Coho salmon Oncorhynchus kisutch strain differences in disease resistance and non-specific immunity, following immersion challenges with Vibrio anguillarum.

Two strains of freshwater-reared coho salmon Oncorhynchus kisutch were compared for differences in the activity of selected non-specific immune factors before and after lethal and non-lethal immersion challenges with the marine bacterial pathogen Vibrio anguillarum (Vang). Two disease challenge experiments were performed. The first experimental challenge resulted in no mortality; however, significant strain and challenge treatment effects were detected at Day 16 post-challenge. Strain differences in plasma lysozyme activity were found in pre-challenge samples. The second challenge experiment compared the same strains of coho salmon following immersion challenges in different doses of Vang. The fish were sampled at Days 0, 2, 7, and 18 post-challenge and mortality, plasma lysozyme, and anterior kidney phagocyte respiratory burst activity were compared. There were significant strain differences in mortality in the high dose group. The more disease-resistant strain was found to have higher levels of plasma lysozyme and anterior kidney phagocyte respiratory burst activity. These strain differences were detected at various times in the lethal (high dose) and non-lethal challenge groups. There was a clear relationship between the enhanced survival of the more disease-resistant strain and a more sustained, elevated non-specific immune response following the experimental disease challenges. The results of this study suggest that the basis for strain differences in innate disease resistance is related to the ability of the fish to respond quickly to the initial infection and to maintain the response until the infection is quelled.     

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.     

Isolation of salmon pancreas disease virus (SPDV) in cell culture and its ability to protect against infection by the 'wild-type' agent

A Scottish salmon pancreas disease virus (SPDV) has been isolated and its optimum growth conditions determined. Although several fish cell lines have been tested, successful culture was achieved only with CHSE-214 cells. Cytopathic effects were observed after 5 days. The highest virus titres, calculated by microtitration assay, were reached at 15 degrees C. After 7-9 days post-inoculation, CHSE-214 cell supernatants contained between 10(7)-10(5) TCID50 ml(-1) The cultured isolate is chloroform- and pH 3.0-sensitive, and virions are 50-60 nm in diameter. These characteristics are similar to the Irish SPDV isolates. The culture isolate induced typical pancreas disease (PD) lesions in experimentally infected Atlantic salmon and convalescent fish were resistant to experimental infection with PD-infective kidney homogenates obtained by serial in vivo passages from a PD-infected farmed salmon (termed wild-type SPDV). Furthermore, fish immunised with the inactivated cultured virus were protected against a cohabitation challenge with the wild-type virus. Immunised fish sera showed virus-neutralising activity before challenge (7 weeks post-immunisation) and from 3-6 weeks post-challenge, when sera from non-immunised fish did not neutralise the virus. At 6 weeks post-cohabitation challenge, previously immunised fish had neutralising titres of up to 1:65. Following intraperitoneal (i.p.) challenge, immunised fish showed neutralising titres as high as 1:226 at 8 weeks post-challenge. Non-immunised fish injected i.p. with the wild-type virus developed serum-neutralising activity against the cultured isolate when sampled 8 weeks after infection, confirming an antigenic relationship between the wild-type and cultured virus. The results demonstrate that the tissue culture-adapted isolate of SPDV could be successfully used to protect against challenge by the wild-type virus and could therefore have potential use as an inactivated vaccine against PD.     

Effects of dexamethasone on host innate and adaptive immune responses and parasite development in rainbow trout Oncorhynchus mykiss infected with Loma salmonae

The effects of dexamethasone (dex) treatment on infections with the microsporidian parasite, Loma salmonae and the effects of dex on initiation of the adaptive immune response were investigated in rainbow trout, Oncorhynchus mykiss experimentally infected with the parasite. Dex treatment resulted in significantly higher infections with the parasite in the gills and other internal organs, suggesting that dex inhibits aspects of the innate immune response to L. salmonae; the heavier infections in the gills and organs of rainbow trout resembled infections seen in Chinook salmon. Mean xenoma counts per microscope field in the gills of fish infected with L. salmonae treated with dex or left untreated were 169 and 30, respectively. Although higher numbers of xenomas were observed in dex treated fish, the xenomas were generally smaller in size than in infected control fish. The xenomas in dex treated fish showed morphological signs of degeneration including loss and degeneration of early parasite stages, accumulation of amorphous material in xenomas, and infiltration with phagocytic cells containing degenerated parasites. The xenomas in infected untreated fish had larger xenomas with a more uniform size and contained identifiable parasite stages in the cytoplasm. According to this study, once fish have developed an adaptive immune response to the parasite by previous exposure, then fish have 100% protection to reinfection even when treated with heavy doses of dex. L. salmonae immune fish treated or untreated with dex during reinfection with the parasite developed no xenomas in the gills 6 weeks post reinfection. These results indicate that once the cellular response is primed to L. salmonae, then dex related immunosuppression does not reduce the effectiveness of the adaptive immune response.     

