to previous exposure was consistent across all body surfaces and suggests that CpG can not only enhance innate responses to L. salmonis in Atlantic salmon, but also further stimulate adaptive responses.

Immunophysiology of Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill), and the relationship to parasitic copepod, Dichelesthium oblongum (Abilgaard) infection.

The copepod parasite, Dichelesthium oblongum, is known to infect the Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus, within the area near New York city, USA, known as the NY Bight. The gross pathology associated with the juvenile and adult copepod stages along with the parasite's link in causing changes in sturgeon osmoregulatory capabilities has led us to investigate the host immunophysiology in relation to this host-parasite system. All the host variables, which included gill Na(+) -K(+) -ATPase activity, serum alkaline phosphatase (AP) and white blood cell differential counts, were affected in a non-linear manner by the copepod parasite. The parasites increased the host gill Na(+) -K(+) -ATPase activity and serum AP along with the percentage granulocytes while decreasing the percentage lymphocytes. A new method, developed to sample and preserve white blood cells in the field for future flow cytometry analysis, proved adequate. The effects of fish size, location and time of sampling were accounted for by the use of generalized linear models, and their effects on the host variables are discussed.

Lepeophtheirus salmonis secretory/excretory products and their effects on Atlantic salmon immune gene regulation.

We have previously shown that Lepeophtheirus salmonis produces trypsin and prostaglandin E(2) (PGE(2)) that are most likely responsible for the limited inflammatory response of Atlantic salmon to infection. After removal of the dopamine and PGE(2), the immunomodulatory activity of unfractionated and pools of the fractionated secretions was determined by examining the effects of the secretions on Atlantic salmon immune gene expression. Incubation of macrophage-enriched isolates of Atlantic salmon head kidney cells with the unfractionated secretion + PGE(2) revealed a significant inhibition of interleukin-1beta (IL-1beta) and major histocompatibility class I gene expression. Inhibition of lipopolysaccharide-induced IL-1beta expression in the Atlantic salmon head kidney cell line (SHK-1) was observed when three pools of the secretory/excretory products were tested. Further purification of products within these pools revealed that fraction 1-2 could account fully for the inhibition of IL-1beta expression in SHK-1 cells observed in pooled fraction 1. This study demonstrates that there are other immunomodulatory compounds produced by L. salmonis, in addition to PGE(2) and trypsin, that can inhibit the expression of Atlantic salmon immune-related genes in vitro.

Differential expression of the pro-inflammatory cytokines IL-1beta-1, TNFalpha-1 and IL-8 in vaccinated pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon juveniles.

Laboratory-reared pink and chum salmon juveniles (approximately 2g) received an intraperitoneal injection with a commercial, unadjuvanted Aeromonas salmonicida bacterin or sterile saline. Relative to elongation factor-1A, expression levels of genes encoding the proinflammatory cytokines interleukin-1beta-1 (IL-1beta), tumour necrosis factor-alpha-1 (TNFalpha) and interleukin-8 (IL-8) in pools of kidney and liver were examined 6- and 24-h after injection. Expression of IL-1beta was significantly elevated in pink and chum salmon by 6-h, and declined in pink salmon but not in chum salmon by 24-h. Similarly, expression of TNFalpha was significantly elevated in both species at 6h and only in chum salmon after 24-h. Expression of IL-8 was significantly elevated in both species at 6- and 24-h after injection. Expression of the three proinflammatory cytokine genes differed between salmon species both in the timing and magnitude of their expression. The significance of these differences with respect to immune function in these fish requires further research.

The effects of Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic salmon (Salmo salar).

This study was conducted to determine the effects of a high level of infection of the parasitic copepod L. salmonis on the stress response and immunological status of Atlantic salmon. An initial low-level initial infection was carried out 14d prior to a second infection in which twice as many parasites were introduced. Plasma cortisol and prostaglandin E(2) (PGE(2)) levels were monitored concurrent to the expression of six immune-related genes over five sample times (9, 21, 26, 33 and 40days post initial infection, dpii). The mean lice counts on the infected fish increased significantly from the first infection (16.3+/-1.89 at 9dpii) to the second (142.8+/-12.8 at 26dpii). Plasma cortisol levels increased significantly at 26, 33 and 40dpii in infected fish compared to controls. Plasma PGE(2) levels were significantly higher in infected fish at 9, 33 and 40dpii, when compared to controls. At 9dpii, expression of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNFalpha)-like cytokine, major histocompatibility class II (MH II), transforming growth factor-beta (TGFbeta)-like cytokine and cyclooxygenase-2 genes were increased in infected fish compared to controls. The expression of most of these genes returned to control levels at 21dpii when the highest expression of the MH class I gene was observed in infected fish (significantly higher than controls). Major histocompatibility class I gene expression remained higher in infected fish at 26 and 33dpii compared to controls and this was observed for the TNFalpha-like gene. By 33dpii, MH class II and TGFbeta-like genes had higher expression in infected fish compared to controls. Interleukin-1beta and TNFalpha-like gene were the only genes that showed significantly higher expression in infected fish compared to controls at 40dpii, while MH class I gene expression was significantly depressed in infected fish at this time. The expression of nearly all immune-related genes studied here increased following initial infection with L. salmonis, however, immunological stimulation did not reduce parasite numbers or protect against re-infection.

Prostaglandin E(2) modulation of gene expression in an Atlantic salmon (Salmo salar) macrophage-like cell line (SHK-1).

Following lipopolysaccharide (LPS)-stimulation of Atlantic salmon (Salmo salar) macrophage-like SHK-1 cells, prostaglandin E(2) (PGE(2)) exhibited dose-dependent inhibition of the antigen presenting molecules major histocompatability class I and II and the pro-inflammatory cytokine interleukin-1 beta gene expression. Prostaglandin E(2) was found to be stimulatory towards cyclooxygenase-2 (COX-2) expression at higher concentrations (1 x 10(-6) and 1 x 10(-8)M) and inhibitory at lower concentrations (1 x 10(-10) and 1 x 10(-12)M) after 4h exposure. After 24h exposure, however, LPS-induced COX-2 expression decreased and was completely inhibited by all PGE(2) concentrations (1 x 10(-6)-1 x 10(-10)M). Incubation of SHK-1 cells with LPS alone had no effect on tumour necrosis factor alpha (TNFalpha)-like gene or transforming growth factor beta-like gene expression after 4h, however, LPS and PGE(2) showed a synergistic effect on TNFalpha-like gene expression after 24h. This study provides evidence for the existence of a PGE(2)-mediated negative feedback mechanism in the control of PGs through down-regulation of COX-2, as well as for inflammatory responses by the down-regulation of both COX-2 and IL-1 beta. The differential regulation of immune-related genes under these conditions further demonstrates the usefulness of the SHK-1 cell line for studying aspects of salmonid immunology.

Lepeophtheirus salmonis: characterization of prostaglandin E(2) in secretory products of the salmon louse by RP-HPLC and mass spectrometry.

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.

Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids.

Adult and mobile preadult sea lice Lepophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sealice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis-derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon.

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.