Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout (Oncorhynchus mykiss)

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT‐PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS‐PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.     

Infectious pancreatic necrosis virus of salmonids: biological and antigenic features of a pathogenic strain and of a non-pathogenic variant selected in RTG-2 cells

Abstract. A non-pathogenic cell culture adapted variant was obtained from a normally pathogenic strain of IPN virus (Sp type) after several passages in RTG-2 cells. This cell culture modified (CCA) strain was compared with the original wild (W) strain in various tests. Cross neutralization tests showed no obvious antigenic difference between the two. However the CCA strain was neutralized by a 1:5000 dilution of normal trout serum whereas the W strain was not. In RTG-2 cells, CCA strain gave both large and small plaques, whereas the W strain gave only small ones. Both virus strains were heat sensitive and labile to cyclic freezing and thawing. The CCA virus was more stable in storage at 4°C under different pH conditions and its growth in RTG-2 cells was more rapid. The rt (supraoptimal temperature at which viral yield is depressed by 90%) appeared to be 19-20°C for the CCA strain and 18-19°C for the W strain. Pre-treatment of RTG-2 cells with ultraviolet inactivated CCA virus could inhibit growth of W virus, but had no effect on replication of homologous virus.     

The first report of viral haemorrhagic septicaemia in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), in the United Kingdom

Viral haemorrhagic septicaemia (VHS) was diagnosed in rainbow trout in the UK in May 2006. VHS virus (VHSV) was isolated from fingerlings showing typical histopathological lesions at a single rainbow trout farm site experiencing high mortality. The virus was confirmed as VHSV by serological and molecular biological tests. Phylogenetic analysis based on the complete glycoprotein gene sequence revealed that the isolate was closely related (99% nucleotide identity) to several Danish isolates from 1991 to 2000 and was assigned to VHSV genogroup Ia. The pathogenicity of the isolate was determined in infection experiments using rainbow trout fry. Following waterborne challenge, cumulative mortalities reached 96.67-100% by 12 days post-infection. This represents the first isolation of a pathogenic freshwater VHSV in the UK.     

Challenge studies of European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), with epizootic haematopoietic necrosis virus

A challenge model for comparison of the virulence of epizootic haematopoietic necrosis virus (EHNV) to European stocks of redfin perch, Perca fluviatilis L., and rainbow trout, Oncorhynchus mykiss (Walbaum), was tested. The model investigated intraperitoneal (IP), bath and cohabitation routes at 10, 15 and 20 °C for 5–6 g fish and 15 °C for 20 g perch. In the IP challenges of perch, significant mortality occurred at 15 °C and 20 °C. In challenge trials for rainbow trout, significant mortalities were observed in IP and bath challenges at 20 °C. The mortality observed in IP challenged 20 g perch was not significantly different from that recorded for 6 g fish challenged IP. No significant mortality was observed in any other treatment groups. Re-isolation of ranavirus was confirmed by IFAT and was consistently associated with dead or moribund fish in the trial groups challenged with EHNV. The findings indicate that EHNV does not pose a high risk for wild perch and trout populations in Europe by natural exposure. Mortality appears to be primarily a function of environmental factors, with temperature playing an important role, and not just the presence of the virus in the fish.     

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.     

Infectivity experiments with Cyprinus carpio iridovirus (CCIV), a virus unassociated with carp gill necrosis

Abstract. Experiments to determine Cyprinus carpio iridovirus (CC1V) virulence to fish of different ages and species were performed; viz. carp, Cyprinus carpio L., goldfish, Carassius auratus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum). Neither signs of disease nor mortality occurred in any of the fish species tested. Five passages of the virus in carp resulted in its loss and, not surprisingly, there was no enhancement of virulence. In carp inoculated intraperitoncally, the virus persisted for more than 6 weeks at 18-20°C, more than 12 weeks at a water temperature increasing from 10 to 20°C and more than 27 weeks at 6 5-8°C. The fish cleared itself of the virus after more than 6 weeks maintenance at 18-20°C. The viral infectivity via water is very low. It was impossible to infect the fish with the cell culture grown virus either by water immersion or by prolonged cohabitation with experimentally infected fish. From infectivity data, the authors conclude that the investigated iridovirus is not the aetiological agent of carp gill necrosis.     

The influence of fish age and water temperature on mortalities of rainbow trout, Salmo gairdneri Richardson, caused by a European strain of infectious pancreatic necrosis virus

Abstract. Rainbow trout fry were infected by a pathogenic Sp type of infectious pancreatic necrosis virus isolated in France. Fry held at 10°C and infected at different ages showed a decrease of sensitivity with increasing age and ceased to be susceptible to the disease when 20 weeks old. Mortality was delayed at 6°C and lowered or suppressed at 16°C. When fry were moved from 10 to 16°C before infection mortality was also lowered but not when they were moved from 16 to 10°C. Late infection of siblings kept at different temperatures before infection suggested a relation between the number of degrees x days and the final mortality recorded.     

