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.     

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.     

Optimum Feeding Rate of the Rotifer Brachionus plicatilis on the Marine Alga Nannochloropsis sp.

The optimum feeding rate of the rotifer Bruchionus plicutilis was investigated to determine the best conditions for growth of the rotifer, and also in order to maintain good water quality of the culture. Fifty rotifers of a large size strain were cultured individually at five food density levels of Nannoehloropsis sp. (0.5 ± 10⁶, 1.5 ± 10⁶, 3 ± 10⁶, 5 ± 10⁶, and 8 ± 10⁶ cells/ml). At each level, daily survival and offspring production were recorded until the death of the final individual. The data obtained were analyzed by the life table method. The maximum value of three growth indices (the net reproduction rate, mean life expectancy at age 0, and intrinsic rate of increase) was obtdned at the food density of 1.5 ± 10⁶ cells/ml. The ration size was calculated to be 325% (dry weight)/ day, which is equivalent to about 70% of the saturated feeding rate. It was suggested that the feeding rate should be controlled to lower than the saturated value for an efficient mass culture.     

The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water

Abstract. The infectivity of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida to Atlantic salmon, Salmo salar L., in sea water was investigated and found to be similar to that reported for fresh water. The minimal infective dose in short duration bath exposures (1–3 days) was 10⁴ colony-forming units (cfu) per ml, while prolonged exposure for three weeks, but not for 1 week, produced infection with 10² cfu/ml. Intragastric intubation of A. salmonicida established infection with doses of >10⁵ cfu. Release of bacteria from dead or morbid infected fish was monitored and found to be in the order of 10⁵–10⁸ cfu/fish/h. These results emphasize the importance of removing dead fish from farm sites.     

Serratia marcescens: a potential pathogen for fish

Abstract. Characterization of a red pigmented enterobacterium isolated from natural population of white perch, Morone americanus (Gmelin), during the course of a bacteriological survey in the Back River (Baltimore, Maryland, USA) indicated that the bacterium belonged to the species Serratia marcescents. The virulence properties of this isolate (RB 469), studied in comparison with the reference strain of S. marcescens ATCC 1800 and S. plymuthica K1R, revealed that all the strains were highly pathogenic for fish with LD₅₀s raging from 5 × 10³ to 1 × 10⁵. Similarly, te extracellular products (ECP) from the three isolates were lethal for fish (LD₅₀ ranging from 0·22 to 4·8 μg protein g^-1 fish). However, only ECP from strains with strong proteolytic activity (the white perch isolate and S. plymuthica ) were cytotoxic for both in fish and homoiothermic cell cultures and both activities were completely destroyed by heating at 100°C for 10min. In contrast, only the two S. marcescens strains which possessed phospholipase active were pathogenic for mice and produced enterotoxins. None of the Serratia strains displayed dermonecrotic factor in rabbits. All these lindings indicate that a direct relationship between eytotoxicity and virulence cannot be established.     

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.     

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.     

Studies on the pathogenesis of streptococcal infection in cultured yellowtails Seriola spp.: the fate of Streptococcus sp. bacteria after inoculation

Abstract. The causative agent of streptococcal infection of yellowtails has been isolated but no information is presently available with respect to the mechanism of infection. The present study deals with the growth of the bacteria in the fish body at various periods after either per-oral or per-cutaneous challenge with Streptococcus sp. YT-3 strain at different passage levels. After percutaneous challenge with bacteria of high virulence, the kidneys retained the bacteria with the relatively high count of 10⁷ cells per gram of tissue while in other organs, although high concentrations of 10⁵-10⁶ cells were detected in 10 min, this was followed by a progressive decrease up to 24 h post-inoculation with a subsequent rapid increase during the later stages of the disease process. The highest rate of growth was obtained in the intestine, where 10⁷ cells were detected at 72 h after inoculation. After oral challenge, the bacteria were detected at high levels from organs and blood within 10 min but they were completely removed from all organs except the intestine within 24 h.     

