australian bohle virus black bullhead iridovirus epizootic haematopoietic necrosis virus European catfish virus European sheatfish virus iridovirus of ornamental tropical fish largemouth bass virus turbot iridovirus-like agent
EHN infection in redfin perch is generally lethal (Whittington et al., 1995). Rainbow trout are relatively resistant and only a small proportion of individuals become infected (Whittington et al., 1994; Whittington and Reddacliff, 1995; Whittington et al., 1999). Infections with ESV and ECV can induce high morbidity and mortality in their catfish hosts (Ahne et al., 1989, Pozet et al., 1992). The disease caused by all three iridoviruses is characterised by mortalities due to necrosis in the liver, spleen, haematopoietic tissue of the kidney and other tissues. Experimental exposure studies have shown that rainbow trout can be infected with ECV and ESV without showing morbidity and mortality (Ahne et al., 1998).
Iridoviridae of the genus ranavirus are becoming increasingly important as pathogens of feral, cultured and ornamental teleosts. They are endotheliotropic and can induce severe disease in susceptible fish species, characterized by necrotic lesions in vascular walls and visceral organs. The group consists of the epizootic haematopoietic necrosis virus (EHNV) (Langdon et al., 1986b; Eaton et al., 1991; Hedrick et al., 1992), isolates from two ictalurid fishes in Europe (Ahne et al., 1989; Pozet et al., 1992), turbot (Scophthalmus maximus) iridovirus-like agent (Bloch and Larsen, 1993), an isolate from two freshwater tropical ornamental species (Hedrick and McDowell, 1995) and largemouth bass virus (LMBV) (Plumb et al., 1996). The present discussion considers mainly diseases caused by EHNV and ictalurid viruses, which can cause 100% mortality in young susceptible warm-water hosts. The economic damage is rare but serious.
Epizootic haematopoietic necrosis and EHNV were reviewed by Wolf (1988) and McAllister (1993a). This datasheet describes EHN in redfin perch.
Topics for Further Studies
The development of methods for distinguishing ranavirus isolates requires further attention. Such methods are needed to study epizootiology and taxonomy. Surveys of sheatfish, bullhead, ornamental and other fish production sites for ranaviruses in fish and amphibians are needed to assess the range, extent and significance of currently known and possible other diseases in this group. Studies on host ranges and reciprocal pathogenicity of fish and amphibian ranaviruses for early life stages could help to detect virus sources and reservoirs.
[Based upon material originally published in Woo PTK, Bruno DW, eds., 1999. Fish diseases and disorders, Vol. 3 Viral, bacterial and fungal infections. Wallingford, UK: CABI Publishing.]
Members of the ranavirus genus were isolated from freshwater fishes on three continents. Epizootic haemopoietic necrosis virus affects redfin perch (Percafluviatilis) and rainbow trout (Langdon et al., 1986a, b, 1988; Langdon and Humphrey, 1987) in Australia (Victoria, New South Wales, and South Australia).Sheatfish iridovirus (or IW for iridovirus wels) was isolated in Germany (Ahne et al., 1989). Black bullhead iridovirus (or ICF for iridovirus of catfish) caused mortality of Ameiurus melas (syn. Ictalurus melas) in France (Pozet et al., 1992) and in Italy (Bovo et al., 1993). Iridovirus of ornamental tropical fish (IOTF) was isolated from imported guppy (Poecilia reticulata) and doctor fish (Labroides dimidatus) in California, USA (Hedrick and McDowell, 1995).Largemouth bass virus was isolated from a mortality among adult largemouth bass in a reservoir in South Carolina, USA (Plumb et al., 1996).
The redfin perch and rainbow trout isolates of EHNV are serologically indistinguishable. Langdon (1989) experimentally infected 11 teleosts. The mosquito fish (Gambusia affinis) is the only species besides redfin perch which is susceptible to horizontal transmission. Four native species are highly susceptible to the virus and at least one of them (Macquarie perch, Macquaria australasia) is in decline. Atlantic salmon (Salmo salar) develops clinical disease but the infection is not lethal. Barramundi is refractory and so are two exotic cyprinids, which do not seem to be the original host (Langdon, 1989). Whittington et al. (1996) demonstrated the spread of EHN in redfin perch to river systems and impoundments in Australia. In outbreaks of sheatfish and black bullhead diseases (Ahne et al., 1989; Pozet et al., 1992), the water environment contained several other warm-water species, including cyprinids, pike and perch, but they remained unaffected. The ornamental fish strain is pathogenic for rainbow trout (Hedrick and McDowell,1995). Largemouth bass virus has not induced mortality in infected adult largemouth bass (Plumb et al., 1996).
