Susceptibility of Australian native freshwater fish to epizootic haematopoietic necrosis virus
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1 Susceptibility of Australian native freshwater fish to epizootic haematopoietic necrosis virus RW Whittington, JA Becker, MM Dennis, A Tweedie, M Asmus, D Gilligan Faculty of Veterinary Science The University of Sydney
2 Acknowledgments Murray Darling Basin Commission DPI New South Wales Vanessa Carracher, Ian Wooden, Andrew Bruce, Peter Heath, Peter Boyd, Tony Fowler, and others DSE Victoria Jarod Lyon, Pam Clunie University of Sydney G. Dhand, K. Williams (staff) P. Hick, Z. Ritchie and C. Ho (students)
3
4 Epizootic haematopoietic necrosis virus Found only in Australia Notifiable to the OIE Genus: Ranavirus
5 Epizootic haematopoietic necrosis virus Origin Unrecognized reservoir? Trans-species spread from frogs or reptiles? Aquarium fish trade? mass mortalities * juvenile redfin perch * lakes in Victoria
6 mortalities * rainbow trout * a few farms in NSW and ACT
7 South Australia 1992* Darling R. New South Wales 2003* Australian Capital Territory (annually)* Murray Darling! EHNV is very resistant. Basin Natural fish movement Mount Bold R L. Alexandrina 1990 L. Albert 1990 Fishermen Water Km Spread Piscivorous birds Adelaide.!.! Murray R. Victoria 2004* L. Nillahcootie 1984 L. Learmonth 1990.!! Burrinjuck Dam 1990 Blowering Dam 1986 L. Hume 1986.! Melbourne.!.!.!.!.!.!.!.!.! L. Mokoan 1984 & 1985 Ovens R L. Eildon 1986 Goulburn R Winneke Res. 1988! Sydney L. Ginnindera 1994 L. Burley-Griffin 1991 & 1994 Googong Res * last reported outbreak 1968 Redfin perch Perca fluviatilis 1961 Distribution of reported EHNV outbreaks in Redfin
8 Species of the Murray Darling Basin Silver perch Macquarie perch Murray cod Golden perch Mountain galaxias What other species might be susceptible?
9 Overall Goal: Assess the impact of EHNV to native Basin fish Basin Survey Experimental Infection Trials ELISA Development EHN distribution & spread Potential ecological impact Tool for Basin Survey Direct measures to mitigate disease
10 Immunology + Immune response blood samples Positive for antibody ELISA test
11 ELISA development Murray cod pro A :10_UV Murray cod pro A :10_pH Murray cod pro A :10_Inject Murray cod pro A :10_Logbook mau ml FPLC chromatography curve 5 ml Murray cod serum SDS PAGE gel Lane 1 Molecular marker, Lane 2 Murray cod serum (1:20) Lane 3 flow through (1:5) Lane 4 elution fraction
12 ELISA development Affinity purified immunoglobulin stage 1 completed redfin perch silver perch Murray cod Macquarie perch Perca fluviatilis Bidyanus bidyanus Maccullochella peelii peelii Macquaria australasica
13 ELISA field survey Blood samples Lachlan R. Redfin Murray cod Macquarie Perch Murray R. Redfin Murray cod Silver perch Darling R. Murray cod Macquarie R. Murray cod Barwon R. Murray cod Murrumbigee R. Murray cod Results to follow next year ~290 samples
14 Change of driver Michelle Dennis
15 Virology Field Survey Homogenise organs Inoculate living fish cells Cytopathic effect
16 Virology field survey Tissue samples Lachlan R. Redfin Murray R. Redfin Galaxids (3 spp.) Blackfish ACT (Cotter Dam Area) Redfin Galaxids Gambusia Rainbow trout Burrinjuck Dam Redfin Murray cod All negative so far. Murrumbigee R. Blackfish Buffalo R. Redfin ~890 fish tested so far.
17 Experimental Infection Trials ~30 fish ~30 fish EHNV Bath Challenge Non-exposed 10 3 TCID 50 ml -1 Negative Control ~4 fish Challenged Redfin Positive Control Repeated 2 to 4 times, 20 to 22 C Inspected for up to 5 weeks Tested for EHNV: Infection: Virus isolation, PCR Disease: Pathology Important to have matched samples from the same fish. This requires fish that aren t tiny!
18 Redfin perch
19 Liver, Redfin perch
20 Liver, Redfin perch
21 Anterior kidney, Redfin perch Disease is similar in other species!!
22 Results: Carp gudgeons ~30 fish ~50% lived to 35 days None infected None had disease EHNV Bath Challenge 10 3 TCID 50 ml -1 Carp gudgeons are NOT susceptible (Basin-wide native species) ~4 fish None lived to 11 days All infected Some had disease Challenged Redfin Positive Control
23 Results: Redfin Perch ~4 fish EHNV Bath Challenge 10 3 TCID 50 ml -1 From Blowering ½ die with EHNV-associated disease ½ survive, but are infected (with viable virus in organs - VI and PCR +) This is in contrast to studies of the 1990 s This is evidence that: 1. Susceptibility of Murrumbidgee redfin perch has shifted 2. Redfin perch can be carriers of EHNV
24 Silver perch Common Name Mountain galaxias Macquarie perch Murray cod Redfin perch Unspecked hardyhead River blackfish Carp gudgeon Trout cod Scientific Name Bidyanus bidyanus Currently in trial Galaxias olidus Macquaria australasica Maccullochella peelii peelii Perca fluviatilis Craterocephalus stercusmuscarum fulvus Gadopsis marmoratus Hypseleotris sp. Maccullochella macquariensis Murray-Darling rainbow fish Melanotaenia fluviatilis Southern purple spotted gudgeon Southern pygmy perch Bony herring Mogurnda adspersa Nannoperca australis Nematalosa erebi In trial Resistant
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