Susceptibility of previously untested basin fish species to Epizootic Haematopoietic Necrosis Virus (EHNV) and its epidemiology in the wild

Transcription

1 18 th November 2011 FINAL REPORT Susceptibility of previously untested basin fish species to Epizootic Haematopoietic Necrosis Virus (EHNV) and its epidemiology in the wild Project No. MD th November 2011 Richard Whittington 1, Joy Becker 1, Alison Tweedie 1, Dean Gilligan 2 and Martin Asmus 3 1 Faculty of Veterinary Science, The University of Sydney 2 NSW Industry and Investment, Batemans Bay Fisheries Office 3 NSW Industry and Investment, Narrandera Fisheries Centre

2 1. Foreword Epizootic haematopoietic necrosis virus (EHNV) is a viral pathogen of international concern. The disease it causes is known from Redfin perch and farmed Rainbow trout in some parts of the upper Murrumbidgee catchment and the lower Murray catchment as well as from some parts of Victoria. Outbreaks have been recorded since It was shown in experiments more than 20 years ago that EHNV can kill native freshwater fish species of high conservation value including some that live in the Murray-Darling Basin. Despite its significance, very little is known about the natural distribution of EHNV in Australia, or its real impact on native fish, as no formal surveys have ever been conducted. The objectives of this project were to identify the extent to which EHNV is a risk to native fish in the Murray-Darling Basin and to provide scientific knowledge to aid in the development of effective natural resource management policy. The specific aims were to (1) to validate earlier findings of susceptibility of native fish to EHNV, (2) to determine the susceptibility to infection by EHNV of a range of previously untested fish species found in the Basin, (3) to investigate the epidemiology of EHNV in wild populations of priority fish species and (4) to develop a test to determine exposure of wild populations of priority fish species to EHNV. A preliminary final report was provided on 4 th June MDBA provided additional funding in 2011 to extend the survey to enable a more complete analysis. This updated report contains results for an additional 69 blood samples [for a total of 492] tested by ELISA and an additional 796 fish tissues [for a total of 3622] tested by virus isolation, together with interpretation of these new results 2. List of abbreviations BF-2 bluegill fry cells EHN epizootic haematopoietic necrosis, a disease EHNV epizootic haematopoietic necrosis virus ELISA enzyme-linked immunosorbent assay FHM fat-head minnow cells g force due to gravity h hour IP immunoperoxidase IU international units kb kilobase pair of nucleic acid nm nanometer OD optical density PCR polymerase chain reaction S:N signal to noise ratio SDS-PAGE sodium-dodecyl sulphate polyacrylamide gel electrophoresis TCID 50 50% tissue culture infective dose 2

3 3. Glossary Antibody EHN EHNV ELISA Epidemiology Histopathology Immunoglobulin Pathogen Population Prevalence Serum A protein produced by the immune system after infection. Antibodies circulate in the blood and bind to invading pathogens, leading to their destruction. The disease of fish that is caused by EHNV. A virus in the Family Iridoviridae, genus Ranavirus. Iridoviruses are very large double stranded DNA viruses known from amphibians, fish, reptiles and insects. Enzyme-linked immunosorbent assay, a type of blood test that is used to detect antibodies. A scientific discipline that describes diseases in populations, including who is infected, when are they infected and where are they infected. The discipline uses mathematical principles to conduct surveys for disease, analyse the results of surveys and make recommendations to control diseases. A diagnostic method that requires a samples of tissue which is cut into very thin slices. It is examined by a trained pathologist using a microscope for evidence of damage to cells and other changes indicative of a pathogen. A class of protein in the blood. Equivalent to antibody. An infectious disease causing agent such as a virus, bacterium or parasite. A group of animals defined by specific criteria such as species and geographical origin. The proportion of animals in a sample that are infected with a pathogen at a point in time. The watery component of blood that remains after clotting and removal of blood cells 3

4 4. Executive summary Description of the project Epizootic haematopoietic necrosis virus (EHNV) is a viral pathogen of international concern. The disease it causes is known from Redfin perch and farmed Rainbow trout in some parts of the upper Murrumbidgee catchment and the lower Murray catchment as well as from some parts of Victoria (Whittington et al., 2010). Outbreaks have been recorded since These have often involved tens of thousands of juvenile Redfin perch. In some outbreaks adult Redfin perch were also affected which severely impacted the recreational fishery. EHNV infects fish through the skin, gills or mouth. It then starts to multiply in the blood forming organs and destroys them the spleen and anterior kidney disintegrate first. The liver and some other tissues also become necrotic. Most infected fish quickly succumb and die. It was shown in experiments more than 20 years ago that EHNV can kill native freshwater fish species of high conservation value including some that live in the Murray-Darling Basin: Macquarie perch (Macquaria australasica), Murray cod (Maccullochella peelii peelii) and Silver perch (Bidyanus bidyanus). Despite its significance, very little is known about the natural distribution of EHNV in Australia, or its real impact on native fish, as no formal surveys have ever been conducted. The objectives of this project were to identify the extent to which EHNV is a risk to native fish in the Murray-Darling Basin and to provide scientific knowledge to aid in the development of effective natural resource management policy. The specific aims were to (1) to validate earlier findings of susceptibility of native fish to EHNV, (2) to determine the susceptibility to infection by EHNV of a range of previously untested fish species found in the Basin, (3) to investigate the epidemiology of EHNV in wild populations of priority fish species and (4) to develop a test to determine exposure of wild populations of priority fish species to EHNV. Key results Based on testing 3622 tissues and 492 blood samples from fish it was concluded that EHNV is still endemic in the upper Murrumbidgee River catchment in the Murray-Darling Basin. No sampling was undertaken in some locations where EHNV has occurred in Redfin perch historically; examples include the lower Murray River in SA, and Lakes Mokoan and Nillahcootie (Broken River catchment) in VIC. During the study an outbreak of disease due to EHNV occurred in juvenile Redfin perch in Blowering Dam NSW (December 2009), a dead Redfin perch infected with EHNV was detected in Lake Ginninderra ACT (December 2008) and there was an outbreak at this location in December 2010, consistent with prior observations of outbreaks in both locations (Whittington et al., 1996). However, not all parts of the upper Murrumbidgee catchment (for example Cotter Reservoir) are infected. Although sampling was not as intense or widespread as initially hoped, EHNV appeared to be absent from Redfin perch populations elsewhere in the Murray-Darling Basin as there were no reports of disease outbreaks and neither virus nor antibodies against the virus were detected. Because EHNV is highly infectious in Redfin perch, and because the survey was capable of finding infection if as few as 1 in 10 fish were affected, the virus is unlikely to be present there. 4

5 EHNV appeared to be absent from other species of fish in the Murray-Darling Basin during the study period, although as for Redfin perch sampling was not as intense or widespread as hoped. Sufficient data were obtained from some regions of the Murray-Darling Basin to be 95% confident that EHNV was present in <10% of the population of the following species: River blackfish, brown trout, Mountain galaxias, Eastern mosquitofish, Murray cod, Silver perch, Southern pygmy perch, Rainbow trout and Redfin perch, and in some cases sample sizes were sufficient to conclude that <5%, <2% or <1% of the population. It is possible that the virus has not entered the middle and lower regions of the southern part of the Basin, nor the western and northern parts of the Basin. The findings of Langdon (1989) concerning the susceptibility of Silver perch, Macquarie perch and Eastern mosquitofish to EHNV after exposure via water were confirmed. In addition, two new susceptible species were identified: Murray-Darling rainbowfish and Freshwater catfish. Species which became infected with EHNV following exposure via water, and in which some individuals survived and appeared to carry the live virus were Silver perch, Eastern mosquitofish, and Redfin perch. Species with resistance to EHNV following exposure via water were Murray cod, Golden perch, Un-specked hardyhead, Carp gudgeon, Southern purple-spotted gudgeon, Trout cod and Southern pygmy perch. Redfin perch generally were highly susceptible to EHNV, but there appeared to be differences in susceptibility between populations. Fish from Blowering Dam in the known endemic region appeared to have a greater degree of resistance than others. The combined results of Langdon (1989) and this study suggest that susceptibility of fish species does not appear to correlate with taxonomic relationships, and therefore, there is no basis upon which to predict susceptibility of untested species based on taxonomic relatedness. The experiments to determine the susceptibility of fish to EHNV were conducted under a limited set of controlled conditions. Evidence from bath challenge experiments, even where only a low proportion of fish in a treatment group developed infection, should be accepted as evidence of susceptibility of the species to EHNV. Species found to be resistant may be susceptible under a different set of experimental conditions. It follows that the list of susceptible species is almost certainly much greater than currently known. A new blood test was developed during this project to detect antibody resulting from immune responses in fish against EHNV. A specific test was developed for Silver perch, Murray cod, Macquarie perch and Redfin perch. The tests were shown to be effective in controlled experiments and several samples collected during this study as well as some collected about 20 years ago from Redfin perch from the known EHNV endemic area in Victoria were positive, as were many samples collected from Redfin perch that survived experimental infection with EHNV. One Redfin perch that was positive in the blood test was carrying the live virus in its organs. Together with other results from the experimental challenge trials, we now know that Redfin perch can become infected and then either die, carry the virus for long periods (many weeks) or recover completely. These results indicate that the blood test has application in field surveys for EHNV. The blood test is very versatile and opens up a new range of options to study the health of fish in the Murray-Darling Basin. It has application in a much wider range of species, and it can be adapted to detect antibodies against many other 5

6 pathogens. An advantage of the blood test is that is can reveal past exposure of a population to EHNV. The sensitivity of the test is not yet known, and further research will be needed to determine this. Outcomes and recommendations The findings of this study reinforce a view that EHNV is a factor detrimental to native fish populations in the Murray-Darling Basin. Policies to reduce the risk of exposure of fish in the Murray-Darling Basin to EHNV are justifiable. The upper Murrumbidgee catchment is an endemically infected region based on recurrent outbreaks of EHNV in Redfin perch and Rainbow trout; the infection may also still occur in places where it once occurred that were not included in the survey (for example lower Murray, SA and Lakes Nillahcootie and Mokoan, VIC). Not all parts of the upper Murrumbidgee catchment (for example Cotter Reservoir) appear to be infected. Other parts of the Basin are apparently uninfected. It is possible that the virus has not entered the middle and lower regions of the southern part of the Basin, nor the western and northern parts of the Basin. It is logical to attempt to prevent or reduce the likelihood of spread from the endemic area to the apparent EHNV-free area. Given the lengthy history of EHNV in the endemic region it would appear that long distance spread of EHNV downstream via water flow in rivers is not important. There was evidence of other means of spread including anthropogenic factors. For these reasons policies should be developed to address the potential movement of EHNV through activities of humans. A multijurisdictional agreement is needed to cover NSW, ACT, VIC, SA and the entire Murray-Darling Basin. To improve current awareness about the occurrence of disease outbreaks to enable disease alerts, routine annual surveillance for EHNV in Redfin perch in the endemic area should be considered. While policy is developed, consideration should be given to an education program for the general public concerning biosecurity in rivers and impoundments. Educational materials should include information about risks of translocating live fish and bait, and disinfection of boats and equipment after use in the endemic area if they are to be used elsewhere. Recommendations are made for further research on the epidemiology of EHNV at ecosystem level, sociological research to underpin biosecurity in conservation policy, and studies on the potential impact of other iridoviruses on native fish species in the Basin. Keywords Epizootic haematopoietic necrosis virus; EHNV; iridovirus; ranavirus; antibody; ELISA; serology; survey; virus isolation; prevalence; susceptibility; natural infection; experimental infection; Australian native fish; biosecurity; fish conservation 6

7 5. Table of contents 1. Foreword 2 2. List of abbreviations Glossary 3 4. Executive summary Table of contents List of figures List of tables Introduction Project objectives Identifying native fish that are susceptible to EHNV Methods Findings Field survey to detect EHNV Methods Findings Development of a blood test for EHNV Methods Findings Discussion and conclusions Recommendations Addressing knowledge gaps Project/consultancy briefs Acknowledgements Reference list

8 6. List of figures Figure 8.1. Experimentally infected Redfin perch showing multiple white spots in the liver Figure 8.2. Figure 8.3. The known distribution of EHN in Australia and the localized distribution of documented outbreaks occurring at Rainbow trout farms on various rivers (R.) in south-eastern Australia Geographic distribution of reported EHN outbreaks occurring in wild Redfin perch in lakes (L.), reservoirs (Res.) and rivers (R.) in south-eastern Australia Figure 8.4. Catchments of the Murray-Darling Basin Figure Figure Figure Figure Decision tree to define the status of fish following an experimental bath challenge with EHNV Hepatic necrosis (upper panel) and splenic necrosis (lower panel) in experimentally infected Redfin perch Immunoperoxidase stain of liver of an experimentally infected Redfin perch. The extensive brown stained areas consist of degenerating cells which are laden with EHNV particles that take up the stain Distribution of all fish tissue and serum samples collected from July 2007 to June 2011 for EHNV testing Figure Distribution of Redfin perch tissue and serum samples collected from July 2007 to June 2011 for EHNV testing Figure Moribund and dead Redfin perch collected from Lake Ginnindera, ACT in December 2008 and 2010 and Blowering Dam, NSW in December 2009 were infected with EHNV Figure Distribution of Murray cod tissue and serum samples collected from July 2007 to June 2011 for EHNV testing Figure Distribution of Silver perch tissue and serum samples collected from July 2007 to June 2011 for EHNV testing Figure Figure Distribution of Macquarie perch tissue and serum samples collected from July 2007 to June 2011for EHNV testing Distribution of tissue samples collected from non-target native fish species from July 2007 to June 2011 for EHNV testing Figure Distribution of tissue samples collected from introduced (alien) fish species from July 2007 to June 2011 for EHNV testing Figure Distribution of serum samples collected from both native non-target fish species from July 2007 to June 2011 for EHNV testing. The locations for golden perch and trout cod were coincident Figure The principle of the ELISA blood test for EHNV Figure Format of the ELISA to detect antibodies against EHNV in fish serum Figure Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified immunoglobulin Figure Antibody levels in fish that were immunized with EHNV antigen on day 0 and day 27 or Figure Antibody level and S/N ratio for 142 sera collected from wild Redfin perch Figure Antibody level and S/N ratio for 55 sera collected from wild Silver perch

9 Figure Antibody level and S/N ratio for 76 sera collected from wild Macquarie perch Figure Antibody level and S/N ratio for 213 sera collected from wild Murray cod Figure Figure Antibody level and S/N ratio for 54 sera collected from Redfin perch in experimental infection trials Decision tree to define the status of fish surviving contact with EHNV based on the detection of antibodies

