Development of Management Strategies for Herpes-like Virus Infection in Abalone

May 2009

FRDC Project No. 2006/243
ISSN 1329-7287
ISBN 978-1-74217-390-0

Author: Gavine, F., Ingram, B. and Doroudi, M.

This work is copyright. 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. It is a federal statutory authority jointly funded by the Australian Government and the fishing industry.

Principal Investigator: Dr Brett Ingram

Objectives:

To develop a "Code of Practice" by improving physical, chemical and biological measures of bio-security for abalone farms to prevent the introduction and spread of the virus.

1. To develop a "Code of Practice" for commercial divers to avoid the introduction and further spread of the virus in wild populations of abalone.
2. To develop a "practical bio-security program" for abalone processing plants.

Non technical summary:

Outcomes achieved to date:

The implementation of the Codes of Practice developed under this project will increase bio-security in all sectors of the abalone industry (wild-capture, recreational, aquaculture and processing). 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. In addition, the other Codes of Practice will assist in maintaining the favourable health status of Australia's wild fisheries through protocols that will lead to improved bio-security in the wild catch, in recreational diving and fishing, and in the processing sectors.

In January 2006, a previously unknown herpes-like virus was identified as being the most likely cause of mass mortalities of abalone (Haliotis spp.) in a number of aquaculture farms in south-west and central Victoria (Hardy-Smith, 2006). The disease caused by the virus was named 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 panel included aquatic animal health specialists, representatives from industry and Government officials.

The Risk Assessment Workshop identified the most important issues for each industry sector; control measures were developed, bearing in mind that knowledge of the virus was incomplete. The controls were subsequently developed (in consultation with industry) into Codes of Practice that are comprised of a series of Standard Operating Procedures.

The adoption and implementation of the Codes of Practice by industry will increase bio-security within the Australian abalone industry and minimise the risk of further outbreaks and the spread of abalone viral ganglioneuritis.

Keywords:

Abalone, abalone viral ganglioneuritis, ganglioneuritis, bio-security, aquatic animal health.

Table of Contents

Background

• Abalone industry in Australia
• The abalone viral ganglioneuritis outbreak in Victoria

Need

Objectives

Methods

• Literature search
• Risk assessment process and development of controls

Results/Discussion

• Literature review
  • Mollusc viruses
  • Herpes viruses
  • Abalone herpes and herpes-like viruses
• Current knowledge on the Victorian abalone viral ganglioneuritis
  • Diagnostic tools to identify the virus in the future
• Potential future mechanisms for control
  • Breeding for disease resistance
  • Selection of spawners
• Risk Assessment Process and Workshop
  • Pathways of viral transmission
  • Outcomes of the Risk Assessment Workshop
• Determining control measures for the three industry sectors
  • Wild abalone harvest sector
  • Abalone aquaculture sector
  • Abalone processing sector

Benefits and adoption

Further development

Conclusion

References

Appendix I: Summary of diseases and parasites reported from abalone

Appendix II: List of Participants

Appendix III: Risk analysis process

• Risk Ratings

Appendix IV: Risk ratings associated with spread of abalone viral ganglioneuritis

Background

In January 2006, a previously unknown herpes- like virus was identified as being the most likely cause of mass mortalities of abalone (Haliotis spp.) in a number of aquaculture farms in south- west and central Victoria (Hardy-Smith 2006). The disease caused by the virus was named abalone viral ganglioneuritis. 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. 2006/243 "Development of management strategies for herpes-like virus infection of abalone". 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 Australia

Abalone (family Haliotidae) are marine gastropods found on rocky reefs in temperate areas of Australia. Abalone form the basis of an important and valuable fishery in Australia, with a wild catch of 5,592 tonnes in 2004/05 worth around $220 million (ABARE 2006). 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. These are:

• blacklip abalone (Haliotis rubra Leach)
• greenlip abalone (H. laevigata Donovan)
• brownlip abalone (H. conicopora Péron)
• Roe's abalone (H. roei Gray).

To ensure that the resource is not over-exploited, the abalone fishery is carefully regulated and managed in all states. Key features of the abalone fishery in each state are shown in Table 1. 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). Other states have small, developing industries that have not yet reached commercially reportable quantities.

Table 1: Key features of commercial abalone fisheries production by state in 2004-05 (ABARE 2006).