Neutrophils and B-cells in blood and head kidney of Atlantic salmon (Salmo salar L.) challenged with infectious pancreatic necrosis virus (IPNV)

Salmon B-cells and neutrophils were studied by flow cytometry in IPNV infected salmon. A highly virulent strain of IPNV was used for challenge of parr and post-smolts. The parr were challenged by intraperitoneal (ip) injection while salmon post-smolts were challenged by ip injection or cohabitation. No mortality occurred in the parr groups, but a cumulative mortality of about 50% was obtained in cohabitant infected post-smolt groups and less than 10% in ip challenged post-smolts. The virus levels were low in head kidney (HK) samples from survivors compared to dead fish. The percentages of neutrophilic granulocytes and Ig+ cells (B-cells) were analysed using HK and blood samples from survivors. The cell populations were identified by monoclonal antibodies (MAb) E3D9, recognising neutrophils, and G2H3 recognising Ig+ cells (B-cells). Parr sampling for leucocyte analyses took place about 1.5 weeks prior to and about 4 weeks post challenge. This corresponded to about 8 and 2.5 weeks before the fish were adapted to seawater transfer. In parr head kidney leucocytes (HKL) we observed significantly lower (p < 0.05) levels of neutrophils in ip infected fish compared to non-infected control fish. The post-smolt sampling from infected fish took place 2 weeks prior to and in the fifth and sixth week post challenge. HKL samples from both surviving cohabitants and ip injected fish had significantly (p < 0.05) lower levels of neutrophils than non-infected control fish. The cohabitant fish also had significantly (p < 0.05) higher levels of B-cells in HKL compared to ip injected fish. No significant changes in B-cells in HKL or peripheral blood leucocytes (PBL) was observed in infected parr or ip infected post-smolts compared to control fish. The relative leucocyte levels of the fish prior to challenge and in non-infected control fish are in accordance with earlier findings. The results indicate that non-specific immune cells like neutrophils are highly influenced by IPNV infection of parr and post-smolts several weeks post challenge.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Comparative characteristics of the lipid status of gills of juvenile Atlantic salmon infected with glochidia of the freshwater pearl mussel living in rivers of the European North

A comparative study of the lipid status of gills of juvenile Atlantic salmon infested with glochidia of the freshwater pearl mussel inhabiting the Vuokinyoki (White Sea basin) and Syuskyuyanyoki (basin of Lake Ladoga) rivers in the fall is carried out. The most infested gills of juvenile salmon and the lower water temperature (1.3°C) in the Vuokinyoki River reveal higher cholesterol levels and cholesterol/phospholipids ratios, which are due to the influence of infection factors; thereby they slow down biochemical processes. It is noted that variations in the lipid composition in juvenile salmon the gills of which are infected with glochidia reflect the development of adaptive responses that maintain homeostasis of juveniles under invasion.     

Expression of Intracellular Enzymes During Hyphal Aggregate Formation in a Fruiting-Impaired Variant of Agaricus bisporus

The specific activity and enzyme protein concentration of the developmentally regulated enzyme glucose 6-phosphate dehydrogenase (G6PD) were measured in the developing aggregates and supporting mycelium of a fruiting-impaired variant strain of Agaricus bisporus. The nonregulated enzymes mannitol dehydrogenase (MD) and hexokinase (HK) were assayed for comparison. G6PD activity was higher in aggregates than in the mycelium, whereas MD and HK activities varied little between mycelium and aggregates. Enzyme protein levels varied in a way different from enzyme activity, suggesting the presence of inactive enzyme at times during development. The raised level of G6PD in aggregates provides a possible mechanism for the increased mannitol concentration previously observed in aggregates. There was no parallel to the rapid increase in G6PD activity associated with primordium development of normally fruiting strains growing on compost.     

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.     

