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Tasmanian abalone biosecurity project: implementation phase 1: biosecurity strategies for abalone processors.

ABSTRACT The Tasmanian abalone biosecurity project was initiated by the Department of Primary Industries, Parks, Water and Environment (Wild Fisheries) and the Tasmanian wild abalone industry in response to an outbreak of abalone viral ganglioneuritis in the Victorian abalone fishery during late 2005. A formal risk assessment of abalone production activities in Tasmania was undertaken, the results of which were used to guide implementation strategies of the Tasmanian abalone biosecurity project. The risk assessment concluded that movements of live abalone in the wild fishery sector represented an unacceptable risk for spread of abalone herpes virus (AbHV) in Tasmanian State waters. Live-holding facilities in Tasmania routinely hold wild-caught live abalone in tanks preceding export. Prior to November 1, 2011, live-holding water was discharged to the marine environment without any treatment or consideration of potential pathogens. Currently, all processors that hold abalone that have been transported long distances must treat their live-holding discharge water to specifications set by the Department and outlined herein. There is limited information available on inactivation of AbHV; however, considering the magnitude of the risk and the value of the Tasmanian abalone fishery, the Department and industry have put measures in place to minimize the risk of potential spread of disease. The decontamination standards presented are based on available information relating to morphology, relative susceptibility, and hardiness of aquatic herpes viruses. The aim of treating water outflow from abalone processors is to avoid the spread of AbHV in Tasmanian State waters. Treatment, however, will also limit spread of other potential novel pathogens that may arise in the future.

KEY WORDS: abalone viral ganglioneuritis, herpes virus, inactivation, disinfection, abalone herpes virus, wastewater treatment

INTRODUCTION

When determining appropriate decontamination standards for abalone live-holding facilities, the Department of Primary Industries, Parks, Water and Environment considered it too restrictive simply to prescribe a list of systems or processes that should be installed. It was recognized that the design of decontamination systems was likely to change with advances in technology, and systems needed to be matched to specific requirements of each live-holding facility. It was therefore considered more appropriate to allow processors to choose systems most suited to their needs, and instead to develop performance standards with which each system needed to comply.

To date, there has been a paucity of published literature regarding the correlation of bacterial and viral loads specifically relating to abalone live-holding effluent. Therefore, a range of published water treatment literature is drawn on for this article.

WHY IS THERE A NEED TO TREAT EFFLUENT AND WATER DISCHARGE FROM ABALONE PROCESSORS?

Outflows from fish processing facilities containing large numbers of microorganisms have always presented a high risk for the spread of pathogens into the receiving marine habitats, particularly where there are no controls over entry of live animals into facilities discharging such water.

Abalone in Tasmania are often caught in waters significant distances from where they will be held live and/or processed. Such movements, although identified as a risk, are considered necessary by industry for ongoing economic sustainability. Hence, there is a need to continue moving wild-caught abalone between regions while at the same time protecting abalone populations.

In ideal conditions, it is possible that abalone may host abalone herpes virus (AbHV) and not express clinical symptoms. However, after abalone are taken from their natural environment and exposed to a potentially stressful environment in live-holding facilities, it is possible that abalone can develop disease. When this occurs, abalone can shed virus in significant amounts, and above the infectious dose. Live-holding water that has previously contained abalone shedding virus has potential implications when being discharged directly to the marine environment, and it is highly likely that adjacent populations of abalone will become infected.

WHAT GAPS ARE THERE IN OUR KNOWLEDGE REGARDING ABHV?

There is currently only limited information available on decontamination methods that specifically address inactivation of AbHV (Corbeil et al. 2012), but such data on minimum inhibitory levels for various treatment types are essential for ongoing assessment of strategic management.

Corbeil et al. (2012) discuss using chemical disinfectants to deactivate AbHV. This may not always be practical in commercial applications because of the large volumes of water to be decontaminated, the associated chemical by-products that can possibly be produced, and the environmenta! legislation that governs the use of chemicals and discharge to waterways. Most abalone processors find it more feasible to use UV light and ozone as large-volume treatment methods. However, there is no information that relates specifically to AbHV and the use of UV or ozone for inactivation.

Unlike many bacteria and viruses of vertebrates, culture techniques for AbHV have not yet been developed. Any assessment of decontamination processes must use inoculation of live abalone to test efficacy--a process that is slow, costly, and must be undertaken within a biosecure laboratory.