Table 2: Production of farmed abalone by state (ABARE 2006).

The commercial harvesting and aquaculture sectors are supported by a vibrant abalone processing sector that prepares the abalone for sale to domestic or export markets. Processors also add value to the product through canning, or the production of other products (e.g. abalone sauce). Around 90% of abalone is sold to Asian markets. 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 Victoria

In 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). A previously unknown herpes-like virus was implicated as the cause. 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 Consultative

Committee 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. On 9 June 2006, a Control Area was declared for 60-days around Port Fairy. The aim was to minimise the risk of human activity transferring the disease to unaffected abalone populations elsewhere in the state. The declared Control Area was later replaced by a Fisheries Order. 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. Figure 2 shows the results of that monitoring up to January 2007. 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. In February 2007, abalone viral ganglioneuritis was also detected and confirmed in Portland Harbour west of this area.

Table 3: Herpes-virus outbreak in Victoria - summary of key events (Source: Hardy-Smith 2006, Abalone Virus - Scientific Forum, 21^st September 2006, Melbourne, and other sources)

Figure 1: Bays near to Warrnambool and Port Fairy in Western Victoria that were monitored for the presence of diseased abalone (courtesy of Fisheries Victoria).

Figure 2: Results of monitoring for the virus - to January 2007 (courtesy of Fisheries Victoria).

Need

In order to protect Australia's valuable abalone industry, there is a need to develop specific management strategies which incorporate disease monitoring, detection and response and control measures in both farmed and wild stocks. 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. To minimise the impact of the virus, the application of effective detection and exclusion (control) methods should also be incorporated into a workable bio-security plan.

Objectives

1. To develop a "Code of Practice" by improving physical, chemical and biological measures of bio-security for abalone farms to prevent the introduction and spread of the virus.
2. To develop a "Code of Practice" for commercial divers to avoid the introduction and further spread of the virus in wild populations of abalone.
3. To develop a "practical bio-security program" for abalone processing plants.

Methods

Literature search

A search of literature was undertaken to review current information on molluscan viruses, specifically herpes viruses and herpes-like viruses, and shellfish health management, particularly related to viruses. Databases searched included:

• Aquatic Sciences and fisheries Abstracts (ASFA)
• Scopus
• Agriculture and Natural resources (ANR-Index and ANR-research)
• Environment and Natural resources (as part of ANR)
• Elixir (DPIʹs Electronic Library Exchange for Information Resources)
• Current Contents (2006 only)
• Oceanic abstracts.

General internet searches were also conducted during the review. There were 65 records related to Haliotis in ANR, of which two referred directly to aspects of health, herpes virus and herpes-like virus of molluscs with a particular emphasis on abalone.

Risk assessment process and development of controls

The risk assessment process was conducted in the following stages:

• Identification of pathways of transmission, vehicles of transmission and key risk issues. This was conducted by the project team prior to the Risk Assessment Workshop;
• Risk Assessment Workshop. Experts from all over Australia were invited to participate on the panel. The Risk Assessment was conducted according to AS/NZ 4360:2004 Standard for Risk Management.

Results/Discussion

Literature review

The recent emergence of a world-wide abalone aquaculture industry has raised awareness of various infectious diseases that are present in both wild and cultured populations of abalone. A number of international reviews have been conducted on diseases associated with abalone (Bower et al. 1994; Hine 1996; Bower 2000; 2003).

In general, relatively few abalone diseases have been identified world-wide, but some authors have suggested that this is due to a lack of examination rather than absence of disease (Handlinger et al. 2006). It is expected that as the global aquaculture industry develops more diseases will emerge, particularly if commercially important species are introduced to new geographic areas (Renault 1996).

A synopsis of currently known diseases of abalone world-wide is contained in Appendix I.

The World Organisation for Animal Health (OIE) (oie.int) recognises seven mollusc diseases that are internationally notifiable, two of which are reported from abalone (Appendix I):

• Perkinsosis caused by the protozoan Perkinsus olsoni, and
• Withering syndrome due to Candidatus Xenohaliotis californiensis, a Rickettsia-like prokaryote.

