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Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission.The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. As a result, the risks of further outbreaks and the spread of abalone viral ganglioneuritis will be minimised, and all industry sectors will be better prepared to respond to and control and manage the negative impacts associated with the further emergence of herpes-like virus.
From the perspective of the Australian abalone aquaculture industry, the increased attention to aquatic bio-security that is required to implement their Code of Practice will result in enhanced production and sustainability because it will assist in minimising the risks of any further emergence of abalone viral ganglioneuritis.
The disease was subsequently found in marine waters adjacent to one of the infected farms resulting in mortalities in wild abalone populations. The outbreak of the disease sparked concern about possible long-term consequences for the abalone industry not only in Victoria, but also in the rest of Australia.
There was a clear need to develop specific management strategies which incorporated disease monitoring, detection, response and control measures in both farmed and wild stocks.This project aims to develop Codes of Practice to standardise and improve the bio-security measures currently in place in aquaculture farms and in commercial harvesting operations and processors.
For completeness, recreational divers and fishers are also included.In developing these Codes of Practice, a robust and defensible process was undertaken to identify the key issues that need to be addressed by the wild harvest, recreational, aquaculture and processing sectors.
These issues were assessed and prioritised at a Risk Assessment Workshop conducted by an expert panel assembled from all Australian states with an abalone industry.
The disease was also apparent in the marine waters adjacent to one of the infected farms and led to mortalities in wild abalone populations.
The outbreak of disease sparked concern about possible long- term consequences for the abalone industry not only in Victoria, but also in the rest of Australia. Investigations were conducted into the likely source and mode of transmission of the virus. A key conclusion was that Codes of Practice should be developed to standardise and improve the bio-security measures currently in place in aquaculture farms, commercial harvesting operations and processors.
This document reports the process through which the Codes of Practice were developed as part of FRDC Project No.
Although the Victorian industry will be used as a case study for the development of the Codes of Practice, the method used and key elements of the Codes of Practice are equally applicable to other states with an abalone industry.Abalone industry in AustraliaAbalone (family Haliotidae) are marine gastropods found on rocky reefs in temperate areas of Australia.
The abalone fishery comprises around 11% (in 2004-05) of the total value of wild fisheries production in Australia (ABARE 2006).
It also represents 55% (from 2001 figures) of the world wild abalone catch (Love and Langenkamp 2003).
There has been a dramatic decline in global abalone production over the last three decades due to overfishing and changes in environmental and ecological dynamics (Gorfine 2002). Australia has one of the last sustainable wild abalone fisheries in the world.There are thirteen species of abalone found in Australian waters, of which four are commercially harvested. Tasmania has the most licence holders and the largest commercial fishery in both tonnage and value, followed by Victoria, then South Australia. Fisheries in Western Australia and NSW are relatively small compared with the other states.In recent years, an abalone aquaculture industry has developed in several Australian states, notably Victoria, Tasmania and South Australia (Table 2). Processors also add value to the product through canning, or the production of other products (e.g. In 2004-05, a total of 4,004 tonnes of abalone were exported with a value of $263 million (ABARE 2006).
This included 2,032 tonnes of fresh, chilled or frozen abalone and 1,972 tonnes of canned abalone (ABARE 2006).As the mollusc aquaculture industry has grown in volume and importance world-wide, the incidence of disease in farming systems has also increased (Elston 1984). There are a number of factors behind this observation, including that the animals are subject to closer scrutiny when held in aquaculture farms and so diseases can be more readily identified and described. The recent outbreak of abalone viral ganglioneuritis in abalone in south-western Victoria has highlighted that Australia is not immune to such disease incursions into developing aquaculture industries.The abalone viral ganglioneuritis outbreak in VictoriaIn late 2005 and early 2006 mass mortality events occurred in abalone at several aquaculture farms in south-west and central Victoria. By May 2006, mortalities were being reported from wild abalone in the area (Hardy-Smith 2006). Table 3 summarises the key events in the outbreak of abalone viral ganglioneuritis in Victoria.Mortalities were first observed in two on-shore abalone farms (Farms 1 and 2) and one sea cage farm (Farm 3). The alarm was raised in early January 2006 and samples of moribund animals were submitted for diagnostic testing.
