Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production

The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to : lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities. Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined. Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs. Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested. An extracellular metallo-caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect. Furuncular-like lesions were produced by ECPs containing AsaP1 activity. One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity. The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases.     

Sequential study of antigen persistence and concomitant inflammatory reactions relative to side-effects and growth of Atlantic salmon (Salmo salar L.) following intraperitoneal injection with oil-adjuvanted vaccines

The persistence of antigens at the injection site (area around pyloric caeca and spleen), concomitant inflammatory reaction and granuloma development were monitored at 3, 6 and 12 months following intraperitoneal injection with multivalent, oil-adjuvanted vaccines in Atlantic salmon. Parallel assessment of side-effect profiles and growth rate were also performed. Antigen persistence was examined by use of a monoclonal antibody that recognises Aeromonas salmonicida lipopolysaccharide in an immunohistochemical method for in situ identification of bacteria or bacterial fragments. The inflammatory reaction was monitored using standard histological techniques. The amount of persistent antigens and size of inflammation/adhesions were estimated semi-quantitatively. A steady decrease in the quantity of antigens at the injection site was observed from 3 to 12 months. Antigens were consistently found in inflamed tissues located in the pancreatic region. The size of inflammation increased during the first 6 months but declined thereafter. These findings suggest that persistent antigens at the injection site may act as inflammatory stimulants that induce and perpetuate the inflammatory reaction, eventually leading to adverse side-effects.     

Time-course study of injection site inflammatory reactions following intraperitoneal injection of Atlantic cod (Gadus morhua L.) with oil-adjuvanted vaccines

The inflammatory response of Atlantic cod (Gadus morhua L.) following vaccination with oil-based vaccines has not been previously characterized in any detail. In this study, groups of Atlantic cod were intraperitoneally injected with commercial oil-adjuvanted vaccines ALPHA JECT 3000 (AJ 3000) and AJ 6-2. A water-based vaccine ALPHA MARINE Vibrio (AVM), an experimental liposome vaccine and physiological saline (placebo) were also included for comparison. Histopathological changes at the injection sites were evaluated semi-quantitatively at 1, 2, 4, 8, 12, 16, 20 and 25 weeks post-vaccination (p.v.), parallel with the examination of vaccine antigen retention. Gross intra-abdominal lesions were only examined at 12 and 25 weeks. The results show that the onset of inflammation in all vaccinated groups was rapid to develop, with intense cellular infiltrations predominated by mononuclear cells especially in groups injected by oil-based vaccines. Inflammation induced by AVM and liposome vaccines resolved within 12 weeks. In contrast, oil-adjuvanted vaccines produced mild, persistent but ultimately decreasing reactions. Persistent antigens were observed in oil-based and liposome vaccines. The results show that the cod inflammatory response is similar to other bony fish species. The findings also suggest that cod has an efficient innate immune system that is able to rapidly remove or sequester antigens from the injection site leading to the down-regulation of inflammation. Oil-adjuvanted vaccines appear to be well-tolerated by this species and show promise as a possible approach for disease control.     

Comparative susceptibility of Atlantic salmon (Salmo salar) to the enteric redmouth bacterium and Aeromonas salmonicida

The bacterium causing enteric redmouth (ERM) and Aeromonas salmonicida were found to be equally pathogenic for fingerling Atlantic salmon (Salmo salar). Injection of 5 x 10(5) cells of ERM or A. salmonicida killed all salmon within 96 h. After a 30 min exposure to water-borne cells of the two test bacteria about one-half of the test salmon died within 14 days. Both ERM and A. salmonicida were transmitted horizontally. Results indicate that efforts should be made to prevent introduction of ERM into watersheds where Atlantic salmon occur.     

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.     

Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.     

The quantitative relationship of lethality between extracellular protease and extracellular haemolysin of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.)

An additive relationship of lethality between purified protease and haemolysin of the extracellular products (ECP) of Aeromonas salmonicida was demonstrated by i.p. injection in Atlantic salmon (Salmo salar L.). The lethal toxicity of the combinations of protease and haemolysin follow a linear regression line y = -54.54x + 2400. The LD50 of protease and haemolysin when injected separately was 2400 ng/g fish and 44 ng protein/g fish, respectively.     

Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.     