Diseases of Australian abalone have been most recently reviewed and described in a comprehensive state-by-state survey undertaken through the Abalone Aquaculture Subprogram of the Fisheries Research and Development Corporation (FRDC) (Handlinger et al. 2006). As part of this project, a national survey of diseases of commercially important abalone species was undertaken to support trade and translocation issues. The most important diseases recorded during this study were:

• Bacterial. Particularly Vibrio harveyi which has been a problem in some farms in Tasmania and South Australia (Reuter and McOrist 1999; Handlinger et al. 2005);
• Protozoans. The most important being Perkinsus olsoni. Goggin and Lester (1995) reviewed research on Perkinsus spp. in Australia and determined it is found in over 30 species of molluscs. 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 viruses

Viruses 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. avian influenza. 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. Renault and Novoa (2004) described two distinct pathological infections in adult oysters in France:

• Gill necrosis virus (GNV) which caused mass mortalities in Portuguese oysters (Crassostrea angulata) in France; and
• Hemocyte infection virus (HIV) which was associated with mortalities of oysters in France.

In addition, oyster velar virus disease (OVVD) has caused occasional mortalities in hatchery reared Pacific oysters on the west coast of North America (Renault and Novoa 2004).

Herpes viruses

Herpes viruses (Herpesviridae) are widespread and have been recorded from a wide range of species over 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). Most agents appear to exhibit strong host specificity (Ahne 1994). 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. 2005). 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. In crustaceans, herpes-like viruses have been reported from several marine crab species. 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. 1992; Nicolas et al. 1992) as well as several other species.

Ostreid herpesvirus (OsHV-1)

Renault and Novoa (2004) reviewed herpes virus in Pacific oysters (Ostreid herpes virus OsHV-1 and OsHV-1 var) which exhibited a summer mortality syndrome was first described in Japan in the early 1940s. 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. 1994b; Renault et al. 2001a). 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. 1992). Larvae show reduced feeding and swimming activities and eventually mortality rates of up to 100% can rapidly occur (Hine et al. 1992; Nicolas et al. 1992). Outbreaks often occur during summer (Renault et al. 1994a; Renault 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. 1996; Arzul et al. 2002). Indeed, vertical transmission studies on successive generations of Pacific oysters indicate that parents pass the virus to offspring (Barbosa-Solomieu et al. 2005). 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. Nuclei in ovoid cells (haemocytes) are highly condensed (Hine et al. 1992; Nicolas et al. 1992; Renault et al. 2001a). The ultrastructure of herpesvirus in Pacific oysters has been described (Hine et al. 1992; Nicolas et al. 1992; Renault et al. 2001a).

The genome sequence for OsHV-1 has been completed and analysed (Davison et al. 2005) (GenBank No. AY509253 and NC005881). 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. These groups may contain a large number of invertebrate herpes viruses (Davison 2002; ICES, 2003; Davison et al. 2005).

Table 4: A summary of herpes and herpes-like viruses of aquatic invertebrates.

Molecular and immunological techniques for detection of herpes virus in Pacific oyster have been developed. These include polymerase chain reaction (PCR) and in situ hybridisation (ISH). Specific polyclonal and monoclonal antibodies have been developed and immunoassays have been validated for detection and identification of OsHV-1 (Renault et al. 2000b; Arzul et al. 2001b; Arzul et al. 2002; Lipart and Renault 2002; Renault 2003; Renault et al. 2004; Batista et al. 2005). 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. 1994; Arzul et al. 2001c; Arzul et al. 2001b; Renault et al. 2001a; ICES, 2006). Indeed, Arzul et al. (2001c) suggested that intensive culture of bivalves in hatcheries, where there is large numbers and a variety of species in close proximity, may promote interspecies transmission.

In summary, herpes viruses have caused massive losses in oyster hatcheries in Europe, USA and New Zealand and the relevant literature is summarised in Table 4.

Herpes viruses are also reported from fish, notably Channel catfish herpes virus (CCV), Anguilla herpes virus, carp herpes virus (Ahne 1994) and pilchard herpes virus, which on a number of occasions has caused high mortalities of pilchards (Sardinops sagax neopilchardus) over their entire geographical range on the Australian coastline (Crockford et al. 2006).

Abalone herpes and herpes-like viruses

The Australian abalone herpes-like virus (which caused the disease known as abalone viral ganglioneuritis) is not limited to one species. It has been confirmed in two species (H. rubra and H. laevigata) and their hybrids. 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. gigas) and mussels (Mytilus edulis). A summary of the viruses reported from abalone is presented in Table 5. Prior to the outbreak of abalone viral ganglioneuritis, herpes viruses had been recorded in abalone in Taiwan.

