FAO
FISHERIES
TECHNICAL
PAPER
395
DNA-based Molecular
Diagnostic Techniques
Research Needs for Standardization and
Validation of the Detection of Aquatic
Animal Pathogens and Diseases
DEPARTMENT FOR INTERNATIONAL DEVELOPMENT NETWORK OF AQUACULTURE CENTRES IN ASIA-PACIFIC COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZATION AUSTRALIAN CENTRE FOR INTERNATIONAL AGRICULTURAL RESEARCH
DNA-based Molecular
Diagnostic Techniques:
Research Needs for Standardization and
Validation of the Detection of Aquatic
Animal Pathogens and Diseases
Edited by
Peter Walker
CSIRO, Australia
and
Rohana Subasinghe
FAO, Rome
Report and Proceedings of the Expert Workshop on DNA-based Molecular Diagnostic Techniques: Research Needs for
Standardization and Validation of the Detection of Aquatic Animal Pathogens and Diseases.
Bangkok, Thailand, 7-9 February 1999
FAO
FISHERIES
TECHNICAL
PAPER
395
DFID
Department for International DevelopmentThe designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the prior permission of the copyright owner. Applications for such permission, with a statement of the purpose and extent of the reproduction, should be addressed to the Director, Information Division, Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracalla, 00100 Rome, Italy.
PREPARATION OF THIS DOCUMENT
This document contains the report, including recommendations, and thirteen papers presented at the Expert Workshop on DNA-based Molecular Diagnostic Techniques: Research Needs for Standardization and Validation of the Detection of Aquatic Animal Pathogens and Diseases, held in Bangkok, Thailand, from 7-9 February 1999.
The Expert Workshop was jointly organized by FAO Inland Water Resources and Aquaculture Service, Network of Aquaculture Centres in Asia-Pacific (NACA), Centre for Scientific and Industrial Research for Australia (CSIRO), Australian Centre for International Agriculture Research (ACIAR), and the Department for International Development of the United Kingdom (DFID) and was held at the NACA Headquarters in Bangkok. The editing, publishing, and distribution of the document were undertaken by FAO, Rome.
Distribution:
Aquatic animal health personnel Ministries and Directorates of Fisheries Participants of the Expert Workshop
FAO Fishery Regional and Sub-Regional Officers FAO Fisheries Department
Walker, P. and Subasinghe, R. (eds.)
DNA-based molecular diagnostic techniques: research needs for standardization and validation of the detection of aquatic animal pathogens and diseases. Report and proceedings of the Joint FAO/NACA/CSIRO/ACIAR/DFID Expert Workshop. Bangkok, Thailand, 7-9 February 1999.
FAO Fisheries Technical Paper. No.395. Rome, FAO. 2000. 93p.
ABSTRACT
In efforts to limit trans-boundary movement of pathogens and reduce the economic and socioeconomic impact of disease in aquaculture, there is considerable scope for more effective use of DNA-based methods of pathogen detection. These technologies offer rapid results with potentially high sensitivity and specificity, at relatively low cost. Recognition of these advantages has led to rapid adoption of available DNA-based tests, particularly in shrimp culture for which histological procedures lack specificity and culture-based methods have not been possible. However, few if any of the available tests have been assessed appropriately against other diagnostic methods or standardized and validated for specified applications. In fish and shrimp, type or strain specificity of most tests for pathogens in the Asian region is poorly understood and, in molluscs, there is little information on the significant pathogens and few tests of any kind have been developed. Furthermore, tests presently available are frequently conducted by technicians who may not be sufficiently aware of the need for stringent test protocols or the meaning and limitations of the data generated. Implementation of standardized practices that produce reliable, useful and comparable data will require a significant investment in research, training and infrastructure development. Effective implementation will also be assisted by enhanced communication between aquatic animal health practitioners in the region and scientists with expertise in molecular diagnostic technologies.
This review recommends development by FAO/NACA of 2 programs of managed cooperative research to assist more effective use of DNA-based detection tests. Program A should focus on improving the knowledge base by identification of new and emerging pathogens, relating pathogens in the region to those described elsewhere, and defining the extent of genetic variation between related pathogens in the region. Program B should draw on information currently available or obtained from Program A to develop suitably specific DNA-based diagnostic methods and to evaluate and validate the methods for disease diagnosis and pathogen screening programs.
