Elevation of lipid peroxide level and production of hydroxy lipids in cultured Hepa-T1 cells by oxidative stressors

ABSTRACT:   In order to investigate the relationships between levels of lipid peroxides (LPO), hydroxy lipids (L-OH) and of antioxidative enzymes in live fish, cultured cells from tilapia liver (Hepa-T1) were treated with oxidative stressors, 2,2'-azobios(2-amidino-propane) dihydrochloride (AAPH) and H₂O₂. By treating with 5 mM AAPH, LPO and L-OH of the cells significantly increased time-dependently. In particular, L-OH increased from 7.6 ± 0.7 to 23.6 ± 1.8 nmol/mg protein after 2 h of treatment, and synchronously the glutathione peroxidase (GPx) activity of the cells increased from 259 ± 106 to 1970 ± 135 mU/mg protein. In the case of H₂O₂, however, no elevation of L-OH was observed. In this case, catalase (CAT) and superoxide dismutase (SOD) activities increased time-dependently. Especially, after 2 h exposure, the CAT activity increased remarkably from 217 ± 21 to 2510 ± 120 mU/mg protein, though the GPx activity did not change significantly. These results suggest that the elevation of the L-OH level is closely associated with the elevation of GPx activity, but not with that of CAT or SOD. However, when vitamin E-enriched Hepa-T1 cells were treated with AAPH and H₂O₂, the production of LPO and L-OH was suppressed significantly. The findings suggest that vitamin E also plays an important role in the levels of the oxidation products in vivo .     

Preparation of algal-oligosaccharide mixtures by bacterial agarases and their antioxidative properties

ABSTRACT:   Algal-oligosaccharide-lysates (AOL), derived from six agars and four algal polysaccharide extracts (APE), were treated with 100–500 activity units (AU) of MA103-agarases or MAEF108-agarases, and their antioxidative properties evaluated. Soluble total polyphenols (TP) were between 462.2 ± 1.6 gallic acid equivalents (GAE, µg/mL) and 70.6 ± 17.4 GAE. The DPPH radical scavenging capacity of all AOL went from 68.3 ± 0.7% to 0.5 ± 0.1%. The ferrous ion chelating capacity of all AOL went from 93.1 ± 0.2% to 21.7 ± 0.9%. Evaluation of the H₂O₂ scavenging capacity of all AOL was between 35.9 ± 5.4% and 0.1 ± 0.2%. The reducing power of all AOL went from 51.3 ± 2.6 to 3.2 ± 6.8 expressed as µg/mL ascorbic acid. In DPPH radical scavenging capacity, ferrous ion chelating capacity and reducing power etc., the AOL derived from the APE of Porphyra dentate (digested by 500 AU of MAEF108-agarases) were highest, in all test sets. However, the AOL derived from the APE of Monostroma nitidum (digested by 500 AU of MAEF108-agarases) had the highest H₂O₂ scavenging capacity in all test sets. The order of antioxidative activity performance of all AOL treated in this experiment, by these four antioxidative methods, is as follows: ferrous ion chelating capacity > DPPH radical scavenging capacity > H₂O₂ scavenging capacity > reducing power; this may be related to their polyphenols, small molecular weight polysaccharides or simple sugar constituents. In this study, it is demonstrated that various agarases derived from algal oligosaccharide mixtures possess good potential for use as a health food, due to their antioxidative capacity.     

Photoprotective activity of scallop shell water-extract in keratinocyte cells

ABSTRACT:   The photoprotective activity of the scallop shell water-extract was investigated using cultured rat skin keratinocyte cells. It was found that the scallop shell water-extract protected keratinocyte cells against ultraviolet (UV)-B-induced damage. The scallop shell water-extract had protective effects against hydrogen peroxide (H₂O₂) in skin keratinocyte cells and also showed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and lipid peroxidation inhibitory activity. In addition, the scallop shell water-extract promoted proliferation of keratinocyte cells. The scallop shell water-extract may protect skin keratinocyte cells against UV-B-induced damage through two factors; antioxidant activity and growth-promoting activity in skin keratinocyte cells. These results suggest utilization of the scallop shell water-extract as materials for protecting skin.     