The disease was diagnosed relatively quickly and was identified using standard histology and transmission electron microscopy (TEM) techniques.
The virus appears to be confined to the nervous system and causes inflammation of the nerves. For this reason the disease was initially given the name "ganglioneuritis" and was made a notifiable disease under the Livestock Disease Act 1994. In 2007, the ConsultativeCommittee on Exotic Animal Disease (CCEAD) decided that abalone herpes-like virus and the disease it causes should now be known as "abalone viral ganglioneuritis".Once the virus was identified as the likely cause of the mortalities, voluntary restrictions were placed on movement of animals from the affected farms to other farms.
Clinically healthy animals were sent to processing plants, with the approval of the Chief Veterinary Officer (CVO). On the farms that had mortalities from the virus, stock was culled and facilities were decontaminated. After a period of time sentinel abalone were then placed into the facilities and monitored. In November 2006, both of the land- based farms (Farm 1 and Farm 2) applied to DPI for (and were granted) permission to re-stock with abalone.Infected abalone were first observed in the wild in the vicinity of Farm 2 (located west of Port Fairy) on 7 May 2006.
By the end of May it was reported that sites east and west of the initial site were infected. The aim was to minimise the risk of human activity transferring the disease to unaffected abalone populations elsewhere in the state.
An Incident Control Team was established and a structured surveillance program commenced to monitor wild abalone populations within and adjacent to the Control Area, as well as at processing plants.Professional divers were encouraged to submit suspected cases for diagnosis. To ensure that industry and community were kept informed of the outbreak, a communication program involving regular meetings with industry and regular dissemination of updates was established.
Figure 1 shows the coastal bays between Warrnambool and Port Fairy that were surveyed in the search for diseased animals.
Based on mortality and presence of lesions, abalone viral ganiglioneuritis was no longer detectable in areas where it had previously been present, notably in the closed area (marked in red in Figure 2). However, since tests that detect the virus if it is latent or sub-clinical are not available it is not known whether the virus is still present. Some mortalities were observed in translocated stock during this period.December 19th 2005aquaculture farms. No further signs of the disease.Mid January 2006Inflammation of the nerves (abalone viral ganglioneuritis) identified by histopathological examination and herpes like virus identified within the nerve cells of abalone dying at Farm 1.
Lesion and virus has never been seen before in abalone in Australia.March 17th 2006Unusual mortalities noted at Farm 2.
Mortalities rapidly escalated and spread to other tanks.April 24th 2006Sick and dead abalone submitted from Farm 4.

It should also include the implementation of routine health management procedures and a system for exclusion of the virus. Health management strategies and Codes of Practice, for both farmed and wild abalone harvest sectors and processing plants, must be developed to improve the productivity of the farmed sector and protect the health of wild populations of abalone.
This was conducted by the project team prior to the Risk Assessment Workshop;Risk Assessment Workshop.
A number of international reviews have been conducted on diseases associated with abalone (Bower et al.
As part of this project, a national survey of diseases of commercially important abalone species was undertaken to support trade and translocation issues.
Particularly Vibrio harveyi which has been a problem in some farms in Tasmania and South Australia (Reuter and McOrist 1999; Handlinger et al.
It is recognised as a disease of wild abalone and has been brought into farms through the translocation of broodstock from infected sites.At the time of this survey, which was conducted in 2003 and 2004, no viruses had been recorded in Australian abalone. In order to put the 2006 Victorian outbreak of abalone viral ganglioneuritis in context, a brief review of current knowledge of viruses in molluscs follows.Mollusc virusesViruses are obligate intracellular parasites that depend entirely on a living host cell for essential elements of replication (Lees 2000).
Viruses are the most numerous organisms in the marine environment (Fuhrman 1999) and have caused mass mortalities in a number of commercially important species around the world. Viral pathogens are often highly infectious and easily transmissible (Renault and Novoa 2004) and can cause significant losses of stocks and income in wild and farmed environments.In general, there is limited information on mollusc viruses as research efforts have focused on viruses that are important to human health. In addition, these viruses cannot be grown in cell culture making it difficult to study them and there are other higher priority viruses on which funding has been spent e.g. Since many bivalves are filter feeders, they may bio-accumulate human viruses (such as hepatitis A and viruses associated with gastrointestinal diseases) especially in areas contaminated with sewage (Meyers 1984; Lees 2000).
