Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England

A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.     

Spring viremia of carp (SVC)

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV). Affected fish show destruction of tissues in the kidney, spleen and liver, leading to hemorrhage, loss of water-salt balance and impairment of immune response. High mortality occurs at water temperatures of 10 to 17 degrees C, typically in spring. At higher temperatures, infected carp develop humoral antibodies that can neutralize the spread of virus and such carp are protected against re-infection by solid immunity. The virus is shed mostly with the feces and urine of clinically infected fish and by carriers. Waterborne transmission is believed to be the primary route of infection, but bloodsucking parasites like leeches and the carp louse may serve as mechanical vectors of SVCV. The genome of SVCV is composed of a single molecule of linear, negative-sense, single-stranded RNA containing 5 genes in the order 3'-NPMGL-5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively. Polyacrylamide gel electrophoresis of the viral proteins, and sequence homologies between the genes and gene junctions of SVCV and vesicular stomatitis viruses, have led to the placement of the virus as a tentative member of the genus Vesiculovirus in the family Rhabdoviridae. These methods also revealed that SVCV is not related to fish rhabdoviruses of the genus Novirhabdovirus. In vitro replication of SVCV takes place in the cytoplasm of cultured cells of fish, bird and mammalian origin at temperatures of 4 to 31 degrees C, with an optimum of about 20 degrees C. Spring viremia of carp can be diagnosed by clinical signs, isolation of virus in cell culture and molecular methods. Antibodies directed against SVCV react with the homologous virus in serum neutralization, immunofluorescence, immunoperoxidase, or enzyme-linked immunosorbent assays, but they cross-react to various degrees with the pike fry rhabdovirus (PFR), suggesting the 2 viruses are closely related. However, SVCV and PFR can be distinguished by certain serological tests and molecular methods such as the ribonuclease protection assay.     

Fish rhabdoviruses: comparative study of protein structure.

Proteins from four fish rhabdoviruses have been studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viruses were: trout viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHN), spring viremia virus of carp (SVC), and the pike fry rhabdovirus (PFR). For the two salmonid viruses (VHS-IHN), gel electrophoresis indicated the proteins, with molecular weights estimated to be 190,000, 80,000, 38,000, 25,000, and 19,000, respectively. The electrophoretic profile of the two other viruses (SVC-PFR) revealed four major proteins with molecular weights of 190,000 80,000 42,000 and 21,000, respectively. In this case a minor component with 50,000 daltons was found. For each virus only one protein was found to be glycosylated, i.e., the one with a molecular weight of 80,000. A major protein (molecular weight between 38,000 and 42,000) was found to be associated with the nucleocapsid. All these results revealed marked similarities in protein structure between the four fish rhabdoviruses and the previously well-characterized members of rhabdovirus group. However, one can distinguish two groups of viruses: the first one is composed of salmonid viruses (VHS and IHN) with a protein structure comparable to that of rabies virus and potato yellow dwarf virus; the second one is composed of carp and pike viruses, having a protein structure very similar to that of vesicular stomatitis virus.     

The reproductive success of pike, Esox lucius: aspects of fecundity, egg density and survival

Power-law relationships have been estimated between fecundity and fish length, weight and age for pike from two gravel pit lakes (Main Lake and St Peter's Lake) in Buckinghamshire, England. Relative fecundity, estimated at 17.6 eggs g⁻¹ for Main Lake pike and 19.4 g⁻¹ for St Peter's Lake pike, did not differ significantly between the lakes. Population fecundity for the Main Lake was estimated at 10.6 million eggs and 6.4 million eggs in 1986 and 1987, respectively.
Experimental work involving samples of eggs from 18 artificially fertilized Main Lake female pike showed a significant relationship between egg diameter and female length, and a significant difference between mean egg diameters for 41-cm and 101-cm females. No significant relationships were found between mean egg size and mean fry size, nor between fry length and adult length. Mean size differences between newly hatched, 18-day-old and 41-day-old fry from the 41-cm and 101-cm females were not significant.
Samples of eggs taken at 2-day intervals from 6 April to 5 May in a pike spawning area of the Main Lake revealed clumped distributions of eggs, with average egg densities ranging from 1 to 47 eggs per 0.071 m² (14–671 m ²) on sand and silt substrata. The maximum egg density of 51 per 0.071 m² (729 m⁻²) was found on flooded grass. The stages of development of the eggs were identified over the sampling period. Survival from stage 1 to fry was estimated at 3–6%. Egg losses were estimated at 9–10% day⁻¹.
Egg survival experiments gave overall hatching success rates of 11 % on sand/silt compared with 2.5% on aquatic plants (1986), and 18.9% on undisturbed sand/silt compared with 7.9% on disturbed sand/silt (1987). The effect of siltation on egg hatching is discussed.     