Antigen-induced blastogenesis of salmon, Salmo saluv L., head kidney leucocytes to modified Aeromonas salmonicida antigens: a preliminary evaluation method for potential vaccine antigens

Various Aeromonas salmonicida antigen preparations were tested in an antigen-induced blasto-genesis assay on the kidney leucocytes of Atlantic salmon. The ECP antigens that had been particularized onto polystyrene beads were the most effective in control fish, and this was further enhanced in fish thymectomized 1 week prior to the assay. The use of this assay for screening potential vaccine antigens is discussed.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua).

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV

A naked plasmid DNA encoding the glycoprotein (pCMV4-G) of a 1976 isolate of infectious hematopoietic necrosis virus (IHNV) obtained from steelhead Oncorhynchus mykiss was used to vaccinate Atlantic salmon Salmo salar against IHNV. Eight weeks post-vaccination the fish were challenged with a strain of IHNV originally isolated from farmed Atlantic salmon undergoing an epizootic. Fish injected with the glycoprotein-encoding plasmid were significantly (p < 0.05) protected against IHNV by both immersion and cohabitation challenge. Survivors of the first challenges were pooled and re-challenged by immersion 12 wk after the initial challenge. Significant (p < 0.05) protection was observed in all of the previously challenged groups including those receiving the complete vaccine. Fish injected with the glycoprotein-encoding plasmid produced low levels of virus-neutralizing antibodies prior to the first challenge. Neutralizing antibodies increased in all groups after exposure to the IHNV. Passive transfer of pooled sera from pCMV4-G vaccinates and IHN survivors provided relative survivals of 40 to 100% compared to fish injected with sera collected from fish immunized with control vaccines or left unhandled. In this study, DNA vaccination effectively protected Atlantic salmon smolts against challenges with IHNV.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

The effect of vaccination and sea water entry on immunocompetence and susceptibility to Kudoa thyrsites in Atlantic salmon (Salmo salar L.)

The effect of intraperitoneal (IP) vaccination and sea water entry (SWE) on the immunocompetence of Cascade Atlantic salmon was investigated. Smolts were IP injected with Aqua Health's Forte trade mark vaccine (Listonella (Vibrio) anguillarum, Listonella ordalii, Vibrio salmonicida and Aeromonas salmonicida) at four times (42, 238, 433 and 630 degree days, DD) prior to SWE and were examined for immunocompetence. Immune response measurements included mitogen-driven proliferation of peripheral blood leukocytes (PBL), head kidney leukocyte respiratory burst activity and alternative complement hemolytic titres and were measured 24h prior to SWE, 72 h post-SWE and again 3.5 weeks post-SWE. A 50% reduction in the number of PBL was observed 3 days post-vaccination. At this time LPS-driven proliferation was low (stimulation index, SI, 1.5-2.9) in all groups prior to SWE compared with that of PBL from freshwater-reared Atlantic salmon parr (6.7). By 72 h and 3.5 weeks post-SWE, the LPS-driven SI from unvaccinated salmon and those vaccinated 630 and 433 DD prior to SWE increased 3-fold. In contrast, SI from salmon vaccinated 42 and 238 DD prior to SWE remained low. A similar pattern was observed for cultured PBL stimulated with PHA, although unlike LPS-stimulated PBL, the SI of cells from parr and unvaccinated control smolts remained low following SWE but increased in fish vaccinated 433 and 630 DD prior to SWE. The respiratory burst activity of head kidney leukocytes was not affected by SWE but showed a transient 50% depression 3 days post-vaccination. The alternative complement activity (ACH50) was similar for all treatment groups prior to and at 72h post-SWE. By 3.5 weeks post-SWE, ACH50 values in salmon vaccinated 42 and 238 DD prior to SWE doubled to 874 and 860 U/ml, respectively. The prevalence and severity of Kudoa thyrsites infections, detected in all treatment groups approximately 2400 DD following SWE, were not significantly different among groups. Atlantic salmon parr should be IP vaccinated no earlier than 433 DD before SWE to avoid an enhanced risk of acquiring pathogens because of transient depression in some immune mechanisms.     