Herpes viruses of abalone in Taiwan

The 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. 2005). 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). These mortalities resulted in losses of US$11.5 million to the domestic abalone industry.

As a direct result of the die-off market, value dropped from NT$600 to NT$400 per kilogram (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. 2005 and Pen Heng Chang, pers comm. 2006):

• Highest prevalence occurred during winter months (water temperatures were 16-19°C). 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. Wild abalone beside the ponds also died
• Japanese black abalone (H. discus) and Trochus spp. held in ponds with infected H. d. supertexta remained normal
• Gross appearance: mantle recession and muscle stiffness
• Histopathology showed that nerve tissue was the primary target. Lesions involving nerve tissue and surrounding muscles occurred in the foot. Lesions were also evident in the oesophagus and intestine
• Electron microscopy showed hexagonal viral particles (90-100 nm) within the degenerated cells of the cerebral ganglion
• Laboratory infection trials indicated horizontal transmission and a high degree of virulence with 100% mortality.

Table 5 :Viral diseases reported from Abalone (source: Bower et al. 1994; Hine 1996; Bower 2000; 2003; Handlinger et al. 2006; Jones 2006 and pac.dfo-mpo.gc.ca/sci/shelldis/)

Figure 3: Change in quantity (a) and value (b) of farmed abalone Haliotis spp. (excluding H. rubra, H. midae and H. tuberculata) in Tawain between 2000 and 2004 (Data source: fao.org/figis)

This was apparently the first reported case of herpes virus in abalone. Chang et al. 2005 suggested the disease was an exotic pathogen, perhaps introduced with imported juvenile abalone. There is no documented management strategy for the Tawain abalone virus. Current control measures for the disease focus on prevention (Pen Heng Chang, pers comm. 2006). These include reducing stress factors in farmed stock, such as lower stocking densities, and prompt removal of mortalities from ponds. In the epizootic area, soda lime is applied to the culture ponds following each harvest. Quarantine is important as workers, equipment and tools may spread the virus to different farms.

Other abalone viruses

In terms of viruses that cause mass mortalities in abalone, a number of serious cases have been reported from China's farmed abalone (Table 5). "Crack-shell" disease of H. discus hannai was reported as being caused by a spherical virus of 150-220nm in size (Wang et al. 2000). It existed in the cytoplasm of cells rather than the nucleus. The virus mainly invaded foot muscle, mantle, gonads, liver and gills. The symptoms of sick abalone included: edge of front shells turned inside out, breathing holes melted into each other, decreased activity, and the soft body became slim and gradually died.

Shi Zhengli and Handlinger (2004) described an abalone viral mortality that encompassed both crack-shell disease of Haliotis hannai and viral disease of H. diversicolor. The authors noted that these two syndromes could be distinct diseases, but further information was required to make that determination. The authors described four types of spherical virion, all less than 150 nm in size. 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. discus discus) in western Japan. Mass mortalities occurred in juveniles at temperatures of between 18-20°C (Otsu and Sasaki 1997). H. discus hannai, has since also been found to be vulnerable to amyotrophia (Nakatsugawa et al. 1999). 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. Feeding and adhesion to the substrate is impeded and shell growth is impaired. Laboratory experiments have shown that the disease can be horizontally transmitted through the water column (Nakatsugawa et al. 2000). The etiological agent of amyotrophia is unknown. Virus-like particles have been observed in the cytoplasm of cells near the nerve trunk (Otsu and Sasaki 1997) and haemocytes (Nakatsugawa et al. 1999) of diseased Haliotis discus discus. Virus-like particles from haemocytes were retrovirus-like in size and morphology (Nakatsugawa et al. 1999).

Currently there are no known methods of prevention or control. 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 ganglioneuritis

Abalone 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 virus is noticed most often where there are dense populations of abalone, but the animals may be pre-disposed to becoming infected if other stress factors are present, including:

• High stocking densities
• High water temperatures >17.5-18°C which are at the upper limit of optimal for abalone culture
• Spawning activity
• Concurrent disease (e.g. Vibrio sp.).