Assessment of commercial test kits for identification of spring viraemia of carp virus

Two test kits for the identification of spring viraemia of carp virus (SVCV), one an enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antiserum, and the other an indirect fluorescent antibody test (IFAT) using a mouse monoclonal antibody, were assessed for specificity using a range of virus isolates. The test viruses were selected from 4 recently described genogroups of piscine rhabdoviruses: Genogroup I (SVCV), Genogroup II (grass carp rhabdovirus), Genogroup III (pike fry rhabdovirus) and Genogroup IV ('tench rhabdovirus'). The test viruses included SVCV isolates from all 4 subgroups of Genogroup I. The ELISA was non-specific for these viruses and did not distinguish between SVCV and isolates from the other 3 Genogroups. However, the IFAT was too specific and detected SVCV isolates from only 1 of the 4 SVCV subgroups. Reliance on these test kits alone could result in misidentification of this OIE notifiable disease.     

Hydrocephalus and red-disease in pike fry Esox lucius L.

Two diseases of pike fry have been investigated, both causing serious losses in Dutch pike culture. In one of these diseases the lump developing on the head of the fry was associated with an internal hydrocephalus.
The other one, 'red disease' after the haemorrhagic areas in the trunk, may be identical with red-sore disease of pike.
The etiologies of both diseases are unknown.     

Eel protection measures within the Manapouri hydro-electric power scheme,South Island,New Zealand

Variation in developmental rate from fertilization to swim-up, and body size at swim-up, may affect the growth and survival of young-of-the-year fish. Fish egg size (diameter) is often positively correlated to adult female size, but whether increased egg size equates to higher egg nutrient content and subsequently improved embryo/larval performance, remains unclear. Artificially fertilized northern pike eggs from individual females (total length 400–800 mm) were cultured under temperature controlled laboratory conditions to test the hypothesis that female body size positively influences egg size and the amount of specific nutrients (carbon, nitrogen, phosphorus, and fatty acids) allocated to eggs. We further hypothesized that greater egg nutrient content would positively influence egg survival, developmental rate, and the size of swim-up fry. These hypotheses were investigated in the context of two different northern pike spawning strategies (early season tributary vs. late season deep shoal spawning). Nutrients were allocated conservatively in northern pike eggs, showing very little variation in nutrient concentrations, but the total mass of all egg nutrients increased linearly with egg dry mass. Neither egg dry mass nor nutrient content (concentration or mass), were related to either egg diameter or female body size. The mass of individual egg nutrients was, however, strongly correlated with egg dry mass (r ² range 0.62 to 0.99 for individual nutrients) and positively related to the total length of swim-up fry (r ² = 0.58). The eggs of late spawning pike had significantly greater dry mass (average = 3.02 mg/egg) and developed more rapidly to swim-up (average = 17.89 days) than did those of early spawners (average = 2.28 mg/egg, 19.05 days). Our results indicate that egg quality may be best assessed by egg dry mass, which was correlated with greater nutrient mass and increased swim-up fry body length, as opposed to egg diameter or female body size which showed no correlation to egg nutrient composition and egg/larval performance. Guest editors: J. M. Farrell, C. Skov, M. Mingelbier, T. Margenau & J. E. Cooper International Pike Symposium: Merging Knowledge of Ecology, Biology, and Management for a Circumpolar Species     

Spring viremia of carp virus RNA and virion-associated transcriptase activity

An RNA-dependent RNA polymerase activity has been demonstrated for spring viremia of carp virus (SVCV). The optimal temperature for in vitro synthesis of RNA was 20 to 25 degrees C. The SVCV enzyme activity was stimulated when the methyl donor S-adenosyl-L-methionine was included in the reaction mixture. S-adenosyl-L-methionine was not particularly effective in stimulating the virion RNA polymerase activity of vesicular stomatitis virus or pike fry rhabdovirus. The 5' nucleotide of the SVCV viral RNA is pppAp.     

