Experimental susceptibility of turbot Scophthalmus maximus to viral haemorrhagic septicaemia virus isolated from cultivated turbot

Juvenile pathogen-free turbot were infected with a viral haemorrhagic septicaemia virus (VHSV) isolate recovered from turbot cultivated on the island of Gigha, West Scotland. Mortality of 100% was recorded in fish infected via the intra-peritoneal (i.p.) route. Horizontal transmission of VHSV in sea water was demonstrated by cohabitation of naive fish with i.p. infected fish at a ratio of 1:1. The total cumulative average mortality in cohabiting fish was 60% by 60 d post-infection. Turbot infected via an immersion route exhibited a cumulative average mortality of 71% by the end of the experiment. VHSV identified by enzyme-linked immunosorbent assay (ELISA) was recovered from both organ (kidney and spleen) and brain samples of individual fish that died following infection by all experimental routes. These findings pose significant implications regarding the persistence of VHSV and its role in limiting natural populations of marine fish species. In addition, the establishment of infection models for the transmission of VHSV in sea water is of fundamental importance to the development of anti-VHSV vaccines in important commercial species such as turbot.     

Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.     

Induction of Mx protein in Atlantic cod with poly I:C: immuno-cross reactive studies of antibodies to Atlantic salmon Mx with Atlantic cod

A polyclonal rabbit antiserum directed against the conserved region of the Atlantic salmon antiviral Mx1 protein was used to detect the putative Atlantic cod Mx protein using Western and dot blotting. A doublet band at about 75kDa and 65kDa was detected by Western blotting in kidney and spleen extracts of cod 3 and 4 days after i.p. injection with poly I:C but not in control fish injected with PBS. In blood leucocyte lysates, similar immunostaining could also be detected in Atlantic cod weakly after injection with PBS and more intensely after injection with poly I:C, suggesting some constitutive expression of Mx protein by leucocytes. Dot blot analysis showed that the Mx protein level was significantly higher in spleen, kidney, liver and gill of cod at least up to 4 days after injection with poly I:C when compared with the PBS-injected controls.     

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6 degrees C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.     

Isolation and identification of Viral Haemorrhagic Septicaemia (VHS) viruses from cod Gadus morhua with the ulcus syndrome and from haddock Melanogrammus aeglefinus having skin haemorrhages in the North Sea

During fish disease surveys for marine rhabdoviruses in 1993 and 1995, the cod ulcus syndrome was seen widely in all ages of cod, especially the 2 to 5+ year classes. Viral Haemorrhagic Septicaemia Virus (VHSV) was isolated from a small proportion of the lesion-positive fish and these isolates were identified by immunofluorescence or ELISA. A serendipitous observation of dermal petechiae on haddock was made. VHSV was isolated from this lesion for the first time indicating a new host species for VHSV.     

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.?noatunensis compared to saline formulation. The antigen epitope was a 20-25?kDa component of F.?noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.?noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.?noatunensis in cod.     

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV)

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.     

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.     

Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.     

The ontogeny of complement component C3 in Atlantic cod (Gadus morhua L.)--an immunohistochemical study

The complement system in fish is well developed and plays an important role in the immune response. Very little is known about the ontogeny of C3 in fish and no study has previously been done on the development of C3 in teleosts. In this study we have detected the presence of C3 in cod larvae from the age of 1 day post hatching (p.h.) till 57 days p.h., using immunohistochemistry. The specific primary antibodies used, were produced against the beta-chain of cod C3. Immunostaining on cod larvae sections revealed that C3 is detectable in the yolksac membrane from day 1 p.h., and in liver, brain, kidney and muscle from day 2 p.h. C3 was also detected in other organs such as eye, notochord, stomach, intestines, pancreas, heart and gills at different stages of cod larval development. These findings suggest that complement is not only important in immune defence against invading pathogens but may also play a role in the formation and generation of different organs.     

Herpesvirus sylvilagus in cottontail rabbits: evidence of shedding but not transplacental transmission.

Herpesvirus sylvilagus was inoculated into five cottontail rabbits (Sylvilagus floridanus) at various stages of pregnancy; they subsequently had litters in the laboratory. Three other cottontails chronically infected with the virus were bred and bore young in large outdoor pens. Thirty-four living neonates and dead fetuses were weighed, measured and aseptically necropsied. A total of 31 liver, spleen and kidney samples, 16 lymph node, 28 heart and 10 brain samples were collected and processed for inoculation into rabbit kidney cell cultures to attempt virus isolation. Virus was not detected in the 147 tissue samples tested. Pre-conception viremias ranged from 10-21 plaque-forming units per 0.5 ml. Virus isolation was attempted from 26 oral and lacrymal, 23 genital, nine urine and fecal, and four milk and male ejaculate samples from eight infected rabbits. Virus was recovered from two salivary samples from the same rabbit. Triamcinolone acetonide administered daily for four days to five rabbits did not stimulate excretion of virus.     

Distribution of viral haemorrhagic septicaemia virus in wild fish species of the North Sea, north east Atlantic Ocean and Irish Sea.