To increase the availability of scientists and technicians with skills in pathology and molecular diagnostic technologies, the review also recommends development of FAO/NACA-sponsored training programs for staff from key laboratories in the region. Training priorities should be in: i) the use of standard histopathological methods for health screening of fish and molluscs; and ii) the use of standard DNA-based methods for pathogen detection including sample collection, application of test protocols and the analysis and interpretation of test results. Because of the urgency of disease problems and the availability of suitable tests, training in DNA-based methods should focus initially on detection of shrimp pathogens. The review also recommends the development of a laboratory accreditation program in order to achieve standardization of sampling methods and test procedures. The establishment of reference laboratories will assist accreditation for each of the major pathogens. Laboratory accreditation and training programs should complement the activities of OIE in obtaining internationally agreed test standards for molecular diagnostic technologies.
CONTENTS
1 BACKGROUND... 2
1.1 GLOBAL AQUACULTURE DEVELOPMENT... 2
1.2 IMPACT OF DISEASE... 2
1.3 ROLE OF DNA-BASED TECHNOLOGIES IN DIAGNOSIS AND PATHOGEN DETECTION... 2
1.4 IMPEDIMENTS TO THE USE OF DNA-BASED DIAGNOSTIC TECHNIQUES... 3
2 EXPERT WORKSHOP ... 4
2.1 TERMS OF REFERENCE... 4
2.2 PARTICIPANTS... 4
2.3 WORKSHOP PROCESS... 5
3 PATHOGEN FOCUS GROUP REPORTS... 5
3.1 SHRIMP PATHOGENS... 5
3.1.1 Status of research and identification of research needs... 5
3.1.2 Standardization of DNA-based diagnostic tests... 6
3.1.3 Networking and communication... 7
3.1.4 Training and extension... 7
3.2 MOLLUSC PATHOGENS... 8
3.3 FINFISH PATHOGENS... 9
3.3.1 Diseases requiring application of DNA-based technologies... 9
3.3.2 Evaluation of needs for rapid detection techniques... 9
3.3.3 Status of research... 9
3.3.4 Key constraints to establishment of international standards... 10
3.3.5 Recommendations for research programs... 10
4 ISSUES RELATING TO THE ADOPTION OF INTERNATIONAL STANDARDS FOR DISEASE DIAGNOSIS AND HEALTH CERTIFICATION... 11
5 KEY OUTCOMES AND RECOMMENDATIONS ... 11
5.1 GENERAL COMMENTS... 11 5.2 RESEARCH NEEDS... 12 5.3 TRAINING NEEDS... 13 5.4 COMMUNICATION NEEDS... 13 5.5 INTERNATIONAL STANDARDISATION... 14 6 REFERENCES ... 14
7 CONTRIBUTED PAPERS AND REVIEWS... 16
1 Background
1.1 Global aquaculture development
In 1995, global production from aquaculture reached 27.8 million tones and was valued at US$ 42,300 million. Developing countries contributed over 87% of total production, of which 90.1% was from Asia. China contributed 63.4% of total world production. Over the past decade, global aquaculture production has grown at an average annual rate of 9.6% compared to 3.1% for livestock meat and 1.6% for capture fisheries. Between 1984 and 1995, growth in aquaculture production in low-income food deficit countries (LIFDCs) was over five times faster than in developed countries (Rana, 1997).
1.2 Impact
of
disease
Disease outbreaks are recognized as a significant constraint to aquaculture production and trade, affecting both the economic development and socioeconomic revenue of the sector in many countries in the world. According to Chamberlain (in press), disease is a primary limiting factor for shrimp farming today and the risk of disease losses is likely to increase as the shrimp sector continues to grow. Economic loss attributed to outbreaks of disease in developing countries in the Asian region was estimated to be at least US$ 1,400 million in 1990 (ADB/NACA, 1991). The cost of lost production in China alone was approximately US$ 1,000 million in 1993. In Thailand, the loss in 1996 due to yellow head virus (YHV) and white spot syndrome virus (WSSV) was estimated to be 40% of total production (70,000 tones) valued at over US$ 500 million (Alday-Sanz and Flegel, 1997). Recent estimates, based on farm level surveys in 16 Asian countries, suggest that disease and environment-related problems have caused annual losses of more than US$ 3,000 million to aquaculture production (ADB/NACA, in press). Serious financial losses have also been recorded in other regions of the world. In 1993, Ecuador lost 28,000 tones of shrimp production in due to an epizootic of Taura syndrome virus. Salmon farming in many countries also faced serious disease problems that resulted in significant production losses. Various factors have been related to the apparent increased incidence of disease. Environmental factors and poor water quality, sometimes resulting from increased self-pollution due to effluent discharge and pathogen transfer via movements of aquatic organisms appear to be an important underlying cause of such epizootics.