Nanoinjection as a tool to mimic vertical transmission of Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss

Newly fertilised eggs of rainbow trout Oncorhynchus mykiss were nanoinjected with Flavobacterium psychrophilum in order to mimic vertical transmission. Two bacterial isolates with different elastin-degrading capacity were used. All infected groups (10, 100 and 1000 colony forming units egg(-1)) showed significantly higher cumulative mortalities than the control groups at the end of the experiment, 70 d post-hatching. The total mortalities in the control groups were below 2.5%. In the high-dose groups, 95 to 100% of the eggs died during the eyed stage. In the intermediate group infected with the elastin-negative isolate, the major mortality occurred during the eyed stage of the egg, with a total cumulative mortality of 83% at the end of the experiment. In the intermediate group infected with the elastin-positive isolate, a total mortality of 63% was recorded. In this group, diseased fry showed clinical signs of disease and morphological changes similar to those described in connection with rainbow trout fry syndrome (RTFS) shortly after the beginning of feeding. In the low dose groups, the mortality in the elastin-negative group was 14% and in the elastin-positive group 11%. The bacterium was isolated from dead eggs and fry in infected groups and demonstrated in internal organs of dead and moribund fry by immunohistochemistry. The nanoinjection method used in this study may be a useful method to study pathogens, like F. psychrophilum, that can be vertically transmitted.     

颗粒体病毒在小菜蛾种群中的垂直传播方式的初步观察

在室内观察了小菜蛾颗粒体病毒PlutellaxylostellaGranulosisVirus(RxGV)在小菜蛾种群水平上可能的垂直传播途径。结果表明 :(1 )上代小菜蛾Plutellaxylostella(L .)幼虫期饲毒不能将小菜蛾颗粒体病垂直传递到下代 ;(2 )取食病毒的雌蛾产的卵所孵化的幼虫中 ,没病毒感染出现 ,说明病毒不能经小菜蛾成虫体内传播到下一代。 (3 )用小菜蛾颗粒体病毒处理卵卡时 ,其初孵幼虫有少量被病毒感染死亡 ,浓度高时感染率也较高 ,但感染率均在 1 0 %以下 ,说明小菜蛾颗粒体病毒能经卵表垂直传递给下代 ,但传递效率不高。但当用病毒处理叶片上的卵时 ,初孵幼虫死亡率高达 77 73 % ,而且孵化出的幼虫大多数染病且死于 3龄以前 ,因此田间病毒的施用时间可提早到卵高峰期。     

Reproduction effort versus the environment; case histories of Windermere perch, Perca fluviatilis L., and pike, Esox lucius L.

The theoretical limits of net reproduction rates for perch, Perca fluviatilis L. and pike, Esox lucius L, have been estimated from the upper and lower limits of fecundity, growth, mortality, age of maturity and biomass of parental stock, observed in Windermere over 40 years. Differences in egg numbers due to changes in fecundity were for perch × 1.5, for pike × 2. Assuming the same mortality, a fast growing cohort would produce more eggs during its lifespan than a slow growing, for perch × 2, for pike × 9. Changes in mortality from high to low resulted in more eggs, for perch × 7, and for pike (where high mortality includes natural and fishing mortality) × 10. Change in age at first maturity from 3 to 2 years was unimportant. Biomass of parental stock varied by × 6 for perch and × 3 for pike, and number of eggs laid varied by x2 for perch and × 5 for pike. The number of recruits at age 2 years varied by × 300 for perch and × 7 for pike. It was concluded that temperature in the first summer of life and predation were of major importance in regulating the number of recruits. Ricker recruitment curves and a simple model showed that perch, but not pike, could produce maximum recruitment from minimum parental stock. For perch, but not pike, compensatory density dependent mortality increased with increased parental stock. It was concluded that responses by the perch and pike populations to a changing environment can not be of sufficient magnitude to prevent wide fluctuations in recruitment.     