The factors modulating the susceptibility of fish to EHNV, ESV and ECV infection are poorly understood. Clinical outbreaks due to EHNV are associated with poor water quality. In rainbow trout, infection occurs naturally at water temperatures from 11 to 17°C and experimentally from 8 to 21°C (Whittington et al., 1994). Disease in redfin perch does not occur at temperatures below 12°C. The following fish species were found to be susceptible to EHNV following bath exposure: redfin perch, rainbow trout, Macquarie perch (Macquaria australasica), mosquito fish (Gambusia affinis), silver perch (Bidyanus bidyanus) and mountain galaxias (Galaxias olidus) (Langdon, 1989). Both juvenile and adult redfin perch may be affected in outbreaks, but juveniles may be more susceptible to the disease. EHNV has now been detected in diseased rainbow trout ranging from hatchery fry to table-sized fish, although mortalities are most often seen in 0+ fish up to 125 mm fork-length.
The reported geographical ranges of fish ranaviruses are so far restricted to respective continents of isolation. However, that from exotic ornamental fish is suspected to be a part of putative transcontinental transmission (Hedrick and McDowell, 1995). The Bohle iridovirus, which is present only in Queensland, is geographically distinct from EHNV (Speare and Smith, 1992).
The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.
Focal necrosis is a consistent finding in haematopoietic kidney and liver of naturally and experimentally infected fish. In the spleen and pancreas, this sign is variable. Necrotic haemopoietic cells are disseminated in all vessels. Lesions in other susceptible species are inconstant (Langdon, 1989). The most consistent is the necrosis in haemopoietic kidney and liver. A description of gross lesions in redfin perch by Reddacliff and Whittington (1996) includes haemorrhage around bases of fins, focal haemorrhage in gills, oedema and multiple necrotic foci in liver. Microscopic changes consist of focal to extensive necrosis in haematopoietic kidney, liver, spleen, heart, pancreas and lamina propria of the intestine. Thrombosis, haemorrhage and fibrinous exudate are common in gills. Lesions in rainbow trout are similar but milder.
Sheatfish Iridovirus Disease
Histopathology and electron microscopy in experimentally infected sheatfish of 4-5 cm revealed alterations in all organs (Ogawa et al., 1990). Endothelium and reticuloendothelial cells are the main target. Periarteriolar necrosis of the haemopoietic tissue and degeneration of tubular and duct epithelia are prominent in kidneys. Gill epithelium and chloride cells are hyperplastic and oedematous. The lumen of the circulatory system is congested and the proliferating cells contain eosinophilic inclusions. Alterations in the skin include proliferation and necrosis of epithelial cells, hyperplasia of monocellular glands and zonal haemorrhage in hypodermis. Myocarditis and endocarditis are diffuse. Small necrotic foci are seen in the liver and spleen. Glial proliferation and spongiosis in the brain are also pronounced. The pathology and incubation most closely resemble those of EHN.
Iridovirus of Catfish Disease
The kidney is the principal target organ and both the haematopoietic and the excretory part are severely altered. Blood vessel walls are damaged. Necroses in the spleen and kidney can be severe. Interlamellar spaces in gills are obliterated and lamellar fusion is evident (Pozet et al., 1992).
Fish ranaviruses are endotheliotropic and cause haemorrhagic diathesis, oedema and peripheral circulatory failure.
EHN
Epizootic haematopoietic necrosis appeared during the spring of 1984 in a lake and caused severe mortality among juvenile redfin perch. The disease is less serious in farmed rainbow trout. Experimentally infected 35-45-day-old redfin perch develop depression, skin darkening and erratic swimming and die after 4-5 days (Langdon, 1989). Some have erythema around the brain and nostrils. Clinical signs in disease outbreaks described by Langdon and Humphrey (1987) are identical, except for skin ulcers invaded by fungus in some fish.
