An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

Occurrence of viral haemorrhagic septicaemia in rainbow trout Salmo gairdneri Richardson reared in sea-water

Abstract. Viral haemorrhagic septicaemia (VHS) is reported for the first time in sea-water cultured rainbow trout. Heavy mortalities with typical signs and lesions A VHS virus (serotype 1) was isolated from the diseased fish. The mortalities were caused only by the VHS virus and 80 days post transfer of trout to sea-water the mortalities reached 85%, of the initial population.
The disease was experimentally transmitted to rainbow trout, both in sea-water 3·10⁴ pfu/ml of virus or by intramuscular injection of various doses of VHS₁ (7·10¹ 7·10⁴ or 7·10⁴ pfu per fish). Death occurred in all infected groups and started earlier in sea-water. Typical signs of VHS were observed in moribund fish. Viral multiplication was demonstrated to have occurred in fish organs.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH₅₀) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH₅₀ activity of serum decreased. The CH₅₀ activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH₅₀ activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH₅₀ activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH₅₀ activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.     

Elevated serum levels of alpha foetoprotein (AFP)-like immunoreactivity in rainbow trout, Oncorhynchus mykiss (Walbaum), with aflatoxin B1 induced hepatocellular carcinoma

Abstract. Alpha foetoprotein (AFP)-like immunoreactivity was detected by double immunodiffusion in thesera of adult rainbow trout, Oncorhyachus mykiss (Walbaum) with histologically confirmed hepatocellular carcinoma experimentally induced in microinjection of embryos with aflatoxin B₁ (AFB₁). Partial characterization indicated that this AFP-like immunoreactivity gave immunologic cross-reaction of partial identity with human AFP. had alpha₁ electrophorctic mobility, and an estimated molecular weight of 72kDa. Elevated serum AFP-like immunoreactivity levels were also measured by RIA in seven out of eight adult rainbow trout with hepatocellular carcinoma, and in three out of 17 AFB₁-exposed trout without demonstrable grosws or microscopic evidence for hepatocellular neoplasms. No serum AFP-like immunoreactivity was detected or measured in normal healthy age matched DMSO-control rainbow trout. These data are consistent with the following conelusions: (1) AFP-like immunoreactivity detected and measured in the sera of rainbow trout with hepaloecllular careinoma is an analogue to human AFP; and (2) the elevated serum AFP levels measured in adult rainbow trout wilh hepatocellular careinoma resrmble those found in humans with this same malignancy. These data further suggest that serum AFP measurements might be useful to confirm the appearance of hepatocellular carcinoma in experimental fish carcinogen-assay systems, and to detect hepatocellular neoplasia in high-risk wild fish populations exposed to carcinogenie pollutants.     

Experimental pathogenicity of reovirus 13p2 for juvenile American oysters Crassostrea virginica (Gmelin) and bluegill fingerlings Lepomis macrochirus (Rafinesque)

Abstract. A viral agent (13p₂), isolated from clinically normal juvenile American oysters ( Crassostrea virginica ) and characterized as a new serotype of reovirus, was tested to determine if it could replicate and produce disease in experimental juvenile oysters. Because the virus replicated well in the bluegill fry (BF-2) fish cell line, fingerling bluegills Lepomis macrochirus were included in the pathogenicity experiments. Exposure of oysters to the 13p₂ virus in ambient seawater resulted in no significant mortality and no increased virus titres or histological lesions. Virus particles were not observed in tissues of exposed oysters when examined with transmission electron microscopy (TEM). Intraperitoneal inoculation of the 13p₂ virus into bluegills resulted in 44% mortality associated with a focal necrotic hepatitis. Evidence of virus replication, when evaluated histological or by virus titration, was observed in 94% of 32 inoculated fish. Samples of infected livers examined with TEM revealed typical cytoplasmic arrays of 13p₂ virus particles in affected hepatocytes. Rising virus titres and hepatic lesions also occurred in bluegills exposed to water containing the 13p₂ virus. These results indicated the natural host of the 13p₂ virus was not the American oyster, but that it was a significant pathogen for at least one fish species.     