1.3 Role of DNA-based technologies in diagnosis and
pathogen detection
The effective control and treatment of diseases of aquatic animals requires access to diagnostic tests that are rapid, reliable and highly sensitive. In many cases, post-mortem necropsy and histopathology have been the primary methods for the diagnosis of fish and shellfish diseases. However, these methods often lack specificity and many pathogens are difficult to detect when present in low numbers or when there are no clinical signs of disease (Ambrosia and De Wall, 1990). Direct culture of pathogens is also widely used. However, these methods are time-consuming and costly, and, for shrimp and other crustaceans, cell lines suitable for virus culture have not been available.
Efforts to overcome these problems have led to the development of immunoassay and DNA-based diagnostic methods including fluorescent antibody tests (FAT), enzyme-linked immunosorbent assays
(ELISA), radioimmunoassay (RIA), in situ hybridization (ISH), dot blot hybridization DBH) and
polymerase chain reaction (PCR) amplification techniques. The use of DNA-based methods derives from the premise that each species of pathogen carries unique DNA or RNA sequences that differentiate it from other organisms. The techniques offer high sensitivity and specificity, and
diagnostics kits allowing rapid screening for the presence of pathogen DNA are moving rapidly from development in specialized laboratories to routine application. DNA probes are expected to find increasing use in routine disease monitoring and treatment programs in aquaculture, in field epidemiology and in efforts to prevent the international spread of pathogens (national quarantine and certification programs).
DNA-based methods have been used in diagnosis and for detection of many economically important viral pathogens of cultured finfish and penaeid shrimp. For finfish, tests have been developed for pathogens such as channel catfish virus (CCV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), viral
nervous necrosis virus (VNNV) and Renibacterium salmoninarum (see Muroga, 1997; Plumb, 1997).
PCR has been used in Japan to screen striped jack (Pseudocaranx dentex) broodstock for VNNV,
permitting selection of PCR-negative spawners as an effective means of preventing vertical transmission of this pathogenic virus to the larval offspring (Muroga, 1997).
DNA-based detection methods for detection of penaeid shrimp viruses are now used routinely in a number of laboratories around the world. These include probes for such diseases as white spot syndrome virus (WSSV), yellow head virus (YHV), infectious hematopoietic and infectious hypodermal and haematopoeitic necrosis virus (IHHNV) and Taura syndrome virus (TSV) which pose the greatest threat to world shrimp culture production (Lotz, 1997). DNA probes have also been developed for an intracellular parasites and bacteria infecting shrimp. DNA-based techniques will have an important role to play in efforts to develop sustainable shrimp culture in Asia and elsewhere. Production facilities in Thailand are currently using PCR techniques to screen shrimp post-larvae for WSSV. Culturing such larvae in closed (biosecure) or semi-closed culture systems can prevent or minimize viral infections, leading to a viable shrimp industry. The development of specific pathogen-free shrimp stocks will also depend on the use of such techniques.
The further development and use of DNA-based diagnostic techniques will also assist international efforts to control the introduction of exotic diseases into new geographic areas. Reliable and rapid techniques are needed by national and regional diagnostic laboratories to screen imported fish and shellfish for important pathogens. The Office International des Epizootics (OIE) or World Animal Health Organization, is a veterinary organization with 147 member countries. The OIE (through its Fish Diseases Commission) is responsible for tracking diseases of fish and shellfish that have a serious economic impact on aquaculture and capture fisheries. There is considerable potential to apply DNA-based methods for OIE testing if they can meet the stringent criteria of a standardized, validated, accurate, reliable and accessible diagnostic technique.
1.4 Impediments to the use of DNA-based diagnostic
techniques
Although offering considerable potential, the routine use of DNA-based diagnostic techniques is
hampered by a number of potential problems (Chanratchakool et al., 1998).
x The extreme sensitivity of these methods allows the detection of target DNA present at very low levels. However, positive results provide little quantitative assessment of the infection level, and do not indicate whether the pathogen is replicating or causing disease in the species tested. Thus, carrier status and viability of the pathogen are not determined using DNA-probes.
x The extremely high specificity of these tests, coupled with the ability of many viruses to rapidly change in genetic structure, can result in failure to detect a virus that has altered its genetic profile.
x Large differences in sensitivity are related to the PCR method used (e.g., 1-step PCR or 2-step PCR with nested primers).
x PCR methodologies are highly susceptible to contamination. Contamination during processing may result in false positives, particularly in 2-step PCR methods. PCR tests must be conducted in very well managed, clean laboratories.
x "False negatives" are easily caused by the selection of inappropriate host tissue sources for detection of the pathogen in question, incorrect choice of DNA extraction method, or low pathogen prevalence in the population sampled.
