The adhesion of Vibrio anguillarum to host tissues and its role in pathogenesis

Abstract. Vibrio anguillarum was shown to adhere to excised sections of rainbow trout gut in numbers which were a function of the concentration of bacterial cells and the time for which the tissue was exposed. Using a standardized method for assaying adhered bacteria the course of infection with vibriosis in rainbow trout was studied. It is suggested that the gut may be an important site of bacterial multiplication in slowly developing infections of mature fish. Bacteria adhere to gut sections of fish protected by vaccination substantially less than to sections from non-vaccinated fish.     

Attachment of Vibrio pathogens to cells of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. The attachment of Vibrio pathogens to cells of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied by use of species-specific monoclonal antibodies and indirect FITC- immunofluorescence microscopy. Vibrio anguillarum, V. ordalii and V. parahaemolyticus attached to cultured cells of rainbow trout gonads, various tissues of cryostat sections of whole fish, and smears of gills, intestine, buccal mucosa and skin. The attachment was inhibited by prior incubation of bacteria with monoclonal antibodies at titres of 1:32, or less. Other Vibrio pathogens used in this study did not attach to any trout cells. The research provides approaches to study the mechanisms of bacterial attachment in the onset of Vibrio infections.     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

Surveillance of health status on eight marine rainbow trout, Oncorhynchus mykiss (Walbaum), farms in Denmark in 2006

The health status of eight marine rainbow trout farms was followed from mid-June to mid-September 2006 by sampling both dead and healthy fish approximately every 2 weeks for bacteriological and virological investigation. No fish pathogenic viruses were detected, but all farms experienced disease and mortality as a result of various bacterial infections. Yersinia ruckeri was found on four and Renibacterium salmoninarum on five of the farms, but only during the first part of the surveillance period. This indicates that the fish carried the infection from fresh water, and cleared the infection in salt water. Aeromonas salmonicida subsp. salmonicida caused mortality on five farms, but persisted throughout the sampling period. Although A. salmonicida was probably carried from fresh water, the fish were not able to clear the infection in the sea. Vibrio anguillarum caused mortality on six of the farms throughout the sampling period, O1 being the dominant serovar, and Photobacterium damselae subsp. damselae was found on seven farms as a cause of disease. During the period of highest water temperatures Vibrio parahaemolyticus and Vibrio vulnificus were detected in dead fish in five and two farms, respectively, although their significance as causative pathogens is questionable. Vibrio vulnificus has not previously been found in rainbow trout in Denmark. Both mortality and number of antimicrobial treatments during the period were considerably higher in unvaccinated compared with vaccinated fish. Resistance to commonly used antimicrobials was low or absent.     

Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins

Abstract. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain) bacterins. The duration of protective immunity varied with the bacterin concentration, size and species of fish, but the duration between the two bacterins was comparable. In fish under 1 g duration of protective immunity was longest when the most concentrated bacterin was used. Generally, immunity lasted in 1-g fish for about 120 days, in 2-g fish for about 180 days, but in 4-g fish and larger immunity lasted for a year or longer. Coho salmon ( Oncorhynchus kisutch ) and sockeye salmon ( O. nerka ) retained immunity for a longer time and pink salmon ( O. gorbuscha ) the shortest time. Chinook salmon ( O. tshawytscha ) and rainbow trout ( Salmo gairdneri ) were intermediate. Field data generally followed the laboratory tests, but the duration appeared somewhat shortened. In one test 20-g rainbow trout were vaccinated by the shower method and no loss of immunity occurred after 311 days.     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

The effects of the use of potassium alum adjuvant in vaccines against vibriosis in rainbow trout, Salmo gairdneri Richardson

Abstract. A formalized bacterin of Vibrio anguillarum , with and without alum adjuvant, was administered intraperitoneally to rainbow trout under farm conditions. Compared with the normal bacterin the adjuvanted preparation caused pathological changes indicative of chronic peritonitis, substantial early mortalities in 3 cm fish, and to a lesser extent in 20 cm fish, and depressed growth rate. No additional degree of protection against artificial challenge was obtained from its use.     

