Cryptobia salmositica: an in vitro and in vivo study on the mechanism of anaemia in infected rainbow trout, Salmo gairdneri Richardson

Abstract. There are two basic antigenic components in Cryptobia that are responsible for the anaemia in infected rainbow trout. A 'lytic component', which is dosage-dependent, causes lysis of red blood cells independent of antibody or complement. The second, an 'immune-complex-forming component', attaches to red blood cells, forms immune complexes with specific antibody and activates complement resulting in haemolysis. These two antigenic components, from both live and lysed Cryptobia , were present in the serum of infected fish. When sonicated antigen or heat-inactivated antiserum (from infected fish) was incubated with red cells from uninfected fish, a portion of the red cells was lysed and a positive Coombs' reaction was observed with the remaining intact red cells. The positive Coombs' reaction was due to immune complexes adsorbed onto the red cells and these lysed when incubated with complement. Antibody by itself did not adsorb onto the red cells. From the fourth week post-infection, a positive Coombs' reaction was observed in all infected fish and haemolysis occurred with complement. The authors suggest that, in infected fish, one or more components of the complement cascade is depleted continually during infection and that the anaemia is due to the lytic action of the antigen and immune complex formation on red cells. These lead to intra-vascular haemolysis as well as erythrophagocytosis. In general, the mechanism of anaemia in cryptobiosis appears similar to that in African trypanosomiasis.     

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.     

Light and electron microscope observations on extrasporogonic and sporogonic stages of a myxosporean parasite of the genus Sphaerospora Thelohan, 1892 from Atlantic salmon, Salmo solar L., in Scotland

Abstract. Juvenile Atlantic salmon from a number of freshwater hatcheries in Scotland were found to be infected by a myxosporean parasite of the genus Sphaerospora. Fish first became infected in June by extrasporogonic stages which could be found in the blood and kidney interstitium. These consisted of a primary cell containing one to over one hundred secondary cells. Some secondary cells contained one or two tertiary cells. Sporogony was disporous and occurred later in the kidney tubules. The development of both extrasporogonic and sporogonic stages was studied by light and transmission electron microscopy.     

Plasma protein changes in Atlantic salmon, Salmo salar L., with infectious salmon anaemia

Moderate to severe anaemia and hypoproteinaemia were reported in a Canadian outbreak of 'haemorrhagic kidney syndrome' in Atlantic salmon, later shown to be caused by a variant of infectious salmon anaemia virus (ISAV). The progressive anaemia associated with ISA has been previously reported, but hypoproteinaemia in salmon infected with European isolates of ISA virus has not been well documented. The present study showed a very significant positive correlation between decreasing haematocrit values and total plasma protein concentrations in Atlantic salmon infected with two Canadian and two Norwegian ISA viral isolates. However, variations in the concentration of individual plasma proteins, typical of acute phase responses in higher vertebrates, were not observed.     

Changes in serum parameters of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., infected with Renibacterium salmoninarum

Abstract. Bacterial kidney disease was induced experimentally in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, S. salar L., and selected serum parameters measured over a 35-day period. In those fish which became infected a significant increase in the erythrocyte sedimentation rate and in the serum levels of bilirubin, blood urea nitrogen and potassium were recorded. A significant decrease in total serum protein, cholesterol and sodium also occurred in these fish. Electrophore-tic analysis of serum proteins showed the fast migrating fraction was reduced before the slower migrating material in infected fish.     

Decreased concentration of hemoglobin,accumulation of lipid oxidation products and unchanged skeletal muscle in Atlantic salmon (Salmo salar) fed low dietary vitamin E

Atlantic salmon (Salmo salar) fry, initial weight 0.16 g, were fed a semipurified diet with 0, 15, 30, 60 or 120 mg dl-α-tocopheryl acetate/kg. After 24 weeks, the first two of these groups were extinct, and the fish receiving 30 mg/kg were clearly vitamin E deficient. Vitamin E deficient fish had low hemoglobin levels, characterized by a combination of reduced cellular hemoglobin concentration, red cell volume and red cell number, and an increased number and fraction of immature red blood cells. The hemoglobin concentration decreased over the decreasing range of experimental dl-ga-tocopheryl acetate levels. Therefore, even if 60 mg dl-α-tocopheryl acetate/kg gave good survival, this level was clearly physiologically suboptimal. Ceroid accumulated in the liver of fish fed 30 mg vitamin E/kg, and autofluorescent inclusions were found in the red blood cells of fish fed 30 and 60 mg vitamin E/kg. Degeneration of skeletal muscle was not observed in the present study.     