DNA-based detection and diagnostic methods have the potential for widespread application of in aquaculture. As the technology is already being adopted rapidly in developing countries in Asia, there is an urgent need to address these issues and to develop an action plan for research and training activities that will facilitate more effective utilization.
2 Expert
Workshop
2.1 Terms of reference
The terms of reference were as follows:x Identify and prioritize research areas where the introduction and use of nuclear and related technologies is likely to have the most significant impact on improving disease diagnosis, with emphasis on diseases affecting aquaculture, in developing countries.
x Evaluate needs for rapid diagnostic techniques for the principal diseases of cultured fish and shellfish.
x Review the status of research towards meeting these needs.
x Identify problems and key constraints related to establishing international standards for protocols and procedures for such tests and make recommendations towards their solution
x Make recommendations for programs of research to be developed jointly by IAEA, FAO, and other interested and concerned agencies and institutions, to assist developing countries to develop, standardize and validate nuclear related, DNA-based rapid diagnostic tools for major aquatic animal pathogens.
2.2 Participants
Dr Alexandra Adams, University of Stirling, Scotland. Dr Franck Berthe, IFREMER, France.
Dr Eugene Burreson, Virginia Institute of Marine Science, Virginia, USA.
Dr Pornlerd Chanratchakool, Aquatic Animal Health Research Institute, Bangkok, Thailand. Dr Supranee Chinabut, Aquatic Animal Health Research Institute, Thailand.
Mr Dan Fegan, Natl. Centre for Genetic Engineering and Biotechnology, Bangkok, Thailand. Prof. Timothy Flegel, Maihidol University, Bangkok, Thailand.
Dr Barry Hill, Fish Diseases Commission, OIE, Weymouth, England.
Dr Mike Hine, National Institute of Water and Atmospheric Research, New Zealand. Dr Maura Hiney, National University of Ireland, Galway, Ireland.
Dr Indrani Karunsagar, University of Agricultural Sciences, Mangalore, India. Prof. Donald Lightner, Arizona State University, Tucson, USA.
Dr James Lilley, Institute of Aquaculture, University of Stirling, Scotland. Dr Sharon McGladdery, Gulf Fisheries Centre, New Brunswick, Canada. Dr Gary Nash, Shrimp Culture Research and Development Company, Thailand. Dr Michael Phillips, NACA, Bangkok, Thailand.
Dr Krishen Rana, FAO, Rome, Italy.
Dr Melba Reantaso, NACA, Bangkok, Thailand.
Prof. Mohamed Shariff, University Putra Malaysia, Serdang, Malaysia. Dr Rohana Subasinghe, FAO, Rome, Italy.
Dr Kamonporn Tonguthai, Aquatic Animal Health Research Institute, Bangkok, Thailand. Dr Peter Walker, CSIRO Tropical Agriculture, Brisbane, Australia.
See Annex I for details.
2.3 Workshop
process
The participants assembled a team of experts currently working on the development of DNA-based rapid diagnostic techniques for the detection of aquatic animal pathogens, and representatives from other concerned agencies. With assistance from several cooperating agencies (FAO, NACA, ACIAR, CSIRO, and DFID), all experts participated in a workshop at NACA Headquarters (Bangkok, Thailand) on 7-9 February, 1999. The workshop comprised a series of papers on issues related to the use and limitations of DNA-based diagnostic technologies and related research needs, and a series of selected focus groups considering finfish, mollusc and shrimp pathogens.
In March and April 1999, Dr Franck Berthe (IFREMER, France) and Dr Peter Walker (CSIRO, Australia) conducted consultancies at FAO, Rome to consider the outputs of the workshop and to assemble this report.
3
Pathogen focus group reports
3.1 Shrimp
pathogens
Ponlerd Chanratchakool, Dan Fegan, Tim Flegel, Indrani Karunsagar, Don Lightner, Gary Nash, Mohamed Shariff, Peter Walker.
3.1.1 Status of research and identification of research needs
Shrimp pathogens in the Asia-Pacific region presently listed by NACA include WSSV and YHV (notifiable), INNHV, GAV, MBV, BMNV and SMV (significant pathogens). HPV also may have significant effects on production. The range of histopathological and molecular techniques available for detection of these agents has recently been reviewed (Lightner and Redman, 1998). Research needs pertaining to the application of DNA-based technologies vary, reflecting the stage of development of the technology and the relative importance of the pathogens. In view of their ongoing impact on shrimp aquaculture in Asia and OIE-notifiable status, this report has focussed primarily on the research status and needs for WSSV, YHV and viruses in the YHV complex. The urgency of implementing measures to control these viruses is presently the dominant concern for shrimp health in Asia.