Indeed, there have been a significant number of food poisoning cases in Australia involving viruses from shellfish (Sumner and Ross 2002).Several irido-like viruses have been reported in bivalves, notably oysters, in different countries around the world. Over 100 different herpes viruses have been described from vertebrates alone (Roizman and Baines 1991); the most notable human herpes virus is herpes simplex type 1 (HSV-1). The herpes viruses fall into three major groups - one associated with mammals, birds and reptiles, one with amphibians and bony fish, and a third with invertebrates (Davison 2002; Davison et al. Ahne (1994) argued that most viruses that infect aquatic poikilothermic animals, such as molluscs, do not differ in morphology and biochemical composition to those infecting terrestrial homoiothermic animals.A summary of herpes and herpes-like viruses from aquatic invertebrates is presented in Table 4.
These include Callinectus sapidus, Carcinus maenas, Carcinus mediterraneus and Macropipus depurator (Johnson 1976; Bower 1995), blue crab (Paralithodes platypus) and golden king crab (Lithodes aequispina) (Sparks and Morado 1997), and Rhithropanopeus harrisii (Payen and Bonami 1979) . Ahne (1994) indicated that herpes viruses are highly pathogenic to blue crabs and blue king crabs.In molluscs, herpes-like viruses have been reported from American oysters (Crassostrea virginica) (Farley et al. 1972), flat oysters (Ostea edulis) (Alderman 1980), and Pacific oysters (Crassostrea gigas) (Hine et al. This syndrome continues to cause high mortality in Pacific oyster and other bivalves around the world (Japan, USA, Canada, China and France).
While a multifactorial aetiology is suspected, the herpes virus (OsHV-1) has been mostly detected in juvenile mortality events when temperatures are high and when animals are in a relatively unstable physiological condition due to gametogenesis and spawning (ICES, 2005).Sporadic high mortalities of larval Pacific oysters associated with OsHV-1 occur each year during summer in European hatcheries (Renault et al. The virus infects connective tissue and mantle epithelium; the velum becomes noticeably less extended and lesions appear on the velar and mantle (Nicolas et al. Larvae show reduced feeding and swimming activities and eventually mortality rates of up to 100% can rapidly occur (Hine et al.
1994b).The presence of OsHV-1 has been demonstrated in asymptomatic adult Pacific oysters (Arzul et al. 2002), but adults appear to be less sensitive to infections and no abnormal mortalities have been reported (Renault and Novoa 2004). It has been suggested that the adults may act as a carrier or reservoir for the virus (Le Deuff et al. Indeed, vertical transmission studies on successive generations of Pacific oysters indicate that parents pass the virus to offspring (Barbosa-Solomieu et al. However it has also been suggested that infected females may transmit some kind of protection or resistance against viral infection to their offspring (Barbosa- Solomieu et al.
2005).Histologically, fibroblast-like cells in the connective tissue exhibit hypertrophied (enlarged) nuclei with marginated chromatin and an abnormal cytoplasmic basophilia. DNA sequence data demonstrated that the OsHV-1 is not closely related to herpes viruses in vertebrates (including fish), but that the herpes viruses of invertebrates form one of three major lineages of the herpes virus group. Specific polyclonal and monoclonal antibodies have been developed and immunoassays have been validated for detection and identification of OsHV-1 (Renault et al. Development of immunochemistry and ELISA tests for OsHV-1 will also be possible when cloned sequences, which enable the synthesis of recombinant virus proteins, become available (Renault and Novoa 2004). These tests may have some relevance to diagnosing abalone viral ganglioneuritis.Unlike vertebrate herpes viruses, which are generally confined to a single host species, OsHV-1 can infect several bivalve species and is probably responsible for all the infections observed in European bivalves species (Le Deuff et al. 2006).Abalone herpes and herpes-like virusesThe Australian abalone herpes-like virus (which caused the disease known as abalone viral ganglioneuritis) is not limited to one species. It is not known if the virus is already capable of (or will mutate to a form that is capable of) infecting other molluscan species in the region, in particular other commercially important species such as Pacific oysters (C. Prior to the outbreak of abalone viral ganglioneuritis, herpes viruses had been recorded in abalone in Taiwan.Herpes viruses of abalone in TaiwanThe abalone Haliotis diversicolor supertexta is the main gastropod farmed in Taiwan.
