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).     

In vivo study of phagocytosis, intracellular survival and multiplication of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), spleen phagocytes

The intracellular behaviour of a Flavobacterium psychrophilum strain, ingested by spleen phagocytes of rainbow trout, Oncorhynchus mykiss , of different ages, was assessed in vivo . Three groups of rainbow trout weighing 1 g (aged 10 weeks), 25 g (aged 20 weeks) and 300 g (aged 15 months), respectively, were injected intraperitoneally with 1 × 10⁶ cfu of a F. psychrophilum strain. It was found that only fry, aged 10 weeks, displayed clinical signs and suffered mortality. Bacteriological colony plating of different organs demonstrated that the spleen and to a lesser extent the kidney of only the fry were affected. The number of colony forming units per gram of spleen tissue increased with time. No bacteria were found in the trout aged 5 months and older. Light microscopical examination and epifluorescence microscopy revealed that the fry spleen phagocytes contained an increasing number of viable intracellular F. psychrophilum bacteria over time. Again, no bacteria were encountered in the phagocytes collected from older fish.     

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.     

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.     

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).     

The zooplankton biomass in the Sea of Japan

We examined seasonal, annual variation and horizontal distribution of zooplankton in the Sea of Japan from 1966 to 1990. Zooplankton was most abundant in the spring. The spring maximum appeared in February–March and in April–May in the southern and eastern parts of the study areas, respectively. In the summer and autumn, a secondary peak was most conspicuous in the eastern part. The difference between the estimated biomass at night and day was large in the spring and small in summer and autumn. The biomass in the offshore southern area peaked about every 3 years between 1966 and 1983, and increased abruptly in 1990. The density in the area north of 39°N or 40°N was high. Total biomass estimated in the upper 150 m layer in the Sea of Japan (10⁶ km²) was 9.5 × 10⁶ t in the daytime and 16.6 × 10⁶ t at night.     

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.     

Insight into nucleotide and Ca<Superscript>2+</Superscript> binding to carp α-actin

ABSTRACT:   Nucleotides and Ca²⁺ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca²⁺ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 10⁴/M and 1.2 × 10⁵/M, respectively. Competitive binding of Ca²⁺ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca²⁺ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.     

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.     

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

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

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

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

Correlates of growth in juvenile three-spined sticklebacks: potential predictors of growth rates in natural populations

Abstract - The possibility of using indices to quantitatively predict short-term growth rates of juvenile three-spined sticklebacks ( Gasterosteus aculeatus L.) was assessed. A range of weight-specific growth rates ( G _W) was obtained in experiments with individual sticklebacks fed enchtraeid worms at different daily rations over 21 days. There was a strong, positive correlation between G _W and the RNA : DNA ratio in white muscle ( r ²  = 0.90) and lipid concentration (as percentage dry weight) of the carcass ( r ²  = 0.92). There were smaller, but significant correlations between G _W and the percentage of dry matter in the carcass ( r ²  = 0.67), and the residuals from the weight–length relationship ( r ²  = 0.49). Regressions relating growth to RNA : DNA ratio and lipid concentration offer a means of estimating short-term growth rates in natural populations. A test of such predictions using the results from a 56-day experiment on stickleback growth suggested that predictions from percentage lipid were biased and inefficient, while predictions from the RNA : DNA ratio were unbiased but inefficient. Predictions from percentage lipid were higher than from the RNA : DNA ratio.     

Pathogenicity of Saprolegnia species associated with outbreaks of salmonid saprolegniosis in Japan

The present study was conducted to evaluate the pathogenicity and pathology of Saprolegnia salmonis NJM 9851 and Saprolegnia parasitica NJM 9868, isolated from outbreaks of saprolegniosis, against the immature stages of five species of salmonids. The cumulative mortalities of the tested fish groups that were exposed to 2 × 10 ⁵  spore/L concentrations of S. salmonis NJM 9851 were 90% for brown trout, 93.3% for sockeye salmon and 100% for rainbow tout, masu salmon, and Japanese char. In contrast, all salmonid species exposed to 2 × 10 ⁵  spore/L concentrations of S. parasitica NJM 9868 experienced cumulative mortalities of 100%. The histopathological changes of the saprolegniosis lesions found in all sites of infection were loss of the epidermis, edema of the hypodermis and different degrees of degenerative changes in the underlying musculature. It is clear from our results that S. salmonis NJM 9851 and S. parasitica NJM 9868 are highly pathogenic species to five species of salmonid fishes     

Cryopreservation of YamúBrycon siebenthalae Milt

The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 10⁹ spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 10⁹ spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 10⁹ and 6.4 × 10⁹ spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 10⁹ and 6 × 10⁹ spermatozoa per g of fresh eggs.     

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 10⁵ pfu mL⁻⁻¹) and high (2.4 × 10⁶ pfu mL⁻⁻¹), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.     

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.     

Gonad development of an anadromous fish <Emphasis Type="Italic">Coilia ectenes</Emphasis> (Engraulidae) in lower reach of Yangtze River,China

ABSTRACT:   Anadromous Coilia ectenes was sampled from the Yangtze estuary at Chongming and two of the primary upstream spawning grounds at Jingjiang and Anqing in April, May, June and August 2006. Gonad development was analyzed for females. In April, fish were collected in the estuary and at Jingjiang, but not at Anqing. No female was mature (gonad at stages IV or V) at either location. In May, 45% of the females were mature in the estuary, 9% at Jingjiang and 5% at Anqing. In June, 86% were mature in the estuary, 83% at Jingjiang and 7% at Anqing. In August, C. ectenes was absent at Jingjiang. No female was mature in the estuary, and all females were mature at Anqing. Absolute fecundity ( AF ) increased significantly with standard length ( SL ) by a power function AF  = 2.27 × 10⁻⁶ ×  SL ^2.67 ( r ² = 0.57, n  = 48, P  < 0.05). Mature females in the estuary were smaller than those at Jingjiang and Anqing. Conservation of spawners in the upstream spawning grounds is important because they have a size-related fecundity advantage over the smaller spawners in the estuary.     

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.