10 7. List of tables Table 8.1 Summary of host susceptibility to an experimental challenge with EHNV Table 8.2. Fish species present in the Murray-Darling Basin that were utilised during various components of the project Table Origin, size and current knowledge of EHNV susceptibility for species used in the challenge experiments Table Overview of the challenge trials to determine the susceptibility of fish to EHNV Table The susceptibility of fish from the Murray-Darling Basin to EHNV following experimental challenge Table The susceptibility of Redfin perch to EHNV following experimental challenge Table Summary of host susceptibility EHNV following experimental bath challenge Table Annual schedule of catchments to be sampled for the Sustainable Rivers Audit program Table Summary of results of virus isolation tests for EHNV Table Table Summary of fish serum collected for EHNV testing based on year and catchment area from 2007 to Summary of fish tissue collected for EHNV testing based on species, year and catchment from 2007 to Table Estimated true prevalence of EHNV in various species of finfish Table Source of fish used to obtain blood for purification of immunoglobulins Table Concentration and total yield of purified immunoglobulin from each species of fish Table Table Table Table Estimated molecular weight (MW) of purified immunoglobulin based on observed molecular weights of the heavy chain (HC) and light chain (LC) components and assuming a tetrameric structure Optimal reaction conditions determined by checkerboard titration for Redfin perch, Silver perch and Macquarie perch and those used empirically for Murray cod Number of samples from wild fish of unknown exposure history and wild fish held in captivity but not exposed experimentally to EHNV (in parentheses) Summary of serum collected from Redfin perch that were alive on day 28 following experimental bath challenge with EHNV in two separate experiments

11 8. Introduction In 1985 a new disease of fish occurred in south-eastern Australia. It was named epizootic haematopoietic necrosis (EHN), meaning destruction of blood forming organs, and was due to a virus. The EHN virus (EHNV) was the first iridovirus to cause outbreaks of mortality in finfish (Langdon et al., 1986). EHNV initially caused severe disease outbreaks in Redfin perch Perca fluviatilis but soon after it was found to cause a mild disease in farmed Rainbow trout Oncorhynchus mykiss in the same area (Langdon and Humphrey, 1987; Langdon et al., 1988; Langdon et al., 1986). Because it can cause a severe disease with no cure possible, its occurrence is notifiable to the World Organisation for Animal Health in Paris under an international agreement to minimize the spread of disease. Strategies for detection, prevention and control are being developed in many countries. EHNV causes an infection of the blood forming organs and destroys them the spleen and anterior kidney disintegrate first. The liver and some other tissues also become necrotic, especially in fish that survive for a few days. However, most infected fish are thought to quickly succumb and die. Many infected Redfin perch develop white spots in the liver which are characteristic of necrosis, and bloody fluid can often be seen in the abdominal cavity (Figure 8.1). Figure 8.1. Experimentally infected Redfin perch showing multiple white spots in the liver. Each spot is an area of dead tissue, where cells have become infected with and killed by EHNV. By 1988 several Australian native fish species including Mountain galaxias (Galaxias olidus), Murray cod (Maccullochella peelii peelii) and Macquarie perch (Macquaria australasica) were shown to be susceptible experimentally to EHNV (Langdon, 1989) (Table 8.1). This led to speculation that EHNV may have contributed to the decline of native fish species such as the Macquarie perch and Mountain galaxias (Langdon, 1989; Lintermans, 2007). 11

12 EHNV is known only from Australia (Figure 8.2), but similar viruses have been detected in other countries, usually associated with severe diseases in fish or amphibians. The various viruses can be distinguished with DNA tests (Marsh et al., 2002). These viruses are all grouped in the genus Ranavirus. They are relatively large DNA viruses, about 150 nm in diameter, have a genome of about 105 kb and replicate in the nucleus and cytoplasm of affected cells (Williams et al., 2005). EHNV is extremely resistant and can survive for months or years in water or frozen fish (Langdon, 1989; Whittington et al., 1996). It may persist in sediments and on equipment for lengthy periods. Transmission of EHNV between susceptible fish in the wild probably occurs via water or ingestion of tissues from infected fish. Spread in aquaculture occurs via trade of trout fingerlings (Langdon et al., 1988; Whittington et al., 1994; Whittington et al., 1999). In Rainbow trout the disease can remain hidden with batches of fish visibly unaffected and therefore is easily spread. EHNV in Redfin perch spread upstream in the upper Murrumbidgee River system through New South Wales and the Australian Capital Territory after 1989 (Whittington et al., 1996). Similar spread was seen in the lower Murray River lakes and reservoirs in South Australia (Whittington et al., 1996) (Figure 8.3). The occurrence of EHNV in Redfin perch in distant parts of river systems and in separated dams, and the upstream spread, indicates that the movement of the virus must have been assisted. The virus could have been spread on fishing nets, boats and other equipment. Anecdotally, illegal movement of live redfin perch and frozen redfin for bait between river systems and impoundments by irresponsible recreational fishermen are activities that might readily result in the spread of the virus between river systems and impoundments. Birds may also be potential vectors as Silver gulls Larus novaehollandiae and great cormorants Phalacrocorax carbo feed on affected Redfin, fly away and can regurgitate infected fish hours later, elsewhere in the environment (Whittington et al., 1996). The potential for spread of EHNV in faeces of these birds requires further assessment. 12

13 Figure 8.2. The known distribution of EHN in Australia and the localized distribution of documented outbreaks occurring at Rainbow trout farms on various rivers (R.) in south-eastern Australia (Langdon et al. 1988, Whittington et al. 1996). Figure 8.3. Geographic distribution of reported EHN outbreaks occurring in wild Redfin perch in lakes (L.), reservoirs (Res.) and rivers (R.) in south-eastern Australia (Langdon and Humphrey 1987, Whittington et al. 1996, and unpublished data). * indicates most recent cases in each state and territory. 13

14 Despite its significance, very little is known about the natural distribution of EHNV in Australia, or its real impact on native fish, as no formal surveys have ever been conducted. Although the susceptibility of native finfish species to EHNV has been suspected since the work of Langdon in 1989, no work has been conducted since. This is despite fisheries biologists raising the issue with relevant State and Federal authorities for over 20 years. However, impacts of disease are very difficult, time consuming and costly to measure in natural aquatic systems, which has deterred research investment. The dynamics of hostpathogen relationships and the speed with which microbial pathogens evolve means that the findings of Langdon need to be verified, and additional species of fish examined. Furthermore, there is need to re-examine the susceptibility of Redfin perch to EHNV. This once highly susceptible species may have evolved resistance through natural selection over 25 years, and instead of being a canary in the mine may now be a Trojan horse, itself able to carry and spread the disease. The distribution of Redfin perch is still expanding (Lintermans, 2004) so there is potential for the distribution of EHNV to also expand. Finally, tests for EHNV generally require destruction of fish, and this is not desirable for threatened species. Therefore there is strong justification for development of less invasive diagnostic tests, specifically a test based on blood, as blood can be collected without any long-term harm to fish. The fish species present in the Murray-Darling Basin that were selected for inclusion in each component of this research program are shown in Table 8.2. Throughout this report the various catchments of the Murray-Darling Basin will be referred to. These are illustrated in Figure 8.4. Figure 8.4. Catchments of the Murray-Darling Basin 14

15 Table 8.1. Summary of host susceptibility to an experimental challenge with EHNV by immersion (bath challenge) or intraperitoneal (IP) injection and conservation status of fish species within Australia (adapted from Whittington et al 2010). The numbers in parentheses are the total number of fish tested. * indicates an alien species. Species Common name Percentage (%) of EHNV-associated Natural Infection mortality following exposure 1 Reported Immersion IP injection Conservation status in EHNV endemic region 2,3 Perca fluviatilis* Redfin perch Yes >95 >95 alien species Oncorhynchus mykiss* Rainbow trout Yes <4 <4 alien species Gambusia holbrooki Eastern No 90 (10) unknown alien species (affinis)* mosquitofish Macquaria australasica Macquarie perch No 100 (20) 100 (5) endangered native species (NSW, ACT, VIC, SA, AUS) Bidyanus bidyanus Silver perch No 28 (36) 100 (5) vulnerable (NSW, SA), endangered (ACT), critically endangered (VIC) native species Galaxias olidus Mountain No 100 (10) unknown threatened (VIC), rare (SA) native species galaxias Maccullochella peelii peelii Murray cod No 0 (4) 100 (4) 50% carrier vulnerable (AUS, endangered (VIC), rare (SA) native species Macquaria ambigua Golden perch No 0 (30) 100 (12) vulnerable (VIC) native species ambigua Macquaria Australian bass No 0 (10) 20 (5) native species novemaculeata Retropinna semoni* Australian smelt No 0 (12) unknown widespread native species Lates calcarifer Barramundi No 0 (20) 0 (6) widespread native species Carassius auratus* Goldfish No 0 (8) 0 (3) widespread alien species Salmo salar* Atlantic salmon No unknown 0 (15) 30% carrier alien species with little natural recruitment 1 adapted from (Langdon, 1989) 2 see Figure 8.1 for EHNV endemic region 3 based on federal and/or state legislation: National status under the Environment Protection and Biodiversity Conservation Act 1999 (AUS), New South Wales Fisheries Management Act 1994, Victorian Flora and Fauna Guarantee Act 1988, updated 2007 (VIC), Australian Capital Territory Nature Conservation Act 1980, updated 2007 (ACT), South Australian National Parks and Wildlife Act 1972, updated

16 Table 8.2. Fish species present in the Murray-Darling Basin that were utilised during various components of the project. * indicates an alien species. Scientific name Common name Experimental infections Not previously tested Previously tested 1 Field survey Develop blood test Bidyanus bidyanus Silver perch Yes Yes Yes Craterocephalus stercusmuscarum fulvus Un-specked hardyhead Yes Cyprinus carpio* Common carp Yes Gadopsis marmoratus River blackfish Yes Galaxias brevipinnis Climbing galaxias Yes Galaxias olidus Mountain galaxias Yes Gambusia holbrooki* Eastern mosquitofish Yes Yes Hypseleotris spp Carp-gudgeons (lumped) Yes Yes Macquaria ambigua ambigua Golden perch Yes Yes Macquaria australasica Macquarie perch Yes Yes Yes Maccullochella macquariensis Trout cod Yes Yes Maccullochella peelii peelii Murray cod Yes Yes Yes Melanotaenia fluviatilis Murray-Darling rainbowfish Yes Misgurnus anguillicaudatus* Oriental weatherloach Yes Mogurnda adspersa Southern purple-spotted gudgeon Yes Nannoperca australis Southern pygmy perch Yes Yes Oncorhynchus mykiss* Rainbow trout Yes Perca fluviatilis* Redfin perch Yes Yes Yes Philypnodon grandiceps Flathead gudgeon Yes Rutilis rutilis* Roach Yes Salmo trutta* Brown trout Yes Tandanus tandanus Freshwater catfish Yes 1 (Langdon, 1989) 16

17 9. Project objectives The objectives of this project were to identify the extent to which EHNV is a risk to native fish in the Murray-Darling Basin and to provide scientific knowledge to aid in the development of effective natural resource management policy. The specific aims were to (1) to validate earlier findings of susceptibility of native fish to EHNV, (2) to determine the susceptibility to infection by EHNV of a range of previously untested fish species found in the Basin, (3) to investigate the epidemiology of EHNV in wild populations of priority fish species and (4) to develop a test to determine exposure of wild populations of priority fish species to EHNV. The project consisted of three main components, each with defined methods: identifying native fish that are susceptible to EHNV; field survey to detect EHNV; development of a blood test for EHNV. For this reason the scientific work in the three components will be presented separately in the sections that follow, and then integrated later in the report. 10. Identifying native fish that are susceptible to EHNV A standard approach to determine whether a particular species of fish is susceptible to a particular pathogen is to challenge the fish with a pure strain of a virulent isolate of the pathogen under controlled experimental conditions. This is known as an experimental model, challenge model or infection model. The experimental model is only an approximation of the situation in nature because of the number of variables: the strain or isolate of the pathogen that is chosen; the genotype, age, sex, size and physiological state of the fish, and; the particular environmental conditions including factors such as water temperature. In nature fish are often infected with a range of pathogens and parasites in addition to the pathogen of interest, and interactions are highly relevant. For example, skin parasites may expose blood vessels in the dermis which then allows entry of microbial pathogens. High stocking rates or predators may lead to skin damage with the same result. The experimental model is, however, a vital tool in investigation of fish diseases, and it is useful to review the principles which underlie it in more detail. These can be discussed in terms of the host, the pathogen and the environment. Host, Pathogen and Environment Relationship Diseases are an integral part of the existence of all animals including both cultured and wild fish populations (Hedrick, 1998). Generally, our knowledge of the diseases that affect cultured fish is greater than that of wild populations for logistical and historical reasons. The artificial rearing of fish has led to the exacerbation of certain diseases that previously existed in wild populations (Reno, 1998). Nevertheless, the impact of a disease outbreak regardless of whether it occurs in wild or captive fish is dependent on the interactions of variables defined for the host, the pathogen and the environment (Hedrick, 1998). This relationship is often depicted as three interlocking circles with disease occurring at the intersection, and is studied in the disciplines of epidemiology and pathobiology. Making sense of the complex interactions within and among the domains of host, pathogen and environment requires an 17

18 understanding of many variables, some of which may not be known. An understanding of the epidemiology and pathogenesis of infectious agents through investigating disease events may result in improved management strategies. Experimental infection models Disease is a process characterized by any impairment of normal physiological function affecting all or part of an organism, including responses to environmental factors such as infectious agents, climate, toxicants, congenital defects and nutrition (Hedrick, 1998). Scientific research investigating fish disease, in particular for those pathogens infecting cultured fish species, often begins with the development of efficient experimental challenge models. Generally, this is followed by successive infection trials aimed at identifying basic disease parameters including water temperature, pathogen strain and host immune response, susceptible host species, resistance to re-infection and dose effects. The main types of challenge models used in fish research include injection of the pathogen into the muscle or peritoneal cavity, feeding of infective material (per os) and waterborne exposure (direct and indirect bath or immersion). Each challenge model has its own advantages and disadvantages. For example, an intraperitoneal injection model allows for a measured amount of pathogen to be introduced into the body at a specific point in time but it bypasses the specific and nonspecific immune mechanisms on the body surface, and does not represent a natural route of exposure. Conversely, waterborne exposure through cohabitation with infectious individuals or via immersion in infectious water reflects a more natural pathway of infection, however one cannot be sure how many infectious organisms are taken up by a specific individual, the route by which the agent enters the body, and at what point in time. Transmission factors associated with the host Transmission factors that fall under the umbrella of host factors are considered constantly present, such as species, genotype, fish size and age, developmental and reproductive stage, population size or nutritional status (Hedrick 1998). Adaptive factors which result from a prior exposure to a pathogen, such as acquisition of immunity can affect a host s susceptibility to a particular infectious agent (Hedrick, 1998). The literature is replete with examples of variation in resistance of different fish stocks to infectious diseases. For example, it was recently demonstrated that resistance to bacterial coldwater disease caused by Flavobacterium psychrophilum in Rainbow trout could be increased by nearly 45% after two generations of family-based genetic selection (Leeds et al., 2010). Another example is the intracellular gill parasite Loma salmonae, which infects all members of the genus Oncorhynchus (the Pacific salmon). Chinook salmon and coho salmon are considered to be more susceptible to this protozoan as they have greater parasite burdens and often succumb to infection compared to Rainbow trout, which exhibit subclinical disease and significantly lower parasite burdens (Becker and Speare, 2007). Using experimental models, two other related salmonids, Atlantic salmon (Salmo salar) and arctic charr (Salvelinus alpinus) were resistant to infection with L. salmonae (Speare et al., 1998). Differential host susceptibility has been elegantly demonstrated in the two natural hosts of EHNV, Redfin perch and Rainbow trout. Redfin perch tend to have explosive disease events with high levels of mortality affecting many age classes with very few carriers among survivors. Conversely, Rainbow trout tend to exhibit subclinical disease with few individuals showing any signs of disease, and latent infections can persist in the population (Reddacliff and Whittington, 1996). An intra-muscular injection challenge model for betanodavirus revealed that many freshwater species were susceptible to 18