Duration of protective immunity and antibody titres measured by ELISA after vaccination of rainbow trout, Salmo gairdneri Richardson, against vibriosis

Abstract. The level of protective immunity in rainbow trout, Salmo gairdneri Richardson, following vaccination by intraperitoneal (i.p.) injection or by direct immersion with Vibrio anguillarum was studied over a period of 46 weeks. Antibody titres to V. anguillarum O-antigen were determined with ELISA at the time of each experimental infection to study the relation between specific antibody production and protective immunity. Both vaccination by i.p. injection and vaccination by direct immersion induced a high degree of protective immunity against vibriosis which lasted for at least 46 weeks. At each experimental infection, the proportions of seropositive fish were lower than the proportions of fish with protective immunity. However, seropositive individuals were found in both experimental groups throughout the study.     

Effects of Temperature on Production and Specificity of Antibodies in Rainbow Trout (Oncorhynchus mykiss)

Abstract.— The effect of temperature on production and affinity of antibodies against antigens from the parasitic ciliate Ichthyophthirius multifiliis were studied in rainbow trout ( Oncorhynchus mykiss ). Fish were immunized with I. multifiliis antigens and reared at three different temperatures, 5, 12, and 20 C for 56 d. The production of specific antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that temperature has a pronounced effect on the assay result of the ELISA technique. Plasma samples analyzed at 5 C showed that the specific antibody response of fish reared at 5 C was similar to fish reared at 12 and 20 C. However, when samples were assayed at 12 and 20 C, the measured antibody response tended to be higher for the samples from trout reared at 12 and 20 C. Additionally, it was found that rainbow trout reared at 5 C showed a delayed but not hampered antibody response compared to fish reared at 12 and 20 C, indicating a correlation between low temperature and delayed antibody production.     

Bath-immunization—a practical and non-stressing method of vaccinating sea farmed rainbow trout Salmo gairdneri Richardson against vibriosis

Abstract. A non-stressing easily applicable vaccination method against vibriosis in rainbow trout is described. The vaccine consisted of trypsin digested Vibrio anguillarum cells and was administered through a 2 h bath treatment, giving adequate protection in challenge experiments with the same V. anguillarum strain.     

The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum)

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 x 10(8) cells g(-1) of feed, JB-1 led to a reduction in mortalities to 0-13% after challenge with a range of bacterial pathogens compared to 80-100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0-16% following the challenge compared to 80-100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.     

Evaluation of 100% Fish Meal Substitution with Chicken Concentrate,Protein Poultry By‐Product Blend,and Chicken and Egg Concentrate on Growth and Disease Resistance of Juvenile Rainbow Trout,Oncorhynchus mykiss

The ability of poultry products to replace fish meal in diets for rainbow trout, Oncorhynchus mykiss, depends on their nutrient composition, cost, and consistency. The aim of this study was to assess the ability of three commercially available poultry products (chicken concentrate, CC, poultry by‐product blend, PBB; or chicken and egg concentrate, CE) to maintain growth and disease resistance when substituted for fish meal in a rainbow trout starter diet. A control diet was formulated to contain 48% crude protein and 18% crude lipid; 100% of the fish meal in test diets was replaced with CC, PBB, or CE. At stocking, fry were counted into groups (50 fish/tank) with six replicate tanks for each diet and fed their respective diets four times daily for 8 wk. All the poultry‐based diets supported growth (over 1600% increase over initial weight), nutrient retention, and feed conversion ratios of rainbow trout fry equal to or greater than those observed for fry fed with the fish meal‐based diet. No effect of diet on survival was observed following subcutaneous injection challenge with Flavobacterium psychrophilum. These data suggest that the examined products can be used in place of fish meal for rainbow trout fry without lowering growth and disease resistance.     