Vaccination-induced systemic autoimmunity in farmed Atlantic salmon

Over half of the salmon consumed globally are farm-raised. The introduction of oil-adjuvanted vaccines into salmon aquaculture made large-scale production feasible by preventing infections. The vaccines that are given i.p. contain oil adjuvant such as mineral oil. However, in rodents, a single i.p. injection of adjuvant hydrocarbon oil induces lupus-like systemic autoimmune syndrome, characterized by autoantibodies, immune complex glomerulonephritis, and arthritis. In the present study, whether the farmed salmon that received oil-adjuvanted vaccine have autoimmune syndrome similar to adjuvant oil-injected rodents was examined. Sera and tissues were collected from vaccinated or unvaccinated Atlantic salmon (experimental, seven farms) and wild salmon. Autoantibodies (immunofluorescence, ELISA, and immunoprecipitation) and IgM levels (ELISA) in sera were measured. Kidneys and livers were examined for pathology. Autoantibodies were common in vaccinated fish vs unvaccinated controls and they reacted with salmon cells/Ags in addition to their reactivity with mammalian Ags. Diffuse nuclear/cytoplasmic staining was common in immunofluorescence but some had more specific patterns. Serum total IgM levels were also increased in vaccinated fish; however, the fold increase of autoantibodies was much more than that of total IgM. Sera from vaccinated fish immunoprecipitated ferritin and approximately 50% also reacted with other unique proteins. Thrombosis and granulomatous inflammation in liver, and immune-complex glomerulonephritis were common in vaccinated fish. Autoimmunity similar to the mouse model of adjuvant oil-induced lupus is common in vaccinated farmed Atlantic salmon. This may have a significant impact on production loss, disease of previously unknown etiology, and future strategies of vaccines and salmon farming.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua)

In 1988, a new plasmid profile was observed for Vibrio salmonicida isolated from cod (Gadus morhua) and Atlantic salmon (Salmo salar) in fish farms in northern Norway. This new plasmid profile, which consisted of plasmids of 61, 21, 3.4, and 2.8 megadaltons, is 1 of 11 plasmid profiles which have so far been observed for V. salmonicida. Plasmid profiling and plasmid DNA hybridization were used in epidemiological studies of cold-water vibriosis. Our results indicate that V. salmonicida was transmitted from Atlantic salmon to cod and vice versa. The 61-megadalton plasmid was found exclusively in V. salmonicida strains originating from northern Norway, which is the only area in which this plasmid has ever been observed. Plasmid DNA hybridization and restriction endonuclease analysis show that the plasmid DNA of V. salmonicida remained stable throughout a 7-year survey.     

Ontogeny of the Digestive System of Atlantic Salmon (Salmo salar L.) and Effects of Soybean Meal from Start-Feeding

Despite a long history of rearing Atlantic salmon in hatcheries in Norway, knowledge of molecular and physiological aspects of juvenile development is still limited. To facilitate introduction of alternative feed ingredients and feed additives during early phases, increased knowledge regarding the ontogeny of the digestive apparatus in salmon is needed. In this study, we characterized the development of the gastrointestinal tract and accessory digestive organs for five months following hatch by using histological, biochemical and molecular methods. Furthermore, the effects of a diet containing 16.7% soybean meal (SBM) introduced at start-feeding were investigated, as compared to a fishmeal based control diet. Salmon yolk sac alevins and fry were sampled at 18 time points from hatch until 144 days post hatch (dph). Histomorphological development was investigated at 7, 27, 46, 54 and 144 dph. Ontogenetic expression patterns of genes encoding key digestive enzymes, nutrient transporters, gastrointestinal peptide hormones and T-cell markers were analyzed from 13 time points by qPCR. At 7 dph, the digestive system of Atlantic salmon alevins was morphologically distinct with an early stomach, liver, pancreas, anterior and posterior intestine. About one week before the yolk sac was internalized and exogenous feeding was started, gastric glands and developing pyloric caeca were observed, which coincided with an increase in gene expression of gastric and pancreatic enzymes and nutrient transporters. Thus, the observed organs seemed ready to digest external feed well before the yolk sac was absorbed into the abdominal cavity. In contrast to post-smolt Atlantic salmon, inclusion of SBM did not induce intestinal inflammation in the juveniles. This indicates that SBM can be used in compound feeds for salmon fry from start-feeding to at least 144 dph and/or 4-5 g body weight.     

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.