19 this internationally important pathogen, which was initially thought to infect only marine species (Furusawa et al., 2007). Four fish species were defined as being susceptible to betanodavirus based on clinical signs of mortality, erratic swimming in some individuals, and re-isolation of the virus from tissues. Another 10 species of freshwater fish were classified as less susceptible as they demonstrated no betanodavirus-associated mortality and no clinical signs of disease (i.e. no erratic swimming), however live virus was re-isolated from at least one individual from each species (Furusawa et al., 2007). Resistant species were those that showed no mortality or clinical signs and from which virus could not be isolated from any individual following injection of pathogen. Finally, infectious hematopoietic necrosis virus (IHNV) causes epizootics in populations of wild and cultured salmonids around the world (Lapatra et al., 1990). Marked differences in host species susceptibility to this virus have been observed. For example, sockeye salmon (O. nerka) exposed to a particular isolate of IHNV can exhibit 95% mortality compared to only 5% in Chinook salmon (O. tshawytscha) (Lapatra et al., 1990). Observations from natural and experimental infections with IHNV revealed that younger salmonids (up to 2 months of age) were most susceptible with acute clinical infections associated with high levels of mortality; adult fish can become infected, although this does not usually result in clinical disease (Lapatra et al., 1990). Transmission factors associated with the virus Transmission factors associated with the pathogen generally include the infective dose or the number of pathogens available, how they are delivered to the host and duration of exposure, which directly influence the severity of the resulting infection (Hedrick, 1998). The virulence or the ability of pathogens to cause disease depends on the strain, biotype, serotype, or genotype of the agent (Engelking et al., 1991). Different sources (or isolates) of specific infectious agents can lead to varying disease outcomes. This is particularly true for viral and bacterial pathogens affecting wild and farmed fish. For example, Rainbow trout exposed to three strains of IHNV demonstrated a high level of susceptibility to one strain (62% mortality) compared to a lower susceptibility for the other strains (5% mortality) (Lapatra et al., 1990). Transmission factors associated with the environment The environment is perhaps the least defined element of the host, pathogen and environment triad, especially when considering wild fish populations (Hedrick, 1998). In laboratory models, the critical environmental parameters, including temperature and flow rate, can be closely controlled and monitored (Hedrick, 1998). Water temperature is considered to be an important environmental variable in the transmission of many fish pathogens because it can act directly on the development of the pathogen, on the immune system of the fish or both (Antonio and Hedrick, 1995). Temperature is often the first factor investigated after a standardized challenge model has been developed. Redfin perch were not susceptible to EHNV when challenged at water temperature below 12 C, but were fully susceptible at higher temperatures (Whittington and Reddacliff, 1995). Typically, an increase in water temperature leads to a reduction in the number of days until disease onset, increased disease prevalence and intensity of disease for many key pathogens, including the bacterium, Aeromonas salmonicida, the causative agent for furunculosis (Nordmo and Ramstad, 1999) and the protozoan L. salmonae, the causative agent for microsporidial gill disease (Beaman et al., 1999; Becker and Speare, 2004). This was the kind of pattern observed for EHNV (Whittington and Reddacliff, 1995). 19

20 These examples show that it is not possible to cover all possible scenarios in a single experimental infection model. Even if only a low proportion of fish in a treatment group develop infection after bath exposure, this is sufficient evidence that the species is susceptible. Furthermore, species found to be resistant in bath challenge experiments, may be found to be susceptible under a different set of experimental conditions. Caution should be used when comparing disease outcomes from experimental models using a differing set of experimental conditions. Development of experimental models for EHNV One of the fundamental aspects of optimal experimental models is that they are well defined, both in terms of experimental parameters and expected levels of infection and disease during a predictable time course in a susceptible host. Typically an initial screening study is completed to test a range of potential host species with a low number of replicates using a few individuals tested from each host species. For example, to test a range of freshwater species for betanodavirus susceptibility, one tank per species was used with a range of two to six individuals in total tested from each (Furusawa et al., 2007). The first EHNV experimental model was devised by Langdon and published in 1989; it appeared to be an initial screening for host species susceptibility and early development of a challenge model. Langdon used few replicates and 4 to 36 (mean 16) individuals per species. Little detail was provided about fish husbandry, other than that the fish were housed in 40 L aquaria and held at water temperatures of 18 to 24 C. No information was provided regarding the source populations, the length of holding prior to experimentation, feeding, other water quality parameters or water changes. In this model, fish were exposed to EHNV for 1 hour at TCID 50 /ml and then added to a 40 L aquarium, presumably with the virus-bath water as the diluted titre was calculated to be TCID 50 /ml (Langdon, 1989). The observation period was restricted to 21 days, or approximately 7 days after the last Redfin perch died. This experimental approach was modified in later studies, by adding virus directly to the aquarium containing the fish; it was serviced by a canister-type biofilter containing a defined substrate. The new model was used to show that temperature can greatly influence the development of EHNV infection in Redfin perch and Rainbow trout, with individual Redfin perch succumbing to infection as late as 28 days post exposure when held at lower temperatures (Reddacliff and Whittington, 1996; Whittington and Reddacliff, 1995). The experimental infection model used in the present study of Basin species was based on the same approach and it used the same strain of EHNV. It differed from the model of Langdon (1989) in husbandry of fish, water temperature, dose of virus, duration of exposure to virus, length of trial and probably also in many other ways that cannot be identified. It is important to note that in previous trials Rainbow trout were highly resistant to EHNV when exposed by the natural route in water, and yet this species is an efficient host and carrier of the virus, capable of spreading it between catchments when fish are traded (Whittington et al., 1999). The intraperitoneal route of infection was required to produce disease experimentally in Rainbow trout (Reddacliff and Whittington, 1996; Whittington and Reddacliff, 1995). This knowledge informed the assessment of the outcomes of infection trials in the present study. 20

21 In summary, the experiments to determine the susceptibility of fish to EHNV were conducted under a limited set of controlled conditions. It was not possible to test a wide range of species using other than under one water temperature and light regime, standard water quality, an intended absence of other diseases and parasites, simple nutrition and fish of a specified size range regardless of age. It is well recognised that susceptibility to disease is not a fixed parameter and is influenced by all of these factors. For EHNV, water temperature, poor water quality and skin lesions due to parasites have been associated with outbreaks of the disease (Whittington et al., 1994; Whittington and Reddacliff, 1995; Whittington et al., 1999). These factors were not included in the experimental model. It is important to use a dose of virus that is not likely to circumvent natural host defences or an unrealistic assessment of host susceptibility might be the result. The experiments conducted in this project most often involved a low to moderate dose of virus administered to the water to encourage a natural route of entry of the virus to the body. However, in some experiments an intraperitoneal injection was also used Methods Source of Fish Candidate fish species identified for inclusion in the laboratory challenges were selected based on their conservation status, current knowledge of EHNV susceptibility, distribution across the Basin, juvenile size ranges and likelihood of acclimatization to artificial feeds and holding tanks, and to maximise the representation of taxonomic families present in the Murray-Darling Basin. The ideal size range was specified to be between 60 to 110 mm (total length) to enable matched tissue samples for testing in both virus isolation and histology. The age of fish at this size will vary substantially among species. The source and description of the fish used in each trial is listed in Table There was no history of EHN from hatcheries from which fish were sourced. Virus isolation was completed on at least three apparently healthy individuals from each consignment of fish; additionally, any mortalities from transportation or during the holding period prior to experimentation were examined for the presence of EHNV. The virus was not detected in these samples. Fish husbandry Individuals of each species to be tested for EHNV susceptibility were acquired either as hatchery reared fingerlings or from wild caught stock. Murray cod, Golden perch, Silver perch and Southern purple-spotted gudgeon were sourced from Narrandera Fisheries Centre (NSW), and were hatchery bred in Fingerlings were harvested from outdoor earthen ponds and held for two days in 2500 L concrete tanks prior to transport. Fingerlings were given a prophylactic treatment by bath for 24 hr in 5 ppt NaCl and were inspected for gill and skin parasites. Southern pygmy perch and Un-specked hardyhead were collected from the wild and transported in aerated 20 L drums to Narrandera Fisheries Centre where they were similarly held in concrete tanks. Following the two day quarantine in concrete tanks, fish were placed into heavy plastic bags containing approximately 12 L of water, aerated, sealed, boxed and sent via courier to Camden overnight. Trout cod and Macquarie perch fingerlings were obtained from Snobs Creek Hatchery (VIC) and were hatchery bred in Macquarie perch 21

22 captured from Cataract Dam were transported to Camden in a 600L aerated, transport tank on the same day. Upon arrival at Camden, the fish were removed from the plastic bag and placed in a tank (50 L) with 50% aerated shipping water and 50% clean de-chlorinated water for one hour, then hand netted into their holding tank, awaiting experimentation. Prior to experimental challenge, fish were housed in variably sized glass or Perspex aquaria (average 100 L habitable volume), plastic tanks (320 L), or 1000 L tanks, one species per tank. Water quality was monitored and maintained within acceptable ranges throughout the duration of the holding period with daily to weekly records of temperature, ph ( ), salinity (1-3 ppt), ammonia (<0.50 mg L -1 ), nitrite (<0.25 mg L -1 ) and nitrate (<40 mg L -1 ). Water quality was maintained with high capacity biological filtration units and weekly to fortnightly (or more often as required) exchange of 10-30% of total water volume. Artificial lighting was provided as 12:12 h light:dark with the changes occurring at 0700 and Fish were fed to satiation daily or up to five times weekly with their preferred feed type selected from frozen blood worms, commercial flakes, native fish commercial pellet, earthworms and lamb liver. All laboratorybased animal experimentation was carried out with the prior approval of the University of Sydney Animal Ethics Committee. 22

23 Table Origin, size and current knowledge of EHNV susceptibility for species used in the challenge experiments Species Common name EHNV status based Date arrived Source 1 Total length at Camden (mm) 2 on previous literature 3 Bidyanus bidyanus Silver perch November 2007 NFC hatchery reared ± 1.4 susceptible and carrier Craterocephalus wild from the Murray River, near Un-specked hardyhead June 2009 stercusmuscarum fulvus Robinvale, NSW 33.3 ± 0.70 unknown Gambusia holbrooki Eastern mosquitofish February 2009 wild from NFC pond 31.8 ± 0.59 susceptible Hypseleotris spp. Carp gudgeon October 2007 wild from the Murray River (Lock 7 & 8) 33.3 ± 0.65 Unknown Maccullochella macquariensis Trout cod February 2010 DPI Victoria hatchery 54.7 ± 0.61 Unknown Maccullochella peelii peelii Murray cod March 2008 NFC hatchery reared ± 1.4 susceptible and carrier Macquaria ambigua May 2009 NFC hatchery reared 34.5 ± 0.50 Golden perch ambigua January 2010 NFC hatchery reared 37.7 ± 0.35 susceptible and carrier Macquaria australasica May 2009 wild from Cataract Dam, NSW ± 3.1 Macquarie perch February 2010 DPI Victoria hatchery 55.4 ± 0.45 Susceptible Melanotaenia fluviatilis Murray-Darling rainbowfish February, April 2009 NFC hatchery reared 46.5 ± 2.6 Unknown Mogurnda adspersa Southern purplespotted gudgeon February, April 2009 NFC hatchery reared 59.4 ± 0.66 Unknown Nannoperca australis Southern pygmy perch June 2009 wild from Coppabella Creek, NSW 50.2 ± 0.77 Unknown Tandanus tandanus Freshwater catfish June 2009 private hatchery reared 98.3 ± 1.3 Unknown 1 NFC, Narrandera Fisheries Centre 2 mean ± s.e. 3 based on Langdon,

24 Experimental Challenge Model In a climate-controlled, restricted-access, animal house with isolated drainage that was capable of containing infectious organisms, experimental infection trials were conducted in 100 L aerated aquaria (habitable volume) with an established external biological filtration unit which was seeded with mature biofilter material created and maintained elsewhere using Common carp Cyprinus carpio. The canister-type or external hang-on-type biofilter units also contained activated charcoal, crushed limestone and filter wool or sponge. Artificial lighting was provided as 12:12 h light:dark with the changes occurring at 0700h and 1900h. Water temperatures were maintained at 18 to 24 C by controlling the air temperature of the room. Water quality was monitored twice weekly and maintained at ph and NH 3 +, NO 2-0 mg L -1, with water changes as needed. Waste water and all equipment was disinfected with 200 mgl -1 hypochlorite solution or immersion in Virkon (1% solution) prior to disposal. Solid waste was autoclaved Fish in the holding tanks were transferred to the high-containment facility and randomly allocated to the experimental tanks. Fish were allowed to acclimate to their new tank for 3 to 7 days prior to virus exposure. Ideally, approximately 30 individuals per tank were used. Each species was tested with a minimum of two replicates exposed to EHNV by bath immersion and up to two replicates of controls given a sham exposure (Table 10.2). All mortalities in the control tanks during the trial were tested for evidence of EHNV infection and approximately three to five individuals from each control tank were selected for testing on the final observation day. The EHNV challenge inoculum was grown in cell culture (see below), harvested and added either as a bolus (total volume of approximately 4 to 6 ml) or delivered as a bath challenge in a smaller volume, with the actual dose calculated retrospectively using end point virus titration (Table 10.2) (Langdon, 1989; Whittington and Reddacliff, 1995). As required, individual fish were given an intraperitoneal (IP) injection with 50 to 100 µl of viral inoculum. Up to 12 Redfin perch exposed to EHNV in the same way in at least one tank were used as a positive control for the viral inoculum in each experiment. All fish were monitored once to twice daily for up to 35 days following exposure for clinical signs of EHNV. Depending on their size, moribund and dead fish were sampled for virus isolation and/or histopathlogy for evidence of EHNV infection. Classification of the infection status and susceptibility of individual fish and species The status of individual fish following challenge with EHNV was defined based on test results using a decision tree (Figure 10.1). If one or more fish in a species was infected or diseased the species was classified as susceptible. If one or more fish in a trial was classified as a carrier, the species was classified as being a carrier host. A species could be both susceptible and a carrier host. 24

