Spring viraemia of carp virus (SVCV): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen

Abstract. The sensitivity of the immunoperoxidase (IP) and fluorescent antibody (FA) techniques applied to frozen sections of organs of carp infected with spring viraemia virus (SVCV) was similar, both in respect of the intensity of the reaction and in the detection rate of the antigen. Seven days after a waterborne infection both methods detected the antigen in the kidneys and to a lesser extent in the liver and spleen. Quantification of virus gave a titre of 10^2.8 to 10^5.5 TCID₅₀/g of kidney, whereas the agent could not be isolated from liver and spleen tissue. In contrast, at 24¹/₂ days post–infection the viral antigen could be readily detected in liver and spleen but only occasionally in the kidneys using the IP and FA techniques. At this time, liver and spleen showed infectivity titres of 10^3.8 to 10^7.2 TCID₅₀/g tissue, whereas the kidneys were found to be free of infective virus. It is concluded that using the IP and FA techniques SVCV can be detected most frequently in the kidneys from 7 to 14 days post–infection and in the liver and spleen from 14 to 24 days post-infection.     

Enzyme-linked immunosorbent assay (ELISA) detection of infectious pancreatic necrosis virus (IPNV) in culture fluids and tissue homogenates of the rainbow trout, Salmo gairdneri Richardson

Abstract. A double antibody enzyme-linked immunosorbent assay (ELISA) for the detection of infectious pancreatic necrosis virus (IPNV) is described. The sensitivity of the assay reached 10² TCID₅₀ per 0·1 ml of culture fluid. The specificity of anti-IPNV sera and of the assay was confirmed by agar-gel immunodiffusion, by the direct immunoperoxidase technique for the deletion of IPNV in tissue cultures and by the ELISA inhibition test. High values of specific inhibition (over 90% at serum dilutions 1:40–1:2560) and low values of non-specific inhibition (8·4% at serum dilution 1:160) demonstrated the quality of the rabbit anti-IPNV serum. The results of ELISA agreed well with those of virological examinations. The potential of ELISA for investigations of a large series of field samples is discussed.     

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.     

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.     

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.     

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.     

The specific detection of infectious pancreatic necrosis virus in infected cells and fish by the immuno dot blot method

Abstract. The immuno dot blot technique is a rapid, sensitive and quantitative method for the diagnosis of infectious pancreatic necrosis virus ( ipnv ) in both infected tissue culture lysates and tissue homogenates of fish. Uninfected tissue culture lysates and fish homogenates do not react in the peroxidase dot blot system after appropriate treatment. The immuno dot blot method takes 4h to complete, and the dot blot membrane can be stored in the dark for a long period. The viral proteins in the infected tissue culture cells could be detected at 3h post-infection if an initial high MOI was used. The minimum dose of ipnv-ab strain which could be detected using homologous serum was 2·5 ng purified ipnv protein/dot. The smallest infectious dose of VR-299, SP, AB, EVE and CY-1 ipnv which could be detected by the immuno dot blot system was 10⁴ to 10⁵ TCID₅₀. When one μg of purified ipnv was used, a 1·6 × 10⁴ dilution of rabbit anti-AB antisera could be detected.     

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).     

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.     

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.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

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.     

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.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10⁷ copies μg⁻¹ DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10⁶–10⁸ copies of CyHV-2 μg⁻¹ DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg⁻¹ DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.     

The growth rate of two species of microalgae used in shellfish hatcheries cultured under two light regimes

Abstract. The production potential of any shellfish hatchery depends on the capacity of its algal system. The hypothesis that microalgal cultures grown under 12:12h light:dark photoperiod may produce the same cell densities as those using constant light is tested.
Two species of marine microalgae used in shellfish hatcheries. Chaetoceros gracilis Schütt and Isochrysis galbana clone T-iso, were grown in cultures at 18°C, under continuous light (1.43 × 10⁷μ^-2day^-1) and 12:12h light:dark cycles (2.87 × 10⁷μE m^-2 day^-1). Both light regimes provided equal amounts of light per day. In the continuous light cultures the mean doublings per day for exponentially growing cells were 1.37 and 1.49 for C. gracilis and I. galbana respectively and for the 12:12h light:dark cycles were 1.47 and 1.56 respectively. After 14 days of growth, the numbers of cells per unit of volume showed no significant differences between the two light regimes. The results are discussed in terms of a review of other authors' findings and in terms of the usefulness of the continuous light method in producing algae to be used in shellfish hatcheries.     