Fertilization efficiency of cryopreserved sperm from striped catfish, Pangasius hypophthalmus (Sauvage)

The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 10⁷ to 6.94 × 10¹⁰ to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 10⁸. Increasing the sperm dose to 3.47 × 10⁹ did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 10⁶ and 3.47 × 10⁷ sperm per egg. Both highest (6.94 × 10¹⁰) and the lowest (6.94 × 10⁷) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 10⁶ fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 10⁶ (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.     

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.     

Influence of photoperiod, frequency of stripping and presence of females on sperm output in turbot, Scophthalmus maximus (L.)

Abstract. The effect of four environmental conditions was investigated upon sperm output in turbot, Scophthalmus maximus (L.), submitted to three different rhythms of stripping. Males kept under a natural light cycle and under a 6-month contracted light programme released a similar sperm output in terms of total volume of semen produced per fish during the experimental period (4·9 ± 0·9ml), mean sperm concentration (29·4 ± 2·8 × 10⁹ spermatozoa/ml) and total sperm number (163·2 ± 40·5 × 10⁹ spermatozoa). Attempts to stimulate spermiation for a second time just after the end of the natural reproduction period resulted in the release of low sperm output (total volume of semen: 1·6 ± 0·4 ml; mean sperm motility: 2 min 36s ± 0 min 47s; mean sperm concentration: 47·6 ± 10·2 × 10⁹ spermatozoa/ml; total sperm number: 84·5 ± 25·3 × 10⁹ spermatozoa). Stripping frequency had no effect on total volume of semen, mean sperm motility and total sperm number. Monthly collection did not modify sperm samples in relation to stripping rank. However, decreasing volume, motility and sperm concentration were observed when males were stripped fortnightly and weekly. During the natural spawning period, the presence of females in the tank enhanced mean sperm motility (from 3 min 27s + 0 min 52s to 6 min 38s ± 1 min 38s).     

Enrichment of Rotifers Brachionus plicatilis with Eicosapentaenoic Acid and Docosahexaenoic Acid Produced by Bacteria

Abstract— Two bacterial strains, rich in either eicosapentaenoic acid [EPA, 20:5(n-3)] ( Shewanella gel-idimarina ACAM 456) or docosahexaenoic acid [DHA, 22:6(n-3)] ( Colwellia psychroeryrhrus ACAM 605) were tested for their ability to enrich rotifers Erachionus plicatilis in these polyunsaturated fatty acids. Rotifers were exposed for 24 h to each bacterial strain and to a mixture of the two strains. They were then harvested and their fatty acid compositions were analysed and compared to those of rotifers that had been either starved or fed yeast Saccharomyces cerevisiae or microalgae Tetraselmis suecica in 2-L glass flasks. Exposure to 1.4 × 10⁹ cells/ml of the EPA-producing bacterium only resulted in rotifer EPA levels increasing from 0.1% to 1.2% of total dry weight (%dw). Similarly, following exposure to 1.0 × 10⁹ cells/mL of the DHA-producing bacterium only, rotifer DHA levels increased from below detection to 0.1% dw. When exposed to a mixture of the two bacterial strains, containing 7.0 × 10⁸ celldml of the EPA producer and 5.0 × 10⁸ cells/mL of the DHA producer, the rotifers'final EPA and DHA levels were 0.5% dw and 0.3% dw respectively. Although feeding strategies need refining, these results show, for the first time, that rotifers can be enriched with DHA from bacteria, and that rotifers can be enriched simultaneously with both DHA and EPA from different bacterial strains.     

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).     

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.     