Replacement of fish meal by potato protein concentrate in diets for rainbow trout, Oncorhynchus mykiss (Walbaum): growth, feed utilization and body composition

Five experimental diets (PPC1, PPC2, PPC3, PPC4 and PPC5) containing about 465 g kg⁻¹ crude protein were formulated to contain graded levels of potato protein concentrate (PPC) (a by-product of potato starch manufacturing) by replacement of fish meal protein. The contents of PPC were 0, 94, 200, 304 and 510 g kg⁻¹ respectively. The diets were prepared isonitrogenously and isocalorically. A 6-week growth trial was conducted with rainbow trout, Oncorhynchus mykiss (Walbaum), at about 14°C to evaluate the potential use of the potato protein concentrate in trout diets.
The results showed that fish fed diets containing PPC showed decreased growth, feed utilization and condition factor, and increased mortality when PPC constituted the sole protein source. Incorporation of PPC in the diets resulted in decreased dry matter, protein and fat and increased ash contents of the fish body. In conclusion, the type of potato protein concentrate used, especially at high levels, was not suitable as an ingredient in trout feed.     

The Effects of Substituting Selected Oilseed Protein Concentrates for Fish Meal in Rainbow Trout Oncorhynchus mykiss Diets

Three oilseed protein concentrates (soybean, canola, and sunflower) were evaluated to determine their potential, when supplemented with deficient essential amino acids, to partially or completely replace fish meal in diets fed to rainbow trout Oncorhynchus mykiss . Triplicate aquaria of juvenile trout (average weight of 12 g) were fed the experimental diets for 10 wk, at which time the average weight of the fish was approximately sixfold higher than the initial weight. Average fish weight gains on diets in which the protein component was 100% fish meal; 75% fish meal, 25% soybean protein concentrate; 50% fish meal, 50% soybean protein concentrate; and 75% fish meal, 25% sunflowerseed protein concentrate were not significantly different ( P < 0.05). The average weight of fish fed a commercial feed was significantly lower than that of fish fed the 100% fish meal diet, but not significantly different from fish fed the three other formulations mentioned above. Fish fed diets containing 50% fish meal, 50% canola protein concentrate; and 25% fish meal, 75% sunflowerseed protein concentrate had significantly lower average final weights than those of fish fed the other diets. Feed conversion ratio patterns among the dietary treatment groups reflected those of weight gain. Fish survival exceeded 95% on all diets. Apparent protein digestibility coefficients ranged from 79.5% (75% soybean protein concentrate, 25% canola protein concentrate) to 90.6% (100% soybean protein concentrate). The results of this study demonstrated that certain oilseed protein concentrates have good potential as protein sources in rainbow trout feeds when properly supplemented with essential amino acids.     

Total fish meal replacement with rapeseed protein concentrate in diets fed to rainbow trout (Oncorhynchus mykiss Walbaum)

The potential of rapeseed protein concentrate as fish meal alternative in diets for rainbow trout (initial average weight 37.8?±?1.4?g) was evaluated. Nine experimental tanks of a freshwater flow-through system were stocked with 12 fish each. Triplicate groups of fish received isonitrogenous (47.9?±?0.5% CP) and isoenergetic (22.4?±?0.2?kJ?g^?1) experimental diets with 0, 66 and 100% of fish meal substituted with rapeseed protein concentrate (71.2% CP), thereby providing 0, 29 and 43% of dietary protein. As the amino acid profile of rapeseed protein concentrate was comparable to fish meal, there was no need to supplement experimental diets with synthetic amino acids. At the end of the 84?days of feeding period, fish growth performance, feed intake and feed efficiencies were not compromised, when 100% of fish meal in the control diet was replaced with rapeseed protein concentrate, revealing a SGR of 1.19 or 1.10, a FCR of 1.09 or 1.18 and a feed intake of 78.5 or 74.7?g in fish fed on the control diet or fed the diet devoid of fish meal, respectively. Intestinal morphology did not reveal any histological abnormalities in all dietary groups. Blood parameters including haematocrit and haemoglobin as well as glucose, triglycerides and total protein in the plasma were not different between treatment groups. Thus, the rapeseed protein concentrate tested here has great potential as an alternative to fish meal in rainbow trout diets.     

