Studies on the pathogenesis of vibriosis in coho salmon Oncorhynchus kisutch (Walbaum)

Abstract. Juvenile coho salmon Oncorhynchus kisutch (Walbaum), cultured in fresh water, were experimentally infected with highly virulent Vibrio anguillarum , and various haematological and biochemical parameters were compared between moribund and non-infected control fish. In addition, endotoxin, culture supernatant, and cell lysate preparations of V. anguillarum were tested separately and in combination to assess their roles in mediating pathological effects.
The haematocrit, haemoglobin, numbers of red and white blood cells and plasma, total protein, albumin, β₂ globulin, chloride, sodium, osmolality and alkaline phosphatase levels were all decreased in infected fish compared to controls. Erythrocyte fragility and plasma glucose, potassium, lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (GOT) levels were increased in infected fish.
No mortality nor significant changes in haematocrit or plasma protein occurred in fish injected intraperitoneally with 35 mg/kg (1 mouse LD₅₀) of V. anguillarum endotoxin, with culture supernatant fluid, cell lysate, or with combinations of the three preparations. There was, however, a decrease in plasma protein concentration in coho injected with 355 mg/kg (10 mouse LD₅₀s) of endotoxin, but no mortality nor significant change in haematocrit.     

Tolerance to Formalin by a Fluidized-Bed Biofilter and Rainbow Trout Oncorhynchus mykiss in a Recirculating Culture System

Formalin tolerances of a fluidized-bed sand biofilter and rainbow trout Oncorhynchus mykiss were tested in a semi-closed freshwater recirculating culture system. Progressively higher formalin levels were tested until fish mortality (±1.9%) occurred. A 1-h exposure to 167 ppm formalin, followed by flushing, was safe for trout (no mortality) at 15.0 C but not at 16.5 C. A 110-ppm indefinite treatment (no flushing) was safe at 17.3 C, but 120 ppm at 17.3 C and 100 ppm at 17.8 C were not. Biofilter nitrification was not impaired by 1-h formdin treatments up to 167 ppm at 16.5 C, and not, usually, by indefinite treatments up to 120 ppm at 17.3 C. However, a find indefinite treatment of 70 ppm was followed by abnormally high nitrite levels for 9 d. Formaldehyde remained detectable in the system for 11 h during indefinite treatment at 120 ppm formalin. Tests on the system's Co₂ stripper did not indicate that it removed formaldehyde.     

Acute LD50 and kidney histopathology following injection of erythromycin (Erythro-200) and its carrier in spring chinook salmon, Oncorhynchus tshawytscha (Walbaum)

We evaluated the toxicity of commercial injectable erythromycin (Erythro-200) and the injectable drug carrier alone (PEG-400, ethyl alcohol and ethyl acetate) in sub-adult spring chinook salmon, Oncorhynchus tshawytscha (Walbaum). Both compounds were toxic to salmon when administered in high dosages. The 50% lethal dose (LD₅₀) of the erythromycin within its carrier (Erythro-200) at 12 °C was estimated at 429 mg erythromycin kg^–1 or 2.145 mL kg^–1 when measured by volume. The LD₅₀ of the drug carrier alone was 2.95 mL kg^–1 by volume or equivalent to 3.21 g kg^–1 of the carrier. We evaluated and scored the histopathology in kidney sections removed from fish 96 h after injection with Erythro-200, the drug carrier alone, or sterile phosphate buffered saline (PBS) that served as a reference control. Kidneys from fish injected with saline had lowest average pathology scores; those injected with the carrier had moderate scores; and the pathology score was highest in tissues from erythromycin-injected fish. In tests at 12 °C, vacuoles were more prominent in proximal and distal tubules of fish injected with higher dosages of 403 and 545 mg erythromycin kg^–1 than in tissues from fish injected with 299 mg kg^–1.     