Clinical signs

Infected abalone are lethargic and may have enlarged mouth-parts and a curled foot. The presence of enlarged mouthparts in isolation is not sufficient to diagnose abalone viral ganglioneuritis and is not shown by all infected abalone. In some cases, the radula (a band of sharp hooked teeth) may protrude from the mouth. 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 identification

Abalone 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 virus

Trials 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). The virus remains virulent and pathologic after being frozen at -80°C. 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 gaps

In 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. As an outcome of the workshops, the following key knowledge gaps on abalone viral ganglioneuritis were identified:

• Origin: It is not known whether the virus is exotic or endemic to Australian waters, but the panel strongly suspected that it is endemic as no probable linkages with an exotic source had been identified.
• Range: If the virus is endemic, it is not known if the outbreak is localised (as a result of translocating infected animals) or if the virus is widespread, but reached a critical dose when concentrated on the farm. Diagnostic techniques (e.g. PCR) must be developed and validated before the distribution of the virus in Australian waters can be determined.
• Mode of action: Very little is known about how this virus infects abalone, what the infectious dose is, how long it survives outside of the host (and in the water column) and if healthy abalone can carry the virus without showing symptoms.
• Characterisation of the virus: The virus has not been fully characterised. Key information on the types of cell infected is limited. 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. Determining the relationship of the Australian abalone viral ganglioneuritis to other herpes viruses of abalone is also important.
• Other species affected: It is not known if the virus is specific to abalone. It is also not known if the virus infects other species or if other species acts as carriers of the virus.
• Deactivation of the virus: There is no specific information on what de-activates the virus. 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. Lancaster, pers. comm.).
• Mechanism of the disease. Once an animal is infected the disease can spread rapidly, particularly if there are dense populations of abalone. It is not known if the rapid time frame is determined by rapid multiplication or an expression of some other response.

Table 6: Some of the International and Australian Experts involved in the gap analysis.

Diagnostic tools to identify the virus in the future

The diagnostic tools currently available to identify the virus are histopathology and electron microscopy (EM). EM is labour intensive, time consuming and can only focus on a very small piece of abalone tissue at a time. However, using EM, pathologists can visualise virus particles in tissues. Histopathology is more suited to screening larger numbers of abalone. 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. inflammatory changes).

For both tools, the tissue will be processed using special chemicals and fixatives. 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. It is highly unlikely that it will be detectable using EM. 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. Two options are available:

Molecular tests

A range of molecular diagnostic technique have been developed for fish and shellfish including polymerase chain reaction (PCR) amplification of nucleic acids, restriction enzyme analysis, probe hybridisation and nucleotide sequencing (Cunningham, 2002).

PCR is a rapid diagnostic technique that will provide a result within days. 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). The test will indicate the presence of the viruses' DNA. 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 agent

Some 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. No immortal marine invertebrate cell lines have been established. There are no cell lines for abalone. 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. Crane, pers com.).

Potential future mechanisms for control

Breeding for disease resistance

Through various selective breeding programs a number of mollusc species have improved resistance to several important diseases. 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 spawners

Another 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 Workshop

A 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. The Workshop was held on 3 October 2006 at Attwood, Victoria. A panel of experts and industry stakeholders from all over Australia was invited to participate (Appendix II). The risk assessment process followed is outlined in Appendix III and is consistent with the AS/NZ 4360:2004 Standard for Risk Management.

Issues were assessed using a consequence - likelihood score. The issues were assigned a risk category according to the score given by the panel:

• High risk - scored between 16 and 25.
• Significant risk - scored between 11 and 15.
• Moderate risk - scored between 5 and 10.

Pathways of viral transmission

Prior to the workshop, the project team identified potential pathways through which the herpes- like virus could be transmitted between the three key sectors under investigation (Figure 4).

Figure 4: Potential pathways of transmission of abalone viral ganglioneuritis.

For the purposes of this exercise, "wild" is primarily activities associated with commercial diving activities. However, this could also incorporate other commercial fishing and recreational activities (e.g. recreational diving, fishing) as well as illegal take of abalone². There may also be some overlap with sea-cage farming, ranching and stock enhancement programs. There are nine primary pathways of transmission of abalone viral ganglioneuritis between the three industries sectors (Table 7).

Table 7: Potential pathways of transmission for abalone viral ganglioneuritis.