Pharmacokinetics of oxolinic acid in gilthead sea bream, Sparus aurata L.

This is the first study on the pharmacokinetic parameters of oxolinic acid (OA) in gilthead sea bream, Sparus aurata L. The kinetic profile of OA was studied after a single intravascular injection (20 mg kg⁻¹) in 100 g fish at 20 °C. The distribution half-life ( t _1/2α) and the elimination half-life ( t _1/2β) of the drug were found to be short (0.51 and 12.60 h, respectively). The drug penetration from the plasma to the tissues was adequate as the apparent volume of distribution of the drug at steady-state ( V _d(ss)) was found to be 2.11 L kg⁻¹. The mean residence time ( MRT ) of OA was short (14.25 h) and the total clearance rate ( Cl _T) of the drug was low (0.15 L kg⁻¹ h⁻¹). The bioavailability ( F %) of OA following oral administration (30 mg kg⁻¹) was also low (14%). Maximum values were observed for muscle at 0.5 h after injection, with levels declining as with subsequent sampling. At the first two time points (0.5 and 1 h) plasma levels of OA were higher than muscle, however, the reverse was evident for subsequent samples. Following oral administration, highest muscle levels were found at 16 h and, with the exception of the 24-h sampling, muscle OA concentrations were higher than plasma at all time points. The fast elimination of OA suggests short withdrawal times with reference to human consumption of treated fish.     

A pharmacokinetic study of flumequine in sea bass, Dicentrarchus labrax (L.), after a single intravascular injection

The pharmacokinetic properties of flumequine following a single intravascular injection (10 mg kg^–1 fish) were studied in sea bass, Dicentrarchus labrax (L.), 120 g held at 18 °C. The absorption half life ( t _1/2α) and the elimination half life ( t _1/2β) of the drug were calculated to be 1.05 and 10.71 h, respectively. Tissue penetration of flumequine seemed to be moderate because both the apparent volume of distribution of the drug at steady-state ( V _d(ss)) and the extensive apparent volume of the central compartment ( V _c) were found to be small (1.51 and 0.626 L kg^–1). The mean residence time ( MRT ) was short (09.73 h) and the total clearance (CL_T) of the drug was rapid (0.156 L kg^–1 h^–1).     

Isolation of a viable but non-culturable suppression mutant of Vibrio vulnificus: role of antioxidant enzymes in surviving stationary phase and low temperatures

ABSTRACT:   The viable but non-culturable (VBNC) suppression mutant was isolated. Suppression subtractive hybridization was used to identify differentially expressed genes among cDNA of the VBNC suppression mutant and the wild-type strain. RpoS-dependent catalase was highly expressed from the VBNC suppression mutant during oxidative stress in the stationary phase and at low temperatures. In experiments of H₂O₂ sensitivity, the VBNC suppression mutant showed a peroxide-resistant phenotype in the stationary phase and at low temperatures. These results suggest that VBNC suppression mutant cells use catalase for protection against oxidative stress in the stationary phase and at low temperatures.     

Digestibility in goldfish fed diets with and without chromic oxide and exposed to sublethal concentrations of cadmium