Temporal variation of seasonality of egg production and the spawning biomass of Pacific anchovy, Engraulis japonicus, in the southern waters of Korea in 1983–1994

Embryonic mortality, egg production and the spawning stock biomass of Pacific anchovy, Engraulis japonicus , off Southern Korea during 1983–1994, and their biological response to oceanographic features in spring and summer, were analysed. The instantaneous mortality rate (IMR) of embryonic stages decreased in spring and increased in summer, with a range of 0.33–1.23 day^–1 in spring and 0.78–1.69 day^–1 in summer. Egg production in summer was three times that during spring and production was low in the late 1980s. Mean lengths of yolk-sac larvae and adult females were greater in spring than in summer, whereas spawning fraction and spawning stock ratio (spawning biomass:adult biomass) were lower in spring than summer. Estimated mean spawning stock biomass ranged from 141 × 10³ to 380 × 10³ MT in spring and from 221 × 10³ to 557 × 10³ MT in summer. Statistically, the seasonal and long-term trends of embryonic mortality, egg production and spawning stock biomass of Pacific anchovy can be explained largely by spring warming, summer cooling and by less abundant zooplankton in the late 1980s.     

Effects of temperature, salinity and irradiance on the growth of the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae) isolated from Hiroshima Bay, Japan

ABSTRACT: Temperature and salinity ranges in which Gymnodinium catenatum (Hiroshima Bay strain) showed specific growth rates higher than 0.2/day were approximately 20–30°C and 20–32. The specific growth rate (μ), expressed as a polynomial equation as functions of temperature ( T ; °C) and salinity ( S ) were μ = (−6.84 × 10⁻⁴ T ² + 0.0354 T – 0.213) × (−1.03 × 10⁻³ S ² + 0.0579 S – 0.548)/0.31; the maximum growth rate (0.31/day) was obtained at 25°C and 30. From a comparison with field data recording temperature, salinity and light intensity, this species may be expected to bloom from summer to autumn in Hiroshima Bay.     

Changes in skin colour and cortisol response of Australian snapper Pagrus auratus (Bloch & Schneider, 1801) to different background colours

A two-factor experiment was carried out to investigate the change in skin colour and plasma cortisol response of cultured Australian snapper Pagrus auratus to a change in background colour. Snapper (mean weight=437 g) were held in black or white tanks and fed diets containing 39 mg unesterified astaxanthin kg⁻¹ for 49 days before being transferred from white tanks to black cages (WB) or black tanks to white cages (BW). Skin colour values [ L ^* (lightness), a ^* (redness) and b ^* (yellowness)] of all snapper were measured at stocking ( t =0 days) and from cages of fish randomly assigned to each sampling time at 0.25, 0.5, 1, 2, 3, 5 and 7 days. Plasma cortisol was measured in anaesthetized snapper following colour measurements at 0, 1 and 7 days. Fish from additional black-to-black (BB) and white-to-white (WW) control treatments were also sampled for colour and cortisol at those times. Rapid changes occurred in skin lightness ( L ^* values) after altering background colour with maximum change in L ^* values for BW and WB treatments occurring within 1 day. Skin redness ( a ^*) of BW snapper continued to steadily decrease over the 7 days ( a ^*=7.93 × e^{−0.051 × time}). Plasma cortisol concentrations were highest at stocking when fish were held at greater densities and were not affected by cage colour. The results of this study suggest that transferring dark coloured snapper to white cages for 1 day is sufficient to affect the greatest benefit in terms of producing light coloured fish while minimizing the reduction in favourable red skin colouration.     

Absorption of immunomodulating β(1,3)-glucan in yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

A soluble immunomodulating β(1,3)-glucan, laminaran, was given in drinking water to yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus L. The larvae absorbed laminaran when the concentration was 25 mg l^–1. After 5 days exposure to laminaran, each larva contained 2.3 ng and 46 ng of ³H- and ¹²⁵I-labelled laminaran, respectively. After 10 days exposure, each larva contained 4.5 ng and 47 ng of ³H- and ¹²⁵I-laminaran, respectively. The absorption was confirmed by fluorescence microscopy of larvae exposed to fluorescein-labelled laminaran (FITC-laminaran). The fluorescence was localized to cells in the intestinal epithelial layer and the skin epithelial layer.     

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.