Brook charr, Salvelinus fontinalis (Mitchill), and cryptobiosis: a potential salmonid reservoir host for Cryptobia salmositica Katz, 1951

Abstract. Laboratory-raised Cryptobia -susceptible brook charr, Salvelinus fontinalis (Mitchill), and rainbow trout, Oncorhynchus mykiss (Walbaum), were vaccinated intraperitoneally with a live Cryptobia salmositica vaccine (250000 parasites per fish), and 4 weeks later were challenged with the pathogen (250000 parasites per fish). Unvaccinated and infected brook charr had high parasitaemias but no clinical signs of disease, while unvaccinated and infected rainbow trout had anaemia and general oedema. Vaccinated and challenged fish had very low parasitaemias compared to unvaccinated and infected brook charr and rainbow trout. Complement fixing antibodies were detected in vaccinated and challenged fish 2 weeks after challenge. Unvaccinated and infected brook charr had consistently higher litres of complement fixing antibody than unvaccinated and infected rainbow trout. Parasitaemias were lower in all fish in which titres of complement fixing antibody were high. In a second experiment, brook charr inoculated intraperitoneally or intramuscularly with 100000 C. salmositica per fish had high parasitaemias but no anaemia or other clinical signs. The results show that susceptible brook charr do not suffer from cryptobiosis and may serve as reservoir hosts for C. salmositica in areas where the disease is prevalent. Vaccination to reduce the parasitaemia when fish become infected may be a control strategy in these areas.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the pathogenic haemoflagellate, Cryptobia salmositica Katz, and protection against cryptobiosis in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated with a live vaccine

Abstract. An enzyme-linked immunosorbent assay using dried blood on filter paper, was developed for the detection of antibodies against the haemoflagellate Cryptobia salmositica in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Each fish (average weight about 5g) in three experimental groups was either inoculated with 20000 attenuated live C. salmositica vaccine, or inoculated with 2000 or 20000 pathogenic parasites per fish. The vaccine was effective in protecting juvenile trout 4 weeks after vaccination and antibody titers were higher in vaccinated and challenged fish than in unvaccinated and infected ones. Specific antibodies were detected one week post-infection (w.p.i.) with the pathogen and declined to low levels at 6 w.p.i. The high-dose group (20000 per fish) had antibody titres comparable to those of the vaccinated and challenged fish.     

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum)

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.     

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.     

In vivo and in vitro cell-mediated immune responses of rainbow trout, Oncorhynchus mykiss (Walbaum), against Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida)

Abstract. Delayed-type hypersensitivity (DTH) reaction (an in vivo manifestation of cell-mediated immunity) was detected in Oncorhynchus mykiss maintained on a pantothenic-acid-supplemented diet 2 weeks after infection with Cryptobia salmositica. The reaction was similar to that in mammals with mononuclear cell infiltration into the dermis and muscle layers and the presence of oedema. DTH reaction was also displayed by fish on a pantothcnic-acid-supplemented diet that had recovered from the infection and were protected against further infection. The reaction was less marked in infected or protected fish on a pantothenie-acid-deficient diet. Inhibition of macrophage migration (an in vitro expression of cell-mediated immunity) was observed when head kidney cell suspensions from protected fish maintained on either pantothenic acid supplemented or deficient diets were incubated with Cryptobia antigen. No inhibition of migration was evident when head kidney cell suspensions from the above fish were incubated without antigen, nor was it evident when cells from uninfected fish were used. The occurrence of a typical DTH reaction in rainbow trout and the feasibility of assessing it by measuring the thickness of the induration provides a simple and practical method for assessing cell-mediated immunity in large scale vaccination programmes against pathogens.     