Effects of three sublethal levels of cadmium (40, 120 and 260 μg L⁻¹) on apparent total dry matter (TDM) and apparent protein (PD) digestibility in goldfish, Carassius auratus (L.), over an 8-week experimental period were investigated using diets with and without chromic oxide (0.5%) marker. The %TDM and %PD were estimated based on crude fibre as the marker for the diet without Cr₂O₃, and Cr₂O₃ and crude fibre as markers for the diet with Cr₂O₃. Generally, digestibility estimations were lower in goldfish fed the diet with the Cr₂O₃ marker. Similarly, for the diet with Cr₂O₃ marker, digestibility estimates based on crude fibre were higher than those based on Cr₂O₃. In all treatments (2 diets × control and 3 cadmium levels), the weekly estimations of %TDM and %PD did not differ significantly ( P > 0.05). The effect of cadmium on weekly %TDM and %PD was not that obvious in all treatments. However, the overall mean %TDM and %PD differed significantly ( P < 0.05) between diets and between markers, at all cadmium concentrations. The present study raises doubts regarding the validity of the use of Cr₂O₃ as a marker in digestibility studies in fish, as had been recently suggested by other authors but for different reasons.     

Nutrients apparent digestibility coefficients of selected protein sources for juvenile Siberian sturgeon (Acipenser baerii Brandt), compared by two chromic oxide analyses methods

Apparent digestibility coefficients (ADCs) of dry matter (ADC_d), crude protein (ADC_p), energy (ADC_e) and amino acids in selected feedstuffs were determined for juvenile Siberian sturgeon (8.38 ± 0.20 g). The tested feedstuffs were fishmeal (FM), meat and bone meal (MBM), poultry by-product meal, hydrolysed feather meal, fermented feather meal solvent-extracted cottonseed meal and soybean meal. ADCs were determined using a reference diet and test diets at 7 : 3 ratios with 5 g kg⁻¹ chromic oxide (Cr₂O₃) as an inert marker. Fish were reared in a recirculating system and fed to apparent satiation five times daily. Cr₂O₃ in diets and faeces samples were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) and acid-digestion colorimetry (AC) methods, respectively. The results showed that ICP-AES method was more accurate for Cr₂O₃ determination than AC method, and the results determined by ICP-AES method were used in this study. ADC_d and ADC_p of seven tested ingredients were lowest for MBM (59.1 and 84.5%) and highest for FM (79.9 and 94.5%); ADC_e of tested ingredients were from 71.8% for SECM to 93.2% for FM. ADCs of amino acid in test ingredients followed similar trend to the ADC_p. The ADCs of individual amino acids varied from 61.6% (histidine in MBM) to 98.8% (valine in FM).     

Evaluation of carbohydrate rich diets through common carp culture in manured tanks

Four diets (T₀–T₃) were formulated reducing the fishmeal (Indian) component by 100 g kg^–1 from 300 to 0 g kg^–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg^–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m^–2 in mud bottomed cement tanks (18 m²), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P  > 0.05). PER was lowest for the 300 g fishmeal kg^–1 diet treatment (diet T₀), increasing with decrease in dietary fishmeal content (diets T₁–T₃). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T₂ and T₃. The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed.     

A study on Flexibacter columnaris infection in loach, Misgurnus anguillicaudatus (Bleeker, Günther)

Abstract. Infectivity of Flexibator columnaris to loach, Misgurnus anguillicaudatus (Blecker, Günther), was studied with variation conditions and the presence of other bacteria in the water medium. Invasion of fish by the poathogen varied with different water conditions. The highest prevalence of infection was observed in an experimentally formulated water containing 0·03% NaCl 0·01% KCl, 0·002% CaCl₂2H₂O and 0·004% MgCI₂ 6H₂O. The percentage of infection in tap water was similar to that of the formulated water. The infeetion rate was low in the individual salt solutions, and no infection occurred in distilled water. Even in the formulated water, the pathogen failed to infect the fish when Citrobacter freundii, Aeromonas hydrophila or Pseudomonas fluorescens were present at densities 100 times that of F. columnaris. However, in such cases, the numbers of F. columnaris in the mucus of the fish body surface did not increase much throughout the experimental period. In contrast, F. columnaris suceessfully invaded the fish in the presence of either Streptococcus sp., Flavobacterium flavescens or Flavobacterium fuscum , even when the numbers of these bacteria were 1000 times that of F. columnaris. In this ease, F. columnaris gradually increased its numbers on the fish body surface.     