Once the pathways of transmission had been identified, it became clear that within each pathway there were common mechanisms through which the virus could be spread. These included:

1. Movements of live infected abalone.These movements may be intentional (e.g. an abalone farm procuring new broodstock from the wild, a commercial diver transferring harvested abalone to a processor) or unintentional (e.g. escape from an abalone farm). It should be noted that abalone may carry the virus but show no symptoms. 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).
2. Movements of dead infected abalone, abalone by- products and wastes. This includes mortalities from abalone farms, shells and processing wastes (viscera etc).
3. Personnel and equipment. Transfer of the virus on contaminated personnel and equipment. This includes transport bins and tanks, boats, wetsuits, buckets, nets and boots. This category also includes abalone diets. As noted above the virus cannot multiply on personnel or equipment, hence the quantity of viable virus will diminish over time on such vectors.
4. Water. Transfer of the virus in contaminated water. This may include water pumped from an infected reef to an abalone farm, water discharged from an abalone farm, boat and ship bilge and ballast water, water used in the holding and transport of live abalone and water used in processing plants. Laboratory experiments have shown that transmission of the abalone viral ganglioneuritis can occur through the water column (horizontal transmission). Viability of the virus in the environment outside the host is unknown.
5. Other animal species. A number of other species may act as carriers or vectors of the abalone viral ganglioneuritis. These include other gastropods, crayfish, vermin (e.g. rats), predators (e.g. birds, fish) and scavengers.

The relationships between the mechanisms of virus movement and the three industry sectors are complex and interlinked. In undertaking the risk analysis, each pathway represented a risk category while the specific issues within each pathway are represented by mechanisms of virus movement. Specific risks also take into account geographic scales of movement within and between the industry sectors. While the current risk assessment focused on the above nine pathways, it is likely there are other pathways through which the viable virus particles may be spread (Figure 5). It is also possible that the current pathways and associated risks will change as knowledge and understanding of the virus (e.g. infective dose, environmental stability) increases.

Figure 5: Additional pathways of transmission of abalone viral ganglioneuritis in abalone and associated industries.

Outcomes of the Risk Assessment Workshop

Appendix IV shows that a total of 84 specific risks were identified for the nine pathways. Error! Reference source not found. Some issues raised at the workshop that did not fall within any of the nine pathways were summarised in a category named "Other". Figure 6 illustrates that the highest number of specific risks were in Pathway 7 (20%) (movement within abalone farms) and the least in Pathway 8 (5%) (movement from processors to the wild).

The outcome of the risk assessment process was as follows:

• 24 risks rated as High
• 20 rated as Significant
• 28 rated as Moderate.

More than half (52%) of the specific risks were associated with movement of infected live abalone (Figure 7), which occurred in all pathways, but especially within Pathway 7. Approximately 50% of the specific risks were associated with local-regional (i.e. within and between management zones) movements. A complete list of specific risks for each pathway, including likelihood, consesquence and ratings scores and associated comments, is presented in Appendix IV.

Herpes-like virus risk ratings

Figure 6: Number of risks recorded for each Pathway of Transmission of abalone viral ganglioneuritis.

Figure 7: Proportion of risks for each of the primary vehicles of transport of abalone viral ganglioneuritis.

Determining control measures forthe three industry sectors

Wild abalone harvest sector

The "wild harvest" abalone sector as defined in this document is primarily associated with commercial harvesting activities. However, this could also incorporate other commercial fishing and recreational activities (e.g. recreational diving, abalone poaching and fishing). There may also be some overlap with sea-cage farming, ranching and stock enhancement programs. The pathways of transmission over which the wild sector has control are shown in Figure 8 and detailed in Table 8.

Figure 8: Pathways of transmission associated with the wild abalone harvest sector.

Table 8: Pathways of transmission associated with the wild abalone harvest sector.

High risk issues related to the wild abalone harvest sector (all pathways)

Table 9 shows that six "High" risk issues were identified across all of the relevant pathways at the workshop. Possible controls for these risks are also shown in Table 9.

Significant risk issues related to the wild abalone harvest sector (all pathways)

Table 10 shows that three "Significant" risk issues were identified across all of the relevant pathways at the workshop. Possible controls for these risks are also shown in Table 10.

Moderate risk issues related to the wild abalone harvest sector (all pathways)

Table 11 shows that nine "Moderate" risk issues were identified across all of the relevant pathways at the workshop. Possible controls for these risks are also shown in Table 11.

Table 9: "High" risk issues associated with the wild abalone harvest sector.