WSSV. A number of PCR, nested-PCR and hybridization tests has been developed for WSSV
detection. The tests use a range of different PCR primers and hybridization probes targeted to different and poorly defined sites in the WSSV genome. Methods of DNA sample preparation and PCR test protocols vary and there has been no objective comparison of the sensitivity and specificity of the tests. A recent analysis of PCR products amplified from WSSV samples obtained from a wide geographic range has indicated remarkable uniformity. However, the data suggested that at least one
WSSV-related virus in the USA may be distinct (Lo et al., 1999). Other reports also suggest some
variability in the detection specificity of PCR tests (Park et al., 1998; R.A.J. Hodgson and P.J. Walker,
unpublished data). There is evidence that a range of crustaceans and other arthropods test positive by
PCR (Lo et al., 1996; Alday-Sanz and Flegel, 1997; Maeda et al., 1998), but the significance of these
results for the epidemiology WSSV infection in shrimp is unclear. A more extensive analysis of WSSV variation should be conducted to determine the implications for detection and disease diagnosis.
The proliferation of PCR tests and protocols for WSSV detection also presents problems for
comparative validation. The one-step and nested PCR primers and procedures described by Lo et al.
(1996) are well documented in the published literature and the utility of the tests has been demonstrated for a range of applications. The test appears to be reliable and not subject to commercial restrictions, and would be suitable as a primary reference for standardization purposes. Estimation of the level of WSSV infection may have important applications in disease management strategies and this should be more clearly defined. It may be useful to adopt a quantitative PCR test as a secondary reference standard.
YHV. The YHV complex constitutes a group of related agents which includes yellow head virus
(YHV), gill-associated virus (GAV) and lymphoid organ virus (LOV). YHV and GAV are closely related but distinct pathogens; LOV is a variant of GAV which occurs in healthy shrimp. Yellow head disease has been reported from many countries in the Asian region but, in most cases, the agents have not been clearly defined. To date, YHV has been shown to occur only in Thailand and GAV/LOV only in Australia.
An RT-PCR test is available for YHV but the test does not detect GAV. RT-PCR and nested RT-PCR tests are available for GAV. The GAV RT-PCR also detects at least some isolates of YHV but the test
will not distinguish GAV and LOV. In situ hybridization probes have also been developed for YHV
and GAV. The YHV probe detects both YHV and GAV. All tests developed to date have targeted sequences in the polymerase gene.
More research is required to determine the range and distribution of YHV complex viruses in the region, to identify other possible members of the complex and to develop both pan-specific and type-specific detection tests. If possible, tests should also be developed to differentiate pathogenic from non-pathogenic variants. There may be value in the adoption of multiplex PCR tests for YHV that allow discrimination of viruses in the complex.
3.1.2 Standardization of DNA-based diagnostic tests
A program of laboratory accreditation is proposed in order to achieve reliability and comparability of DNA-based test results in the Asia-Pacific region. The program should seek primarily to provide an improved regional capacity for effective disease control. However, if adequate and common levels of test performance can be obtained, the program may also provide a mechanism for eventual adoption of molecular-based methods for health certification by OIE.
It is proposed that FAO/NACA accreditation should be issued by reference laboratories designated for each pathogen. The reference laboratory should publish a detailed standard protocol including procedures for sample collection, DNA/RNA preparation, PCR reagent preparation and storage, PCR amplification and sample analysis, and preparation and use of control reagents. Adoption of the standard protocol will be facilitated by publication in local languages. The reference laboratories should also be responsible for:
x maintaining an agreed test as the primary reference standard against which other testing protocols should be assessed;
x maintaining standard PCR reagents including primers and suitable positive and negative controls; x monitoring standards and providing technical advise to accredited laboratories;
x providing definitive diagnosis in difficult or unusual cases; x retaining and archiving virus isolates for reference;
x ongoing assessment and Research and Development of DNA-based testing protocols.
Accreditation should initially focus on WSSV diagnosis. Because of the proliferation of WSSV PCR testing protocols, a primary reference standard should be identified. It is proposed that the one-step
and nested WSSV PCR tests described by Lo et al. (1996a; 1996b) should be adopted as the primary
standard. Implementation of a regional accreditation program should proceed following the issue of standard protocols. A relative evaluation of the diagnostic capabilities of participating laboratories should be conducted using standard coded samples of extracted DNA and shrimp tissue. An epidemiologist should be involved in the design of the evaluation and data analysis. The evaluation should be used as a basis for the development of an accreditation protocol.
Standardization and accreditation for YHV diagnosis is considered to be premature, as there is presently insufficient information available on the relationship of viruses that constitute the YHV complex. The establishment of a WSSV accreditation program will facilitate future accreditation of laboratories for diagnosis of YHV and other shrimp pathogens.