A surveillance programme was initiated on the occurrence and distribution of viral haemorrhagic septicaemia virus (VHSV) in wild marine fish. Six research cruises were undertaken in an 18 mo period during 1997 and 1998, covering the North Sea, the Atlantic waters off the north and west coasts of Scotland and the Irish Sea. A total of 19,293 fish were sampled from 23 different species including cod, haddock, Norway pout, herring and sprat. Individual fish lengths were recorded and the fish were checked for lesions, haemorrhaging and other signs of disease. Pools of organ samples were taken for virus assay. The majority of fish sampled did not display clinical signs indicative of viral haemorrhagic septicaemia. A small number of cod were found with skin lesions and haddock with skin haemorrhaging. Of the 2081 organ and skin sample pools collected, 21 tested positive for VHSV by tissue culture and enzyme-linked immunosorbent assay. Seventeen of the isolates originated from Norway pout Trisopterus esmarkii, one from cod Gadus morhua (skin lesion), one from herring Clupea harengus, one from whiting Merlangius merlangus, and one from a previously unreported host species, poor cod Trisopterus minutus.     

An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan.

A rhabdoviral disease occurred in farmed populations of market sized Japanese flounder (hirame) Paralichthys olivaceus in the Seto Inland Sea of Japan in 1996. The causative agent was identified as viral hemorrhagic septicemia virus (VHSV) based on morphological, immunological, and genetic analyses. Diseased fish that were artificially injected with a representative virus isolate showed the same pathological signs and high mortality as observed in the natural outbreak. This is the first report of an outbreak of VHSV infection in cultured fish in Japan. Clinical signs of diseased fish included dark body coloration, an expanded abdomen due to ascites, congested liver, splenomegaly, and a swollen kidney. Myocardial necrosis was most prominent and accompanied by inflammatory reactions. Necrotic lesions also occurred in the liver, spleen and hematopoietic tissue, and were accompanied by circulatory disturbances due to cardiac failure. Hemorrhagic lesions did not always appear in the lateral musculature. Transmission electron microscopy revealed many rhabdovirus particles and associated inclusion bodies containing nucleocapsids in the necrotized myocardium. The histopathological findings indicated that the necrotizing myocarditis could be considered a pathognomonic sign of VHSV infection in Japanese flounder.     

鳜鱼病毒病原的检出及组织病理分析

经对患暴发性传染病的鳜组织及病变组织超微切片的电镜观察,在细胞质,核及间隙中都有病毒颗粒,对该病毒进行了分离提纯,这是一种直径约为２８０ｎｍ球形病毒,将粗提病毒液接种到培养细胞中,会引起细胞病变;病毒通过人工感染健康鳜,会引起鳜发病和死亡,对患病獗进行解剖和组织病理观察,表明病鳜的５种器官都发生了病变,研究结果表明分离的鳜病毒（ＳｉｎｉｐｅｒｃａｃｈｕａｔｓｉＶｉｒｕｓ,ＳＣＶ）是引起鳜流４行的病     

Viral load of various tissues of rainbow trout challenged with viral haemorrhagic septicaemia virus at various stages of disease

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID50) or real-time RT-PCR (copy number) were > 10(4) in preclinical fish, > 10(3.8) in clinically affected fish, > 10(3.9) in mortalities and > 10(1.2) in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

Flow cytometry detection of infectious pancreatic necrosis virus (IPNV) within subpopulations of Atlantic salmon (Salmo salar L.) leucocytes after vaccination and during the time course of experimental infection

In the present study, intracellular infectious pancreatic necrosis virus (IPNV) in salmon leucocytes was detected by flow cytometry after experimental cohabitant challenge. IPNV vaccinated, non-vaccinated and intraperitoneally (i.p.) infected salmon (virus shedders) were analysed at different times throughout the period when mortality occurred. Fish that had survived 61 days post challenge (carriers) were also analysed. In particular, we analysed the presence of IPNV in B-cells (C7G7+cells) and in neutrophils (E3D9+ cells) in head kidney leucocytes (HKL) and in peripheral blood leucocytes (PBL).IPNV was present in HKL and PBL from all challenged fish groups at all samplings, including carriers. IPNV was also found intracellular in other leucocytes than B-cells and neutrophils. During the time course of infection there were changes in proportion of B-cells and neutrophils and in proportions of IPNV+ cells. In vaccinated fish, a delay in the changes observed in the proportion of IPNV+ cells and in the proportions of the two subpopulations was identified. The vaccinated fish were protected against disease as no fish died compared to 30.8% of non-vaccinated cohabitant fish. All i.p. infected fish, except one, survived the challenge. This is consistent with previous studies and confirmed that the routes of infection can influence mortality. The analyses in this study could not identify any factors enlightening this absence of mortality in i.p. infected fish, but both flow cytometry and qRT-PCR showed that i.p. infected fish were carriers of IPNV. The present study also found that IPNV was present in both B-cells and neutrophils as well as in other leucocytes in all carriers after cohabitant challenge. These fish had survived 9 weeks post challenge and 4 weeks after mortality has ceased. The fish harbouring virus within their leucocytes might become life long carriers and represent a risk for disease outbreaks, being virus shedders. Such fish are protected from later infections if the virus exposure has resulted in protective immunity. Flow cytometry was found to be very suitable for detection of intracellular virus after in vivo challenge and the sensitivity was demonstrated by the detection of virus in carriers.