The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida

Abstract. Mutants of Aeromonas salmonicida strains lacking either the A-protein, O-antigen or both of these major surface antigens were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), for their suitability as live vaccines (LV). All of these mutants were shown to be attenuated, as fish receiving ∼5 × 10⁷ of the respective strains showed no clinical signs of furunculosis. Immersion vaccination of fish in 5 × 10⁷ cfu ml^-1 of these strains with an identical immersion dose 14 days later resulted in significant protection by all strains from challenge with a heterologous virulent strain of A. salmonicida 5 weeks later. The levels of protection conferred were all greater than or equal to that provided by an injected bacterin using the same vaccination schedule. With one exception, all LV strains that still possessed a functional O-antigen provided protective indices (PI) four- to seven-fold greater than the PI for the fish injected with bacterin. When antibody responses of vaccinated fish were compared, it was found that only vaccination by bacterin gave rise to a measurable agglutinating litre. Western immunoblots using the immune fish sera failed to reveal any major differences in antigen recognition in fish that received any of the vaccines tested. These data suggest that the immune response generated by the use of live vaccine strains is different from that generated by a bacterin, and that these useful mutations may be incorporated into existing furunculosis LVs for further attenuation.     

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.     

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.     

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.     

Potential benefit of clove oil sedation on animal welfare during salmon smolt, Salmo salar L. transport and transfer to sea

The purpose of this study was to determine the effect of clove oil (4.0 mg L⁻¹) sedation, compared with non-sedation, on the primary (plasma cortisol), secondary (osmoregulation) and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. Clove oil sedation during on- and off-loading sufficiently reduced the primary stress response to lower mortality (2.1%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 12.2%. The unsedated fish experienced an acute mortality that stabilized only 6 days after the transport. None of the secondary stress responses measured in this experiment could contribute towards explaining this phenomenon, with the possible exception of plasma magnesium (Mg²⁺). Plasma Mg²⁺ differed between the groups; while plasma Mg²⁺ in the clove oil sedated group returned to pre-stress levels 72 h after transport, the unsedated group showed no such recovery even 1 week after transport, which may indicate a disturbance in the hydromineral balance, and provides a plausible explanation for the delayed mortality in this group. Eugenol-based anaesthetics appear to be promising as a stress-reducing sedative for Atlantic salmon smolts, and, if used properly, this chemical could improve animal welfare and survivability during and after common aquaculture-related incidents.     

Effects of digestible protein levels in isonitrogenous diets on growth performance and tissue composition of juvenile Atractoscion nobilis

A study was undertaken to estimate the effects of isonitrogenous diets (ca. 604 g kg⁻¹ crude protein) containing formaldehyde-treated (FT) fish meal and graded levels of digestible protein (DP) (541, 491, 372, 347 and 247 g kg⁻¹) on growth performance and tissue composition of juveniles white seabass. Five diets were formulated to contain increasing levels of FT fish meal (from 0 to 384 g kg⁻¹) and decreasing levels of non-treated fish meal (from 480 to 96 g kg⁻¹). Each dietary treatment was fed in triplicate to apparent satiation to groups of 25 fish for 50 days. Significantly higher growth performance and feed conversion ratio were obtained in fish-fed diets containing 491 or 541 g kg⁻¹ DP, compared with all other treatments. Apparent digestibility coefficient of protein in the diets was not significantly affected by the inclusion of treated fish meal in the diets. Estimation of protein requirements using a broken-line regression analysis indicated that maximum weight gain would be obtained with a diet containing 503 ± 23 g kg⁻¹ DP. The results from this study suggest that a single-diet formulation using protein treated with formaldehyde as filler might be useful to estimate the requirement of DP for fish.     