Nutritional evaluation of waste date fruit as partial substitute for soybean meal in practical diets of juvenile Nile tilapia, Oreochromis niloticus L.

The potential of waste date meal (WDM; low-quality date palm, Phoenix dactylifera L.) as a carbohydrate source in formulated diets for Nile tilapia was evaluated. Four isocaloric-practical diets (15.7 kJ g⁻¹) were formulated incorporating WDM at 0, 100, 200 and 300 g kg⁻¹ levels as partial substitutes for soybean meal (SBM). These were designated D₀ [284 g crude protein (CP) and 383 g carbohydrate (CHO) kg⁻¹ diet], D₁ (279 g CP and 446 g CHO kg⁻¹ diet), D₂ (207 g CP and 495 g CHO kg⁻¹ diet) and D₃ (175 g CP and 578 g CHO kg⁻¹ diet). Each diet was fed to three replicate groups of 30 fish [20.20 ± 0.09 g (±SE)] for 75 days. No feed-related mortality was observed during the entire experimental period. Final body weight (FBW) and specific growth rate (SGR) in the different treatments were statistically not significantly different ( P  > 0.05). Protein efficiency rate (PER) was lowest in diet D₀ and increased with decrease of SBM content (D₁–D₃). A significant increase in whole body lipid content was recorded in fish fed diets D₂ and D₃. Results showed that WDM could be a substitute for SBM up to 300 g kg⁻¹ in practical Nile tilapia diets without compromising growth.     

Effects of Aeration, Alum Treatment, Liming, and Organic Matter Application on Phosphorus Exchange between Soil and Water in Aquaculture Ponds at Auburn, Alabama

Application of readily-oxidizable organic substrate to laboratory soil-water systems and fish ponds caused anaerobic conditions in bottom soil and water, and concentrations of soluble reactive phosphorus (SRP) increased. Aeration of ponds increased total phosphorus (TP) concentrations by suspending soil particles in the water, but SRP concentrations declined because of increased oxy- genation of bottom water and soil, Alum [Al₂(SO₄)₃·14H₂O] treatment of ponds reduced SRP and TP concentrations in ponds, but the low concentration of alum used, 20 mg/L, had little residual effect on phosphorus concentration. Application of agricultural limestone at 0.2 kg/m² to ponds with soil pH of 5.5 and Ca²⁺ concentration of 5 mg/L did not affect SRP and TP concentration. Unless pond soils were anaerobic at their surfaces, a condition not acceptable in thermally-unstratified fish ponds, soils released little phosphorus to the water. Strong adsorption of phosphorus by soils in intensive ponds with feeding is beneficial, because removal of phosphorus by aerobic soils is a control on excessive phytoplankton growth. In fertilized ponds, phosphorus must be applied at frequent intervals to replace phosphorus removed from the water by soils.     

Role of prostaglandin in the control of ovulation in the Japaneseeel Anguilla japonica

ABSTRACT:   The in vitro effects of 17,20β-dihydroxy-4-pregnen-3-one(DHP) and prostaglandins (PGE₁, PGE₂, PGF_1α,PGF_2α) on ovulation in the Japanese eel Anguillajaponica were examined. Oocytes with follicle layers at themigratory nucleus stage (approximately 850–900 µmdiameter) were removed using a polyethylene cannula from artificiallymatured fish. At concentrations of 10 and 100 ng/mL,DHP was found to induce both germinal vesicle breakdown and ovulation.The prostaglandins, except for PGE₁, effectively inducedovulation of previously matured oocytes by DHP treatment in vitro .Prostaglandin F_2α was the most effective. Asignificant increase in ovulation rate was observed even at a concentrationof 0.01 µg/mL PGF_2α.Indomethacin blocked the in vitro ovulation induced by DHPand addition of PGF_2α reversed indomethacin-blockedovulation. Actinomycin D and cycloheximide blocked DHP-induced ovulationand PGF_2α reversed the effects of both inhibitors. Theseresults indicate that DHP induces ovulation through endogenous prostaglandinsynthesis in the follicle layers of the Japanese eel.     