25 Individual fish experimentally challenged with EHNV Died or euthanized with clinical disease Alive at day 35 post exposure Positive histopathology and virus isolation result Positive histopathology (virus isolation not tested) Positive virus isolation (histopathology not tested) Negative histopathology and/or virus isolation (all tests negative) Positive histopathology or virus isolation result Negative histopathology and/or negative virus isolation result (all tests negative) Infected and diseased Infected and diseased Infected and presumed diseased Not infected and presumed to have died due to other causes Carrier or incubation period >35 days Not infected or recovered from infection Figure Decision tree to define the status of fish following an experimental bath challenge with EHNV Virus EHNV isolate 86/8774, originally isolated from clinically affected Rainbow trout, was propagated in bluegill fry (BF-2) cells or fathead minnow (FHM) cells and incubated at 22 C. After destruction of the monolayer, the culture medium was collected, centrifuged at 1000 x g for 10 min to remove cell debris, and then used as inoculum on the same day (except in initial trials where the inoculum was kept at 4 C for several weeks prior to use). An end point titration assay (50% tissue culture infective dose, TCID 50 ) was completed retrospectively to determine the quantum of virus in each inoculum. Virus isolation Moribund or dead fish were sampled for virus isolation. Kidney, liver and spleen were target organs, pooled either after dissecting out the viscera, or by using the whole fish with head and tail removed (for fish <40 mm total length). Tissues were placed in sterile microcentrifuge tubes and stored at -80 C, if not processed immediately. Each sample was weighed and a 9 x weight/volume of homogenising medium (HM) (minimum essential medium supplemented with 200 IU/ml penicillin, 200 µg/ml streptomycin and 5 µg/ml Fungizone) was added. Tissues were prepared by grinding in a chilled mortar and pestle with sterile sand and HM then clarified by centrifuging at 900 x g for 10 minutes in a microcentrifuge. A 200 µl aliquot of the clarified homogenate was removed for DNA extraction and a second 500 µl aliquot was prepared for virus isolation. It was further diluted 1:4 v/v in HM, passed through a 0.22 µm low protein binding syringe-end filter and used to inoculate bluegill fry (BF-2) cells in 25

26 suspension in 24-well tissue culture plates. The cells were prepared by resuspending 80-90% confluent cell monolayers in minimal essential medium supplemented with 10% foetal bovine serum (FBS), 200 IU/ml penicillin, 200 µg/ml streptomycin and 5 µg/ml Fungizone, to 2 x 10 5 cells/ml. In duplicate, 150 µl of each sample was inoculated directly into a 1.5 ml cell suspension. Cells were incubated at 22 C and examined for development of a cytopathic effect (CPE). If CPE developed, the infected tissue culture supernatant (TCSN) was harvested, 150 µl was passaged into a fresh cell suspension and a 200 µl aliquot was reserved for DNA extraction to confirm the presence of EHNV by PCR. Any wells with cells that exhibited no CPE after 7-10 days were freeze thawed at -20 C overnight and 150 µl of TCSN was passaged by well to well transfer into fresh cells. This was repeated after a further 7-10 day incubation to confirm samples as negative. Confirmation of virus isolation by PCR The DNA was extracted from 200 µl of TCSN using the HighPure Viral Nucleic Acid Extraction Kit (Roche) and examined as described (OIE, 2003). 26

27 Histopathology For larger fish, portions of posterior kidney, liver and spleen were excised and fixed in 10% neutral buffered formalin for at least 24 h. Small whole fish were fixed in 10% neutral buffered formalin, placed in a 12.5% w/v ethylene-diamine-tetra-acetic acid ph 7 decalcification solution for 24 h, and transferred back to formalin. Tissues were processed routinely through graded ethanol solutions, embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin (H & E). The sections were viewed under a light microscope. Sections containing evidence of characteristic tissue degeneration and necrosis with or without viral inclusion bodies were recorded as positive (Figure 10.2). Figure Hepatic necrosis (upper panel) and splenic necrosis (lower panel) in experimentally infected Redfin perch. 27

28 Immunohistochemistry Immunoperoxidase stains were conducted on selected tissue sections (Reddacliff and Whittington, 1996), using a commercial kit (DAKO ) containing peroxidase-labelled streptavidin and a mixture of biotinylated anti-rabbit/anti-mouse/anti-goat immunoglobulins as link antibodies. The primary antibody (affinity purified rabbit anti-ehnv Lot No. M708, OIE Reference Laboratory, University of Sydney) and a negative control reagent (non-immune rabbit serum) were used at a dilution of 1:1500 (Whittington and Deece, 2004). Positive results were characterised by brown stain deposition associated with areas of necrosis (Figure 10.3). Figure Immunoperoxidase stain of liver of an experimentally infected Redfin perch. The extensive brown stained areas consist of degenerating cells which are laden with EHNV particles that take up the stain. 28

29 Table Overview of the challenge trials to determine the susceptibility of fish to EHNV Trial ID Lab No. Trial Date Species Challenged Challenge Type Water Temperature C Source of Redfin perch for positive control Virus Titer 1 (TCID 50/ ml) 1 07/132 November 2007 Carp gudgeon 1 hr bath in 12 L none 20 µl 1 x /133 December 2007 Redfin perch 1 hr bath in 12 L 7.5 ml 1 x 10 5 or 50 µl IP Blowering Dam, NSW & 1 x 10 6 injection 3 08/003 January 2008 Carp gudgeon, Redfin perch 1 hr bath in 4 L & 3 ml 1 x 10 9 in tank bolus Blowering Dam, NSW & 8 ml 1 x /069 April 2008 Murray cod, Redfin perch in tank bolus Blowering Dam, NSW 4 ml x /101 May 2008 Redfin perch in tank bolus Blowering Dam, NSW; Upper Tarcutta, NSW; 4 ml 1 x Mulwala Canal, NSW 6 08/164 August 2008 Silver perch, Murray cod, Redfin perch in tank bolus Upper Tarcutta, NSW 6 ml 1 x /250 November 2008 Silver perch, Murray cod, Redfin in tank bolus 2 Western Australia, perch Upper Tarcutta, NSW 4 ml 1 x 10 6 Eastern mosquitofish, Southern 8 09/054 March 2009 purple-spotted gudgeons, Murray-Darling rainbowfish, in tank bolus Bethungra Dam, NSW 6 ml 1 x 10 6 Redfin perch 9 09/092 May 2009 Eastern mosquitofish, Southern purple-spotted gudgeons, Murray-Darling rainbowfish, Redfin perch in tank bolus Bethungra Dam, NSW 5 ml 1 x /138 July /044 February amount of EHNV added to bath volume 2 tank volume was 100 L Golden perch, Un-specked hardyheads, Freshwater catfish, Southern pygmy perch, Macquarie perch, Redfin perch Golden perch, Macquarie perch, Trout cod, Freshwater catfish, Redfin perch in tank bolus 2 & 100 µl IP injection in tank bolus Bethungra Dam, NSW private dam near Lithgow, NSW 5 ml 1 x 10 5 & 1 x ml 1 x

30 Table The susceptibility of fish from the Murray-Darling Basin to EHNV following experimental challenge Species Common name Challenge method Total no. of replicate tanks No. of deaths/ total number exposed Proportion of fish with an EHNV positive result (no. of samples collected) challenged fish that died challenged survivors 1 control fish 3 histology virus isolation 2 histology virus isolation no. dead/ no. observed no. positive/ no. tested Bidyanus bidyanus Silver perch bath 4 43/107 0/4 (4) 2/39 (39) 2/64 (64) 15/163 0/63 Craterocephalus Un-specked insufficient stercusmuscarum bath 2 9/96 hardyhead tissue (1) fulvus 0/8 (8) not done (6) 0/80 (80) 3/32 0/13 0/5 + 3 Gambusia holbrooki Eastern mosquitofish bath 5 47/167 insufficient 17/39 (39) 0/3 (15) 7/105 (105) 7/98 0/37 tissue (8) Hypseleotris spp. Carp gudgeon bath 4 50/95 0/4 (25) 0/25 (25) 0/4 (19) 0/26 (26) 21/59 0/59 Maccullochella macquariensis Trout cod bath 4 4/115 0/1 (1) 0/3 (3) not done (8) 0/103 (103) 4/34 0/4 Maccullochella peelii peelii Murray cod bath 6 9/61 0/9 (9) 0/52 (52) 5/55 0/38 Macquaria ambigua insufficient Golden perch bath 5 34/109 ambigua tissue (2) 0/32 (32) not done (8) 0/67 (67) 1/27 0/7 Macquaria australasica Melanotaenia fluviatilis Mogurnda adspersa Nannoperca australis Tandanus tandanus Macquarie perch Murray-Darling rainbow fish Southern purplespotted gudgeon Southern pygmy perch Freshwater catfish bath 5 26/135 IP 1 3/3 1/1 + 6 insufficient tissue (7) 1/1 + 2 insufficient tissue (3) 0/21 (21) not done (12) 0/92 (92) 32/132 0/32 1/3 (3) 1/2 6 0/2 6 bath 5 27/166 1/3 (3) 5/24 (24) 0/3 (17) 0/112 (122) 19/74 0/23 bath 4 3/71 0/2 (2) non-viable sample (1) 0/3 (12) 0/56 (56) 3/40 0/10 bath 2 2/68 0/2 (2) not done (6) 0/60 (60) 10/48 0/10 bath 3 0/65 not done (8) 0/35 (35) 0/80 0/3 IP 2 13/15 4/7 (11) 2/12 (12) not done (1) 0/2 (2) 1/13 6 0/

31 1 survivors were euthanized at least 28 days following exposure 2 positive virus culture was confirmed using a DNA-based diagnostic test (quantitative PCR) 3 conspecifics given a sham exposure and the EHNV status confirmed by virus isolation 4 4 to 6 ml of media containing EHNV (isolate 86/8774) at a concentration > 10 6 TCID 50.mL -1 was added to a 100 L aquarium holding the challenge fish µl of EHNV (isolate 86/8774) at a concentration of 10 6 TCID 50.mL -1 was injected in the peritoneal cavity of fish that survived the bath challenge 6 control fish were survivors of the bath challenge that received a sham injection 31

32 Table The susceptibility of Redfin perch to EHNV following experimental challenge Collection Site (EHNV status) Blowering Dam, NSW 5 Age & Size (mm) 1 juveniles No. of Total no. deaths/ of total replicate number tanks histology virus (no. that isolation3 histology virus isolation died) exposed 4 bath /40 2/10 (13) 11/16 (16) 1/11 (11) 0/13 (13) Challenge method Proportion of fish with an EHNV positive result (no. of samples collected) challenged fish that died challenged survivors 2 control fish IP 7 1 7/7 not done (4) 3/3 (3) 0/7 (7) Upper Tarcutta, NSW 8 adults bath 8 11/26 11/11 (11) 11/11 (11) 2/13 (14) 4/14 (14) 0/2 (2) Mulwala Canal, NSW 8 adults bath 2 7/10 7/7 (7) 7/7 (7) 0/3 (3) 2/3 (3) juveniles & Bethungra Dam, NSW 8 adult bath 6 30/43 27/29 (30) 9 28/30 (30) 9 not done (13) 0/13 (13) 0/122 (122) 10 Western Australia 8 adults bath 2 9/12 8/9 (9) 9/9 (9) 0/3 (3) 1/3 (3) Lithgow, NSW 8 juveniles bath 2 4/9 1/1 (1) 3/3 (3) not done (1) 0/4 (4) - 1 generally fish < 50 mm were sampled for either histology or virology and larger fish were sampled for both tests when possible 2 survivors were euthanized at least 28 days following exposure 3 positive virus culture was confirmed using a DNA-based diagnostic test (quantitative PCR) 4 conspecifics given a sham exposure and the EHNV status confirmed by virus isolation 5 water body with documented outbreaks of EHNV 6 4 to 6 ml of media containing EHNV (isolate 86/8774) at a concentration > 10 6 TCID 50.mL -1 was added to a 100 L aquarium holding the challenge fish 7 50 µl of EHNV (isolate 86/8774) at a concentration of 10 6 TCID 50.mL -1 was injected in the peritoneal cavity of fish that survived the bath challenge 8 water body with no reports of EHNV at the time of fish collection 9 all 30 fish that died were virus isolation and/or histology positive 10 mortalities from field collection and transfer 32

33 Table Summary of host susceptibility EHNV following experimental bath challenge Previous research 1 Current project Fish Species Fish source Age or size Fish source Age or size EHNV Susceptibility (total EHNV Susceptibility (total no. tested) 2 no. tested) 2 Bidyanus bidyanus fish farm 3 3 months, juvenile ++ (36) NFC hatchery 1 year +, juvenile + (107) Carassius auratus farm dams free of < 1 year, juvenileadult EHNV unaffected (8) not tested Craterocephalus stercusmuscarum fulvus not tested previously Murray River juvenile - adult unaffected (96) Galaxias olidus streams above Lake Nillahcootie 3 1 year +, adult ++++ (10) not tested Gambusia holbrooki farm dam free of EHNV 1 year +, adult ++++ (10) NFC pond adult + (167) Hypseleotris spp. not tested previously Murray River adult unaffected (95) Lates calcarifer fish farm 3 4 months, juvenile unaffected (20) not tested Maccullochella DPI Victoria not tested previously macquariensis hatchery ~5 months, juvenile unaffected (115) Maccullochella peelii peelii fish farm 3 2 months, juvenile unaffected (4) 4 NFC hatchery <1 year, juvenile unaffected (61) Macquaria ambigua ambigua fish farm 3 2 months, juvenile unaffected (30) 4 NFC hatchery ~4 months, juvenile unaffected (109) Cataract Dam, NSW 1 year +, juvenile + (59) Macquaria australasica fish farm 3 3 months, juvenile ++++ (20) DPI Victoria hatchery ~5 months, juvenile unaffected (76) Macquaria novemacuelata fish farm 3 6 months, juvenile unaffected (10) 4 not tested Melanotaenia fluviatilis not tested previously NFC hatchery juvenile + (166) Mogurnda adspersa not tested previously NFC hatchery adult unaffected (71) Nannoperca australis not tested previously Coppabella Creek, NSW adult unaffected (68) Tandanus tandanus not tested previously fish farm juvenile unaffected (65) 4 Retropinna semoni Lake Nillahcootie after EHNV detected 1 year +, adult unaffected (12) not tested 1 adapted from Langdon based on a bath challenge to EHNV: + equals <20% population exposed succumb to EHN, ++ equals 21 to 50, +++ equals 51-79, ++++equals >80 3 the reported water source was considered free of Redfin perch 4 determined to be susceptible to EHN following injection challenge with the virus 33

