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Figure 2. Electron micrograph of icosahedral virions within asplenic cell.

SHORT CONTRIBUTIONS

Iridovirus-associated mortality infarmed Murray cod(Maccullochella peelii peelii)

MJ LANCASTER Victorian Institute of Animal Science,MM WILLIAMSON 475 Mickleham Road,CJ SCHROEN Attwood, Vic 3049

Aust Vet J 2003;81:633-634

Iridoviruses are large double-stranded DNA viruses whichhave been isolated from insects, fish, amphibians and rep-

tiles.1 Severe systemic infections characterised histologically bylarge cytoplasmic basophilic inclusions and ultrastructurally byaggregates of icosahedral virions have been described in manywild, farmed and ornamental fish species.1,2 In Australia,Epizootic Haematopoietic Necrosis Virus (EHNV) has causedhigh morbidity and mortality rates in redfin perch (Perca fluvi-atilis)3 and low morbidity but high mortality rates in rainbowtrout (Oncorhynchus mykiss).4 This short communicationdescribes high mortality rates associated with an iridovirus-likeagent, apparently not EHNV, in two age groups of farmedMurray cod.

Some 9,000 of 10,000 (90%) 4 to 6 cm fingerling Murraycod died over a 3 to 4 week period in February 2003, whenwater temperatures peaked at 26 to 27oC. Approximately 140 of560 (25%) 10 to 15 cm fingerlings died during the same timeperiod. Inappetence was the first sign noted, followed bylethargy and death within 4 to 7 days. Older fish remained clin-ically normal.

Significant gross changes were not detected in fingerlingsfrom either group. Histological examination revealed apoptoticcells in renal haematopoietic tissue, gill capillaries, gill intersti-tial tissue and spleen. Cells with a large basophilic cytoplasmicinclusion were seen in the spleen, renal haematopoietic tissue,gill connective tissue (Figure 1) and heart. Areas of focal necro-sis and inflammatory cell accumulation associated with bacteriawere present in the liver and spleen of several dead fish. An indi-rect immunoperoxidase test incorporating rabbit polyclonalanti-EHNV antiserum stained EHNV inclusions in redfinperch tissue used as a positive control, but did not stain inclu-sions in any cod tissue. Significant bacteria were not culturedaerobically from livers or spleens.

Splenic inclusions detected histologically were examined byelectron microscopy. Formalin-fixed splenic tissue wasreprocessed from wax blocks, post-fixed in 2% osmium tetrox-ide, dehydrated and embedded in araldite resin. Ultrathin sec-tions were cut, mounted on formvar-coated copper grids andstained with uranyl acetate and lead citrate. Many clusters ofnon-enveloped icosahedral virus particles were identified incytoplasmic remnants of splenic cells (Figure 2). The minimumdiameter of 25 virions ranged from 132 to 165 nm, mean 148+/- 8 nm.

This viral morphology and associated tissue changes are char-acteristic of the group of systemic piscine, amphibian and rep-tilian iridoviruses.1,5,6 Viral diameters fitted the reported sizerange of 153 +/-10 nm.1 Other intracytoplasmic icosahedral

Figure 1. Histological section of gill tissue with several intracyto-plasmic inclusions (arrows). Haematoxylin and Eosin x 400.

viruses reported in fish are much smaller: birnaviruses at 60 nm7

and nodaviruses at 25-30 nm.8 Three iridoviruses have beenreported from wild or farmed fish in Australia: lymphocystisvirus,9 EHNV,3 and Bohle virus. Lymphocystis virus infectionresults in massive hypertrophy of fibroblastic cells, not evident

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in these cod, and its virions are large (225 +/- 6 nm).9

Experimental infection of barramundi (Lates calcarifer) withBohle virus, originally isolated from the ornate burrowing frog(Limnodynastes ornatus) in Queensland,10 resulted in 100%mortality,11 but reports of experimental infections of Murraycod with Bohle virus were not found. Juvenile Murray cod havebeen infected with EHNV by bath and intraperitoneal inocula-tion.12 Only the latter route produced clinical disease, and pan-creatic necrosis was the only consistent histological feature.

Polyclonal antibodies raised against EHNV detect the sys-temic fish and amphibian iridoviruses.1 EHNV shares antigenswith the Bohle iridovirus,13 European Sheatfish Virus andEuropean Catfish Virus.14 Similarities with the frog viruses ofEurope has resulted in these viruses being grouped together inthe Ranavirus genus of the family Iridoviridae.1 Our negativeresults from the immunoperoxidase test suggests that this virusis not EHNV and may not be a member of the genus Ranavirus.