Cryptobia salmositica: cortisol increases the susceptibility of Salmo gairdneri Richardson to experimental cryptobiosis

Abstract. Intraperitoneal implants of cortisol (cortisol suspended in hydrogenated coconut oil) were used to induce a graded hypercortisolism in rainbow trout, Salmo gairdneri Richardson. There was no obvious reduction in circulating lymphocytes in cortisol-implanted rainbow trout (70, 140 or 210μg/g body weight). Cortisol-implanted fish infected with Cryptobia salmositica had significantly higher parasitaemia and lower antibody litres compared with controls infected with haemonagellate but given coconut oil implants. These confirm the immunodepressive effects of the steroid. The parasite was also more readily detected at the early stage of the infection (shorter prepatent period, more infected fish and higher parasitaemia) in cortisol-implanted fish (140 and 210 μg/g body weight) than in controls. The mortality of the infected cortisol-implanted fish was higher than that of the infected fish implanted with only coconut oil, or the cortisol-implanted but non-infected fish. This in vivo study suggests that protective immunity against C. salmositica is, in part, due to a humoral response.     

Acclimation of rainbow trout to sea water

In some fish farms it is advantageous to transfer rainbow trout from fresh water to sea water at as small a size as possible. It has been shown that a relatively small reduction in salinity at the marine site can give appreciable improvements in survival rates during acclimation by smaller fish, thus rainbow trout of 15 g have routinely been put directly into sea cages at a salinity of 22% with mortality levels of from 1 to 8%.     

Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout (Oncorhynchus mykiss)

A trial was conducted to determine the effect of a combination of dietary glucan and vitamin C on the immune response of rainbow trout. After 3 weeks of adaptation on a control diet (without glucan but containing 150 ppm vitamin C), rainbow trout were fed the experimental diets containing yeast glucan and vitamin C at 150, 1000 and 4000 ppm for 2 weeks and then switched back to the control diet for the following 4 weeks. Macrophage activity (chemiluminescence response), complement activation and lysozyme levels were monitored just after feeding the experimental diets (week 0) and 2 and 4 weeks later. The kinetics of antibody response after vaccination against enteric redmouth disease were determined. The fish were immunised at week 0, at the end of the experimental feeding.Tissue ascorbic acid contents were monitored every 2 weeks and reflected the dietary treatments. No differences were observed in complement levels activated via the classical pathway. Two weeks after feeding the experimental diets, alternative pathway of complement activation and chemiluminescence response were enhanced by high doses of vitamin C. Significant enhancement of macrophage activity was still evident at week 2. No significant differences were observed in lysozyme levels. The specific immune antibody response was enhanced following vaccination, when glucan was also present in the diet.     

A comparative epizootiologic study of the two fish‐pathogenic serovars of Vibrio vulnificus biotype 2

Vibrio vulnificus biotype 2 is subdivided into two main serovars, serovar E, able to infect fish and humans, and serovar A, only virulent for fish. Serovar E emerged in 1976 as the causative agent of a haemorrhagic septicaemia (warm‐water vibriosis) affecting eels cultured in brackish water. Serovar A emerged in 2000 in freshwater‐cultured eels vaccinated against serovar E, causing warm‐water vibriosis with fish showing a haemorrhagic intestine as the main differential sign. The aim of the present work was to compare the disease caused by both serovars in terms of transmission routes, portals of entry and host range. Results of bath, patch‐contact and oral‐anal challenges demonstrated that both serovars spread through water and infect healthy eels, serovar A entering mainly by the anus and serovar E by the gills. The course of the disease under laboratory conditions was similar for both serovars in terms of transmission and dependence of degree of virulence on water parameters (temperature and salinity). However, the decrease in degree of virulence in fresh water was significantly greater in serovar E than in serovar A. Finally, both serovars proved pathogenic for tilapia, sea bass and rainbow trout, but not for sea bream, with significant differences in degree of virulence only in rainbow trout. In conclusion, serovar A seems to represent a new antigenic form of V. vulnificus biotype 2 with an unusual portal of entry and is better adapted to fresh water than serovar E.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Comparison of brown trout (Salmo trutta) reared in fresh water and sea water to freshwater rainbow trout (Oncorhynchus mykiss): II. Phosphorus balance