Sheatfish Iridovirus Disease
The sheatfish iridovirus disease (iridovirous wels disease (IWD)) is characterized by loss of appetite, apathy, ataxia (including rapid spiral swimming), petechial haemorrhage in skin and internal organs and generalized destruction of haematopoietic tissues in the kidney and spleen. The cumulative fry mortality in a recirculation system was 100% (Ahne et al., 1989). Fry infected by bath in virus suspension and by cohabitation also succumbed to high mortality within 8 and 11 days, respectively (Ahne et al., 1990). Adult fish are also susceptible but mortality does not exceed 30% (Ahne et al., 1991).
Iridovirus of Catfish Disease
Iridovirus of ornamental tropical fish was isolated from carriers but the experimental infection of rainbow trout induced low mortality, considerable lesions in the kidney and spleen and virus titres greater than 108 TCID50 g-1 (Hedrick and McDowell, 1995).
Experimental infection of barramundi with Bohle iridovirus resulted in mortality and focal necrosis in the liver (Moody and Owens, 1994).
Procedures recommended by the OIE Manual (1995b) for EHNV are applicable to other viruses in the ranavirus group considered in this chapter. Presumptive diagnosis is based on clinical signs, virus isolation in BF-2 or other susceptible cells and electron microscopy. Identification of EHNV is based on IFAT or ELISA tests (OIE Manual, 1995b) and PCR (Gould et al., 1995). Other viruses in the group show antigenic relatedness to EHNV and FV3 demonstrable by IFAT (Hedrick et al., 1992; Ahne et al., 1995; Hedrick and McDowell, 1995), Western blotting and nucleic acid hybridization (Hedrick and McDowell, 1995).
Epizootic haematopoietic necrosis virus is detectable by ELISA, immunohistochemistry and electron microscopy (Hyatt et al., 1991). An improved antigen-capture ELISA (Whittington and Steiner, 1993) can recognize EHNV in clarified fish tissue and in cell-culture supernatant. The lowest detectable level of the virus in supernatant is 103.5 TCID50 ml-1. Sensitivity and specificity of this method for tissue samples are about 81 and 99%, respectively, and for cellculture supernatant 96 and close to 100%. Manual grinding with a pestle in a tube, followed by vortexing in the same tube with 3 mm glass beads and clarification in a microcentrifuge, is the most efficient method for releasing EHNV from tissue samples (Whittington and Steiner, 1993).
The viruses do not induce differentiating neutralising antibodies in mammals or fish (Hyatt et al., 2000). The viruses can be identified by immunofluorescence, enzyme-linked immunosorbent assays (ELISAs) or immunoelectron microscopy (Hedrick et al., 1992; Hengstberger et al., 1993; Hyatt et al., 1991; Hyatt et al., 2000; Steiner et al., 1991). EHNV has several antigens in common with ESV and ECV from Europe and at least one antigen in common with the amphibian iridoviruses from North America (frog virus 3) and Australia (Bohle iridovirus) (Ahne et al., 1995; Hedrick et al., 1992; Hengstberger et al., 1993). Antigen-detection tests with polyclonal antibodies against EHNV detect each of these viruses. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and polymerase chain reaction (PCR) can differentiate to varying degrees between the different viruses (Hyatt et al., 2000; Mao et al., 1997).
The screening and diagnostic procedures for EHNV, ESV and ECV are based on isolation of virus in cell culture, ELISA, indirect fluorescent antibody tests (IFAT), and electron microscopy (Ahne et al., 1989; Eaton et al., 1991; Hedrick et al., 1992; Hyatt et al., 1991; Langdon et al., 1986; Pozet et al., 1992; Steiner et al., 1991). Antigen-capture ELISA has a sensitivity of 60-80% depending on the stage of infection when used to test tissues from both rainbow trout and redfin perch (Hyatt et al., 1991; Steiner et al., 1991; Whittington et al., 1995; Whittington and Steiner, 1993). Antigen-capture ELISA is the method of choice for confirming the cause of cytopathic effect in cell culture; IFAT and electron microscopy are also useful. IFAT or immunoperoxidase staining may also be used for diagnosis on formalin-fixed tissues. PCR may be used to detect Ranavirus DNA in infected tissues (6). Western blots, SDS-PAGE and PCR sequencing can be used to specifically identify EHNV, ESV and ECV (Hyatt et al., 2000; Mao et al., 1997).