34 10.2 Findings A total of seven previously untested native fish species and an additional six species with reported EHNV susceptibility were included in the experimental challenge studies. At least one positive control Redfin perch was determined to be infected with EHNV and/or diseased for each of the challenge trials (except for the initial pilot trial where no perch were available) using techniques including virus isolation, histopathology and immunohistochemistry. This indicated that all of the viral inocula used throughout the 11 trials were viable and able to infect fish and that these trials to determine the susceptibility of native and alien species found in the Murray-Darling Basin were valid. There was no evidence of EHNV infection in any individuals prior to experimentation or in any of the individuals sampled from the shamexposed control tanks. Detailed results are provided in Tables Silver perch (Bidyanus bidyanus) Mortalities in the juvenile silver perch over the duration of the experiment were 43/107 (40%) in the challenged group. The virus was re-isolated from two fish in the challenged group that died and from two surviving silver perch. The virus was not isolated from any dead or surviving fish in the control group. These findings indicated that the challenged fish may have become carriers of the virus or had an incubation period greater than 35 days (the duration of the trial). Although the virus may have caused the deaths in the challenged fish from which virus was re-isolated, it cannot be confirmed due to EHN, as only virological samples were obtained. Thus, under the experimental conditions described, silver perch were found to have a low level of susceptibility to EHNV, at least in the carrier state. Previously, 28% (10/36) of bath challenged three month old silver perch succumbed to EHN (Langdon 1989) (Table 8.1) Unspecked hardyheads (Craterocephalus stercusmuscarum fulvus) Mortalities in the Un-specked hardyheads over the duration of the experiment were 9/96 (9%) in the challenged group. All of the exposed and sham-exposed Un-specked hardyheads tested were negative for EHNV. It appeared that this species was not susceptible to EHNV, following a bath challenge or the animals were able to recover during the challenge period. Eastern mosquitofish (Gambusia holbrooki) Mortalities in the adult Eastern mosquito fish over the duration of the experiment were 47/176 (28%) in the challenged group. The virus was re-isolated from 17 fish in the challenged group that died and from seven surviving animals, for a total of 14% of the challenged fish. The virus was not isolated from dead or surviving fish in the control group. These findings indicate that the challenged fish may have become carriers of the virus or had an incubation period greater than 35 days. Although the virus may have caused the deaths in the challenged fish from which virus was re-isolated, this cannot be concluded as only virological samples were obtained. Under the experimental conditions described, Eastern mosquito fish were found to be susceptible to EHNV, at least in the carrier state. Previously, Langdon (1989) reported that 90% (9/10) of bathchallenged Eastern mosquitofish died with evidence of EHNV infection (Table 8.1). Carp gudgeons (Hypseleotris spp.) Overall mortalities in the adult carp gudgeon over the duration of the experiment were 50/95 (53%) in the challenged group. All of the exposed and sham-exposed Carp gudgeons tested 34

35 were negative for EHNV. It appears that this species was not susceptible to EHNV, following a bath challenge or the animals were able to recover during the challenge period. Trout cod (Maccullochella macquariensis) Overall mortalities in the juvenile Trout cod over the duration of the experiment were 4/114 (6%) in the challenged group. All of the exposed and sham-exposed trout cod were negative for EHNV. It appeared that this species was not susceptible to EHNV, following a bath challenge or the animals were able to recover during the challenge period. Murray cod (Maccullochella peelii peelii) Overall mortalities in the juvenile Murray cod over the duration of the experiment were 9/61 (15%) in the challenged group. All of the exposed and sham-exposed Murray cod tested were negative for EHNV. Under the described conditions, it appeared that this species was not susceptible to EHNV following a bath challenge or the animals were able to recover during the challenge period. Previously, it was reported that two month old Murray cod were not susceptible to EHNV following a bath exposure but the virus was re-isolated from a few fish (2/4) at 21 days post exposure, suggesting carrier potential (Langdon 1989). Moreover, the disease was induced in 100% (4/4) of two month old cod following an IP injection with EHNV (Langdon 1989). From the current study, it appears that larger Murray cod fingerlings were not susceptible to the virus or were able to recover from the infection within the 35 days challenge period as no carriers were identified Golden perch (Macquaria ambigua ambigua) Mortalities in the juvenile Golden perch over the duration of the experiment were 34/109 (31%) in the challenged group. All of the exposed and sham-exposed golden perch tested were negative for EHNV. It appeared that this species was not susceptible to EHNV following a bath challenge or the animals were able to recover during the challenge period. This was in agreement with previous research with no observed mortality in two month old Golden perch following a bath challenge (Langdon 1989). However, 100% mortality (12/12) was observed in Golden perch following an IP injection (Langdon 1989) (Table 8.1). Macquarie perch (Macquaria australasica) Overall mortalities in the juvenile Macquarie perch derived from Cataract Dam, NSW and fingerlings derived from Victoria over the duration of the experiment were 26/135 (28%) in the groups challenged by bath exposure. The virus was not isolated from any fish in the challenged group that died, from the surviving challenged fish or from any of the sham-exposed control group. Of the 59 juvenile Macquarie perch sourced from Cataract Dam, NSW and bath challenged with EHNV, just over half (58%) of the fish survived to Day 35. One Macquarie perch of this group that died on day 35 had microscopic lesions characteristic of an EHNV infection (note: only the histopathology sample was obtained from this individual). Further, of the remaining Macquarie perch, three individuals were injected IP with 100 μl of virus culture and two were given a sham injection and monitored for up to 35 days. One injected Macquarie perch that died was infected with the virus and displayed microscopic lesions indicative of the infection. These findings showed that under the conditions of the present study, and even though virus isolation was negative following bath exposure, EHNV was capable of causing characteristic lesions and death in a small proportion of fish. Previously, it was reported that all (20/20) Macquarie perch died with EHNV following bath exposure (Langdon 1989) (Table 8.1). 35

36 Murray-Darling rainbow fish (Melanotaenia fluviatilis) Overall mortalities in two separate challenge trials on juvenile Murray-Darling rainbow fish over the duration of the experiment were 27/166 (16%) in the group challenged by bath exposure. None of the rainbow fish that were bath challenged in two separate tanks from one trial were EHNV positive. However, in a subsequent trial where Murray-Darling rainbow fish were held in three separate tanks, the virus was re-isolated from five fish that died in two different tanks. Additionally, one of three dead rainbow fish that was examined microscopically had lesions consistent with EHNV infection. None of the exposed survivors showed any evidence of EHNV infection and were considered to be not infected or to have recovered from infection within the 35 day period. Also, the virus was not isolated from any of the control fish. Under the experimental conditions described, Murray-Darling rainbow fish had a low level of susceptibility to EHNV, with a small proportion (3%) of the population developing an infection or succumbing to the disease. Southern purple-spotted gudgeon (Mogurnda adspersa) Overall mortalities in the adult Southern purple-spotted gudgeons over the duration of the trial were 3/71 (4%) in the challenged group. All of the exposed and sham-exposed Southern purple-spotted gudgeons tested were negative for EHNV. It appeared that this species was not susceptible to EHNV following a bath challenge or the animals were able to recover during the challenge period. Southern pygmy perch (Nannoperca australis) Overall mortalities in the adult Southern pygmy perch over the duration of the trial were 2/68 (3%) in the challenged group. All of the exposed and sham-exposed Southern pygmy perch tested were negative for EHNV. It appeared that this species was not susceptible to EHNV following a bath challenge or the animals were able to recover during the challenge period. Freshwater catfish (Tandanus tandanus) A total of 65 juvenile Freshwater catfish were bath challenged with EHNV, with no observed mortality during the observation period. At day 35 following bath challenge, 35 fish were sampled for detection of EHNV infection, with no evidence of the virus and all of the sham-exposed fish were negative for the virus. Of the remaining Freshwater catfish, 15 individuals were subjected to an IP injection with 100 ul of virus culture and 13 were given a sham injection. Four of the IPexposed fish that died had microscopic lesions consistent with an EHNV infection and were classified as infected and diseased. The virus was re-isolated from one additional Freshwater catfish that was exposed IP. Under the experimental conditions described, there was evidence that Freshwater catfish can be infected with EHNV and develop characteristic hepatic lesions associated with EHNV infection. Redfin perch (Perca fluviatilis) Juvenile Redfin perch were initially sourced from Blowering Dam, NSW to be used as positive controls for the challenge studies. However, previous results had suggested that Redfin perch from Blowering Dam were no longer susceptible or had reduced susceptibility to EHNV (unpublished data). A small trial (SVC 07/133) concluded that at least some Redfin perch from Blowering Dam were susceptible to the virus, based on the isolation of EHNV from these fish, the clinical findings (death) and histopathology. However, inconsistencies between the current challenge trial results and published reports continued to be observed with regards to the proportion of Redfin perch susceptible to EHNV. Previous reports indicated 36

37 nearly 100% mortality in Redfin perch following exposure to EHNV (Langdon 1989, Whittington and Reddacliff, 1995). Therefore a series of challenge trials were designed to elucidate differential susceptibility between wild populations of Redfin perch (Table 10.4). Redfin perch from an EHNV-endemic area, Blowering Dam A total of 40 juvenile Redfin perch wild caught from Blowering Dam were exposed over six separate trials, with 28% of individuals surviving the bath challenge. The virus was re-isolated or characteristic microscopic lesions were found in thirteen perch that died and these fish were classified as infected and (presumed) diseased. Moreover, EHNV was re-isolated from one surviving perch, which was declared a carrier fish or the incubation period was greater than 35 days. Overall 35% (14/40) of the Redfin perch from Blowering Dam became infected with EHNV after bath exposure. Redfin perch from areas with no previous history of EHNV Redfin perch were sourced from other parts of NSW with no history of EHNV: the Upper Tarcutta region (lower Murrumbidgee catchment), Mulwala Canal (lower Murray catchment), Bethungra Dam (lower Murrumbidgee catchment) and a farm dam near Wallerawang (Hawkesbury-Nepean catchment) containing young of the year. Over a series of eight challenge trials, 11 Redfin perch (42%) from the Upper Tarcutta region succumbed to EHNV infection with characteristic histopathological lesions and virus was isolated from all fish. Furthermore EHNV was isolated from six of the surviving Redfin perch from the Upper Tarcutta region, which were therefore classified as carrier fish, or had an incubation greater than 35 days, while an additional three surviving fish showed evidence of sero-conversion in the antibody ELISA (Table 12.7). Similarly, seven Redfin perch (70%) from Mulwala Canal were classified as infected and diseased, with an additional two fish (20%) classified as carriers. A total of 30 fish (70%) from Bethungra Dam, NSW succumbed to the disease with evidence of EHNV-associated microscopic lesions or positive virus isolation; none of the surviving fish showed any evidence of infection with EHNV or had recovered from infection during the study period. Redfin perch sourced from Western Australia showed a high level of susceptibility with nine of the fish (75%) classified as infected and diseased while one of survivors developed a carrier state. Finally, an experiment with young of the year Redfin perch collected from a farm dam resulted in EHNV being re-isolated or characteristic microscopic lesions were found all four fish that died (44%). There was no evidence of any carriers among the survivors. Redfin perch were not known to be in this farm dam prior to 2010 and it was unknown if their occupation was a result of a recent translocation. Background mortality levels Many of the fish used in the experimental infections were sourced from the wild, and so had unknown history and health status. Mortality that was not associated with EHNV was observed in some species. For example, Eastern mosquitofish were wild collected in February 2009 and held for up to six months in the laboratory. The mortality in fish that were exposed to EHNV but not due to EHNV was nearly 18% (30/167), which was similar to the mortality in the control fish sampled for testing (19% of 35 fish). Some mortality may have been due to the short natural lifespan of these fish. Some of the wild-sourced species did not feed well or developed strong social hierarchies. As an example, Macquarie perch sourced from Cataract Dam experienced a high level of post-transfer mortality within four days of arriving in the laboratory and subsequently the surviving fish fed poorly. This was despite being offered a 37

38 variety food types, such as blood worms, beef liver, live earthworms and freshwater shrimp. No post-transfer mortality occurred in the subsequent challenge trial in which Macquarie perch from a state hatchery were used; these fish fed well on blood worms and had a mortality level below 2% in the challenge trial. The majority of the mortality observed in the wildsourced Carp-gudgeons was attributed to difficulties in feeding using an assortment of prepared diets. However, once a live diet of Artemia was provided, the fish began to feed and mortality levels were similar in exposed and non-exposed tanks. Finally, a high level of mortality was observed in exposed Golden perch due to a fungal infection. In a subsequent trial there were no mortalities in either the exposed or non-exposed tanks of Golden perch. It is important to note that all individual fish that died during the observation period in all trials, regardless of exposure to EHNV or apparent cause of death, were sampled for evidence of EHNV infection. Each fish was classified using the decision tree (Figure 10.1) to determine its EHNV status. Summary Several species of fish have been identified as being susceptible to EHNV through laboratory challenges in Australia, while natural outbreaks have been reported only in the introduced species, Redfin perch (in the wild) and Rainbow trout (on farms). The current research confirmed an earlier report (Langdon, 1989) that Silver perch, Macquarie perch and Eastern mosquitofish were susceptible to EHNV following an experimental bath challenge (Table 10.5). In both Silver perch and Macquarie perch these results differed quantitatively from those in prior reports, probably due to difference in the experimental model. For example there were differences in age of fish between different trials. Older life stages may become less susceptible to the virus. Langdon (1989) reported that all 20 three month old Macquarie perch died with EHNV following bath exposure while in the present study less than 1% of older fish were susceptible. This same trend was observed for Silver perch with just under 30% of 3 month old fish dying with EHNV (Langdon, 1989) compared to less than 4% of older fish greater than 100 mm total length in the present trial. Differences in source of fish may also explain differences in results between the two studies. In the current study Eastern mosquitofish sourced from Narrandera, NSW had a lower susceptibility to EHNV with 14% of fish succumbing to infection or developing a carrier state, compared to 90% percent mortality observed in Eastern mosquitofish from a farm dam presumably in Victoria (Langdon, 1989). Eastern mosquitofish have a relatively short life cycle compared to Silver perch and Macquarie perch and are not considered to be migratory (Lintermans, 2007). Interestingly, throughout their natural range in North America, Eastern mosquitofish have some of the highest genetic variability observed in vertebrate species (Smith et al., 1989), although Australian populations have been shown to be considerably less diverse with unidirectional gene flow from headwaters downstream depending on barriers and flood events (Congdon, 1995). The combination of the strong potential for genetic variability and geographical distance of these two Eastern mosquitofish populations may explain differences in resistance to challenge with EHNV. Another factor applying to Eastern mosquitofish is the lengthy period (>20 years) between the trials of Langdon (1989) and the present trials, during which natural selection may have played a significant role to increase the level of resistance of fish populations to EHNV. 38

