Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III). The similarity between another PFRV-like virus isolated from grass carp and representatives of Genogroups III and IV was also <80%, indicating that this virus belonged to a third group (Genogroup II). All of the putative SVC viruses were assigned to a 4th group (Genogroup I), sharing <61% nucleotide identity with viruses in Genogroups II to IV.     

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.     

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could détéct SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was détécted by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of détéction of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was détécted in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.     

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.     

Invasive fish species in the largest lakes of Scotland,Northern Ireland,Wales and England: the collective UK experience

An invasive species is defined as an alien (or introduced or non-native) species whose establishment and spread threaten ecosystems, habitats or species with harm. Such threats to UK lake fish communities have long been appreciated and this review assembles case histories, including new data, from the largest lakes of Scotland, Northern Ireland, Wales and England to examine the hypothesis that at least some of these introductions have become invasive. Loch Lomond in Scotland has experienced six introductions [chub (Leuciscus cephalus), common bream (Abramis brama), crucian carp (Carassius carassius), dace (Leuciscus leuciscus), gudgeon (Gobio gobio) and ruffe (Gymnocephalus cernuus)], of which the most significant has been that of the percid ruffe, which has been implicated in a recent decline of the native coregonid whitefish (Coregonus lavaretus). In Northern Ireland, the introduction of the cyprinid roach (Rutilus rutilus) to Lough Neagh has apparently had a negative impact on some overwintering waterfowl, although the native coregonid pollan (Coregonus autumnalis) remains abundant. Llyn Tegid in Wales has received three introductions [rudd (Scardinius erythrophthalmus), ruffe and silver bream (Blicca bjoerkna)], although no impacts on the native whitefish or other fish populations have been observed. In England, individuals of at least 12 native and non-native fish species have been brought to Windermere for the purpose of live-baiting, although only those of the cyprinids roach and common bream have established abundant populations. At the same time, the native salmonid Arctic charr (Salvelinus alpinus) has declined markedly while the native esocid pike (Esox lucius) has shown changes in abundance, distribution and individual condition, although these developments have not been shown to be causally linked. None of these introductions were sanctioned by appropriate fisheries or other regulatory bodies and almost all of them probably arose from the release or escape of live-bait used by pike anglers. Of the 10 species introductions documented here, four (common bream, gudgeon, roach and ruffe) have established abundant populations and two of these (roach and ruffe) have apparently caused or currently threaten harm, supporting the hypothesis that at least some of these introductions have become invasive.     

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.     

An in situ estimate of food consumption of five cyprinid species in Lake Balaton

Daily rations of five cyprinid species, bream Abramis brama , silver bream Blicca bjoerkna , roach Rutilus rutilus , gibel Carassius auratus gibelio and carp Cyprinus carpio , in Lake Balaton, a large shallow lake, estimated by the Eggers model differed from that of the Elliott – Persson model by only − 4.3 to +7.3% (the differences were insignificant). Daily rations varied within the range of 0.23–0.69 in bream, 0.55–3.61 in silver bream, 0.69–4.65 in roach, 0.38–3.16 in gibel and 0.50–9.74 g dry 100 g wet fish mass⁻¹ day ⁻¹ in carp at temperatures ranging from 8.7–25.8% C. Daily ration was related exponentially with temperature in silver bream, roach, gibel and carp. For bream, a significant relationship was obtained only when a daily ration value was excluded from the analysis. Annual rations were assessed using the relationships between the daily ration estimates from the Elliott—Persson model and water temperature, and the long-term averages of the monthly water temperature data. From these estimates the bream population consumed 104%, silver bream 424%, roach 487%, gibel 363% and carp 913% dry mass of food of its wet biomass annually.     

Growth and production of O+ bream (Abramis brama), O+ roach (Rutilus rutilus) and O+ carp (Cyprinus carpio) in 10 drainable 0.1 Ha ponds

In ten 0.1 ha drainable ponds O⁺ cyprinids were stocked to assess their impact on the aquatic community. Bream and roach were stocked in early June, 1986, and carp in early July, 1986. The ponds were drained mid November, 1986. The relationship between the fish community and its food resource was estimated in terms of production and consumption with use of the data on growth and mortality of the fish. The average gross production of the fish species in the ten ponds (48.4 kg during the experiment) was dominated by the carp (58.5%). A minimum estimate of the food consumption of the O⁺ cyprinids is 760 kJ.m^–2. The average production of bream and roach was much lower than that of carp due to a higher mortality and a more specialized feeding pattern. The gross productions of carp, bream and roach in the ten ponds were not significantly related, although stomach analysis showed diet overlap between the carp and the bream.     

Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus.

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus. Nevertheless, the sea bream appeared to be a potential carrier of nodavirus, as juvenile sea bass became infected when maintained in a tank containing experimentally contaminated sea bream.     

Experimental infection of rainbow trout Oncorhynchus mykiss with viral haemorrhagic septicaemia virus isolates from European marine and farmed fishes

The susceptibility of rainbow trout Oncorhynchus mykiss to infection with various isolates of viral haemorrhagic septicaemia virus (VHSV) was examined. A total of 8 experiments with rainbow trout ranging from 0.6 to 6.2 g was conducted for 139 isolates originating from wild marine fishes in European waters (115 isolates), farmed turbot from Scotland and Ireland (2 isolates), and farmed rainbow trout (22 isolates). The isolates were tested by immersion and/or intraperitoneal injection either as pooled or single isolates. The isolates from wild marine fishes did not cause mortality by immersion while some of the isolates caused mortality when injected. All VHSV isolates from farmed rainbow trout caused significant mortality by immersion. Currently, pathogenicity trials are the only way to differentiate VHSV isolates from wild marine fishes and farmed rainbow trout. The 2 farmed turbot isolates did not cause mortality by immersion, supporting the view that they originated from the marine environment.     

Fertility of roach × bream hybrids, Rutilus rutilus (L.) ×Abramis brama (L.), and their identification

Adult roach, bream and their presumed F₁ hybrid from an Anglian Water reservoir were identified on the basis of morphological and meristic characteristics. The hybrid was clearly intermediate. Four hybrid breeding crosses were induced to spawn by hypophysis. A bream × roach cross (female named first) failed to produce fertile eggs, whereas F₁ hybrid × roach, roach × F₁ hybrid and F₁ hybrid × F₁ hybrid all produced fry. Fertility (defined as survival of eggs to hatching) was high for the F₁ hybrid × roach back-cross (56%) but low for the others (<2%), in comparison to the pure species controls (roach 69%, bream 76%). Progeny from these crosses were reared until anal fin rays could be counted. These counts indicated intermediacy between the parents and back-crossed individuals, and similarity between F₁ hybrids and their F₂ progeny.     

鲤春血症病毒中国分离株糖蛋白基因和氨基酸序列的初步解析

2002～2004年间从国内养殖鲤科鱼类和观赏鱼类中分离出8株鲤春血症病毒(SVCV)。根据SVCV参考株 全序列,设计引物,用逆转录聚合酶链式反应扩增出8株SVCV糖蛋白编码基因片段,并对扩增产物进行了克隆和 序列测定。用生物信息学方法对测得的序列进行分析,结果8个国内分离株的糖蛋白基因序列与参考株的基因序 列相似性均在92％以上,8个国内分离株之间基因序列相似性均在97．7％以上;8个国内分离株之间糖蛋白推导 出的氨基酸序列相似性均在94．5％以上,与参考株氨基酸序列相似性在92．9％-94．9％之间。系统发育树分析结 果表明,SVCV国内分离株与USA株、980451株、980528株和970469株的进化方向一致,与其它SVCV毒株在进 化方向上不同。8个毒株有19个共同的酶切位点,推导出的氨基酸序列中有10个亲水区、10个可能的抗原位点 和10个跨膜蛋白区域,其峰值基本一致。对SVCV国内分离株的糖蛋白6个功能位点(天冬酰胺糖基化位点、精 氨酸-甘氨酸-天冬氨酸序列、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸磷酸化位点和肉豆蔻酰 基化位点)进行了初步分析。     

Molecular Analysis of Spring Viraemia of Carp Virus in China: A Fatal Aquatic Viral Disease that Might Spread in East Asian