Early this century, the annual production value of the industry was estimated to be in excess of US$200 million (Chang et al.
In January 2003, high mortalities of abalone occurred among both sea and land farms in north-eastern Taiwan (Chang et al.
2005), though die-offs apparently first occurred two years prior to this event (Chiu Yu- Tzu 2003).
The impact of this outbreak is evident in production figures for famed abalone in Taiwan leading up to 2003 (Figure 3).Key features of the Taiwanese virus included (after Chang et al. High mortalities also occurred in 2004 at some previous virus-free farms where water temperature was 18°C or less in February (Pen Heng Chang, pers comm. 2006)Both juveniles and adults were affected, mortalities up to 80% and death within 3 days of onset of clinical signs. Causes necrosis and disorders of connective tissues of all organs (see Wang and Li 1997; Li et al.
2005 suggested the disease was an exotic pathogen, perhaps introduced with imported juvenile abalone. These include reducing stress factors in farmed stock, such as lower stocking densities, and prompt removal of mortalities from ponds. Quarantine is important as workers, equipment and tools may spread the virus to different farms.Other abalone virusesIn terms of viruses that cause mass mortalities in abalone, a number of serious cases have been reported from China's farmed abalone (Table 5). The authors noted that these two syndromes could be distinct diseases, but further information was required to make that determination.
The type 1 virus was present in the cytoplasm of haemocytes and connective tissues whereas types 2-4 were usually present in the cytoplasm of epithelial and connective cells of liver and intestines (Shi Zhengli and Handlinger 2004).

Infection with type 1 virus resulted in low activity, lost appetite, unresponsive to light, thin shell with the edge turned down and decreased growth rate. Virus type 2-4 is characterised by the secretion of mucus, loss of appetite and low activity, contracted feet and mantle, black and hardened feet.Amyotrophia (Epizootic fatal wasting disease) was first reported in the early 1980s from a number of culture facilities growing Japanese black abalone (H. Mass mortalities occurred in juveniles at temperatures of between 18-20°C (Otsu and Sasaki 1997). Infected abalone develop tumours and muscle atrophy in the mantle and foot, especially near the nerve trunk of the pedal ganglia and their transverse commissures. Laboratory experiments have shown that the disease can be horizontally transmitted through the water column (Nakatsugawa et al. Virus-like particles have been observed in the cytoplasm of cells near the nerve trunk (Otsu and Sasaki 1997) and haemocytes (Nakatsugawa et al. Virus-like particles from haemocytes were retrovirus-like in size and morphology (Nakatsugawa et al. Management focuses on avoiding the risk of introducing this pathogen to other culture facilities or natural stocks. Only animals certified to be free of infection should be considered for transplantation from areas where the disease occurs.
In addition, imported animals must be held in quarantine and assayed for cryptic or subclinical infections prior to release into the new environment (Bower 2001).Current knowledge on the Victorian abalone viral ganglioneuritisAbalone viral ganglioneuritis causes damage to the nerve tissues of abalone (the ganglion) and results in death in most cases.
It is known to affect greenlip, blacklip and hybrid abalone and it has been observed that young stock are more seriously affected than adult stock. The incubation period of the virus is between 2-14 days with significant mortalities occurring after 10-14 days. The presence of enlarged mouthparts in isolation is not sufficient to diagnose abalone viral ganglioneuritis and is not shown by all infected abalone. When clinically affected by the virus, the abalone are weak and are easily removed or fall off the substrate.
In tanks on aquaculture farms, where the abalone can be more closely monitored than animals in the wild, mortalities of 5% to 90% have occurred.Initial identificationAbalone viral ganglioneuritis was initially observed in infected abalone using light microscopy (Hardy-Smith 2006).