Branchial lesions associated with intermittent formalin bath treatment of Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum)

Two trials were conducted to assess the effects of repeated prophylactic formalin treatments on the gill structure of salmonids. In trial 1, which involved Atlantic salmon, Salmo salar L., approaching smoltification in a commercial facility, fish were treated with either 167 or 250 mg l^-1 formalin for 90 min every 2 weeks for 12 weeks. Formalin-treated salmon had slight, but not significant, increases in the frequency of lamellar fusion, numbers of lamellar mucous cells, and numbers of an endemic gill ciliate, Trichophyra piscium, after 6 and 12 weeks of treatment. In trial 2, which involved juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), fish were treated with 200 mg l^-1 formalin for 60 min twice weekly for 12 weeks. Significant effects were limited to an increase in the numbers of mucous cells present on gill lamellae. In both trials, there was no evidence of lamellar oedema or necrosis of lamellar epithelial cells.     

Retention of formaldehyde in the tissues of two tropical fish species following exposure to therapeutic levels

Abstract. Formalin is an effective fish chemotherapeutant which is used to control certain ectoparasitic and bacterial infections. It is toxic to fish and its pathological and physiological effects have been reported. An experiment was conducted to elucidate the possibility of retention of formaldehyde in the muscles of two tropical food fish species, following exposure to therapeutic levels of formalin. The results indicated that Puntius gonionotus (Bleeker), retained 5·75 ppm formaldehyde following exposure to 50 ppm formalin for 24h. In contrast, Clarias batrachus (L.) retained 14·03 ppm and 18·72 ppm formaldehyde following exposure to formalin at 50 ppm and 100 ppm for 24 h respectively. However, no formaldehyde was detected after keeping the exposed fish in clean running water for 24h.     

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.     

Supplementing copper to a fish meal based diet fed to Atlantic salmon parr affects liver copper and selenium concentrations

The present experiment was conducted to study growth and tissue responses in Atlantic salmon, Salmo salar L., fed a fish meal based diet supplemented with copper (Cu). The findings of the experiment were used to evaluate the need for dietary Cu supplementation. Atlantic salmon parr, initially weighing ≊ 7.5 g, were randomly distributed among 10 tanks, with 300 fish in each tank. Duplicate groups of fish were fed a fish meal based diet containing 3.5 mg Cu kg⁻¹, or this diet supplemented with 5, 10, 50 or 100 mg Cu kg⁻¹ (as CuSO₄*5 H₂O) for 12 weeks. Growth was recorded and blood haemoglobin measured. The Cu concentrations in whole body, liver, serum and selected muscle samples were measured, as was liver selenium (Se) concentration.
There were no difference in growth among the dietary treatments. There were, however, significant differences among the dietary groups in liver Cu and Se concentrations. The fish fed the diet supplemented with 5 mg Cu kg⁻¹ had increased liver Cu concentration compared with the other groups. Similar trends were found for serum Cu concentration and whole-body Cu concentration, but these effects were not significant. Liver Cu and Se concentrations were positively correlated and liver Se concentrations were inversely correlated to dietary Cu concentration, confirming an interaction between these two elements in salmon.
There may be a positive effect of a modest Cu supplementation level, and we suggest that a small amount of Cu (5–10 mg kg⁻¹) should be added to fish meal based diets.     

Oxytetracycline Residue in Hybrid Striped Bass Muscle

Hybrid striped bass ( Morose saxatilis male × M. chrysops female) were injected with 25 and 50 mg oxytetracycline (OTC) per kg of fish. Rate of elimination of OTC from muscle tissue and inhibition of bacteria by muscle injected with OTC were studied. OTC residue in muscle of fish injected with 50 mg OTC/kg fish was much higher (P < 0.01) than that in fish injected with 25 mg/kg and took a longer time to clear from the muscle. In fish injected at 25 mg OTC/kg of fish, OTC was completely eliminated from muscle in 24 d; the rate of elimination constant (β) was 0.278/day and the half-life (t_1/2) 2.5 d. In fish injected at 50 mg OTC/kg of fish, the OTC was not depleted in fish muscle until 32 d after injection; the rate of elimination constant 13 was 0.265/day and the half-life was 2.6 d. No statistical difference was found in muscle OTC residue between male and female fish. Size of fish (16.5 to 21.5 cm and 21.6 to 25.5 cm) made little difference in OTC residue in muscle. Muscle removed from fish injected with 25 mg/kg OTC 12 d after injection inhibited the growth of Vibrio anguillarum, V. ordalii and Aeromonas hydrophila in cultures.     