39 In agreement with Langdon (1989), Murray cod and Golden perch were not susceptible to the virus following a bath challenge. Although we did not undertake intraperitoneal trials in either of these two species, Langdon demonstrated that both of these species were highly susceptible (100% mortality) following an intraperitoneal injection with the virus. These species should be considered susceptible to EHNV, as the trials were limited to investigate a narrow thermal range and particular husbandry conditions (high levels of dissolved oxygen, low stocking densities, fed to satiation with a high protein diet). Environmental variables can have a profound influence on susceptibility to viruses such as EHNV (Whittington and Reddacliff, 1995), and only a one set of environmental conditions could be tested in this project. Murray-Darling rainbowfish and Freshwater catfish were newly identified in this project as being susceptible to EHNV. Murray-Darling rainbowfish are of high conservation value and their main potential threats include predation from two other EHNV host species, Redfin perch and gambusia (Lintermans, 2007). Similar to Murray cod and Golden perch, Freshwater catfish were unaffected by EHNV following a bath challenge. However, one-third (5/15) of the intraperitoneally-injected catfish succumbed to EHNV. Different life stages of Freshwater catfish (i.e. younger juveniles) or animals under differing levels of environmental stress may display increased susceptibility to the virus. Native fish species that were considered to be unaffected by EHNV following a bath challenge include Un-specked hardyhead, Carp gudgeon, Trout cod, Southern purple-spotted gudgeon and Southern pygmy perch. Although, the probability of a natural outbreak occurring in these species in the Basin is probably low, it is important to reiterate that these results apply only to the experimental conditions and life stages tested for each species. From above, there is some evidence that younger age classes and geographically restricted populations of fish may show differential susceptibility to EHNV. It was reported previously that in both natural outbreaks (Langdon and Humphrey, 1987) and experimental challenges (Langdon, 1989; Whittington and Reddacliff, 1995) over 90% mortality due to EHNV occurred in all age groups of Redfin perch. However, Langdon (1989) reported that adult Redfin perch (14 20 cm) collected from Lake Nillahcootie (Broken Catchment Lower Murray region) following the discovery of EHNV were unaffected by the virus following bath challenge and the virus could not be isolated from any of the surviving fish. Some juvenile Redfin perch apparently survived an outbreak in Lake Nillahcootie in Victoria and two carriers were identified among 40 apparently healthy adult Redfin perch (Langdon and Humphrey, 1987). Furthermore EHNV was isolated from one adult and one immature fish from Lake Mokoan (Broken Catchment Lower Murray region) Victoria around the time of juvenile mortalities (Langdon and Humphrey, 1987). Several ranavirus isolates have since been obtained from Redfin perch in Victoria at times when there was no obvious epizootic (unpublished data). However, a cautious interpretation of such data is required. For example the incubation period in Redfin was up to 28 days (Whittington and Reddacliff, 1995) so a cross-sectional sample of wild fish is not appropriate to infer carrier status. Nevertheless several apparently healthy Redfin in Victoria had serum antibodies against EHNV or a related virus when blood was collected from them about 20 years ago (see section 12 this report). 39

40 The current study was the first to confirm experimentally that Redfin perch may develop a carrier state. Approximately, 18% of the survivors (8/44) of bath challenge were declared carriers of EHNV or to have an incubation period greater than 35 days. Although we were not able to differentiate these possibilities with the design we used, the species is generally susceptible and incubation periods >28 days were not observed in the early studies (Langdon, 1989; Whittington and Reddacliff, 1995). The relatively high proportion of Redfin perch that may develop an EHNV carrier state and perhaps shed live virus into the environment may aid in the dispersal of EHNV. In the past six years, unpublished results from our laboratory and those in the present study have suggested that Redfin perch from the original endemic area (e.g. Blowering Dam) have markedly decreased susceptibility to the virus compared to 16 years ago. In contrast, Redfin perch from neighbouring and distant water bodies displayed moderate to high susceptibility when challenged with the virus. The mechanism(s) responsible for this change in the host s ability to recognize and contend with this viral pathogen are unknown and warrant investigation. Changes in the host-parasite relationship, intense selection pressure for resistant fish following EHN outbreaks, and subsequent attenuation of virulence of the virus in resistant fish are potential factors. 40

41 11. Field survey to detect EHNV Surveys to detect pathogens in the broad scale environment are difficult to design and implement. EHNV is already known to lack host specificity so a range of fish species logically would be targeted, but very little is known about other aspects of its behaviour in nature. For example it is unknown whether fish can become infected and remain as carriers for life, and therefore whether testing would need to be conducted during an outbreak of disease. It is also unclear whether an environmental reservoir of virus may exist in river sediments or indeed in species other than fish. As the virus in a laboratory is highly resistant to drying, in water and on surfaces, and persists in frozen fish tissues it has been assumed that it would also be highly resistant in the environment. Whether environmental sources of EHNV are available to fish is completely unknown. It may for example persist in river sediments but be tightly bound to sand particles and unavailable to fish. A field survey to identify whether a pathogen is present in a population requires special consideration of several parameters including the likely prevalence of the pathogen if it is in fact present (the design prevalence), the sensitivity and specificity of diagnostic tests, the degree of confidence in the results that is required, and an ability to obtain representative samples, generally through random sampling. The present survey was conducted assuming that EHNV would be present in at least 10% of fish in a population. This is based on observations in Redfin perch where the virus tends to affect a large proportion of the population (Langdon and Humphrey, 1987; Langdon et al., 1986). It would be desirable to have much greater power of detection, down to 5, 2 or 1% prevalence, because at those levels claims about freedom from infection could then be made with more certainty. Other things being equal, a greater sample size is needed for greater confidence or to have a lower design prevalence. 41

42 11.1 Methods Collection of fish tissue and serum Samples provided for laboratory testing were collected during established research programs by various collaborating agencies. Most of the tissue and serum samples were collected by Industry & Investment NSW during sampling for the MDBAs Sustainable Rivers Audit (SRA), a basin-wide assessment of the health of the river ecosystems in the Murray-Darling Basin designed to cover the entire Murray-Darling Basin over three years (Table 11.1) (Davies et al., 2008). Industry & Investment NSW also collected samples from its Effectiveness of Stocking project which is restricted to major impoundments in NSW that received an annual stocking of native fish and trout, and several other independent research projects. The Arthur Rylah Institute VIC conducted annual fish sampling across three catchments, representing three to four altitudinal zones, and in other projects. Territory Municipal Services (ACT) undertook sampling under its Urban Lakes Monitoring Program as well as specific collections for this project. Overall, the additional time required to process all samples suitable for this project was not always available. Table Annual schedule of catchments to be sampled for the Sustainable Rivers Audit program Year Catchments 2008 Border Rivers, Broken, Darling, Loddon, Lower Murray, Mitta, Murray- Riverina and Upper Murray catchments Avoca, Goulburn, Kiewa, Lachlan, Macquarie-Bogan, Namoi, Paroo and Warrego catchments Campaspe, Castlereagh, Condamine-Culgoa, Gwydir, Murrumbidgee, Ovens and Wimmera-Avon catchments. The collection method was predominantly boat or backpack electrofishing and/or a combination of gill, seine and fyke nets. Fish were collected according to appropriate animal ethics permits. Fish were euthanased and were kept whole or dissected in the field, individually bagged (as practicable) and labeled with collector, date, species and location details. These samples were held on ice or in portable refrigerators until return to a research station where some were dissected. Alternatively, whole fish were frozen at -20 C until dissection at the University of Sydney, Camden usually within six months of collection. For transport, frozen fish or tissues were packed with ice bricks and transported to Camden by road. During dissection, the spleen, posterior kidney and liver was removed and placed into individual sample vials. All fish tissue was kept frozen at -20ºC until it was tested for EHNV. For serum collection, fish were placed in plastic holding tubs and anaesthetised with 20 ml of AQUI-S per 100 L water until there was deep sedation evidenced by limited movement and 42

43 complete loss of equilibrium. Blood was extracted from the caudal vein using an appropriate needle for the size of the fish (25-19 gauge) and was expelled into a 1.5 ml polypropylene tube (Eppendorf). Tubes were labeled and placed on ice or in a portable fridge until return to a research station. Whole blood samples were allowed to clot and were kept chilled for up to seven days in the field. Samples of clotted blood were centrifuged at 2,000 to 10, 000 rpm for 10 minutes. The separated serum was removed to a labelled tube (Eppendorf) and placed at - 20ºC. Serum samples were transported to Camden at -20 C, thawed at room temperature, diluted 1:10 in 50% v/v glycerol in 25 mm Tris, 150 mm NaCl ph7.4 with 0.02% v/v merthiolate (TSGM) and stored at -20ºC. Virus isolation and confirmation The procedures were carried out as described in section Serology The procedures are described in section Mapping The generation of the field collection maps was completed using ArcMap (ESRI, Redlands, California, USA). Prevalence of EHNV infection and likelihood of freedom of infection Populations were defined as groups of fish restricted by species, year of collection, catchment and zone, and if samples arose from an impoundment (for example samples derived from Burrinjuck Dam were regarded as having been drawn from a separate population than those from the upper Murrumbidgee River). Otherwise, samples were aggregated into populations. A fish was considered positive if either the virus isolation or ELISA assay was positive. The prevalence of EHNV was defined as the proportion of test positive fish in that population and 95% exact binomial confidence intervals were determined using Minitab Statistical Software. The diagnostic sensitivity and specificity of virus isolation were both assumed to be 100% because it is the gold standard test, therefore apparent prevalence equals true prevalence (Whittington et al., 2010). For ELISA, true prevalence and Blaker s exact confidence limits were calculated assuming test sensitivity of 70% and specificity of 100% using the calculator at For tissue samples tested by virus isolation where all test results were negative, the sample size required to be 95% certain that the population was free of EHNV at a specified design prevalence was determined using software at specifically the modified hypergeometric exact calculation within the FreeCalc analyse results of freedom testing function provided in the Survey Toolbox package (Cameron, 2002). Sample size was evaluated to obtain 95% confidence in detecting infection at specified design prevalences. The following were specified as input parameters: type I and II error levels 0.05; sensitivity 99.9%; specificity 99.9%; population threshold for a binomial calculation 10,000. For a population size of 10,000, the sample sizes required for design prevalences of 10%, 5%, 2% and 1% were 29, 58, 145 and 270, respectively. Samples sizes were slightly lower for smaller populations. 43

44 For example, those for a population of 500 were 28, 55, 124 and 213. Where sample size was > 30, these values were used to infer the prevalence that is not exceeded with 95% confidence at population sizes of 10,000 and 500. For blood samples tested by ELISA, the same method to estimate sample size was used, but test sensitivity was assumed to be 70%. For a population of 10,000, the sample sizes for 95% confidence at design prevalences of 10%, 5%, 2% and 1% were 41, 84, 211 and 421, respectively. For a population of 500, the sample sizes were 40, 78, 175 and Findings Tissue samples were collected and tested from a total of 3622 fish while blood samples were from 492 fish over the duration of project. A summary of the blood samples collected for the project is provided in Table 11.3 while a summary of the tissue samples collected for the project is provided in Table The geographic distribution and intensity of sampling is illustrated in Figures 11.1 to The overall coverage of the Murray-Darling Basin was reasonable, but sample sizes from the required species and from many locations were less than hoped and often small (Figure 11.1), which limited the power of the survey. Fewer samples of any species were collected from northern catchments. This was partly because of the composition of the species assemblage sampled for the SRA. The majority of samples from Redfin perch came from the known EHNV-endemic area in the south eastern part of the Basin but substantial numbers were collected from the Macquarie River at Dubbo and the upper Gwydir (Copeton dam) (Figure 11.2). There are few populations of Redfin perch in any of the northern catchments. Redfin are only present in the upper Gwydir catchment and in a very small area of the upper Beardy River sub catchment of the Border Rivers catchment. Therefore, only one major population from NSW was not sampled. Some additional sites outside the Basin were tested, such as Catarract Dam in the Nepean catchment because of the significance of the species present there, in particular Macquarie perch. It was not intended to obtain samples from South Australia or Queensland during this study. 44

45 Figure Distribution of all fish tissue and serum samples collected from July 2007 to June 2011 for EHNV testing. 45

46 Figure Distribution of Redfin perch tissue and serum samples collected from July 2007 to June 2011 for EHNV testing. 46

47 Figure Moribund and dead Redfin perch collected from Lake Ginnindera, ACT in December 2008 and 2010 and Blowering Dam, NSW in December 2009 were infected with EHNV. 47

48 Figure Distribution of Murray cod tissue and serum samples collected from July 2007 to June 2011 for EHNV testing. 48

49 Figure Distribution of Silver perch tissue and serum samples collected from July 2007 to June 2011 for EHNV testing. 49

50 Figure Distribution of Macquarie perch tissue and serum samples collected from July 2007 to December 2009 for EHNV testing. 50

51 Figure Distribution of tissue samples collected from non-target native fish species from July 2007 to June 2011 for EHNV testing. 51

52 Figure Distribution of tissue samples collected from introduced (alien) fish species from July 2007 to June 2011 for EHNV testing. 52

53 Figure Distribution of serum samples collected from both native non-target fish species from July 2007 to June 2011 for EHNV testing. The locations for golden perch and trout cod were coincident. 53

54 Blood samples A total of 492 blood samples were tested. None of the blood samples from native fish in the Murray-Darling Basin were positive for EHNV. Samples were also available from other regions such as the Yarra River in Victoria and are included in Table 11.3 for completeness. In general only small numbers of samples were available for testing. However, there were sufficient samples collected from Macquarie perch in the Yarra River to be 95% confident that if present, EHNV infected <10% of fish in the population. For Murray cod, there were sufficient samples collected to be 95% confident that prevalence of EHNV was less than 5% in the lower Murray and <10% in the lower Lachlan rivers. These were not sufficiently low design prevalences to be certain that EHNV is absent from these populations, but do suggest the virus is not common. There were sufficient samples collected from Redfin perch in the lower Murray river to be 95% confident that if present, EHNV infected <10% of fish in the population. As EHNV would be expected to infect a large proportion of Redfin perch over time, this may be sufficient evidence to conclude that EHNV was absent from this population. Several samples from Redfin perch from the endemic area in Victoria that were collected about 20 years ago were positive for EHNV antibodies as were two blood samples collected from Redfin perch in the present study (see section 12.2). The latter came from fish from Lake Ginninderra which is within the known endemic area in the upper Murrumbidgee catchment, and a site from which EHNV-infected Redfin perch were identified during the project (see below). The fish were sampled in February 2011 and were 146 mm and 160 mm FL suggesting that they were at least 1 year old and not young of the previous year. They were from a sample of 10 adult fish (range mm FL) collected on one day. Tissue samples A total of 3622 tissue samples were cultured during this study, representing multiple species and geographic locations in the Murray-Darling basin and several other sites (Table 11.4). EHNV was not isolated from species other than Redfin perch during this study. There were several positive tissue samples from Redfin perch; all were from fish in the known endemic area in the upper Murrumbidgee River catchment impoundments (Figure 11.4). One positive Redfin perch was collected from Lake Ginninderra, ACT in December 2008 (laboratory reference SVC 09/028). This sample was one of two dead Redfin perch collected on 4 th December 2008 near a boat ramp in Lake Ginnindera; the other dead fish and four that were electrofished at the site were negative for EHNV; it is unclear whether these fish were involved in an outbreak of EHN. A further three positive Redfin perch were from a group of 12 collected from Lake Ginninderra on 15th December 2010 (SVC11/076). The fish ranged in size from mm FL g body weight (n=12) and were young of the year. The collections undertaken by Territory and Municipal Services in 2008, 2010 and 2011 in Lake Ginninderra were progressive and provided useful information about the detectability of EHNV in an endemically infected impoundment. The virus was detected only in young of the year prior to mid summer as shown in Table