WHAT APPROACHES ARE USED FOR MONITORING DISINFECTION EFFICACY?

Because of the inherent constraints associated with pathogen monitoring, indicator organisms are used routinely as surrogates for pathogens (Payment et al. 1985). Although indicator organisms are generally not harmful themselves, they indicate the possible presence of pathogenic (disease-causing) bacteria, viruses, and protozoans that also reside in effluent systems. Because it is difficult, time-consuming, expensive, or simply not possible to test directly for the presence of many pathogens of interest, water is usually tested for particular types of microorganisms that have been established as indicators of specific pathogens.

The concept of using indicator microorganisms as a proxy measure for viral inactivation has been proposed by others for various wastewater treatment applications (Payment & Franco 1993, Payment et al. 2001, Harwood et al. 2005). Indicators in water treatment facilities are typically bacteria and include total coliforms, fecal coliforms, or Clostridium perfringens. However, these commonly documented indicator species for assessing freshwater effluent systems are not considered relevant for testing efficacy of wastewater treatment in abalone live-holding systems.

A variety of simple culture-based tests that are intended to recover a wide range of marine microorganisms from water are collectively referred to as a heterotrophic plate count test. This count is an attempt to provide a single value that expresses the number of aerobic and anaerobic microorganisms or total marine heterotrophic bacteria (TMH) in a water sample (Edberg & Smith 1989).

HOW DOES ABHV FIT ON THE SCALE OF RESISTANCE TO DISINFECTION?

Viruses vary in their susceptibility to inactivation by disinfecting agents. For disinfection purposes, viruses have been classified into 3 basic categories within the AQUAVETPLAN Operational Procedures Manual on decontamination (Department of Agriculture, Fisheries and Forestry (DAFF) 2008):

* Category A: These viruses have a lipid-containing envelope and are intermediate to large in size. Category A viruses are the easiest group of viruses to inactivate because the lipid envelope is sensitive to disinfecting compounds.

* Category B: These viruses are the most difficult to inactivate. They include small, nonlipid-containing viruses and those protected within a protein layer.

* Category C: These viruses are intermediate in their case of inactivation by disinfecting agents. They do not contain lipids, but are usually larger than the viruses in category B.

Virus particles, morphologically similar to herpes viruses, were first reported in an invertebrate host (the Eastern oyster, Crassostrea virginica) by Farley et al. (1972). The virus has been isolated from infected Ostrea and Crassostrea larvae and formally classified as a member of the Herpesviridae under the name Ostreid herpes virus 1 (OsHV-1) (Le Deuff & Renault 1999, Friedman et al. 2005, Batista et al. 2007). Investigations now support the view that AbHV also belongs to the Malacoherpesviridae family (Savin et al. 2010) and is 1 of only 2 members in this family, the other being OsHV-1.

For decontamination purposes, both AbHV and OsHV-1 have been classified as category A viruses based on their morphology (DAFF 2008). As indicated in Table 1, being larger viruses and having a lipid envelop that is easily disrupted, they may be assumed to be relatively fragile. High temperature, chemicals, ozone, and UV (Schikorski et al. 2011) may all be used to destroy the lipid envelope.

WHAT LEVEL OF TREATMENT IS REQUIRED TO DEACTIVATE ABHV?

Wastewater treatment should aim to reduce the number of microorganisms to an acceptable level (Feachem et al. 1983, Leong et al. 1984), but this level can be different according to the proposed use of the receiving waters (Payment et al. 2001) and the risk posed to susceptible species. In the case of abalone live-holding water outflows, treatment should be at a level required to reduce the concentration of viable AbHV discharged to below the infective dose.

Guidelines outlined here are designed to achieve an average 3 [log.sub.10] or 99.9% reduction of TMH bacteria in discharge water from abalone processors. This kill rate is normally used as a benchmark for highly resistant viruses and bacteria (Payment et al. 2001), and is expected to have a significant impact on susceptible enveloped viruses such as AbHV.