An Alternative Procedure for Isolation of Rainbox Trout,Oncorhynchus mykiss,Serum Immunoglobulin

A method for purification of rainbow trout, Oncorhynchus mykiss, serum immunoglobulin (Ig) that relies on specific retention of rainbow trout Ig in the presence of other serum proteins, by an ion-exchange chromatography matrix (ABx resin) previously developed for the isolation of mammalian Ig is described. This protocol allows for rapid and substantial purification of total Ig including antigen-specific antibodies from the serum of rainbow trout. The procedure does not utilize antibody-derivatized matrices for immunoabsorption so it is suited to situations where fish Ig-specific antisera or monoclonal antibody is unavailable. Furthermore, total Ig can be isolated from pre-immune sera since no antigen-derivatized affinity matrices are used, eliminating the requirement for lengthy immunization regimens. With minor modification, ABx-chromatography can be adapted for the isolation of Ig from other species of fish.     

Host-specific pathogenicity of strains of Vibrio anguillarum isolated from rainbow trout Salmo gairdneri Richardson and saithe Pollachius virens (L.)

Abstract. Vibrio anguillarum strains isolated from diseased rainbow trout were found to be pathogenic to salmonids and probably to a very low extent to saithe. Strains isolated from saithe were pathogenic to saithe and non-pathogenic to salmonids.     

Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (∼93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi . Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of ∼93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.     

Stable isotopes and gut content show diet overlap among native and introduced piscivores in a large oligotrophic lake

Abstract – In past dietary studies kokanee Oncorhynchus nerka were prominent in the diet of Pend Oreille Lake's large piscivores: native bull trout Salvelinus confluentus, cutthroat trout O. clarki and northern pikeminnow Ptychocheilus oregonensis, and introduced lake trout S. namaycush and Kamloops rainbow trout O. mykiss gairdneri. However, kokanee have declined to 10–20% of their former abundance. We therefore initiated this study to understand current predation demands on kokanee and diet overlap among piscivores, using gut content samples and analysis of stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotopes from the lake's fish and invertebrate community. In gut content samples, kokanee were the main prey item of large [i.e., ≥400 mm total length (TL)] bull and lake trout; a conclusion that was affirmed by stable isotope analysis. Rainbow trout >500 mm TL consumed mostly kokanee, thus there was a high degree of diet overlap among large bull, lake and rainbow trout. Small (i.e., <400 mm TL) rainbow and cutthroat trout diets overlapped, and were composed mostly of littoral benthic invertebrates. However, gut content and stable isotope analysis did not accord for 400–500 mm TL rainbow trout, small lake trout, and large cutthroat trout. In these instances, a linear mixing model using stable isotope results predicted kokanee consumption for each species, but no kokanee were identified in rainbow or lake trout gut content. Gut content and stable isotope analysis of native northern pikeminnow indicated a diet of mostly littoral benthic invertebrates at smaller (100–150 mm TL) lengths, with kokanee becoming more prominent in the diet of individuals >300 mm TL. Percent of kokanee in the diet of northern pikeminnow has declined from a prior study; otherwise piscivore diets have apparently remained unchanged. In this study, judgments as to the feeding of some piscvores, based on gut content alone, would be tenuous because of small sample sizes, but stable isotope analysis provided an efficient means for confirming diets.     

Salmonid macrophages: separation, in vitro culture and characterization

Abstract. A method is described for the separation of fish leucocytes and the establishment of pure monolayers of fish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90–99% of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.