Inactivation of infectious pancreatic necrosis virus for vaccine use

Abstract. Formalin and β-propiolactone (BPL) were compared for efficacy in inactivating infectious pancreatic necrosis virus (IPNV) for vaccine use. Incubation with a 1:200 dilution of formalin at 20°C for four days or longer, or a 1:200 dilution of BPL at 4°C for six days or longer, completely inactivated the virus infectivity. However, whereas treatment with formalin caused only a slight reduction in anti-genicity as titrated by in vitro tests, treatment with BPL destroyed over 50% of the antigenicity of the virus. Virus inactivated with formalin also proved highly effective at inducing high titres of neutralizing antibody in trout.     

Thermal tolerance of a rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> strain selected by high-temperature breeding

ABSTRACT:   Thermal tolerance was studied in a rainbow trout strain successively selected through high temperature breeding at 20–27°C since 1966 in Miyazaki Prefecture, Japan. The hatching rate and fry mortality at high temperatures were examined in the selected strain along with normal strains cultured at water temperature of 9–17°C. The hatching rate of embryos fertilized at either 10 or 14°C and subsequently subjected to high temperatures in the blastula or neurula stage of the selected strain, was marginally higher than that of the normal strain counterparts. The upper 50% lethal temperatures (LT₅₀) for embryos in the early segmentation, blastula and neurula stages of the selected strain were also higher than those of the normal strain counterparts. Death temperatures and LT₅₀ of fries acclimated to 20°C of the selected strain were significantly higher than those of the normal strains. However, no difference in the critical thermal maximum was detected between the different strains. These results suggest that the selected strain of rainbow trout established by selecting successively for many generations at high temperatures acquired a degree of thermal tolerance.     

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.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

Selective breeding provides an approach to increase resistance of rainbow trout (Onchorhynchus mykiss) to the diseases, enteric redmouth disease, rainbow trout fry syndrome, and viral haemorrhagic septicaemia

In this study, we reasoned that if we challenged rainbow trout with the causative agents of enteric redmouth disease (ERM), rainbow trout fry syndrome (RTFS), and viral haemorrhagic septicaemia (VHS), we would: 1) detect additive genetic variation for resistance to ERM, RTFS, and VHS; and 2) find that resistance of the trout to ERM and RTFS are favourably correlated genetically, while resistance to VHS is unfavourably correlated with resistance to ERM and RTFS. We tested these premises by challenging 63 full-sib families of rainbow trout (50 sires, 38 dams) with Yersinia ruckeri, Flavobacterium psychrophilum, and VHS virus, the causative agents of ERM, RTFS, and VHS. Resistance to each disease was assessed as both a binary trait (i.e., died/survived) and a longitudinal trait (i.e., time until death following challenge). Additive genetic variation and genetic correlations for resistance to ERM, RTFS, and VHS were estimated by fitting a threshold liability model to resistance assessed as a binary trait. As a longitudinal trait, additive genetic variation and genetic correlations were estimated by fitting a Weibull frailty model to the times until death. Our findings support the first of our premises as we detected additive genetic variation for resistance to ERM, RTFS, and VHS. The heritability for resistance to ERM, RTFS, and VHS ranged between 0.42 and 0.57 on the underlying liability scale when resistance was assessed as a binary trait. As a longitudinal trait, the heritabilities ranged between 0.07 and 0.21 for time until death on the logarithmic-time scale. We were, however, unable to support our second premise as we found that resistance to each of the diseases tended to be weakly correlated genetically. The genetic correlations between the resistances ranged between −0.11 and 0.15 when resistance was assessed as a binary trait, and between −0.23 and 0.16 when resistance was assessed as a longitudinal trait. These findings are encouraging for commercial trout production. The additive genetic variation detected for resistance demonstrates that selectively breeding trout for resistance to ERM, RTFS, and VHS will be successful, providing a complementary approach to control these diseases. The weak genetic correlations suggest that it should be relatively easy to improve resistance to each of the diseases simultaneously.     

Transmission of some fish pathogenic viruses by the heron, Ardea cinerea

Abstract. Various possibilities of transmitting fish pathogenic VHS, SVC and IPN viruses by the heron, Ardea cinerea , were investigated. Shedding of IPN virus in the faeces could be demonstrated for a period of 7 days when about 1 g of IPN virus-infected trout fry was fed to the birds once only, whereas feeding larger quantities of infected fish on 5 consecutive days resulted in virus excretion for a further 5 days only. Infectious IPN virus could not be isolated from samples of blood and virus specific neutralizing antibodies could not be demonstrated in serum samples. A waterborne infection of trout fry was established by adding very small (0·2 g) quantities of faeces from herons fed IPN virus-contaminated fish to 20 1 water. Infectious SVC and VHS virus was also re-isolated from samples of food which were regurgitated by the herons at different times up to 120 min after feeding of contaminated fish. It was concluded that herons are able to act as mechanical vectors for IPN, VHS and SVC viruses and therefore, may be a potential source of infection and spread of the diseases.     