Effect of marine Eubothrium sp. (Cestoda: Pseudophyllidea) on the growth of Atlantic salmon, Salmo salar L.

Atlantic Salmon, Salmo salar L., experimentally infected with marine Eubothrium sp. were kept together with uninfected salmon in the laboratory for 11 months in two tanks, 80 infected and 80 uninfected in each tank. The infected fish had a reduced growth rate compared with the uninfected fish. Significant differences in growth between infected and uninfected fish were not observed until several months post-infection. There was no correlation between the number of Eubothrium sp. and fish weight, indicating that even low intensity alters the growth rate of the salmon. The cestode had the same effect on both sexes of salmon. Haematocrit level was found to be significantly lower in infected compared with uninfected salmon in one of the samples during the experiment.     

Effects of adrenaline on ionic equilibria in red blood cells of rainbow trout (Salmo gairdneri)

Effects of adrenaline on the equilibrium distributions of Na⁺ , K⁺ , H⁺ , Cl^– , and H₂O across the cell membrane of rainbow trout (Salmo gairdneri) erythrocytes were determinedin vitro, as a function of P CO₂ (1.76–7.77 torr). CO₂-carrying capacity of the blood was also examined. Plasma catecholamine concentrations inunanaesthetized, unrestrained trout were 3.1 nM adrenaline and 1.2 nM noradrenaline. Elevation of the plasma adrenaline concentrationin vitro to 4.6 × 10³ nM resulted in net gains of Na⁺ , Cl^– and H₂O by red cells, a net loss of H⁺ from red cells, and a pronounced red cell swelling. Adrenaline also reduced the CO₂-carrying capacity of trout bloodin vitro. The magnitudes of these effects increased with P_CO2 and, thus, were sensitive to blood HCO₃ ^– concentrations. The distribution of K⁺ between red cells and plasma was unaffected by adrenaline. Adrenergic-mediated ion movements and red cell swelling were sensitive to both propranolol and SITS. These results are consistent with the symport NaCl uptake model for adrenergic-mediated swelling of Baroinet al. (1984). The adrenergic response of fish erythrocytes may function to ameliorate the effects of blood acidoses on O₂-carrying capacity by maintaining red cell pH in the face of a decrease in plasma pH.     

Control of Aeromonas hydrophila Infection in Spotted Snakehead, Channa punctatus, by Solanum nigrum L., a Medicinal Plant

The present study describes the potential of black nightshade, Solanum nigrum , to control Aeromonas hydrophila infection in spotted snakehead, Channa punctatus . Similar sizes of Ch.   punctatus were grouped into three categories as follows: control (injected with sterilized water), Ae.   hydrophila infected   +   untreated, and Ae.   hydrophila infected   +   treated with S.   nigrum leaf extract. Hematological parameters (white blood cell [WBC], red blood cell [RBC], hemoglobin [Hb], and packed cell volume [PCV]) were analyzed in these fishes once every 5   d from Days 5 to 30 postexposure. Preliminary antibacterial assay reported that leaf extracted with ethyl acetate had the maximum inhibition compared to other parts of the plant (i.e., berries and whole plant) or when extracted with other solvent extracts (chloroform and ethanol). Except for WBC count, all other parameters showed a declining trend from Day 5 onward and reaching to a minimum on Day 30 in Ae.   hydrophila -infected fish, whereas the WBC count increased. The infected fish treated with S.   nigrum leaf extract reported a significant reversion to normal RBC, WBC, Hb, and PCV levels and the skin ulceration recovered within a short period. The antiulcer potential of the medicinal plant as described in humans may be extended to the fish population.     

Herpesvirus salmonis: pathological changes in parenterally-infected rainbow trout, Salmo gairdneri Richardson, fry

Abstract. After fry of rainbow trout, Salmo gairdneri Richardson, had been infected parenterally with Herpesvirus salmonis , moribund or freshly dead specimens were examined histopathologically. The virus produced a generalized infection, the first signs of which appeared after 2–3 weeks. Visceral and respiratory organs and the heart showed major pathological changes, and pancreatic syncytia were judged to be pathognomonic. Kidneys were prime targets for the virus and showed the highest levels of infectivity; lesser amounts of virus were present in the stomach, liver, and intestine. The virus did not spread by contact to produce clinical disease nor could disease or viral replication be induced by parenteral inoculation of yearling Oncorhynchus nerka (Walbaum).     