Impact of a pathogenic haemoflagellate, Cryptobia salmositica Katz, on the metabolism and swimming performance of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Oxygen consumption of juvenile rainbow trout (5 g at 13°C) at moderate swimming speeds did not change significantly when infected with Cryptobia salmositica. However, significant reductions of as much as 44% of the maximum aerobic scope for activity and 24% of the critical swimming speed were observed when the parasitaemia reached a maximum of 57.6 × 10⁶ ml⁻¹ fish blood at 3 weeks post- infection. Blood haematocrit was significantly reduced from the initial 34.1 to 19.7% at 4 weeks post- infection, probably as a result of haemolysis by the parasite. The destruction of red blood cells clearly led to lower oxygen carrying capacity, and reduced respiratory and swimming performance.     

The susceptibility of salmonid fish to an atypical strain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), in Australia

Abstract. An enzootic, Australian, atypical strain of Aeromonas salmonicida isolated from diseased goldfish, Carassius auratus (L.), was inoculated into Atlantic salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, S. gairdneri Richardson, and brook trout, Salvelinus fontinalis (Mitchill), fingerlings by intraperitoneal injection (i.p.) and by bath challenge, the latter with and without prior abrasion of skin. The 10-day LD₅₀ (i.p.) was estimated to be 7·4 × 10^-3 colony forming units (cfu) for Atlantic salmon, 3·0 × 10^-2 cfu for brown trout, 3·7 × 10² cfu for brook trout and 6·4 × 10³ cfu for rainbow trout. Brown, rainbow and brook trout succumbed to bath challenges with between 10⁵–10⁶ cfu/ml, developing ulcers of the skin and septicaemia. The organism was trasmitted from inoculated fish to five of 195 within-tank control fish via water and established a carrier state in one of 14 Atlantic salmon. It was concluded that the organism poses a significant threat to the salmonid farming industry and wild salmonid fisheries in Australia.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

Epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection in farmed rainbow trout, Oncorhynchus mykiss (Walbaum): findings based on virus isolation, antigen capture ELISA and serology

Abstract. The epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection was studied in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Estimates of mortality during five outbreaks on a commercial farm from 1986 to 1992 ranged from 0033 to 0.2% per day and total mortality did not exceed 3–4% in any outbreak. Affected fish were 0+ and less than 125 mm forklength. Clinical signs were non-specific, and laboratory examination was required to confirm the diagnosis. At the height of the outbreak in 1992, EHNV was demonstrated by virus isolation and antigen capture ELISA in 89% of clinically affected fish and 51% of dead fish, while the prevalence of infection in apparently healthy in-contact fish was 4%. Two and 4 months later the virus was not detected in a group of apparently healthy fish that had been affected earlier. Antibodies specific for EHNV were not found in rainbow trout from the infected farm; however, strong humoral responses were detected by ELISA in two immunized fish, indicating that the virus was immunogenic. These data suggested that EHNV was poorly infective but highly virulent in rainbow trout. Clinical EHNV infection was positively correlated with high rearing density and a low rate of water exchange, and therefore, with presumed poor water quality. Water temperature, which ranged from 11 to 17°C during outbreaks, did not appear to determine the incidence of clinical infection. EHNV infection in farmed rainbow trout was preceded by infection in free-living redfin perch, Perca fluviatilis L., in the water catchment, but it was uncertain whether this represented the source of infection for rainbow trout.     

Haemorrhagic areas in the mouth of farmed turbot, Scophthalmus maximus (L.)

Abstract. An epizootic in juvenile turbol reared on a farm located in The Ria de Vigo, Galicia, Northwest Spain, is described. The diseased turbot did not display unusual swimming behavior; the external signs of the disease were abdominal distension and haemorrbagic areas in the mouth. Internal examination of the fist showed an accumulation of reddish fluid in the peritoneal cavity. Microbiological analysis of the diseased fish revealed the presence, in pure culture in all the organs and lesions examined, of a bacterium which was characterized biochemically as Vibrio splendidus biotype I. The virulence tests showed that the V. splendidus biotype I isolate was pathogenic for rainbow trout (LD₅₀: 2.2 × 10⁴) and also for turbot (LD₅₀: 1.2 × 10⁴). The treatment of the fish using flumequine incorporated into the feed was effective in offsetting the mortality rate.     