鸡传染性法氏囊病病毒超强毒株GX8/99株的致病性

鸡传染性法氏囊病病毒(IBDV)超强毒株GX8/99,系1999年从广西一自然发病鸡群采集到.用原始病鸡法氏囊悬液连续3次人工感染SPF鸡后,再取其法氏囊制备悬液,分装,在-70℃保存.以此悬液经卵黄囊接种10日龄SPF鸡胚,测定鸡胚的半数致死量(ELD50).随着鸡的日龄和接种剂量的不同,其致死率有很大差异.对28～30日龄SPF鸡,病毒接种量为200个ELD50时,感染后7日内死亡率最高可达73%～90.5%(11/15和19/21);感染量为20个ELD50时,死亡率亦可达53%～92%(8/15和23/25).以500个ELD50感染28～104日龄的SPF鸡,死亡率均在55.1%～67.2%;甚至113～120日龄的SPF鸡,感染后仍有10%～15%致死率.但128日龄的SPF鸡感染后既不引起死亡也不表现任何症状,但抗体全部转阳.人工接种发病死亡的鸡,其法氏囊的出血程度也随感染量和年龄而异.50日龄鸡接种2000个ELD50后,死亡的鸡100%(12/12)法氏囊严重出血;而40日龄鸡感染200个ELD50后,死亡鸡中仅17%(3/18)发生出血.在2、3、4周龄带有母源抗体的商品代蛋鸡,以2000个ELD50病毒接种后,只引起10%(2/20)、35%(7/20)和35%(7/20)的死亡率.但在5周龄商品代蛋鸡,仅接触感染的致死率可达61.3%(98/160).另一批商品代蛋鸡,在4周龄和5周龄人工接种200个ELD50病毒后,死亡率分别是81.6%～94.3%(62/76～33/35)和93.9%～94%(31/33～47/50).通过总共1200多羽鸡的试验表明,GX-8/99株是一个超强毒IBDV毒株,表现为高死亡率(最高可达94%),易感年龄延长至4月龄,中枢性免疫器官法氏囊出血严重和胸腺明显萎缩.     

The carotenoids of eggs of wild and farmed Atlantic salmon, and their changes during development to the start of feeding

The only carotenoid detected in newly fertilized eggs of wild Atlantic salmon, Salmo salar, from western Scotland was astaxanthin at a concentration [μg carotenoid g^?1 wet wt of eggs, mean ±S.D. (number of parental females)] of 6.2±1.2(7) in 1982, 6.4±1.8(20) in 1983, and 7.6 ± 13(6) in 1984. In eggs of farmed Atlantic salmon the only carotenoid detected was canthaxanthin at concentrations which varied significantly between farms depending on the level of synthetic canthaxanthin in the broodstock diet. Thus on two farms using feed with 50 μgg^?1, the levels were 11.8 ± 3.4(7) and 12.3 ± 2.9(6), while on two farms using 75μgg^?1 the levels were 18.7 ± 5.0(9) and 21.2 ± 2.7(21). The levels in eggs of one-seawinter fish (grilse) did not differ from those of two-seawinter fish reared on the same farm and diet. During development from newly fertilized egg to fry at the end of yolk-sac absorption, the quantity of carotenoid present per individual decreased, presumably as a result of metabolism. Despite large differences in quantity present, the quantity so metabolized was fairly constant at 2–4 μg carotenoid g^?1 original egg weight for eggs from two-seawinter farmed and wild salmon, except that in eggs from farmed grilse it was 7 μg g^?1. In fry from wild eggs, 99.14% of the remaining carotenoid was present in the integument (skin and fins) as astaxanthin, astaxanthin monoester and astaxanthin diester. In fry from farmed salmon eggs, 47 ± 8% of the carotenoid present was found in the unused yolk oil droplets and in the liver, and 37 ± 6% was found in the integument as canthaxanthin and an unidentified metabolite of canthaxanthin. These findings explain visible colour differences between fry from wild parents and fry from canthaxanthin-fed farmed parents, particularly in the fins, liver and residual oil droplets. The canthaxanthin metabolite was also found, together with canthaxanthin, in the skin of farmed adults fed canthaxanthin. Preliminary tests showed it to be unchanged by saponification but reduced by sodium borohydride. For eggs from the three farms incubated under the same conditions in the same season, percentage mortality both to the eyed stage and between hatching and first feeding varied significantly between farms, but percentage mortality between the eyed stage and hatching did not do so. Results combined from two seasons for eggs from three farms and one wild source showed that egg mortality between fertilization and the eyed stage was not significantly different between wild and farmed salmon, but mortality between the eyed stage and hatching, and between hatching and first feeding, were both significantly higher in farmed salmon than in wild salmon. Such differences could not be explained simply by the large differences in egg carotenoid content, but were almost certainly due to factors such as broodstock nutrition, broodstock management, and stripping and fertilization procedures.     