Water samples from Tasmanian abalone live-holding facilities were collected by the Department of Primary Industries, Parks, Water and Environment between December 2010 and March 2011. Results in Table 2 demonstrate that TMH levels varied considerably across processors. Processor 5 had the highest average TMH count of 1.2 x [10.sup.6] cfu/mL, whereas processor 9 had the lowest recorded value of 4.4 x [10.sup.3] cfu/mL. This represents a substantial difference between potential system capacity, management regimes, and husbandry practices across processors tested. The average value for TMH concentrations listed in Table 2 is approximately [10.sup.5]. This is to be expected considering the intensive nature of processing and live-holding wild-caught abalone in high densities.

In general, sewerage treatment facilities use a 3 [log.sub.10] reduction as a guideline to effectively remove 99.9% microorganisms (Leong et al. 1984, Payment et al. 2001). However, because of the variability in pretreatment levels within live-holding tanks, a 3 [log.sub.10] reduction alone may not be sufficient in all cases. Systems operating with a below-average pretreatment level less than [10.sup.5] cfu/mL would be at a significant disadvantage if a single reduction value were applied to all processors. It is easier and presumably more cost-effective to achieve a 3 [log.sub.10] reduction from a system averaging pretreatment values of [10.sup.6] cfu/mL than from one operating at [10.sup.4] cfu/mL. The preferred approach is to set discharge limits with which processors must comply. A final emission limit of 9.99 x [10.sup.2] cfu/mL for TMH in abalone live-holding discharge water effectively represents an average 99.9% (3 [log.sub.10]) reduction in microbial loading and does not burden those few operators with low residual bacterial levels. This level must be maintained at all times as an auditable requirement.

In addition, of those processing facilities sampled, some contained TMH counts [greater than or equal to] [10.sup.5] cfu/mL. This constitutes a significant abalone health risk, as heightened bacterial levels within systems is considered to correlate with increased disease risk such as vibriosis, fungal infections, or outbreaks of AbHV. Clinical outbreaks of disease in abalone are associated routinely with poor environmental conditions. It is recommended that live-holding systems be managed to ensure that TMH counts are maintained to less than [10.sup.5] TMH cfu/mL.

CONCLUSIONS

* The best indicator for assessing decontamination of water outflows from abalone live-holding facilities is TMH.

* AbHV, the causative agent of abalone viral ganglioneuritis, is considered to be a relatively fragile virus classified within the category A group of viruses by DAFF (2008).

* A 3 [log.sub.10] reduction in TMH is equivalent to a 99.9% reduction.

* It is recommended that a 3 [log.sub.10] reduction be combined with a maximum emission limit of 9.99 x [10.sup.2] TMH cfu/mL in abalone live-holding water outflows.

* Emission levels for each facility should be monitored routinely throughout the year by facility operators and will be audited by an independent organization.

* A 5 [log.sub.10] cap on bacterial levels within live-holding tanks (pretreatment) is recommended, and should be monitored by processors as part of normal abalone health management.

* The recommendations made are based on available data and may be subject to change as additional data become available.

LITERATURE CITED

Batista, F. M., I. Arzul, J. F. Pepin, F. Ruano, C. S. Friedman, P. Boudry & T. Renault. 2007. Detection of ostreid herpesvirus 1 DNA by PCR in bivalve molluscs: a critical review. J. Virol. Methods 39:1-11.

Corbeil, S., L. M. Williams, J. Bergfeld & M. St. J. Crane. 2012. Abalone herpes virus stability in sea water and susceptibility to chemical disinfectants. Aquaculture 326-329:20-26.

Department of Agriculture, Fisheries and Forestry. 2008. AQUAVETPLAN operational procedures manual: decontamination. Version 1.0. Canberra: DAFF. 122 pp.

Edberg, S. C. & D. B. Smith. 1989. Absence of association between total heterotrophic and total coliform bacteria from a public water supply. Appl. Environ. Microbiol. 55:380-384.

Farley, C. A., W. G. Banfield, J. R. G. Kasnic & W. S. Foster. 1972. Oyster herpes-type virus. Science 178:759-760.

Feachem, R. G., D. Bradley, H. Garelick & D. D. Mara. 1983. Sanitation and disease: health aspects of excreta and wastewater management. New York: Wiley. 501 pp.

Friedman, C. S., R. M. Estes, N. A. Stokes, C. A. Burge, J. S. Hargrove, B. J. Barber, R. A. Elston, E. M. Burreson & K. S. Reece. 2005.

Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes. Dis. Aquat. Organ. 63:33-41.