Histopathology, electron microscopy and isolation of channel catfish virus in experimentally infected European catfish, Silurus glanis L.

Abstract Two groups of European catfish, Silurus glanis L., fingerlings were infected with channel catfish virus (CCV) by either intraperitoneal injection with 10⁵ TCID₅₀ of CCV, or bathing in water containing 10⁵ TCID₅₀ of CCV per 1·0 ml. The virus was isolated from spleen, intestine and brain of CCV-injected fish at day 1 and the titres ranged from 10^2·1 to 10^3·3 TCID₅₀/g. However, the tissue distribution of CCV was irregular and no virus was isolated after day 3 post-exposure. In CCV-bathed fish, the virus was isolated only from the liver of one specimen at day 3 post-exposure. No clinical signs of CCV disease developed in any of the fish. Specimens in each regime from all sampling periods showed some minor histopathological changes, but there were no differences between treatments. Lesions included oedema and focal haemorrhage in the liver and the spleen was congested. Electron micrographs of tissue samples showed the presence of a few virus particles around the nuclei of kidney, spleen and intestinal cells, and in or around a myelinated nerve within the optic lobes of infected fish during the first 4 days of infection.     

Effects of hydrogen peroxide on clearance of formalin-killed Flavobacterium branchiophilum from the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

The effect of hydrogen peroxide (H₂O₂) treatment on the clearance of formalin-killed Flavobacterium branchiophilum from the gills of rainbow trout was examined. In untreated control fish, clearance was characterized by a rapid initial phase, with 50% reduction in bacterial antigen in the first 12 h after exposure. The bacteria then cleared more gradually, with total clearance being achieved by 40 h. Treatment of fish with 100 p.p.m. H₂O₂ did not influence bacterial clearance compared to untreated controls. Exposure for 1 h to H₂O₂ concentrations ≤ 100 p.p.m. caused no detectable clinical signs or evidence of ultrastructural damage to the respiratory epithelium. However, levels in excess of this caused significant gill damage.     

Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture

A selection of 16 field isolates of Photobacterium damselae from marine rainbow trout farms in Denmark was subjected to phenotypic and genotypic characterization and pathogenicity to fish. All isolates belonged to the subspecies damselae , being positive for haemolysis, motility and urease. There were considerable differences in haemolytic properties, some isolates presenting a broad zone of haemolysis and others only a narrow zone. Pulsed-field gel electrophoresis revealed a high diversity indicating that P. damselae subsp. damselae is an opportunistic, not clonal pathogen in Danish marine rainbow trout. Virulence of the strains to rainbow trout was highly variable with LD₅₀ values ranging from 3.9 × 10³ to 1.5 × 10⁸ cfu at 20 °C. The virulence was significantly higher at 20 °C than at 13 °C. The strains with the strongest haemolytic properties were the most virulent suggesting a strong involvement of haemolysin in the pathogenesis. The pathological changes were consistent with a bacterial septicaemia and the haemorrhages were more pronounced than for most other bacterial infections.     

A comparison of the effect of silicone coated ascorbic acid and ascorbyl phosphate on the course of ichthyophthiriosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. The aim of the present study was to compare the effect of high and low doses of different vitamin C formulations on the course of ichthyophthiriosis ( Ichthyophthirius multifiliis infection) in rainbow trout. After a depletion period, trout fingerlings were fed diets deficient in vitamin C(AA 0), or containing different levels of silicone coated ascorbic acid (AA; 50/200 mg kg feed⁻¹) or ascorbyl phosphate (AP; 50/2000 mg AA equivalent kg feed⁻¹). Fish infected with I. multifiliis demonstrated a significant reduction in mortality when fed high levels of AA or AP. A correlation between the parasite burden and the vitamin C content in the diet was not found. Specific immunity—measured as immobilization litre of sera—was not influenced by the level or the formulation of vitamin C in the diet. The most probable mechanism of high level doses of vitamin C is an increase of the general health status expressed as an improved nonspecific resistance and ability to cope with different stressors. In rainbow trout infected with I. multifiliis , the beneficial effect of ascorbyl phosphate is equal to that of silicone coated ascorbic acid, and therefore, the more stable AP may be a valuable alternative for use in fish diets.     