55 Table Summary of results of virus isolation tests for EHNV conducted on Redfin perch collected from Lake Ginninderra ACT. Date collected No. of fish Total length EHNV 15 th October not detected 5 th November not detected 4 th December * infected 18 th March * not detected 17 th September not detected 15th December * infected not detected not detected 19 th December not detected 21 st December not detected 21 st February not detected *young of the year; 1 three sites were fished Eight positive Redfin perch were identified from Blowering Dam, NSW collected during a reported fish kill in December 2009 (laboratory reference SVC 10/038). In many cases only a small number of samples of each species were available in any year from any site. In order to overcome the effect of low sample size on the statistical confidence in the findings of the study, samples were aggregated into populations based on conservative assumptions about what constitutes a population of fish (Table 11.5). It was defined to be fish of a given species collected in the same year from a given catchment and zone, with fish collected from impoundments and lakes considered to be in populations distinct from those in rivers. As a rule of thumb, where sample size is less than 30 and infection is not detected, it is not possible to be very confident that the true prevalence of infection is <10%. For this reason the prevalence of EHNV infection in populations was calculated only where > 30 samples were taken. Australian bass (Hunter River) were from outside the Basin, but are included for completeness, as are Rainbow trout (Snowy River), Refin perch (Yarra River), and Macquarie perch (Nepean River, Yarra River) as they are potential hosts for EHNV; other non-basin collections are shown in Tables 11.3 to For some populations of Basin species such as Eastern mosquitofish in the upper Murrumbidgee, Southern pygmy perch in the upper Murray, and Redfin perch in Lake Buffalo and the upper Gwydir (Copeton Dam), the results suggested that EHNV is absent. Technically, the results showed that it is highly unlikely that EHNV, if present, exists in more than 1 to 2% of fish in each of these populations. For many other populations the figure was <5%, which is still a reasonable level. International standards for surveys to show that an infectious disease is not present have been proposed (Anon, 2009). For diseases which are present in a small section of the population, for example because they are transmitted slowly or are thought to be at an early stage of an outbreak, a design prevalence of 1 to 2 % is recommended. For diseases which are highly transmissible, a design prevalence >5% can be used. As EHNV is highly infectious in some 55

56 species, particularly Redfin perch, if it was present it would likely affect >5% of fish, and probably a much higher proportion. Therefore it was highly likely that the virus was absent from those populations in this study. An overarching assumption was that the samples were random, but this cannot be guaranteed and so the confidence limits may be wider than shown in Table The data for Lake Ginninderra in Table 11.2 confirm that EHNV infection is highly clustered temporally and may be detected readily only in juvenile Redfin perch. In both 2008 and 2010 the virus emerged in the population between September/October and the end of December. The failure to detect EHNV in samples of 44 and 36 adult Redfin perch in October 2008 and February 2011, respectively, is consistent with <10% of those populations being infected, even though within 2 months of these dates the virus was actively replicating in juvenile Redfin perch. It is unknown whether the virus was truly absent from adults, or whether it was present at a very low level, but sufficient to initiate an outbreak in juvenile fish when conditions are appropriate for transmission. Alternatively, there may be another reservoir host in Lake Ginninderra. These results show that even when present in a given body of water, the virus can be difficult to detect in Redfin perch. Where EHNV was detected in Redfin perch, the upper confidence limits for prevalence were 7.1 to 14.0% (Table 11.5). Overall, there was reasonable confidence that EHNV was not present during the study period in species other than Redfin perch and in regions of the Murray-Darling Basin other than the known endemic region which is centered on impoundments in the ACT and Blowering Dam in NSW, both of which are in the upper Murrumbidgee catchment. The survey did not include Rainbow trout on farms within the endemic region. 56

57 Table Summary of fish serum collected for EHNV testing based on year and catchment area from 2007 to 2011 Species Collection Year Compartment Zone 1 Site Location Number Golden perch* 2008 Murrumbidgee Upper ACT 4 Golden perch* 2010 Murrumbidgee Upper Lake Ginninderra 3 Golden perch* 2011 Murrumbidgee Upper Lake Ginninderra 2 Golden perch* 2011 Murrumbidgee Upper Yerrabi Pond 1 Macquarie perch 2008 Lachlan Upper Abercrombie River, Lachlan River 29 Macquarie perch 2009 Yarra 2 Wonga Park, Jumping Creek 48 Murray cod 2007 Lachlan Lower Euabalong 40 Murray cod 2008 Murray Lower Colligen Creek., Edwards River, Murray River, Wakool River, Niemur River, Mulwala Canal 102 Murray cod 2008 Murray Upper Clarkes Reserve 3 Murray cod 2008 Darling Lower Minda, Menin Court, Cuthero Point, Whurlie 17 Murray cod 2008 Murrumbidgee Lower Hay boat ramp, Markey's Beach, Buckingbong 7 Murray cod 2008 Lachlan Lower Booligal Weir 4 Murray cod 2008 Macquarie Lower Dubbo boat ramp, Fred Firth boat ramp 4 Murray cod 2009 Namoi Lower 7 Murray cod 2009 Namoi Upper 5 Murray cod 2011 Murrumbidgee Upper Lake Ginninderra 10 Murray cod 2011 Murrumbidgee Upper Yerrabi Pond 10 Redfin perch 2008 Murray Lower Mulwala Canal 42 Redfin perch 2008 Lachlan Upper Reidsdale, Little Narraway 5 Redfin perch 2010 Murrumbidgee Upper Lake Ginninderra 8 Redfin perch 2011 Murrumbidgee Upper Lake Ginninderra 14 Redfin perch 2011 Murrumbidgee Upper Yerrabi Pond 21 Silver perch 2007 Murrumbidgee Lower NFC (Buckingbong) 32 Silver perch 2008 Murray Lower Colligen Creek, Edwards River, Murray River, Wakool River, Niemur River 23 Trout cod 2008 Murrumbidgee Upper ACT 4 TOTAL Upper (>400 m) and lower (<400 m) zones were designated based on altitude from Lintermans (2007); * tested with antisera against Redfin perch, Murray cod and Silver perch no positive control available; 2 not in Murray-Darling Basin 57

58 Table Summary of fish tissue collected for EHNV testing based on species, year and catchment from 2007 to 2011 Species Collection Year Compartment Zone 1 Site Locations Number of fish VI Collected Tested Positive Australian bass 2009 Hunter 2 Upper Glenbawn Dam River blackfish 2008 Murray Lower Mulwala Canal River blackfish 2008 Murrumbidgee Lower Tarcutta Creek 9 9 Brown trout 2009 Macquarie Upper Lake Oberon 7 7 Brown trout 2009 Snowy 2 Upper Swamp/Hughes Creek, Thredbo River (near Gaden Hatchery) Common carp 2009 Murray Lower Yarrawonga to Tocumwal 1 1 Flat-headed gudgeon 2009 Murray Lower Mulwala Canal Flat-headed gudgeon 2009 Murrumbidgee Upper Blowering Dam 1 1 Climbing galaxias 2008 Murray Upper Geehi River 1 1 Mountain galaxias 2008 Murray Upper Geehi River 5 5 Mountain galaxias 2008 Murray Upper Upper Mannus Creek Mountain galaxias 2008 Murrumbidgee Upper Pierces Creek, ACT Mountain galaxias 2009 Macquarie Upper Winburndale Rivulet Eastern mosquitofish 2008 Murrumbidgee Upper Cotter River, ACT Eastern mosquitofish 2011 Murrumbidgee Upper Yerrabi Pond 1 1 Golden perch 2008 Murrumbidgee Upper Blowering Dam Golden perch 2008 Murrumbidgee Upper Burrinjuck Dam Golden perch 2008 Murrumbidgee Upper ACT 7 7 Golden perch 2009 Murrumbidgee Upper Burrinjuck Dam Golden perch 2009 Gwydir Upper Copeton Dam Golden perch 2009 Murray Lower Yarrawonga to Tocumwal 3 3 Golden perch 2009 Unknown Victoria 2 2 Golden perch 2010 Gwydir Upper Copeton Dam 8 8 Macquarie perch 2009 Nepean 2 Upper Cataract Dam Mountain galaxias 2009 Lachlan Upper Retreat River Mountain galaxias 2009 Lachlan Upper Flyers Creek Murray cod 2007 Murrumbidgee Upper Burrinjuck Dam

59 Species Collection Year Compartment Zone 1 Site Locations Number of fish VI Collected Tested Positive Murray cod 2007 Murrumbidgee Lower Buckingbong 3 3 Murray cod 2008 Murray Lower Mulwala Canal Murray cod 2008 Murrumbidgee Upper Blowering Dam Murray cod 2008 Murrumbidgee Upper Burrinjuck Dam Murray cod 2009 Murrumbidgee Upper Burrinjuck Dam Murray cod 2009 Murray Lower Yarrawonga to Tocumwal, Lake Mulwala to Ovens River, Lake Hume to Lake Mulwala Murray cod 2009 Murrumbidgee Lower Biagee 1 1 Murray cod 2009 Namoi Lower East Yarral, Miloo 3 3 Murray cod Unknown Lachlan 1 1 Murray cod Unknown Unknown Murray cod 2009 Gwydir Upper Copeton Dam Murray cod 2010 Murray Lower Yarrawonga 7 7 Murray cod 2010 Murrumbidgee Lower Narranderra Oriental weatherloach 2009 Hawkesbury 2 Lower Little River 7 7 Southern pygmy perch 2009 Murray Upper Coppabella Creek Rainbow trout 2008 Murrumbidgee Upper Cotter River and Condor Creek, ACT Rainbow trout 2009 Lachlan Upper Retreat River, Boree Creek 5 5 Rainbow trout 2009 Macquarie Upper Lake Oberon, Turon River, Campbells River, Duckmaloi River Rainbow trout 2009 Snowy 2 Upper Gang Gang Creek Rainbow trout 2009 Namoi Upper McDonald River 4 4 Redfin perch 2007 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2008 Goulburn Lower Tahbilk Lagoon Redfin perch 2008 Lachlan Upper Reidsdale, Little Narraway 5 5 Redfin perch 2008 Lachlan Upper Boorowa River site Redfin perch 2008 Ovens (Murray) Lower Lake Buffalo, VIC Redfin perch 2008 Murray Lower Mulwala Canal Redfin perch 2008 Murrumbidgee Upper Cotter River and Murrumbidgee River ACT Redfin perch 2008 Murrumbidgee Upper Burrinjuck Dam

60 Species Number of fish Collection Compartment Zone 1 Site Locations Year VI Collected Tested Positive Redfin perch 2008 Murrumbidgee Upper Lake Ginnindera, ACT Redfin perch 2008 Murrumbidgee Lower Columbo Creek 6 6 Redfin perch 2008 Unknown Redfin perch 2009 Broken Redfin perch 2009 Gwydir Upper Copeton Dam Redfin perch 2009 Goulburn Seven's Creek Redfin perch 2009 Lachlan Upper Boorowa River, Belubula River, Reidsdale, Narrawa Bridge, Abercrombie River Redfin perch 2009 Macquarie Upper Fish River, Campbells River Redfin perch 2009 Murray Lower Neds Anabranch, Yarrawonga to Tocumwal, Lake Victoria, Mulwala Canal Redfin perch 2009 Murrumbidgee Upper Cotter Dam 2 2 Redfin perch 2009 Murrumbidgee Upper Cotter River 1 1 Redfin perch 2009 Murrumbidgee Upper Molonglo River 7 7 Redfin perch 2009 Murrumbidgee Upper Yerrabi Pond 2 2 Redfin perch 2009 Murrumbidgee Upper Point Hut Crossing 1 1 Redfin perch 2009 Murrumbidgee Upper Googong Reservoir Redfin perch 2009 Murrumbidgee Upper Lake Ginnindera Redfin perch 2009 Murrumbidgee Upper Blowering Dam Redfin perch 2009 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2009 Murrumbidgee Lower Bethungra Dam 6 6 Redfin perch 2009 Murrumbidgee Lower Columbo Creek, Hay, Maude 9 9 Redfin perch 2009 Snowy Upper Bombala 2 2 Redfin perch 2009 Yarra 2 Wonga Park, Jumping Creek Redfin perch 2010 Gwydir Upper Copeton Dam Redfin perch 2010 Macquarie Lower Dubbo Redfin perch 2010 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2010 Murrumbidgee Upper Lake Ginninderra Redfin perch 2011 Murrumbidgee Upper Yerrabi Pond Redfin perch 2011 Murrumbidgee Upper Lake Ginninderra

61 Species Collection Year Compartment Zone 1 Site Locations Number of fish VI Collected Tested Positive Roach 2009 Yarra 2 Wonga Park 1 1 Silver perch 2007 Murrumbidgee Upper Burrinjuck Dam 4 4 Silver perch 2009 Gwydir Upper Copeton Dam 4 4 Silver perch 2009 Murray Lower Yarrawonga to Tocumwal 7 7 Silver perch 2009 Gwydir Upper Copeton Dam Silver perch 2010 Gwydir Upper Copeton Dam 4 4 Trout cod 2009 Murray Lower Yarrawonga to Tocumwal Trout cod 2008 Murrumbidgee Lower Buckingboing boat ramp 1 1 TOTAL Upper (>400 m) and lower (<400 m) zones were designated based on altitude from Lintermans (2007); 2 not in Murray-Darling Basin 61

62 Table Estimated true prevalence of EHNV based on testing tissue and serum samples, in various species of finfish. Where sample size was > 30 per population, and the prevalence which results indicate was not exceeded for population sizes of 10,000 and 500. A population was defined as a group of fish restricted by species, year of collection, catchment, zone, and impoundment. Species Collection Year Population Total no. No. of Positive Compartment Zone Site restriction samples Point estimate Prevalence % 95% lower confidence limit 95% upper confidence limit Prevalence level not exceeded with >95% confidence Population of 10,000 Prevalence level not exceeded with >95% confidence Population of 500 Australian bass 2009 Hunter 2 Upper Blackfish 2008 Murray Lower Brown trout 2009 Snowy 2 Upper Golden perch 2009 Gwydir Upper Copeton dam Macquarie perch 2009 Yarra Mountain galaxias 2009 Macquarie Upper Mountain galaxias 2008 Murrumbidgee Upper Mountain galaxias 2009 Lachlan Upper Mountain galaxias 2008 Murray Upper Eastern mosquitofish 2008 Murrumbidgee Upper Murray cod 2007 Lachlan Lower >10 10 Murray cod 2008 Murray Lower Murray cod 2008 Murrumbidgee Upper Blowering dam Murray cod 2009 Murrumbidgee Upper Burrinjuck Dam Rainbow trout 2008 Murrumbidgee Upper Rainbow trout 2009 Snowy 2 Upper Redfin perch 2009 Broken Redfin perch 2009 Goulburn Redfin perch 2010 Gwydir Upper Copeton dam Redfin perch 2008 Ovens (Murray) Lower Lake Buffalo Redfin perch 2010 Macquarie Lower Redfin perch 2008 Murray Lower Redfin perch 2008 Murray Lower Redfin perch 2009 Murray Lower Redfin perch 2009 Murrumbidgee Upper Blowering Dam na Na 62