3.1.3 Networking and communication
NACA should be the conduit for maintaining communication between regional diagnostic laboratories, preferably through an email network. The establishment of an accreditation program should involve an initial meeting of participating laboratories to set up comparison protocols.
3.1.4 Training and extension
There is a need for training both at farm-level and of diagnostic practitioners. Practitioner training should be through intensive short courses which include both theory and practice of DNA-based technologies and high quality tertiary courses for veterinarians and fish pathologists. Farm-level training is best achieved through in-country training of extension officers to meet local needs and should include: i) instruction on sample collection and preparation methods; ii) accurate interpretation of PCR results; iii) limitations of PCR technology; and iv) basic epidemiology.
Standard procedures for the application of DNA-based technologies for disease prevention should also be documented. These should include the use of PCR in hatcheries and on-farm, and for the selection of SPF broodstock.
3.2 Mollusc
pathogens
Franck Berthe, Eugene Burreson, Mike Hine, Sharon McGladdery, Mike Phillips, Melba Reantaso.
With the possible exception of Australia and New Zealand, there is a lack of published or readily available information on molluscan parasites, pests and diseases in the Asia-Pacific region. Most countries lack dedicated expertise, facilities and infrastructures for molluscan health examination. However, in many countries, mollusc production (subsistence and aquaculture) is established and growing. Species under cultivation for food or secondary products include pearl oysters, edible oysters, mussels, scallops, abalone and capiz shell. Although there is no immediate application for use of DNA-based diagnosis methods on a routine basis for molluscs, tools already developed could be used for cross-checking the specific/generic identities of emerging parasites which appear related to
known pathogens e.g. Haplosporidium spp., Marteilia spp. and Perkinsus spp. Unpublished data from
Australia indicates the presence of Haplosporidium sp. in Pinctada maxima, and Perkinsus sp. in
Saccostrea commercialis. In view of the presence of potential pathogens, the reported growth of mollusc aquaculture industries and the increasing pressure for live introductions and transfers, there is an urgent need for a survey of normal and diseased animals to obtain more extensive data on mollusc health.
There is also a need to establish national and regional expertise through training. As a prerequisite to implementation, NACA should contact National Coordinators to: i) confirm support for a mollusc health program; ii) identify commercially significant species; and iii) designate at least two technicians who would be dedicated to the project. NACA should also establish a panel of experts (from within and outside the region) who will support the project and provide reagents and reference material. Initially, training will aim to establish an initial baseline of expertise in husbandry, sampling, gross observations, fixation, anatomy, and histology. This first step would provide the basis for national programs for mollusc health monitoring. Where resources are limited, this could include market samples. Initial training would be followed (3-4 months later) by an advanced session for the same participants covering histopathology and technical problem solving. During this session, each participant would bring examples of their own material for joint consultation and evaluation. In addition, contacts with mollusc pathology specialists and reference material from other collections (slides and guides) should be available for these participants. The goal of this training program would be to establish a capability for independent monitoring of mollusc health in each country.
As a result of this training program, a clearer picture of the health status of molluscs and the diagnostic needs in the region should emerge. The connection between this initiative and the supporting network (comprising specialist mollusc pathology laboratories) will enable access to the advanced diagnostic tools discussed in detail in the full workshop forum (for finfish, shrimp and molluscan pathogens). The mollusc pathogen Focus Group determined that, in the Asia-Pacific region, these methods should be reserved for cross-checking material containing organisms resembling known pathogens for which
advanced diagnostic methods are available in supporting laboratories (e.g., Marteilia spp.
DNA-probes). They could also be used to determine the geographic and host distributions of these pathogens as they emerge. DNA sequence analysis should also be conducted (e.g. to confirm positive probe results) to validate application of available probes to pathogens in the region. The need to develop additional molecular diagnostic tools and other pathogen/pathogen-group research needs will be determined as pathogens emerge from this surveillance program.
3.3 Finfish pathogens
Alexandra Adams, Supranee Chinabut, Barry Hill, Maura Hiney, James Lilley, Kamonporn Tonguthai.
3.3.1 Diseases requiring application of DNA-based technologies
There are 3 areas for which the introduction and use of DNA-based techniques is likely to have a significant impact on improving disease diagnosis in developing countries.
Mycobacteriosis. As there is a zoonosis risk associated with M. marinum, there is a special need to
identify mycobacterium infections to the species level. Currently available PCR methods should be standardized and validated to the extent that stocks can be certified (see paper by Adams, Appendix 1).