Samples of infected tissues were subsequently submitted to the Australian Animal Health Laboratory (AAHL) where Electron Microscopy was used to examine the specimens. Viral particles were visualised in pleuropedal ganglion and, morphologically, these particles resembled viruses from the family Herpesviridae (Hardy- Smith 2006).Transmission of the virusTrials at AAHL have shown that the virus is transmitted horizontally through the water column and does not require direct contact for transmission (Hardy-Smith 2006). The virus is highly pathogenic, killing all healthy experimental animals within six days of exposure to the virus (by injection and co- habitation). An experiment in which healthy animals were placed in water spiked with various dilutions of virus inoculum indicated that the virus remained infectious even after a 1 in 100 dilution. There is a marked decrease in virulance with dilution.Knowledge gapsIn September 2006, a series of workshops were held with international and Australian experts on aquatic animal diseases (Table 6) and other stakeholders in the abalone industry. These workshops were organised by the Victorian Department of Primary Industries and the commercial abalone harvesting sector in a process separate to this project. It is suspected that mucous cells can carry the virus, but it is not known how important mucus and physical strands of decaying tissue are in disease transmission. It is not currently possible to conduct research on which detergents or disinfectants are effective against this virus. The thermal tolerance range of the virus is unknown although it is known to survive freezing to - 80°C (M.
Once an animal is infected the disease can spread rapidly, particularly if there are dense populations of abalone.
EM is labour intensive, time consuming and can only focus on a very small piece of abalone tissue at a time. This is a field of pathology which specialises in the study of the structure of diseased tissue. If the animal is clinically infected with the virus then signs of the virus can be detected within the nerve cells (e.g. Using histopathology, the tissues will be examined under a light microscope by an experienced pathologist. However, if the animal is infected but ganglioneuritis has not developed or the virus is latent, then it cannot be detected using histopathology.
The development of more sensitive and specific tests will be required to learn more about the nature of abalone viral ganglioneuritis, and diagnosis of it in animals not exhibiting clinical signs of disease.Options for detection of the virus in abalone need to be rapid, cost effective and ideally non- destructive. PCR amplifies DNA, is very specific and very sensitive and has been used to investigate herpes virus infections in Pacific oyster larvae (Renault and Arzul, 2001).
However, because the test cannot determine whether the virus is viable or not, there is a risk of false positives. PCR needs to be done in conjunction with other testing methods such as in-situ hybridisation and immunohistochemistry.Culture agentSome viruses are maintained in the laboratory on cells lines of the host. Marine invertebrate cells are maintained in primary cultures with limited or no growth, surviving for periods ranging from a few hours to several months. Attempts to grow OsHV-1 in the laboratory on fish cell lines and a gastropod cell line have failed. There are gastropod cell lines, but it is uncertain if herpes virus can be cultured on these lines. Abalone viral ganglioneuritis virus so far cannot be grown on available fish cell lines at AAHL (M. Some examples of resistance include: Sydney rock oysters to QX disease and winter mortality (Nell and Perkins 2006), eastern oysters (Crassostrea virginica) to MSX (Allen 1998), and European flat oysters (Ostrea edulis) to Bonamia (Naciri-Graven et al. 1998).Selection of spawnersAnother potential control measure involves collecting and using virus-free broodstock based on the detection of the pathogen by PCR.
This has served as an effective control measure against vertical transmission of viral nervous necrosis (VNN) in Japanese parrotfish and striped jack and panaeid acute viremia (PAV) (Muroga 2001).Risk Assessment Process and WorkshopA Risk Assessment Workshop was convened to identify and prioritise the risks associated with the transfer of abalone viral ganglioneuritis between the abalone aquaculture industry, commercial fishing and processing sectors. A panel of experts and industry stakeholders from all over Australia was invited to participate (Appendix II). However, this could also incorporate other commercial fishing and recreational activities (e.g. There may also be some overlap with sea-cage farming, ranching and stock enhancement programs. It should also be noted that a live abalone infected with abalone viral ganglioneuritis may be constantly or intermittently secreting the virus particles (as compared to dead abalone, personnel or equipment where the defined quantity of virus will diminish over time).Movements of dead infected abalone, abalone by- products and wastes. This includes mortalities from abalone farms, shells and processing wastes (viscera etc).Personnel and equipment.

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