The immune response of rainbow trout, Salmo gairdneri Richardson, to the haemoflagellate, Cryptobia salmositica Katz, 1951

Abstract. Rainbow trout that recovered from experimental Cryptobia salmositica infection 6 and 10 weeks earlier were protected against multiple intraperitoneal challenges of 50 000 and 10 000 parasites isolated from infected fish. The immunity was non-sterile; low parasitaemias were detected following a larger challenge (112 000 parasites). The indirect haemagglutination test was used to detect C. salmositica -specific agglutimns. Antibody titers increased during the first 18 weeks of infection. The infectivity of cultured C. salmositica was neutralized by incubation in heat-inactivated immune plasma. Infectivity of C, salmositica from infected fish was not neutralized by similar treatment. Complement fixing antibody was detected using the in vitro immune lysis test. Immune lysis occurred when cultured C. salmositica were used. Adoptive transfer of both leucocytes and plasma from immune fish conferred partial protection against the parasite in naive recipients. Complement fixing antibody may be important during early acute infection while phagocytosis may be important during the later chronic phase.     

Changes in serum parameters of Saprolegnia-infected brown trout, Salmo trutta L.

Abstract. Fungal infection of both wild and hatchery-reared sexually mature brown trout, Salmo trutta L., was caused by Saprolegnia diclina Humphrey Type 1. The major ions in the serum of infected fish were all significantly reduced in concentration and it is suggested that the primary cause of death was osmoregulatory breakdown resulting in a lethal haemodilution. Severe hypoproteinaemia and a significant reduction in the albumin/globulin ratio were reflected in the electro-phoretogram of the serum proteins from infected fish.     

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, BKD and cold water vibriosis

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, bacterial kidney disease (BKD) and cold water vibriosis was studied by challenge testing one-year-old fingerlings. Fish from 81 full-sib families within 32 sire progeny groups were infected with Aeromonas salmonicida, Renibacterium salmoninarum and Vibrio salmonicida. Estimated heritabilities were relatively low, being highest for BKD (h²= 0.23) and lowest for cold water vibriosis (h²= 0.13). Genetic correlations between the ability to survive the diseases were all positive, but the magnitude of the genetic correlation between furunculosis and BKD may be biased upwards because some of the dead BKD fish were also infected with furunculosis. The application of selection to develop resistant populations of Atlantic salmon is advocated. Challenge testing seems to be a feasible method, with relatively low costs and easy management. The future response to selection will depend on the relationships between results from a challenge test and mortalities under farming conditions and between disease resistance and other traits in the breeding goal.     

Immunoglobulin heterogeneity in the rainbow trout, Salmo gairdneri Richardson

Abstract. Hetereogeneity of rainbow trout immunoglobulins was demonstrated by using monoclonal antibody 1A6 and polyacrylamide-gradient gel electrophoresis. Immunoglobulins defined by elisa using monoclonal antibody 1A6 were about 30% of the total immunoglobulins, detected by elisa using polyclonal antibodies, in healthy rainbow trout. In trout obtained from farms with a previous history of infectious viral diseases, 1A6-immunoglobulins were only about 14% of the total. Several serum pools from infected trout could be totally depleted of 1A6-immunoglobulins (about 12% of total immunoglobulins) by affinity chromatography over Sepharose immobilized monoclonal antibody 1A6. Polyacrylamide-gradient gel electrophoresis under denaturing conditions of total immunoglobulins, 1A6 immunoglobulins and no-1A6 immunoglobulins purified by affinity chromatography, showed a majority heavy chain of 70 KDa and a minority heavy chain of about 60 KDa, two light chains of 24 and 26 KDa, and a 11–14 KDa polypeptide.     

Further consideration of the haematology of proliferative kidney disease (PKD) in rainbow trout, Salmo gairdneri Richardson

Abstract. A study of selected haematological parameters in rainbow trout, Salmo gairdneri Richardson, exposed to proliferative kidney disease (PKD) under field conditions is described. Changes in the blood picture are related to kidney pathology at different stages of the disease. As extensive renal vascular pathology, haemoglobin crystallization and inflammatory lesions developed, there were progressive decreases in haemoglobin and packed cell volume values, erythrocyte numbers declined to less than 50% of those in control fish, and numbers of misshapen erythrocytes increased. Leucopenia, neutrophilia and thrombocytopenia developed, especially in oedematous fish. With recovery, most values were similar to those of control fish. In recovered, re-challenged fish, erythrocyte counts, packed cell volume and haemoglobin values decreased in infected fish compared with uninfected fish. The results of the study are compared with those of other authors and the type of anaemia in PKD is discussed.     