Brown trout and rainbow trout (average weight 100 g) were reared in fresh water at 12 °C under the same conditions before transferring brown trout to sea water, in order to compare phosphorus utilisation in both species. Apparent phosphorus availability, orthophosphate excretion and phosphorus accretion in the fish were directly determined. Thus, actual phosphorus mass balance was built. Rainbow trout raised in fresh water had a higher phosphorus retention coefficient (maximum 50 %) than brown trout reared in fresh water (maximum 45 %). Transferring brown trout to sea water induced a reduction in phosphorus retention (maximum 39 %). Orthophosphate excretion, ranging 7–20 mg phosphorus per kg wet weight per day, represented 10–20 % of ingested phosphorus. Phosphorus availability was lower in brown trout raised in sea water (65 %) than brown trout raised in fresh water (76 %). Phosphorus balance measurements showed that 90 to 98 % of phosphorus flow could be accounted for.     

Effects of Oral Administration of Specific Antibodies to Rainbow Trout Oncorhynchus mykiss

A new product called oralized fish serum concentrate (OFSC) was evaluated for a possible effect against various bacterial pathogens in rainbow trout. The OFSC produced from immune trout sera was found to contain fully functional antibodies and complement component C3. The antibodies detected in the serum concentrate were specific to Vibrio anguillarum (O1 and O2) and Aeromonas salmonicida , which had been used for vaccination of the fish prior to serum collection. The functionality of the specific antibodies in OFSC was not reduced after 6 wk storage at -20 C, 5 C, and 20 C. The serum was mixed with commercial trout feed and used for feeding rainbow trout fry (first feed period). After oral delivery of OFSC to rainbow trout for 1 mo, samples of gut content and gut tissue contained functional antibodies. In gutted fish no functional antibodies were found. This suggests that antibodies from OFSC are unable to be transferred across the gut wall in a functional state. Oral administration of OFSC did not increase survival of rainbow trout in an immersion challenge with Vibrio anguillarum .     

Comparison of growth rate in Atlantic salmon,pink salmon,Arctic char,sea trout and rainbow trout under Norwegian farming conditions

Growth rates of Atlantic salmon, pink salmon, Arctic char, sea trout and rainbow trout were compared under Norwegian farming conditions. During the juvenile, freshwater period, growth was fastest in pink salmon, followed by rainbow trout and Arctic char. Freshwater growth of sea trout and, especially, Atlantic salmon, was slow. After transfer of smolts or fingerling to sea water, Arctic char failed to survive the autumn. Sea water growth of sea trout was slow, but the three species, rainbow trout, Atlantic salmon and pink salmon, all grew rapidly through all seasons. When in sea water, rainbow trout and pink salmon were regularly attacked by vibriosis, while Atlantic salmon were rarely attacked, and sea trout never. It is concluded that, for commercial farming in Norway, rainbow trout are of value for production of fish of any size up to 3–4 kg, and pink salmon for production of small fish of 0.5–1.5 kg. Atlantic salmon is the only species suitable for production of a very large salmonid, i.e., more than 4–5 kg.     

The Migrations of North Esk Sea Trout

The migrations of North Esk sea trout were studied by tagging all stages of sea trout taken in a stationary trap situated on Kinnaber Mill Lade which drains its water from the main river. The majority of recaptures were reported from the Montrose area, and evidence of a great deal of intermixing between the sea trout of the North Esk and neighbouring rivers was found. After their initial migration as smolts a proportion of the tagged sea trout returned to fresh water within the same year. Some tagged fish undertook several journeys back and forth between sea water and fresh water during (his period. Numerous examples of tagged sea trout travelling appreciable distances (>100 km) along the coast were obtained. In addition four tagged fish were recaptured in excess of 500 km from the North Esk, three off the Scandinavian coast and one in the River Barvas, North West Lewis.     