The source of the virus in this outbreak was most likely otherinfected fish (or an infected amphibian), although mechanicaltransfer via water birds, for instance, cannot be ruled out. Thecod were farmed in several tanks in a single recirculatory systemwith water from a chlorinated domestic supply. The fish origi-nated from three different locations in south-eastern Australia.Although reports of natural disease outbreaks in Murray codassociated with intracytoplasmic viral inclusions were notfound, this species may harbour an iridovirus that requires spe-cial circumstances to cause disease. Larger cod fingerlings wereintroduced to the farm 2 to 3 weeks before mortality com-menced in the smaller fingerlings. Minced frozen rainbow troutfillets were fed to the smaller fingerlings during January.Rainbow trout have been demonstrated to carry EHNV, andthis virus is resistant to freezing. A few goldfish (Carassius aura-tus) from a separate dam on the farm were fed to broodstock codin late 2002. Two types of iridovirus have been reported in thisspecies, albeit not in Australia, and their virions were 180 nm indiameter.1 Bohle virus has not been reported in Victoria. Typicaliridovirus inclusion bodies have been detected in ornamentalfish imported into Victoria (MJL unpublished observations)and other parts of Australia15 but a link with these species wasnot established.

In conclusion, an iridovirus, apparently not EHNV, maycause significant mortality in farmed Murray cod. A more defin-

itive conclusion awaits isolation of the virus and its inoculationinto uninfected Murray cod. The source of this virus was notestablished.

We thank Richard Whittington for the gift of rabbit poly-clonal EHNV antisera, and Andrea Howse for undertaking theimmunoperoxidase testing.

References1. Ahne W, Bremont M, Hedrick RP et al. Iridoviruses associated with epizootichaematopoietic necrosis (EHN) in aquaculture. World J Microbiol Biotechnol1997;13:367-373.2. Nakajima K, Maeno Y, Yokoyama K et al. Antigen analysis of red sea breamiridovirus and a comparison with other fish iridoviruses. Fish Pathol 1998;33:73-78.3. Langdon JS, Humphrey JD. Epizootic haemopoietic necrosis, a new viral dis-ease in redfin perch, Perca fluviatilis L., in Australia. J Fish Dis 1987;10:289-297.4. Whittington RL, Philbey A, Reddacliff GL et al. Epidemiology of epizootichaematopoietic necrosis virus (EHNV) infection in farmed rainbow trout,Oncorhynchus mykiss (Walbaum): findings based on virus isolation, antigencapture ELISA and serology. J Fish Dis 1994;17:205-218. 5. Reddacliff LA, Whittington RJ. Pathology of epizootic haematopoietic necro-sis virus (EHNV) infection in rainbow trout (Oncorhynchus mykiss Walbaum)and redfin perch (Perca fluviatilis L.). J Comp Pathol 1996;115:103-115.6. Essbauer S, Ahne W. The epizootic haematopoietic necrosis virus(Iridoviridae) induces apoptosis in vitro. J Vet Med B Infect Dis Vet Public Health2002;49:25-30.7. Murphy FA, Gibbs EPJ, Horzinek MC et al. Birnaviridae. In: Veterinary virol-ogy. 3rd edn. Academic Press, 1999:405.8. Glazebrook JS, Heasman MP, de Beer SW. Picorna-like viral particles asso-ciated with mass mortalities in larval barramundi, Lates calcarifer (Bloch). J FishDis 1990;13:245-249.9. Pearce M, Humphrey JD, Hyatt AD et al. Lymphocystis disease in captivebarramundi Lates calcarifer. Aust Vet J 1990;67:144-145.10. Speare R, Smith JR. An iridovirus-like agent isolated from the ornate bur-rowing frog Limnodynastes ornatus in northern Australia. Dis Aquat Org1992;14:51-57. 11. Moody NJG, Owens L. Experimental demonstration of the pathogenicity ofa frog virus, Bohle iridovirus, for a fish species, barramundi Lates calcarifer. DisAquat Org 1994;18:95-102.12. Langdon JS. Experimental transmission and pathogenicity of epizootichaematopoietic necrosis virus (EHNV) in redfin perch, Perca fluviatilis L., and 11other teleosts. J Fish Dis 1989;12:295-310. 13. Hengstberger SG, Hyatt AD, Speare RS et al. Comparison of epizootichaematopoietic necrosis virus and Bohle iridoviruses, recently isolatedAustralian iridoviruses. Dis Aquat Org 1993;15:93-107.14. Hedrick RP, McDowell TS, Ahne W et al. Properties of three iridovirus-likeagents associated with systemic infections of fish. Dis Aquat Org1992;13:203–209.15. Anderson IG, Prior HC, Rodwell BJ et al. Iridovirus-like virions in importeddwarf gourami (Colisa lalia) with systemic amoebiasis. Aust Vet J 1993;70:66-7.

(Accepted for publication 18 August 2003)