Predation of Northern Pike (Esox lucius L.) Eggs: A Possible Cause of Regionally Poor Recruitment in the Baltic Sea

Northern pike (Esox lucius) spawning habitat and egg mortality were studied in three spawning areas in 2001 along the Swedish coast of the Baltic proper: the Blekinge Archipelago, Kalmar Sound and coastal freshwater streams. Spawning peaked during the last week of April in streams, at temperatures ranging from 7.7 to 8.9 °C and during the first week of May in brackish waters, at temperatures ranging from 8.9 to 13.8 °C. Spawning occurred in shallow waters, at depths between 0.2–1.5 m, but generally most of the spawn was found in the shallowest areas. In streams, eggs were mainly attached to emersed vegetation, while in brackish sites pike eggs were well scattered among flooded emersed plants, submersed plants and filamentous algae. Mean egg density varied between 469–1829 m^–2 with the lowest density observed in Kalmar Sound. The calculated egg loss occurring from approximately one day after spawning to one day before hatching ranged from 41±7% in coastal streams to 67±6% in the Blekinge Archipelago and 100% in Kalmar Sound. The significant removal of eggs from spawning sites in Kalmar Sound and Blekinge was most likely due to predation from several fish species. In situ observations and stomach analyses suggested that many pike eggs in Kalmar Sound were lost to the three-spined stickleback (Gasterosteus aculeatus), a species that presently dominate the littoral small fish community. This study therefore suggests that egg predation by sticklebacks and other fishes may be a possible cause of the reported poor recruitment of coastal pike populations in the Kalmar Sound region.     

Predicting fluctuations in the size of newly emerged sea-trout fry in a Lake District stream

The objective was to predict interannual fluctuations in the size of sea-trout fry when they emerged from the redd, using models developed from field data for 70 excavated redds (≥three per year), and from experimental data on egg and alevin development at 30 constant temperatures in the laboratory (range 1·5—10·5) C with 100 naturally fertilized eggs at each temperature). Egg weight increased with female length and also with the number of eggs laid in a redd, both relationships being well described by a power function. Early spawners were the largest females laying the largest and most numerous eggs, whilst late spawners were the smallest females laying the smallest and least numerous eggs, with middle spawners being intermediate between these two extremes. Mean values for egg weight and number of eggs per redd were obtained for these three groups. The numbers of early, middle and late spawners for each year of a 30-year study and the mean values from the excavated redds were used to estimate weighted means for the number of eggs per unit area and egg weight. Mean values varied considerably between years (30-year ranges: 518–7964 eggs per 60 m2; 112–138 mg wet weight). In the laboratory, mean weights of newly hatched alevins and newly emerged fry were both related positively to mean egg weights. Alevin and fry mean weights were independent of the number of days required for 50% of the eggs to hatch or fry to emerge. Models described in a previous paper formed the basis of those used to predict fry weights over the emergence period. Model predictions were validated by field data for the whole emergence period in 8 years (1967–1971, 1974, 1975, 1980), and by pre-fry weights on single dates in 21 years (1967–1987). As pre-fry densities on these single dates were very similar to egg densities for the same year class, mortality in the egg and alevin stages was very low. The chief objective was therefore fulfilled, and the extent of interannual fluctuations for the 30-year study showed some variation in mean fry weight (30-year ranges: 153–193 mg for both the whole emergence period and the date on which 50% of fry emerged) but a progressive decrease in fry weight through the emergence period. Possible reasons for this variation are discussed, and it is concluded that the size of the female spawners is the dominant factor.     

Radioactive strontium (85Sr) in marking newly hatched pike and success of stocking

Immersion of newly hatched pike fry in an aqueous solution of 1·85 MBq ml⁻¹⁸⁵Sr did not affect their growth or survival over 3 weeks. Cannibal pike fry retained only l.2–3·3% of the ⁸⁵Sr from their labelled prey. Newly hatched ⁸⁵Sr-marked fry stocked into two lakes in 3 years were distinguishable from wild fish 4–6 weeks later. Stocked and wild fry grew equally well, and stocked pike survived best where density of wild pike was highest. There was no evidence of increase in density as a result of Stocking.