Viral nervous necrosis (VNN). VNN is a potential threat to an important grouper industry in the
region. DNA probes are available to particular strains in Europe and possibly Australia. A comparative study of available probes is required and a validated method for screening wild grouper broodstock should be developed.
Epizootic ulcerative syndrome (EUS). EUS infections cannot presently be distinguished from
occurrences of ulcerative mycosis in USA. There is a need for a standardized, validated in situ
hybridization test that is specific for A. invadans. The development of PCR primers and/or
hybridization probes for the EUS-associated rhabdovirus would also assist in understanding the disease syndrome (see Lilley and Chinabut, Appendix I)).
In addition to these 3 areas, there is a need for basic research on potentially emerging diseases, including red spot disease and streptococcal infections.
3.3.2 Evaluation of needs for rapid detection techniques
The majority of pathogens causing the principal diseases of fish in the Asia-Pacific region can be detected and identified using existing methodologies. These include visual observation and light microscopy for parasitic infections, and routine bacteriology and histology for bacterial pathogens. Specific staining methods and antibody-based techniques are also utilized. Cell culture is used for grown and identification of viral pathogens. The primary fungal infection (EUS) is detected by histology. However, DNA-based technologies would assist in controlling the spread of fish diseases
disease if used for confirmation of diagnosis and for screening fish for specific pathogens such as M.
marinum, VNNV and A. invadans.
3.3.3 Status of research
Research is already underway on all three areas:
Mycobacterium spp. Monoclonal antibodies (MAbs), PCR primers and DNA probes are available.
The MAbs can discriminate M. marinum at species level but do not detect all isolates. New MAbs are
currently being developed. Two PCR-based methods have been developed, one employing enzyme restriction analysis of the PCR product, and the other employing reverse cross-blot analysis. The later
appears to be more specific and can detect all 3 species infecting fish (M. marinum, M. fortuitum and
M. chelonei). This test is presently used to detect aquatic mycobacteria in fish tissue samples, water samples and in human clinical biopsies but it requires further standardization and field validation.
VNNV. VNN is present in Australia and has recently been detected in grouper in Thailand.
Diagnostic antibodies, PCR primers and DNA probes have been developed for VNNV isolates from the Mediterranean region where the disease causes widespread mortalities. These reagents should be used to determine their specificity for VNNV isolates from the Asia-Pacific region.
EUS. PCR and in situ hybridization tests are currently being developed to detect A. invadans from
Asia. These should be utilized to screen samples from different geographical locations for the
presence of A. invadans. Primers should also be developed against the rhabdovirus which is often
isolated in association with EUS.
3.3.4 Key constraints to establishment of international standards
The need for a more formal set of protocols and procedures for PCR-based diagnostic assays has been recognized for a number of years. However, as in many other areas of diagnostics, there has been no serious attempt to set agreed international standards. For a technique with the potential power PCR, the lack of clear guidelines for the correct performance is a serious constraint to its routine use. A second and more serious constraint is the almost complete absence of validation studies undertaken on field samples for any of the currently available assays (see Hiney, Appendix I). Although validation programs are expensive and time consuming, and carefully designed laboratory studies can provide much of the data required to ensure that PCR-based assays perform with acceptable precision, information on the meaning of the results generated by non-culture-based diagnostic techniques can only be obtained through field validation programs. Therefore, collaborative projects to assess currently available PCR-based diagnostic techniques through comparative and predictive field validation studies are urgently required and should be actively supported by funding agencies.
3.3.5 Recommendations for research programs
Adoption of PCR-based diagnostic assays, to replace more established methods is an attractive option for many laboratories. These assays are seen as more sensitive, rapid and logistically simple, and have the allure of high technology. However, in the absence of adequate internal (laboratory) and external (field) validation, interpretation of the meaning of the results generated by the PCR-based assays will remain problematic. While an assay may be acceptable in a research context it will not be acceptable in either a diagnostic or regulatory context and could generate misleading data. Therefore, the priority of any research program that aims to replace current diagnostic methods with PCR-based assays must be a carefully considered validation program that addresses the issue of interpretation of meaning. As a first approach, comparative validation programs should be conducted in which PCR-based assays are performed in parallel with established methods over a reasonable period of time and using statistically significant number of samples. This type of validation program would also allow standardization of the performance of the assay in the laboratory. Standard reference material (both positive and negative controls) supplied from a central reference laboratory would be important to ensure the precision of the results generated by such a study.
More importantly, it is essential that results generated by PCR-based diagnostic assays can be related to field situations. Positive or negative results generated by the assay should be reliably related to actual disease episodes. It is only through such ‘predictive validation’ that interpretation of the results in relation to disease diagnosis is meaningful. Some components of a predictive validation study could be conducted retrospectively if field data of adequate quality were available.