Spring viraemia of carp (SVC) is a fatal viral disease for cyprinid fish, which is caused by spring viraemia of carp virus (SVCV). To date, no SVC outbreak has been reported in China. Between 1998 and 2002, outbreaks of SVC were reported in ornamental and wild fish in Europe and America, imported from multiple sources including China. Based on phylogenetic analysis, the viral strain isolated from America was shown to be originated from Asia. These outbreaks not only resulted in huge economic losses, but also raise an interesting question as to whether SVCV really exists in China and if so, is it responsible for SVC outbreaks? From 2002 to 2006, we screened 6700 samples from ornamental fish farms using the cell culture method of the Office International des Epizooties (OIE), and further verified the presence of SVCV by ELISA and real-time quantitative RT-PCR. Two infected samples were found and the complete genome of SVCV was sequenced from one of the isolates, termed SVCV-C1. Several unique hallmarks of SVCV-C1 were identified, including six amino acid (KSLANA) insertion in the viral RNA-dependent RNA polymerase (L) protein and ten nucleotide insertion in the region between glycoprotein (G) and L genes in European SVCV strains. Phylogenetic tree analysis of the full-length G protein of selected SVCV isolates from the United Kingdom and United States revealed that G proteins could be classified into Ia and Id sub genogroups. The Ia sub genogroup can be further divided into newly defined sub genogroups Ia-A and Ia-B. The isolates derived from the United States and China including the SVCV-C1 belongs to in the Ia-A sub genogroup. The SVCV-C1 G protein shares more than 99% homology with the G proteins of the SVCV strains from England and the United States, making it difficult to compare their pathogenicity. Comparison of the predicted three-dimensional structure based on the published G protein sequences from five SVCV strains revealed that the main differences were in the loops of the pleckstrin homology domains. Since SVCV is highly pathogenic, we speculate that SVC may therefore pose a serious threat to farmed cyprinid fish in China.     

Salmonella enterica serotype Bredeney: antimicrobial susceptibility and molecular diversity of isolates from Ireland and Northern Ireland.

Salmonella enterica serotype Bredeney has emerged as the third most commonly identified serotype among human clinical isolates referred to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. A collection of 112 isolates of S. enterica serotype Bredeney collected during the period 1995 to 1999 from animal, food, and human sources from both Ireland and Northern Ireland were studied. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and DNA amplification fingerprinting (DAF) were performed on all isolates. Plasmid profiles were examined on a subset of 33 isolates. A high proportion (74%) of isolates were susceptible to all antimicrobial agents tested. Resistance to both sulfonamide and trimethoprim was observed in 21% of isolates, and resistance to multiple (five) antimicrobial agents was observed in a single isolate (0.9%). Eight different PFGE patterns were obtained, with 87% of isolates grouping as PFGE type A. PFGE type A was predominant in animals, food, and humans. There was good overall concordance between the groups identified by PFGE and DAF. Overall results indicate that most S. enterica serotype Bredeney isolates in Ireland and Northern Ireland from animal and human sources are clonally related.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

In situ estimates of gut evacuation and its dependence on temperature in five cyprinids

Gut evacuation and its dependence on the temperature in bream Abramis brama , silver bream Blicca bjoerkna , roach Rutilus rutilus , gibel Carassius aurtaus gibelio and carp Cyprinus carpio , held in mesh cages in Lake Balaton, was best fitted by an exponential model in 18 of 36 trials, while in 14 and four trials, a linear and a square-root model, respectively, gave a better fit. Adjusted r ² values, however, often did not differ markedly between the three models. The shape parameter, B , was 0.36 for carp and ranged from 0.81 to 1.24 for the other four cyprinids, according to the general evacuation model expanded with the temperature variable, and fitted to whole data sets. Relationships between the food evacuation rates obtained from the exponential models and the temperature were described by exponential functions for bream, silver bream, roach and gibel.     

Demonstration of the shorter flagellin (flaA) gene of urease-positive thermophilicCampylobacter isolated from the natural environment in Northern Ireland

The PCR amplicons (about 1450 bp in length) of flaA gene fragments of 11 isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from the natural environment not including wild birds in Northern Ireland were demonstrated to be shorter than those of C. jejuni 81116 and six isolates of C. jejuni and C. coli (about 1700 bp) isolated in Northern Ireland and Japan. When the nucleotide lengths of the possible open reading frame (ORF) of the flaA genes were determined, those from the 11 UPTC isolates were estimated to be 1464-1503 bp, and those from the six C. jejuni and C. coli isolates and C. jejuni 81116 strain to be 1716-1728 bp. Nucleotide sequence and deduced amino acid sequence alignments of the possible ORFs demonstrated that the ORFs from the 11 UPTC isolates lack about 80 amino acid residues, mainly from the approximate residue numbers 390-470 of the large variable region in the flaA protein of the seven isolates of C. jejuni and C. coli, and do not have any internal termination codons. High amino acid sequence similarity of both amino- and carboxy-termini of the ORFs of the flaA gene was demonstrated between the 11 isolates of UPTC and the 7 isolates of C. jejuni and C. coli. The 11 UPTC isolates examined were strongly suggested to possess a shorter flaA gene without any internal termination codons.