Table 10: Significant risks associated with the wild abalone harvest sector.

Table 11: "Moderate" risk issues associated with the wild abalone harvest sector.

Abalone aquaculture sector

The pathways of transmission over which the abalone aquaculture sector has control are shown in Figure 9 and detailed in Table 12.

Figure 9: Pathways of transmission associated with the abalone aquaculture sector.

Table 12: Pathways of transmission associated with the abalone aquaculture sector.

High risk issues related to the abalone aquaculture sector (all pathways)

Table 13 shows that eight "High" risk issues were identified across all of the relevant pathways at the workshop. No high risk issues were identified for Pathway 4 (Movements from abalone farms to processors). Possible controls for these risks are also shown in Table 13.

Significant risk issues related to the abalone aquaculture sector

Table 14 shows that 10 "Significant" risk issues were identified across all of the relevant pathways at the workshop. Possible controls for these risks are also shown in Table 14.

Moderate risk issues related to the abalone aquaculture sector (all pathways)

Table 15 shows that 11 "Moderate" risk issues were identified across all of the relevant pathways at the workshop. Possible controls for these risks are also shown in Table 15.

Table 13: "High" risk issues associated with the abalone aquaculture sector.

Table 14: "Significant" risks associated with the abalone aquaculture sector.

Table 15: "Moderate" risk issues associated with the abalone aquaculture sector.

Abalone processing sector

The pathways of transmission over which the abalone processing sector has control are shown in Figure 10 and detailed in Table 16.

Figure 10: Pathways of transmission associated with the abalone processing sector.

Table 16: Pathways of transmission associated with the abalone processing sector.

High risk issues related to the abalone processing sector

Table 17 summarises the eight high risk issues associated with the abalone processing sector and the potential controls.

Significant risk issues related to the abalone processing sector

Table 18 summarises the six risk issues that were considered significant for the abalone processing sector. Appropriate controls are also summarised.

Moderate risk issues associated with the abalone processing sector

Three moderate risk issues were identified for the processing sector and are summarised in Table 19.

Table 17: High risk issues associated with the abalone processing sector

Table 18: Significant risk issues associated with the abalone processing sector

Table 19: Moderate risk issues associated with the processing sector

Benefits and adoption

The outputs of the Risk Assessment Workshop were used to develop Codes of Practice for various sectors of the abalone industry. This document, "Biosecurity Control Measures for Abalone Viral Ganglioneuritis: A Code of Practice", has been published by Fisheries Victoria as a separate, stand-alone document³ so that it can be disseminated to stakeholders. Victoria was used as a case-study, but the key elements of the Code of Practice can be adapted for other states with an abalone industry.

All sectors of the abalone industry, including wild-capture, recreational, aquaculture and processing will directly benefit from the increased bio-security that will arise through the implementation of the Codes of Practice. For the Australian abalone aquaculture industry, the implementation of the Code of Practice and the necessary increased attention to aquatic bio- security will result in enhanced production by:

• minimising morbidity and mortality
• obtaining an optimal growth response
• ensuring high survival of stock
• preventing the spread of infectious disease.

In addition, the other Codes of Practice will assist in maintaining the favourable health status of Australia's wild fisheries through protocols that will lead to improved bio-security in the wild catch and processing sectors. All industry sectors will be better protected by being more prepared to respond to, control and manage the negative impacts associated with disease outbreaks.

Social and community benefits will flow from further protection of natural biodiversity and the security of both the recreational and commercial fishery for abalone that provides recreation, jobs and income for community members.

Further development

As outlined in the literature review, current knowledge on the virus is very limited. Future developments in this area need to concentrate on addressing the knowledge gaps identified in this report. In particular, pre-clinical diagnostic techniques must be developed to allow for identification of the virus in otherwise healthy animals. This will allow surveillance programs to be undertaken.

Conclusion

This project has developed Codes of Practice to increase bio-security in the Australian abalone industry and minimise the risk of outbreaks and spread of abalone viral ganglioneuritis. In developing these Codes of Practice, a robust and defensible process was undertaken to identify the key issues that needed to be addressed by the wild harvest, recreational, aquaculture and processing sectors. These issues were assessed and prioritised at a Risk Assessment Workshop by an expert panel assembled from all Australian states with an abalone industry. The panel included aquatic animal health specialists, representatives from industry and Government officials.