Atypical growth of Renibacterium salmoninarum in subclinical infections

Two growth types of Renibacterium salmoninarum were isolated from subclinically infected rainbow trout, one producing the smooth colonies typical of R. salmoninarum and the other forming a thin film on the surface of the agar with no separate colonies. The atypical growth was present on kidney disease medium agar in primary cultures of the kidney but not on selective kidney disease medium (SKDM). Fluorescent antibody staining of the fresh isolate and polymerase chain reaction amplification were the most reliable techniques to identify the atypical growth of R. salmoninarum. The condition was reversible, with growth reverting from atypical to the smooth colony form in experimentally infected rainbow trout and under laboratory conditions. There was no mortality, or any clinical signs of bacterial kidney disease (BKD) in the fish challenged with the atypical growth, although small numbers of smooth colonies of R. salmoninarum were isolated from 8% of these fish. The atypical growth reported here may explain some of the failures of culture, when SKDM agar alone is used for the detection of BKD in subclinically infected fish.     

Infectious pancreatic necrosis in Atlantic salmon, Salmo salar L

A clinical and histopathological review was carried out of 21 outbreaks of acute infectious pancreatic necrosis (IPN) in Scottish Atlantic salmon, Salmo salar L., farms (13 marine and eight fresh water) during 1991-2004. A distinctive syndrome was evident in both post-smolts in sea water and fry in fresh water, where liver lesions, which had not previously been associated with IPN, became a consistent finding in addition to the more typical pancreatic and intestinal changes. Initial cases were described in post-smolts in Shetland, but by the end of the period of investigation this type of pathology had extended down the West coast of Scotland and into Ireland. Limited viral strain analysis suggested that similar strains were involved in both fresh water and sea water and that these differed from earlier isolates from rainbow trout, Oncorhynchus mykiss (Walbaum). In fresh water, recovered fish frequently developed a greatly distended intestine associated with accumulation of undigested food. In sea water, after the initial, often significant (50% or more), losses, there were many fish which failed to grow and became chronically emaciated and prone to sea louse infection. Although use of transfer diets containing immune enhancers and the selection of IPN resistant broodstock has reduced losses the disease remains a serious cause of economic loss.     

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.     

Protection, immune response and side-effects in European whitefish (Coregonus lavaretus L.) vaccinated against vibriosis and furunculosis

As indicated by market demand and cultivation prospects, the whitefish (Coregonus lavaretus L.) has the potential to become an important alternative to rainbow trout in fish farming in Finland. The fish processing industry has called for a fish species having non-pigmented flesh, and there is a long tradition of whitefish farming for stocking purposes in Finland. However, cultivation in net cages in the brackish water of the coastal area exposes fish to vibriosis (Listonella (Vibrio) anguillarum) and furunculosis (Aeromonas salmonicida salmonicida). Hence, profitable whitefish farming requires the efficient control of both of these diseases. The efficacy of vaccination (Apoject 1800®) was studied in the laboratory using challenge tests and by monitoring specific antibody production (ELISA) and the blood leucocyte pattern. The fish (22 g) were vaccinated at 15°C and the immunity developed during 8 weeks prior to challenge with vibriosis (1.8×10⁵ cfu) and 12 weeks prior to challenge with furunculosis (4.9×10² cfu). The challenge was performed by i.p. injection. Side-effects around the injection site were studied 16 weeks post-vaccination. The immune system of the whitefish responded well and vaccination gave good protection against vibriosis and furunculosis. The RPS was 100% following the Vibrio challenge and 99% following the Aeromonas challenge. The antibody levels continued increasing in the vaccinated group throughout the study period. Lymphocyte and neutrophil counts were elevated in vaccinated compared to non-vaccinated fish at 8 weeks post-vaccination, but not after 16 weeks. Vaccination produced weak adhesions around the injection site in 42% and minor pigmentation in 9% of the 65 fish examined.     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.