Differences in performance, flesh composition and water output quality in relation to density and feeding levels in rainbow trout, Oncorhynchus mykiss (Walbaum), farming

Abstract. The aim of this research was lo evaluate the effect of density and feeding level (initially 8 or 16kg/m³ and 1·3% or 2% of present biomass respectively) on productive traits, meat composition and water quality output in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Individual fish growth shows significant differences in relation to density and feeding level; feed conversion rate is positively Influenced by the lower feeding level and low rearing density. At low density, specific growth rate, protein efficiency ratio and coefficient of variation of individual final weights are more favourable than at high density. Dry matter and lipid composition of meat are positively affected by feeding level; whereas no differences are recorded on the amino acid and fatty acid profiles. Water quality shows differences due to density (NO₃ and NH₄⁺) and feeding level (NO₂, NH₄⁺ and P).     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Effects of Dietary Chlorella ellipsoidea Supplementation on Growth, Blood Characteristics, and Whole-Body Composition in Juvenile Japanese Flounder Paralichthys olivaceus

The study was conducted to investigate the effects of dietary Chlorella powder (C) supplementation on growth performance, blood characteristics, and whole-body composition in juvenile Japanese flounder Paralichthys olivaceus . Four experimental diets were supplemented with C at 0, 1, 2 and 4% (C₀, C₁, C₂, and C₄) on a dry-weight basis. Three replicate groups of fish averaging 1.13 ± 0.02 g (Mean ± SD) were randomly distributed in each aquarium and fed one of four experimental diets for 12 wk. After 12 wk of the feeding trial, fish fed C₂ diet had higher weight gain (WG), feed efficiency (FE), and protein efficiency ratio (PER) than did fish fed C₀ and C₁ diets ( P 0.05); however, there was no significant difference among fish fed C₁ and C₄ diets, and among fish fed C₂ and C₄ diets ( P > 0.05). Fish fed C₂ and C₄ diets had a lower serum cholesterol level than did fish fed C₀ and C₁ diets ( P 0.05). Fish fed C₂ and C₄ diets had a lower body fat than did fish fed C₀ ( P 0.05). These results indicate that dietary supplementation of 2% Chlorella powder in the commercial diets could improve growth, feed utilization, serum cholesterol level, and whole-body fat contents in juvenile Japanese flounder.     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Experimental pathogenicity in rainbow trout, Oncorhynchus mykiss (Walbaum), of two distinct morphotypes of long-spined Saprolegnia isolates obtained from wild brown trout, Salmo trutta L., and river water

Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were experimentally infected to investigate the pathogenicity of 20 isolates of two morphotypes of long-haired Saprolegnia obtained from wild brown trout, Salmo trutta L., and river water in Spain. The trout were exposed to 2 × 10⁵ and 3 × 10⁵ L^–1 zoospores. Saprolegnia infection could not occur without 'ami-momi' treatment. Pathogenicity varied greatly among isolates as mortality ranged from 0 to 100% of the fish. There was a statistically significant difference ( P  < 0.001) between the mortality caused by morphotype I isolates and that produced by those of morphotype II. The most pathogenic isolates usually belonged to morphotype II, consisting of isolates which had secondary cysts with bundles of hooked hairs which were shorter and less numerous than those of morphotype I; the morphotype I isolates usually had low pathogenicity. Lesions were most frequently found on the fins. Cultures detected the presence of Saprolegnia in internal organs. Histopathology of the intestine suggests that Saprolegnia may reach this and other organs via the blood stream from surface lesions.