The diagnosis of epizootic haematopoietic necrosis (EHN) is based on direct methods that are either the isolation of virus (epizootic haematopoietic necrosis virus [EHNV], European sheatfish virus [ESV] and European catfish virus [ECV]) in cell culture followed by its immunological identification (conventional approach), or the immunological demonstration of virus antigen in infected fish tissues.
Due to insufficient knowledge of the serological responses of fish to virus infections, the detection of fish antibodies to viruses has not thus far been recognised as a valuable diagnostic method for assessing the virus status of fish populations. In the case of EHNV, preliminary investigations using enzyme-linked immunosorbent assay (ELISA) in known infected rainbow trout populations have revealed up to 1% prevalence of seropositive fish in adult age classes. Further investigation of the epidemiology of EHNV in rainbow trout is needed before serological results can be interpreted.
Adult redfin perch is extremely susceptible to EHNV infection by bath and i.p. inoculation. As little as 0.08 TCID50 ml-1 was lethal at 19-21°C. The incubation period at this temperature is 11 days and at 13-19°C up to 28 days. Disease does not develop below 12°C (Whittington and Reddacliff, 1996). Virus replication in endothelial cells results in necrosis and consequent haemorrhage.
The immunological response in fish and rabbits does not generally include neutralizing antibodies. One survivor of ICFD had neutralizing antibodies (Pozet et al., 1992). However, rabbits react to these viruses by producing antibodies suitable for IFAT and ELISA. Redfin perch survivors from natural disease outbreaks are resistant to challenge with EHNV (Langdon, 1989). The epizootiology of EHN in rainbow trout suggests that this species is incapable of developing long-lasting resistance (OIE Manual, 1995b).
The epidemiology of EHNV in rainbow trout is incompletely understood (Whittington et al., 1994; Whittington and Reddacliff, 1995; Whittington et al., 1999). Due to their extreme susceptibility, it is unlikely that redfin perch are a natural host (Whittington and Hyatt, 1996; Whittington et al., 1995). Infection may recur annually at a rainbow trout production site and this may be due to reinfection from wild redfin perch in the catchment area. A carrier state in naturally infected rainbow trout appears to be very uncommon as neither EHNV antigen nor anti-EHNV antibody are routinely detected in rainbow trout surviving an outbreak (Whittington et al., 1980; Whittington et al., 1999). However, the disease can occur at a very low frequency in an infected population, and mortalities may not exceed the usual background rate. Thus infected fish could easily be included in batches of healthy translocated fish and there is good evidence that this has occurred. No epidemiological data are available for ESV or ECV.
Natural epizootics of EHN in early summer among young redfin perch last for 2-3 weeks. They are recurrent in several major waterways in Victoria, Australia (Langdon, 1989). Adults are rarely affected. Virus isolation from juveniles and adults immediately after an epizootic is infrequent (Langdon and Humphrey, 1987). 100-day-old survivors of disease outbreaks are resistant to challenge (Langdon, 1989). Redfin perch carriers were not detected and there is no evidence for vertical transmission. An unknown reservoir and carrier host are suspected. Silver gulls (Larus novaehollandiae) and great cormorants (Phalacrocorax carbo) can spread EHNV by the regurgitation of ingested material (Whittington et al., 1996). Other means of spread include transportation of fish by humans, transfer on boats, nets and other equipment as well as water flow and migration of carrier fish in a catchment area (Whittington et al., 1996). The epizootiology of other diseases in the group was not studied.
The close relatedness of fish and amphibian ranaviruses and the pathogenicity of Bohle iridovirus for barramundi should be kept in mind in programmes for avoidance of pathogens in aquaculture (Hedrick et al., 1992; Ahne et al., 1995). Frogs are ubiquitous on large farms for warm-water fishes. These could be reservoirs and vectors of fish pathogens. Hedrick et al. (1992), Hedrick and McDowell (1995) and Hedrick (1996) consider transcontinental movements of amphibians and exotic ornamental fish as possible reasons for the appearance of similar viruses in Australia and Europe. It was suggested that control be extended to aquatic amphibians and tropical aquarium fishes (Ahne et al., 1995; Hedrick, 1996).
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