Reproductive Performance of Pigmented and Albino Female Channel Catfish Induced to Spawn with HCG or Ovaprim

Reproductive performance of albino and pigmented channel catfish ( Ictalurus punctarus ) females was compared when spawning was induced with HCG (human chorionic gonadotropin) or Ovaprim ³ (domperidone and [d-Ala⁶, Pro⁹ NEt]-luteinizing hormone-releasing hormone). In 1985, only HCG was used to induce spawning. Albino females required more injections, had a longer latency period from time of first injection to spawning, and had lower spawning success (30%) than pigmented females (77%). No differences were observed between albino and pigmented females for time-of-day of spawning, number of eggs obtained by manual stripping, and number of eggs/kg body weight. In 1988, HCG, Ovaprim, and saline (control) were used to induce spawning. HCG induced spawning in 80% of albino and 71% of pigmented females. Ovaprim induced 67% of pigmented females to spawn, but only 2 of 5 albino females spawned. One of four pigmented females injected with saline spawned. Albino females yielded 40–50% fewer eggs/kg body weight than pigmented females. Other performance characteristics for albino and pigmented females were similar whether injected with HCG or Ovaprim. High temperatures encountered during the 1985 spawning season my have had a differential effect on albino and pigmented fish and may account for some differences in spawning success.     

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.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

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.     

14C-glucose injection in Atlantic cod, Gadus morhua, metabolic responses and excretion via the gill membrane

In the present study cod were separately placed in fish metabolic chambers. Ten individuals were injected with a dose of 1 μCi ¹⁴C-glucose, in addition to 1 g pure glucose per kg live weight, and 10 individuals were kept under the same conditions and sham injected with 9 g L⁻¹ (0.9%) sterile saline solution.
Of the injected ¹⁴C-glucose, 3% was recovered in plasma, 2.9% in liver, 1.7% in red muscle, 18.2% in white muscle, 0.2% in heart, 1.9% in gills and 12.1% in gill-surrounding water. Of the 12.1% in gill-surrounding water, 9.1% was found to be in the form of CO₂, leaving 3% to pure glucose and lactate. These results indicate a relatively high production of CO₂ from glucose showing a utilization for energy, and a possible route of excretion of excess glucose via the gill membrane.
Activity of ¹⁴C-glucose on a concentration basis showed in descending order the following organs to be metabolically active in glucose utilization: gills > plasma (as a transporter) > heart > red muscle > liver > white muscle.
Of the total amount of ¹⁴C-glucose injected, only 0.3% was recovered in the liver lipid fraction, and mainly as triacylglyceroles; minor ¹⁴C-activity was found in mono- and diacylglyceroles and free fatty acids. These results show very low activity of de novo syntheses of lipid from injected glucose in the cod.     

Pharmacological treatments against Ichthyobodo necator (Henneguy, 1883) in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Various antiparasitic drugs belonging to several pharmacological groups were tested by bath administration for in vivo activity against a natural infestation of rainbow trout. Oncorhynchus mykiss (Walbaum), by he flagellate protozoan Ichthyobodo necator. Fish were also monitored for signs of drug toxicity. Complete elimination of infestation in all fish was achieved only by bithionol (25 mgl^-1 for 3h on two consecutive days). Ketoconazole, toltrazuril, amprolium, sulphaquinoxaline, quinacrine, N-metylglucamine, chloroquine, 1,3-di-6-quinolylurea, diminazene aceturate and paromomycin were not effective. Diminazene aceturate (100 mgl^-1, 3h) was the only drug which was clearly toxic. The comparison of the response of symptomatic and asymptomatic infested fish to a formaldehyde bath indicates that poor health caused by I. necator may greatly increase the susceptibility of fish to the toxic effects of a drug or chemical.