Prevalence and pathogenicity of infectious pancreatic necrosis virus (IPNV) associated with feral lake trout, Salvelinus namaycush (Walbaum)

Abstract. A study was undertaken in 1987 to determine the prevalence of infectious panercatic necrosis virus (1PNV) infection in the lake trout population of Cornwall Lake. Alberta, Canada, and its pathogenicity to cultivable salmonid fish. Virological examination indicated that 44.4% of the adult lake trout in the lake, which is situated in a remote northern region of Alberta, were infected with the virus, mainly in the pyloric caeca and intestine. Virus was not detected in kidney, leucocytes, liver or gonads. In experimental immersion infection of brook trout fry, the virus caused a cumulative mortality of up to 74% in 30 days, beginning at 10 days post-infection. Pyloric caeca, intestine and to some extent gills were found to be early sites of viral replication. The virus was less pathogenic to rainbow trout causing a cumulative mortality of 10% and the survivors were IPNV carriers for at least 3 months. The virus did not cause mortality in young lake trout, the natural host, but the infected fish carried the virus during the experimental period of 30 days.     

Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

Infection of mitogen-stimulated trout leucocytes with salmonid viruses

Abstract. The effects of infection with salmonid viruses on mitogen-stimulated cells and colonies from rainbow trout, Oncorhynchus imkiss (Richardson), kidney cell cultures have been studied using culture in clots of fibrin. Phytohaemaglutinin, concanavalin A and lypo-polysaccharides from Escherichia coli have been used as mitogens. The viral haemorrhagic septicaemia (VHS) virus destroyed any mitogen-induced trout kidney colonies or cells, but the infectious pancreatic necrosis (IPN) virus caused no cell or colony death, and even increased their count in the cultures. The results suggested that the target for IPN virus replication is not any trout leucocyte but that among the possible targets of VHS virus are the different types of leucocytes found in the trout kidney and in their in vitro mitogen-stimulated colonies.     

Differences in self-feeding activity between thermally selected and normal strains of rainbow trout Oncorhynchus mykiss at high temperatures

ABSTRACT:   Feeding activity was examined at high temperatures by using a demand feeder for thermally selected and normal rainbow trout Oncorhynchus mykiss strains. When the water temperature was raised in experiment 1 from 17.5 to 25.7°C at 0.3°C/day in 21 days, the average daily food consumption rate in the thermally selected strain rose to 7.1%, which was significantly higher than that of the normal strain (4.1%, P  < 0.05). The corresponding rate was also significantly higher in the thermally selected (0.8%) than in the normal strain (0.2%, P  < 0.05) of fish in experiment 2 where water temperature was raised to 24.1°C in 0.5°C/day increments. When water temperature was raised rapidly in experiment 3 from 16.7 to 21.7°C in one day and gradually to 24.4°C in 28 days at 0.1°C/day, the average daily food consumption rates were 1.0 and 0.1% for the thermally selected and normal strains, respectively, with significant difference ( P  < 0.01). These results suggest that the thermally selected strain has acquired thermal tolerance as a result of artificial selection.     

Epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection in farmed rainbow trout, Oncorhynchus mykiss (Walbaum): findings based on virus isolation, antigen capture ELISA and serology

Abstract. The epidemiology of epizootic haematopoietic necrosis virus (EHNV) infection was studied in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Estimates of mortality during five outbreaks on a commercial farm from 1986 to 1992 ranged from 0033 to 0.2% per day and total mortality did not exceed 3–4% in any outbreak. Affected fish were 0+ and less than 125 mm forklength. Clinical signs were non-specific, and laboratory examination was required to confirm the diagnosis. At the height of the outbreak in 1992, EHNV was demonstrated by virus isolation and antigen capture ELISA in 89% of clinically affected fish and 51% of dead fish, while the prevalence of infection in apparently healthy in-contact fish was 4%. Two and 4 months later the virus was not detected in a group of apparently healthy fish that had been affected earlier. Antibodies specific for EHNV were not found in rainbow trout from the infected farm; however, strong humoral responses were detected by ELISA in two immunized fish, indicating that the virus was immunogenic. These data suggested that EHNV was poorly infective but highly virulent in rainbow trout. Clinical EHNV infection was positively correlated with high rearing density and a low rate of water exchange, and therefore, with presumed poor water quality. Water temperature, which ranged from 11 to 17°C during outbreaks, did not appear to determine the incidence of clinical infection. EHNV infection in farmed rainbow trout was preceded by infection in free-living redfin perch, Perca fluviatilis L., in the water catchment, but it was uncertain whether this represented the source of infection for rainbow trout.