Strawberry disease in rainbow trout, Salmo gairdneri Richardson

Abstract. Strawberry disease is a subchronic, non–debilitating, and non–fatal inflammatory skin disease of rainbow trout that has been recognized for the past 25 years. Morbidity ranges up to 80% and the disease affects fishes from 15cm in length to brood stock size. Epizootiological data indicate that strawberry disease usually appears, first in the autumn and reaches peak incidence during the following winter and spring. Data also indicate that the disease is found only in certain trout farms in the western and Pacific north–western regions of the United States. Although the cause of strawberry disease is unknown, the normal clinical course of 8 weeks can be reduced by oral treatment with oxytetracycline. Clinical signs include appearance of bright red, raised, circumscribed, ulcerated and indurated lesions in the skin that usually range up to 3 cm or more in diameter. Microscopically, the disease is characterized as a subchronic, focal, non–suppurative dermatitis with ulceration and extensive infiltration of mononuclear inflammatory cells.     

Immunization of rainbow trout, Salmo gairdneri Richardson, against bacterial kidney disease: preliminary efficacy evaluation

Abstract. A bacterin for immunization against bacterial kidney disease of salmonid fishes caused by Renibacterium salmoninarum is described. Cultures were grown in Evelyn's KDM2 medium containing 10% calf serum in a fermenter under the following conditions: pH 7.2, 15°C, 800ml/min air, 200 rev/min agitation and 5–15 days of incubation. Possible substitutes for calf serum were 10% horse serum 0.15% starch and leptospira medium. The bacterins were inactivated with 0.3% formalin and no adjuvants were used. Other tests evaluated pH-lysed bacterin, 50% concentrated bacterin and 50% concentrated pH-lysed bacterin. Juvenile rainbow trout, salmo gairdneri Richardson, were vaccinated either by intraperiotoneal (i.p.) injection, 2 min immersion or 2-step hyperosmotic infiltration. Fish were held from four to six weeks at 11°C, then challenged by i.p. injection with the homologous virulent bacterium. Fish died from days 19 to 40 after challenge. The best preparation was pH-lysed bacterin given by a single i.p. injection; hyperosmotic and immersion vaccination were not effective. Typically when 80% or more of unvaccinated controls were infected as detected by Gram stain, 10% or less of the vaccinated fish were infected.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream,Pagrus major (Temminck & Schlegel)

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.     

The replacement of fish meal with Antarctic krill, Euphausia superba in diets for Atlantic salmon, Salmo salar

A total of six isoprotein and isolipid diets for Atlantic salmon, Salmo salar L., were prepared substituting from 0 to 100% of fish meal protein (0–68% of diet by dry weight) with meal from Antarctic krill (Euphausia superba). The feed produced from high inclusion levels of krill meal had lower ability to absorb lipid during vacuum coating than fish meal. Both amino acid and fatty acid compositions of the diets were fairly similar. The experiment commenced using salmon averaging 500 g and ended at a mean weight of 1500–1800 g (140 days of feeding). Moderate amounts of krill meal (20–60% of krill protein) in the diets increased growth during the first 71 days of feeding compared with the fish meal control, while no growth difference was observed during the last 69 days of feeding. This may, at least in parts, be explained by a feed‐attractant function of the krill meal. Muscle dry weight and lipid concentrations were unaffected by the diet. Feed conversion rate increased with high levels of krill meal in the diets (e.g. for the last period from 0.94 in the 0% diet to 1.26 in the 100% diet). This indicates that the fish were able to compensate by eating more to maintain growth. The apparent digestibility coefficients of dry matter and protein were not influenced by diet, but both faecal moisture and lipid had a tendency to increase at the highest inclusion level (all protein from krill meal). This may be related to chitin in the krill diet that is known to decrease lipid absorption and induce diarrhoea (increased water content in faeces). Chitin was not utilized to any major extent. Welfare parameters such as blood haemoglobin, red blood cell counts, plasma protein, cholesterol, triacylglycerols and glucose levels were unaffected by diets. Clinical indicators of cellular damage (alanine aminotransferase and aspartate aminotransferase) were similar indicating no diet‐induced tissue damage during the trial.