The Risk Assessment Workshop identified the most important issues for each industry sector and control measures were subsequently developed (bearing in mind that knowledge of the virus is incomplete). The controls were subsequently developed into a Code of Practice for each industry sector that is comprised of a series of Standard Operating Procedures.

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Appendix I: Summary of diseases and parasites reported from abalone

For further details and related references see Bower et al. 1994; Hine 1996; Bower 2000; 2003; Handlinger et al. 2006; Jones 2006 and pac.dfo-mpo.gc.ca/sci/shelldis/title_e.htm)

Appendix II: List of Participants

Appendix III: Risk analysis process

Risk analysis is a key step in the development of Codes of Practice for the abalone industry sectors. It allows an objective appraisal of individual risks: it puts into context the likelihood that they will occur and the consequences if they do. Prior to the workshop a preliminary list of issues (Appendix IV) was compiled by the project team. It was based on the pathways and vehicles of transmission discussed earlier.

Experts in fish health and bio-security from across Australia participated in the workshop. Participants were drawn from both Government (State and Federal) and industry and are listed in Appendix II. Workshop participants reviewed the list of issues. They identified gaps and then assessed the likelihood and consequences of the identified risks using the ranking system described below.

Risk Ratings

The risk assessment process used at the workshop was based on the Australian/New Zealand Standard for Risk Management (AS/NZ 4360:2004). However, the definitions of "likelihood" and "consequence" were tailored to align with the overarching project objective, which was:

With this objective in mind, "likelihood" and "consequence" rankings were defined as follows:

Likelihood is defined as a general description of probability or frequency of an event occurring. In the case of transmission of abalone viral ganglioneuritis, there are two ways in which likelihood can be interpreted:

• Likelihood that the virus can be transmitted via that pathway and specific risk. In a worst-case scenario with no controls present, it is assumed that the event (virus transmission) could lead to clinical disease at the destination; OR
• Likelihood that the specific risk will lead to the establishment of disease in a previously non-infected population of abalone. In this scenario the likelihood of the event (disease establishment) occurring would depend on many other factors (such as virus being transmitted in sufficient quantity to establish infection; exposure of non-infected abalone to sufficient quantities of the virus to become infected etc.). In most cases there is inadequate information to adequately assess the likelihood of disease establishment.

For the purposes of this risk assessment "likelihood" has been defined as the former, i.e. the likelihood that the virus can be transmitted via that pathway and specific risk. It is further assumed that exposure to the virus will result in establishment of the disease so that the worst-case scenario could be assessed. for likelihood are shown below:

Likelihood risk scores

Consequence is defined as the outcome or impact of an event, in this case the transmission of the virus via a pathway or specific risk. Given that the overarching objective of the project is to protect the Australian abalone, it was decided that consequence rankings should reflect the impact on the industry as a whole rather than the impact on an individual site, farm or processor. That is, although the consequences to the site, farm or processor of abalone viral ganglioneuritis would be catastrophic to the individual, it is the impact on the entire industry that needs to be assessed. As outlined earlier, it was assumed that exposure to the virus would result in establishment of the disease so that the worst- case scenario could be assessed.

Scores for consequences are shown below:

Likelihood risk scores

Workshop participants were split into 3 groups and asked to assess the risks associated with the three sectors individually. All risks were rated under the assumption that the virus was present. The risk rankings were calculated by multiplying the likelihood score and the consequence score to give rankings from low to high.

Appendix IV: Risk ratings associated with spread of abalone viral ganglioneuritis.

Pathway 1: Movement within the wild.

Pathway 2: Movement from the wild to abalone farms.

Pathway 3: Movement from the wild to processors.

Pathway 4: Movement from abalone farms to processors.

Pathway 5: Movement from abalone farms to wild sector.

Pathway 6: Movement from processors to aquaculture farms.

Pathway 7: Movement within and between abalone farms.

Pathway 8: Movement from processors to wild sector.

Pathway 9: Movement within and between processors.

Pathway 10: Other

Notes:

¹ nd=no data.

² Illegal take of abalone is not considered further in this risk assessment as a Code of Practice is not a suitable mechanism to control such activities.

³ This document is available from the Library, DPI Queenscliff Centre, PO Box 114, Queencliff, Victoria 3225.

Acknowledgments:

This Information Note was developed by Gavine, F. Ingram, B. and Doroudi, M., May 2009.