Water Quality and Microbial Dynamics in Shrimp Ponds Receiving Bagasse-Based Feed

Pond water quality and associated microbial biomass were studied in relation to the type of feed applied during the culture of the marine shrimp Penaeus vannamei . The feeds tested included conventional feedlot manure as well as two feeds based on bagasse, a sugarcane waste product. Physical and chemical parameters were studied during a 100 day trial in 200 m² earthen ponds. Both bagasse-based feeds supported a significantly larger microbial community as measured by specific biomass numbers ( P < 0.01), ATP content ( P < 0.001) and amount of the particulate organic matter present on pond bottoms ( P < 0.025). For both bagasse-based treatments, the estimated bacterial cell number in the flocculent layer was 3.11 ± 10¹²/m², compared to the much lower cell number of 7.53 ± 10¹⁰/m² for control ponds. Harvest data suggest that bagasse forms a potential base for feeds when applied to extensive shrimp cultures.     

Determining the optimum doses of Kurasan (ethoxiquinolin) and butylhydroxytoluol (BHT) in dry pellets: effect of both anti-oxidants on some haematological and condition parameters of rainbow trout, Salmo gairdneri Richardson

Abstract. Kurasan and BHT were tested at doses of 100, 200 and 400mg kg^-1 incorporated in dry pellets. The administration of these antioxidants did not influence the red blood count of rainbow trout, Salmo gairdneri Richardson, at body weights of 100 to 400g, reared in flow-through tanks at water temperatures of 7 to 13°C and in cage culture at water temperatures of 15 to 2O°C. In laboratory experiments at a water temperature of 14°C, the highest Kurasan dose increased the red blood cell (RBC) count insignificantly by 14% (1.06 vs 0.93 10¹²1^-1) after 80 days, haematocrit (PCV) was increased by 27% (0.410 vs 0.32511^-1), and haemoglobin (Hb) by 16% (72 vs 62gl^-1), This was seen in the cage culture experiment, but not in the experiments in the flow-through tanks. A trend of diminishing haematological parameters of the red blood count and total blood serum protein (TP) of the fish fed with BHT-stabilized diet was recorded only under laboratory conditions at the water temperature 9°C, The decline of RBC count in the experimental group with 0.04% BHT represented 14% (0.90 vs 1.05 10¹²1^-1), the decline in PCV amounted to 18% (0.328 vs 0.39811^-1), Hb to 17% (57 vs 69g 1^-1), and TP to 11% (39 vs 44g 1^-1).     

Changes in the nutritional parameters of muscles of the common carp (Cyprinus carpio) and the silver carp (Hypophthalmichthys molitrix) following environmental exposure to cyanobacterial water bloom

The present study evaluated the effect of naturally developing cyanobacteria on the composition of muscles of two commercially important freshwater fish species. Fish were exposed to cyanobacterial biomass including Microcystis aeruginosa and Microcystis ichthyoblabe for 4 weeks. Then, they were transferred to dechlorinated potable water without any cyanobacteria for another 4-week period, thus modelling their preparation for consumers. Samples of muscles were collected every week during exposure and subsequent stay in dechlorinated potable water. The cyanobacterial water bloom of 3.9–6 × 10⁵ cells mL⁻¹ (133–383 μg g⁻¹ of total MC DW) induced statistically significant effects only in the content of fatty acids ( P <0.05; P <0.01) in the common carp ( Cyprinus carpio ), while all studied parameters including the content of dry matter and fat ( P <0.01), proteins ( P <0.05), fatty acid composition ( P <0.05; P <0.01) and some amino acids ( P <0.05) were affected in the silver carp ( Hypophthalmichthys molitrix ). This study has shown that cyanobacteria in the environment of commercially produced fish may decrease the dietetic value of fish muscles.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).