63 Redfin perch 2007 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2008 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2009 Murrumbidgee Upper Burrinjuck Dam Redfin perch 2009 Murrumbidgee Upper Googong Reservoir Redfin perch 2008 Murrumbidgee Upper Lake Ginnindera na Na Redfin perch 2009 Murrumbidgee Upper Lake Ginnindera Redfin perch 2010 Murrumbidgee Upper Lake Ginnindera na na Redfin perch 2011 Murrumbidgee Upper Lake Ginnindera Redfin perch 2011 Murrumbidgee Upper Yerrabi pond Silver perch 2007 Murrumbidgee Lower >10 >10 Silver perch 2009 Gwydir Upper Copeton dam Southern pygmy perch 2009 Murray Upper na, not applicable; 1 serum tested by ELISA; remaining tests were by virus isolation; 2 not in Murray-Darling Basin 63

64 12. Development of a blood test for EHNV An important aim of this project was to develop a new test that could be applied to threatened species, so that one did not require fish to be killed to obtain samples. Blood samples can be collected from fish without harming them, so the approach adopted was to develop a serological test in which antibodies are detected. The particular serological assay to be developed is the enzyme-linked immunosorbent assay (ELISA). While the immediate target of this research and development is to provide a means of revealing past exposure to EHNV, the assay can be modified to detect antibodies to a wide range of pathogens, and may be further adapted for general health assessment purposes. Serological assays such as ELISA are formatted to measure the immunoglobulins (antibodies) that are produced by an animal following contact with a foreign substance known as an antigen. In the context of this project the antigen is the pathogen EHNV. Immunoglobulins are long-lived molecules which remain in circulation in blood for months or years. Therefore their detection in blood is a sign that the animal has been exposed to the pathogen (Figure 12.1). Thus serological assays can be powerful tools for pathogen surveillance in animal populations. It is important to note that the discipline of serology as applied to finfish has received little research attention and so there have been very few practical applications anywhere in the world. This is quite surprising because all vertebrates possess the genetic code required to produce immunoglobulins in response to exposure to foreign antigens. Figure The principle of the ELISA blood test for EHNV 64

65 To the authors knowledge this is the first work of this kind that has been attempted in native freshwater fish in Australia. However, the concept to develop a serological assay for ranavirus was previously applied and used successfully in pilot studies in cane toads, Redfin perch and Rainbow trout in Australia (Whittington, 1993; Whittington et al., 1997; Whittington et al., 1994; Whittington et al., 1999; Whittington and Speare, 1996). Based on these earlier studies several factors were identified as being of utmost importance to provide reliable methods for native fish: ensuring that immunoglobulin obtained from fish was highly purified; ensuring that reagents developed to detect immunoglobulins of fish were specific for immunoglobulins, and; ensuring that antibody responses detected in fish against EHNV were specific for EHNV. While the success of earlier work gave confidence that the present project would be successful, some vertebrate species are idiosyncratic in their biochemistry and physiology, therefore a careful approach was essential Methods The strategy for development and application of the ELISA was as follows: 1. Purification of immunoglobulins from the blood of fish 2. Preparation of antibody reagents in rabbits and sheep that could be used to detect immunoglobulins of fish when incorporated into an ELISA, and examination of the specificity of these mammalian reagents for the fish species under examination 3. Evaluation of the titre and specificity of the mammalian antisera for each species of fish 4. Production of positive and negative control blood samples for each species of fish 5. Optimisation of an ELISA for each species of fish to detect immunoglobulin against EHNV in serum 6. Evaluation of blood samples collected from wild fish in the Basin In order to determine whether assays developed for Redfin perch, Silver perch, Murray cod and Macquarie perch might have broader applicability in related and unrelated fish species in the Basin, up to three other species were included in steps 1 to 3: Australian bass, Barramundi and southern bluefin tuna. These species provided wide diversity across families of fish. 1. Purification of immunoglobulins from the blood of fish Source of fish. Serum was obtained from Redfin perch, Australian bass, Silver perch, Murray cod, Macquarie perch, Barramundi, and southern blue fin tuna. Fish were sourced from a variety of locations, depending on the geographical distribution of the species (Table 12.1). Fish were anaesthetised using clove oil or benzocaine and blood was collected from the caudal vein using gauge needles and a syringe (1-5 ml). Blood was allowed to clot overnight at room temperature (RT), and then centrifuged at 2000 g for 20 min. Serum was harvested and stored at -20ºC until used. Serum samples from 8 to 71 individual fish within a species were pooled to obtain sufficient material for purification. 65

66 Table Source of fish used to obtain blood for purification of immunoglobulins Order Family Latin name Common name Source of fish Wild/captive/farmed Perciformes Percidae Perca fluviatilis Redfin perch Perciformes Percichthyidae Perciforme Terapontidae Macquaria novemaculeata Bidyanus bidyanus Mulwala Canal, lower Murrumbidgee River NSW Wild Australian bass Grafton, NSW Farmed Silver perch Perciformes Latidae Lates calcarifer Barramundi Perciformes Percichthyidae Perciformes Percichthyidae Perciformes Scombridae Maccullochella peelii peelii Macquaria australasica Thunnus maccoyii Murray cod Murrumbidgee River, John Lake Centre Narrandera, NSW Darwin Aquaculture Centre, NT Mulwala Canal, lower Murray River NSW Captive Captive Wild Macquarie perch Abercrombie River NSW Wild Southern blue fin tuna Port Lincoln, SA Wild farmed Purification of immunoglobulin from fish serum. Purification of serum immunoglobulin was carried out using methods based on those optimised for Redfin perch (Whittington, 1993). Assessment of the purity of immunoglobulin. Proteins were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using the standard buffer system (Laemmli, 1970). Gels were stained with Coomassie brilliant blue. The molecular weight (MW) of the heavy chain (HC) and light chain (LC) components was estimated for each species using a standard curve constructed from protein standards. Measurement of protein concentration. A Bradford assay kit (BioRad) (Bradford, 1976) was used to determine the protein concentration of purified immunoglobulin using bovine IgG as the standard, according to the manufacturer s instructions. 2. Preparation of antibody reagents in rabbits and sheep New Zealand white rabbits and Merino sheep were housed in pens and at pasture, respectively, at the University of Sydney at Camden NSW. Two rabbits and two sheep were immunised by intramuscular injection with purified fish immunoglobulin. There were 12 rabbits and 12 sheep in total. Rabbits and sheep were boosted at 28 days by injection of the same preparation. Animals were euthanased at day 42. Blood was collected from the ear vein of the rabbits and the jugular vein of the sheep at day 0 (negative control) and then again at day 42. It was left to clot at room temperature for 2 hours before centrifuging at 2000 g to enable collection of the serum. Serum samples were stored at -20 C. Aliquots of serum were freeze dried and stored at 4 C and also 66

67 diluted 1:10 in Tris saline solution (25 mm Tris, 150 mm NaCl ph 7.4) containing 50% v/v glycerol and 0.02% v/v merthiolate (TSGM) and stored at -20 C. Rabbits and sheep were not immunised with purified southern blue fin tuna immunoglobulin. 3. Evaluation of the potency and specificity of the mammalian antisera for each species of fish Sheep and rabbit pre-immune and immune sera were evaluated in ELISA. Pre-immune serum was added at a final dilution of 1:1250. Immune sera were added in doubling dilutions from 1:1250 to 1:256 x The potency was defined as the optical density (OD). 4. Production of positive and negative control blood samples for each species of fish In order to produce known negative and positive control sera for relevant fish species it was necessary to directly expose fish to killed EHNV and allow sufficient time for an immune response. A reference isolate of EHNV (86/8774) was propagated in BF-2 cell monolayers in cell factories to produce a large quantity of infectious virus which was separated from cell culture components by differential and density gradient centrifugation as described (Whittington and Deece, 2004). Purity of the viral protein was confirmed by SDS-PAGE, electron microscopy and immunoblotting using antisera prepared against the cell culture antigens (Steiner et al., 1991). A stock of purified EHNV was used to prepare a vaccine; virus was double heat inactivated. Each fish was anaesthetised in benzocaine 40 mg/l, and injected intraperitoneally with two doses of vaccine, with a 4 to 8 week interval between these doses. Blood was collected from each fish prior to immunization, at the time of revaccination and then at intervals of at least two weeks. Serum was harvested as described above and was stored diluted 1:10 in TSGM at -20 C prior to analysis by ELISA. Two fish of each of the following species were immunized: Redfin perch, Silver perch, Murray cod and Macquarie perch. One Silver perch, Murray cod and Macquarie perch died following immunisation so that data are available for only one fish in these species. Antibodies in each fish specific for EHNV were measured using an indirect antibody capture ELISA (see below). 5. Optimisation of an ELISA for each species of finfish to detect immunoglobulin against EHNV in serum A basic format for detection of antibodies in fish serum that were specific for EHNV was based on published methods (Whittington et al., 1994) and was conducted as follows. All reagents were added to wells in volumes of 50 µl and all incubations were at room temperature unless otherwise stated. A plain 96-well ELISA plate (Immulon) was coated with a capture antibody, being affinity purified rabbit-anti-ehnv antibody (batch V038) at a dilution of 1:12,800 in borate coating buffer (0.1 M boric acid, M Na 2 B 4 O 7.10H 2 O, M NaCl), and was incubated for 90 min. The plate was washed 5 times in reverse osmosis purified water with 0.05% v/v Tween 20 using a plate washer (Wellwash , Thermo Electron Corporation). EHNV antigen, consisting of cell culture supernatant batch V034 with TCID 50 of 10 7 /ml stored at -20 C, was inactivated immediately prior to use by heating at 65 C for 15 min and cooling to room temperature. 67

68 Antigen was added to pairs of alternate columns on each plate to enable all sera to be tested in duplicate in wells with and without EHNV antigen to confirm the specificity of the response, and the plate was incubated overnight at 4 C. After washing as above, remaining binding sites were blocked by adding 1% w/v gelatin in phosphate buffered saline ph 7.2 with 0.05% v/v Tween 20 (PBST) solution and incubated for 30 mins. After washing, fish serum diluted in 0.01% w/v gelatin in PBST (PBSTG) was added and incubated for 90 min. After washing, a sheep anti-fish immunoglobulin reagent appropriate for the particular species of fish being tested was diluted in PBSTG, added and incubated for 90 min. Donkey anti-sheep-horseradish peroxidase (HRP) conjugate (KPL) batch R893 was diluted in PBSTG, added to wells and incubated for 90 min. After washing, 1 mm 2,2 azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) in buffer (0.1 M citric acid, 0.1 M Na 2 HPO 4, ph4.2) was activated with 2.5 mm H 2 O 2 and added to each well. The plate was incubated for 20 min before the reaction was stopped with 25 µl per well of stop solution (0.01% w/v NaN 3 in 0.1 M citric acid). The optical density (OD) was read at 405 nm with a microplate reader (Multiskan Ascent, Thermo Electron Corporation). Signal (S) was defined to be the mean OD for duplicate wells with EHNV antigen, while noise (N) was defined to be the mean OD for duplicate wells without EHNV antigen. Optimal concentrations of reagents were determined by checkerboard titration for each species of fish. A variation of the protocol was used for Macquarie perch serum to reduce putative non-specific reactivity. The block with gelatin solution preceded incubation with EHNV antigen, and incubations were for 60 instead of 90 min. The basic assay format is shown in Figure Figure Format of the ELISA to detect antibodies against EHNV in fish serum Positive ELISA results were defined to be those where OD > 0.4 and signal:noise ratio (S/N) > 2. Additionally, and only for Macquarie perch, where OD was greater than 0.6, a S/N > 1.5 was acceptable and was used to increase sensitivity. 68

69 6. Evaluation of blood samples collected from wild fish in the Basin The panel of blood samples collected from wild fish in the Basin was tested on a batch basis using the relevant optimised protocol for each species Findings 1. Purification of immunoglobulins from the blood of fish Isolation of immunoglobulin was confirmed by SDS-PAGE. Under reducing conditions the native immunoglobulin molecules dissociated into bands consistent with heavy chain (HC) and light chain (LC). The purity of the immunoglobulin preparations was evident in the Coomassie blue stained SDS-PAGE gels. In each case the HC and LC bands were the predominant bands. There were very few non-specific bands observed, but some were present in low concentrations at a MW higher than that of the HC. Purity was estimated to be > 95% for each species based on visual assessment of the relative amount of protein in bands other than the HC and LC, in comparison to the bovine IgM standard (Sigma) which was specified to be 95% pure (Figure 12.3). Murray cod had the highest concentration of immunoglobulin per ml of serum (1.9 mg/ml) while Silver perch and Macquarie perch had the lowest (0.9 mg/ml). The total yields of purified immunoglobulin ranged from 2.3 mg from Macquarie perch to 12.6 mg from Murray cod; these yields were influenced by the variable amount of serum that was available to be processed (Table 12.2). Table Concentration and total yield of purified immunoglobulin from each species of fish Total Total amount of Concentration of Species No. of fish amount of immunoglobulin immunoglobulin in in pool serum used purified fish serum (ml) (mg) (mg/ml) Redfin perch Australian bass Silver perch Barramundi Murray cod Macquarie perch Southern blue fin tuna unknown

70 SDS-PAGE was used to determine the molecular weight of immunoglobulin HC and LC for each species. Concentrated fish immunoglobulin from each species were electrophoresed on the same gel with bovine IgM as a standard (Figure 12.3). Molecular weight was estimated from a standard curve. Assuming a tetrameric structure for the immunoglobulin of each species, i.e. each immunoglobulin molecule is H 8 L 8, the estimated MW of the native molecule was calculated using the observed molecular weights of the HC and LC (Table 12.3). Table Estimated molecular weight (MW) of purified immunoglobulin based on observed molecular weights of the heavy chain (HC) and light chain (LC) components and assuming a tetrameric structure Species MW HC MW LC MW of tetrameric (kda) (kda) immunoglobulin (kda) Redfin perch Australian bass Silver perch Barramundi Murray cod Macquarie perch Southern blue fin tuna Bovine IgM Figure Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified immunoglobulin. M, molecular weight markers; RP, Redfin perch; AB, Australian bass; SP, Silver perch; B, Barramundi; MC, Murray cod; MP, Macquarie perch; SBT, southern blue fin tuna; BOV, bovine IgM standard, 95% purity 70