Beneficial properties of lactic acid bacteria isolated from a Rana catesbeiana hatchery

This work addresses the selection of potentially probiotic lactic acid bacteria (LAB) to be used in raniculture. Thus, strains belonging to the genera Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactococcus lactis and Enterococcus faecium isolated from a Rana catesbeiana hatchery were evaluated for their inhibitory properties against RLS-associated pathogens ( Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis ) and food-borne bacteria. Cell-free supernatants of LAB strains inhibited the growth of at least one of the pathogens by organic acids, but L. lactis CRL 1584 also produced a bacteriocin-like metabolite. The ability of LAB strains to produce H₂O₂ in MRS+TMB medium was also studied. Seventy-eight to ninety six per cent of the strains showed some level of H₂O₂ production. Moreover, different organic solvents were used to determine the hydrophobicity and Lewis acid/base characteristic of LAB strain surfaces. Most of the strains presented hydrophilic properties, but no acidic or basic surface characters. However, some strains isolated from the skin showed a high degree of hydrophobicity and basic components in the cell surface due to their adhesion to chloroform. These properties were not observed in LAB from balanced feed and freshwater. Taking into account general guidelines and the beneficial properties studied, five strains were selected as potential candidates to be included in a probiotic for raniculture.     

Effects of organic acids on growth and phosphorus utilization in red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   A feeding experiment was conducted to investigate the effects of various organic acids and supplements on the growth, phosphorus (P) utilization, and environmental loading of nitrogen (N) and P in red sea bream Pagrus major. Seven diets designated as PA (0.5% inorganic P as Ca[H₂PO₄]₂), PO (no inorganic P), CA (1% citric acid), MA (1% malic acid), LA (1% lactic acid), MHA (1% methionine hydroxy analog) and LTE (1% liquid trace elements) were formulated. Duplicate groups of 25 fish were fed four times daily to near satiation for 75 days. Fish fed CA and LTE diets showed significantly better weight gain and feed conversion ratio compared to fish fed diet PO. Absorption of P was significantly improved in all organic acid-supplemented groups compared to the PA and PO groups. The P retention in CA and LTE groups were significantly higher compared to PA and PO diets. The P excretion was also significantly decreased in all organic acid-supplemented groups. Thus, use of organic acids, in particular CA and LTE, in red sea bream diets can reduce the use of inorganic phosphorus and contribute to the development of much-desired eco-friendly diets.     

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.     

Effect of dietary lysine and methionine supplementation on growth, nutrient utilization, carcass compositions and haemato-biochemical status in Indian Major Carp, Rohu (Labeo rohita H.) fed soy protein-based diet

An 8-week feeding trial was conducted in flow through system to examine the effects of dietary supplementation of lysine and methionine on growth, nutrient utilization, haemato-biochemical status and carcass compositions in Indian major carp, rohu, Labeo rohita fingerlings (average weight 6.32 ± 0.06 g). Four experimental soy protein-based diets D ₀ (without lysine or methionine supplementation), D ₁ (lysine supplementation alone), D ₂ (methionine supplementation alone) and D₃ (both lysine and methionine supplementation) were fed to triplicate groups. l -Lysine and dl -methionine were added to the diets containing 550 g kg⁻¹ soybean meals at 4 and 7 g kg⁻¹ of dry diet respectively. Significant higher weight gain, specific growth rate (SGR), protein efficiency ratio (PER), dry matter retention, nitrogen retention, total ash retention, whole carcass protein, haemoglobin concentration, haematocrit value, total erythrocytic count, total leucocytic count, plasma glucose and plasma total protein and lower FCR, per cent lipid retention and whole body moisture content were observed in fish fed soya protein-based diet supplemented with both lysine and methionine than that of fish of other dietary groups at the end of 8 weeks feeding trial. Although fish fed diet supplemented with either methionine or lysine did not show any significant differences of growth performances, feed utilization, carcass composition and haemato-biochemical status, fish of both of these dietary groups showed significantly better growth performances, feed utilization, carcass composition and haemato-biochemical status than that of fish fed diet without lysine and methionine supplementation.     

Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO₄) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Fish were cutaneously abraded and divided into five treatment groups: (i) challenged by waterborne exposure to F. columnare and not treated with KMnO₄ (positive control), (ii) challenged and simultaneously treated with KMnO_4, (iii) challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge, (iv) not challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge (first negative control) and (v) not challenged and not treated (second negative control). The dosing of KMnO₄ was 2.0 mg L⁻¹ above the potassium permanganate demand for 2 h duration. The survival of the group challenged and simultaneously treated with KMnO₄ (99%) was significantly higher than the positive control (78%) and was not significantly different from the negative control groups. The challenged fish treated with KMnO₄ post-challenge had 7% higher survival than the positive control (85% compared with 78%), but that difference was not statistically significant. The results demonstrate that KMnO₄ has a clear prophylactic value but probably a marginal therapeutic value once the infection has established.     

Use of salt to control ichthyophthiriosis and prevent saprolegniosis in silver perch, Bidyanus bidyanus

The ciliate protozoan, Ichthyophthirius multifiliis , and the fungus, Saprolegnia parasitica , cause the diseases ichthyophthiriosis and saprolegniosis respectively. Both diseases are difficult to control and can cause high mortalities of freshwater fish, including the Australian silver perch, Bidyanus bidyanus (Mitchell). The efficacy of salt (NaCl) in controlling and preventing these diseases in silver perch was evaluated in aquaria and tanks. Low pH levels were also evaluated as a control for ichthyophthiriosis. Concentrations of 2 or 3 g L⁻¹ salt controlled infestations of I. multifiliis , and fish were free of both theronts and trophonts by day 8 at temperatures of 17.3–21.3 °C and by day 6 at 19.2–23.5 °C. Fish treated with 1 g L⁻¹ salt remained infested and all fish in a control treatment (0 g L⁻¹ salt) died. Although the mean survival rates of infested fish at pH levels of 5 or 6 were only 13.9% and 7.6%, respectively, there were no theronts or trophonts on surviving fish after 12 days. Silver perch harvested from a pond and treated with 2 or 3 g L⁻¹ salt did not become infected with S. parasitica and survival was 100%, whereas 16.6% of untreated (0 g L⁻¹ salt) fish became infected and survival was only 66.7%. A concentration of 2 g L⁻¹ NaCl is recommended for the control of ichthyophthiriosis and the prevention of saprolegniosis in silver perch held in tanks, aquaria and re-circulating aquaculture systems.     

Studies on the pathogenesis of streptococcal infection in cultured yellowtails Seriola spp.: effect of the cell free culture on experimental streptococcal infection

Abstract. A previous paper has revealed that experimental streptococcal infection was associated with a rapid growth of the intestinal bacteria, suggesting association of the condition with an exotoxin. The present study was undertaken to see the effect of the exotoxin on the streptococcal infection by administering cell free culture before the bacterial challenge. Cell free culture of Streptococcus sp. strain YT-3 was inoculated intramuscularly at a dosage of 0–5 ml per 100 g body weight 1 h prior to bacterial challenge. Three fish were killed at intervals after challenge for viable counting of bacteria in the viscera and blood. Intramuscular inoculation with either low virulent or virulent bacteria alone at dosages of 10⁶ to 10⁷ cells did not produce the disease in the fish, with almost complete clearance of the bacteria from the viscera and blood within 120 h. When the exotoxin was inoculated intramuscularly prior to the bacterial challenge, however, either low virulent or virulent bacteria at 10⁶ to 10⁷ cells could produce fatal infection with prominent streptococcal clinical signs.