Harwood, V. J., A. D. Levine, T. M. Scott, V. Chivukula, J. Lukasik, S. R. Farrah & J. B. Rose. 2005. Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection. Appl. Environ. Microbiol. 71:3163-3170.

Le Deuff, R. M. & T. Renault. 1999. Purification and partial genome characterisation of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas. J. Gen. Virol. 86:1317-1322.

Leong, L. Y. C., D. G. Argo & R. R. Trussell. 1984. Enterovirus removal by a full scale tertiary treatment plant. J. Am. Water Works Assoc. 75:199-204.

Payment, P. & E. Franco. 1993. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts. Appl. Environ. Microbiol. 59:22418-22424.

Payment, P., R. Plante & P. Cejka. 2001. Removal of indicator bacteria, human enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater primary treatment facility. Can. J. Microbiol. 47:188-193.

Payment, P., M. Trudel & R. Plante. 1985. Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven treatment plants. Appl. Environ. Microbiol. 49:1418-1428.

Savin, K., B. Cocks, F. Wong, T. Sawbridge, N. Cogan, D. Savage & S. Warner. 2010. A neurotrophic herpes virus infecting the gastropod, abalone, shares ancestry with oyster herpes virus and a herpes virus associated with amphioxus genome. Virol. J. 7:308.

Schikorski, D., T. Renault, D. Saulnier, N. Faury, P. Moreau & J. Pepin. 2011. Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpes virus 1: demonstration of oyster spat susceptibility. Vet. Res. 42:27.

TRAVIS BAULCH, (1) * KEVIN ELLARD (2) AND MATT BRADSHAW (1)

(1) Department of Primary Industries, Parks, Water and Environment, Water and Marine Resources Division, GPO Box 44, Hobart, TAS 7001, Australia; (2) Department of Primary Industries, Parks, Water and Environment, Animal Health and Welfare Branch, GPO Box 44, Hobart, TAS 7001, Australia.

* Corresponding author. E-mail: Travis.Baulch@dpipwe.tas.gov.au

DOI: 10.2983/03J.032.0107
TABLE 1.
Microbial groups and associated susceptibility to inactivation.

Susceptibility to chemical
disinfectants and
UV irradiation               Microorganism

Highly susceptible#          Mycoplasmas#

Susceptible                  Enveloped viruses
                             Gram-positive bacteria
                             Gram-negative bacteria
                             Fungal spores

Resistant                    Nonenveloped viruses
                             Mycobacteria

Highly resistant             Bacterial endospores
                             Protozoal oocysts
                             Protozoallike spores

Extremely resistant          Prions

Highlighted in bold are characteristic and susceptibility
of abalone herpes virus, an enveloped virus. Adapted from
the Department of Agriculture, Fisheries and Forestry (2008).

Note: Highlighted in bold are characteristic and susceptibility
of abalone herpes virus, an enveloped virus are indicated with #.

TABLE 2.
Results of heterotrophic plate counts for Tasmanian live
holding processors' effluent.

Processor      Date    Average (cfu/mL)    Ozonation *

1           12/15/10   6.67 x [10.sup.5]       Yes
2           12/16/10   1.06 X [10.sup.4]       Yes
3           12/12/10   6.35 x [10.sup.4]       No
4           12/12/10   2.1 x [10.sup.4]        No
5            1/12/11#  1.2 x [10.sup.6]#       No#
6            1/10/11   3.34 x [10.sup.5]       No
7            3/09/11   1.5 x [10.sup.5]        Yes
8            3/19/11   6.06 x [10.sup.3]       Yes
9            3/23/11#  4.40 x [10.sup.3]#      Yes#
10           3/23/11   1.30 x [10.sup.4]       Yes
11           3/23/11   2.76 X [10.sup.4]       Yes
12           3/24/11   3.00 x [10.sup.5]       No
13           3/24/11   1.73 x [10.sup.4]       No

Highlighted in bold are the highest and lowest total
marine heterotrophic bacteria results. * Water within
live-holding tanks treated with ozone.

Note: Highlighted in bold are the highest and lowest total
marine heterotrophic bacteria results are indicated with #.
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Author:Baulch, Travis; Ellard, Kevin; Bradshaw, Matt
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:8AUST
Date:Apr 1, 2013
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