Functional Feeds Reduce Heart Inflammation and Pathology in Atlantic Salmon (Salmo salar L.) following Experimental Challenge with Atlantic Salmon Reovirus (ASRV)

Heart and Skeletal Muscle Inflammation (HSMI), recently associated with a novel Atlantic salmon reovirus (ASRV), is currently one of the most prevalent inflammatory diseases in commercial Atlantic salmon farms in Norway. Mortality varies from low to 20%, but morbidity can be very high, reducing growth performance and causing considerable financial impact. Clinical symptoms, including myocarditis, myocardial and red skeletal muscle necrosis, correlate with the intensity of the inflammatory response. In the present study, the effects of two functional feeds (FF1 and FF2) were compared to a standard commercial reference feed (ST) in Atlantic salmon subjected to an ASRV challenge. The functional feeds had reduced levels of total lipid and digestible energy, and different levels and proportions of long-chain polyunsaturated fatty acids (LC-PUFA). The objective was to determine whether these feeds could provide effective protection by decreasing the inflammatory response associated with HSMI. Histopathology, viral load, fatty acid composition and gene expression of heart tissue were assessed over a period of 16 weeks post-infection with ASRV. The viral load and histopathology scores in heart tissue in response to ASRV infection were reduced in fish fed both functional feeds, with FF1 showing the greatest effect. Microarray hierarchical cluster analysis showed that the functional feeds greatly affected expression of inflammation/immune related genes over the course of the ASRV infection. Viral load correlated with up-regulation of pro-inflammatory genes at the early-mid stages of infection in fish fed the ST diet. Expression of inflammatory genes 16-weeks after ASRV challenge reflected the difference in efficacy between the functional feeds, with fish fed FF1 showing lower expression. Thus, severity of the lesions in heart tissue correlated with the intensity of the innate immune response and was associated with tissue fatty acid compositions. The present study demonstrated that dietary modulation through clinical nutrition had major influences on the development and severity of the response to ASRV infection in salmon. Thus, HSMI was reduced in fish fed the functional feeds, particularly FF1. The modulation of gene expression between fish fed the different feeds provided further insight into the molecular mechanisms and progression of the inflammatory and immune responses to ASRV infection in salmon.     

Glochidiosis of salmonid fishes. II. Comparison of tissue response of coho and chinook salmon to experimental infection with Margaritifera margaritifera (L.) (Pelecypoda: Margaritanidae)

Coho salmon (Oncorhynchus kisutch) are more resistant to experimental infection with the glochidia of the freshwater mussel (Margaritifera margaritifera) than are chinook salmon (Oncorhynchus tshawytscha). Histological sections made at intervals during the infection showed that coho salmon sloughed the parasites from their gills by 4.5 days postinfection, but the parasites remained encysted in the gills of chinook salmon for 12 weeks, when metamorphosis to juvenile mussels was complete. Coho salmon sloughed the parasites by a well-developed hyperplasia. No such pronounced hyperplastic reaction was seen in the gills of chinook salmon.     

General transducing phages like Salmonella phage P22 isolated using a smooth strain of Escherichia coli as host

A smooth colony strain, resistant to phages λ and P22, was isolated from sewage and identified as Escherichia coli (strain H). Four temperate phages plaquing on strain H were isolated from sewage. The archetype, HK620, does not plaque on strains C and K12 of E. coli nor on the LT2 strain of Salmonella enterica. Bacterial mutants resistant to a clear plaque mutant of HK620 produce rough colonies. Some are also galactose-negative, a few are histidine auxotrophs, and most show sensitivity to λ. HK620 can transduce a wide variety of auxotrophic mutants of E. coli H to prototrophy. It can recombine with λ but its virions resemble those of P22.     

Envelope Determinants of Equine Lentiviral Vaccine Protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection.     

Susceptibility of Baltic and East Atlantic salmon Salmo salar stocks to Gyrodactylus salaris (Monogenea)

The susceptibility of a Baltic salmon stock Salmo salar (Indals?lv, central Sweden) to Norwegian Gyrodactylus salaris (Figga strain, central Norway) was experimentally tested and compared with previously obtained results on East Atlantic salmon (Lierelva, SE Norway). Contrary to expectation, the Baltic salmon, which had no prior exposure to this parasite strain, appeared almost as susceptible as the Norwegian salmon parr that naturally experience G. salaris-induced mortality. Individually isolated salmon of both stocks sustained G. salaris infections with little evidence of innate resistance. A few individuals of the Indals?lv stock controlled their infection from the beginning, but overall there was considerable heterogeneity in the course of infection in both stocks. On individual hosts, G. salaris growth rates declined steadily throughout the infection, a trend which was particularly marked amongst the Lierelva stock. On shoaling Lierelva fish, there was some evidence of reduced parasite population growth towards the end of the infection; this was not apparent in Indals?lv fishes. These results reflect a growing awareness that not all Baltic salmon may be resistant to Norwegian G. salaris, and that Norwegian and Baltic G. salaris strains may differ in virulence. Consequently, management decisions concerning this parasite-host system should be based upon the actual, and tested, susceptibility of stocks under consideration and not upon identification of stocks as either Atlantic or Baltic.     

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.