The 3 fish diseases (mycobacteriosis, VNN and EUS) highlighted in this report are recommended for future research.
4
Issues relating to the adoption of international
standards for disease diagnosis and health
certification
In 1995, the World Trade Organisation (WTO) implemented the Agreement on the Application of Sanitary and Phyto-Sanitary Measures (SPS Agreement) to define the conditions under which countries can impose sanitary conditions on imports of animal and animal and plant products. This Agreement is intended to promote international trade by requiring all member states to use only international standards for reducing disease risks associated with imported products. For animals and animal products, the Agreement identifies the guidelines and recommendations established by the Office International des Epizooties (OIE) as the appropriate and preferred international standard. However, the Agreement also allows governments to use standards developed by other relevant international organisations whose membership is open to all WTO members, or to use higher standards if an appropriate risk assessment provides adequate scientific justification.
In Europe, EU Member States have agreed common standards for the health conditions applying to intra-Community trade in aquaculture animals and their products, based on those recommended by OIE. Countries in the Asia-Pacific Region may also wish to agree on common standards of diagnostic and health assurance tests. However, if international standards established in a particular region differ significantly from those recommended by the OIE, trade with countries outside the region may be
adversely affected. For example, if the standard adopted in a region is lower than the OIE standard,
countries which apply the OIE standards could rightly refuse imports on the basis that they have
insufficient health guarantees. Conversely, if the standard agreed in a region is higher than those of
OIE, the same standard must be applied to imports from other regions, to ensure equivalence and protection of the regional health situation. Furthermore, if an individual country adopts higher health protection standards, the SPS Agreement requires scientific justification based on import risk assessment. For diseases not listed by the OIE, trading countries may agree on any mutually acceptable standards.
If a regional training and laboratory accreditation program for molecular diagnosis is to be established in the Asia-Pacific region, the adoption of tests recommended in the OIE Diagnostic Manual for Aquatic Animal Diseases would assist in achieving universally agreed standards. However, as the Manual is revised only every 3 years or so, better methods may emerge from research before the next edition is published. In considering a case for adoption of new molecular diagnostic methods, the suitability must be rigorously assessed in consultation with independent experts. Acceptance is more likely if the method has been published, has received wide scientific acceptance and has been standardised, and preferably validated, in comparison with other standardised methods.
5
Key outcomes and recommendations
5.1 General
comments
There is considerable scope for more effective use of DNA-based methods of pathogen detection and disease diagnosis in Asia-Pacific aquaculture. However, implementation of standardised practices that produce reliable, useful and comparable data will require a significant investment in research, training and infrastructure development. Effective implementation will also be assisted by enhanced communication between aquatic animal health practitioners in the region and scientists with expertise in disease diagnosis and pathogen detection.
Although there are some common themes, it is also evident that there are significant differences in the current relevance of DNA-based methods of pathogen detection for the different aquaculture sectors. DNA-based methods are particularly suitable for detection and diagnosis of shrimp and mollusc pathogens because of the absence of an antibody response in invertebrates and lack of suitable cell lines for virus cultivation. In shrimp, the primary pathogens are well known and many DNA-based methods have already been developed. However, in molluscs there is very limited knowledge of pathogens and few diagnostic procedures of any kind are being employed in the Asia-Pacific region. In fish, antibody and culture-based diagnostic methods are available and considered to be robust and effective for routine diagnostic applications. As such, DNA-based methods in fish appear to be most suitable for confirmatory diagnosis and rapid screening of low level or unapparent infections. To achieve maximum impact, it is essential that research and training programs recognise these differences and are tailored to reflect current levels of knowledge and sector-specific needs.
Where DNA-based tests are available and/or suitable, the most significant impediment to effective implementation is the lack of standardised methodologies that are validated for specific applications. There is a need for international agreement on methodologies that have been rigorously evaluated and accredited for specific applications in disease diagnosis and pathogen screening. There is also a need to ensure that tests are performed by trained staff with access to standardised reagents and suitably equipped laboratories.
Because of existing limitations on the reliability and accessibility of the methods, international standards recommended by OIE do not presently include DNA-based methodologies. However, the potentially high sensitivity and specificity and relatively low cost of these tests has resulted in a surprisingly rapid adoption rate in Asia, particularly for shrimp pathogens. Therefore, it is essential that DNA-based tests are assessed on their merits against existing technologies and that programs to achieve improved performance and international standardisation should be developed. It is also essential that these programs should assist and complement the activities of OIE in obtaining internationally agreed test standards.
5.2 Research
needs
There are a number of pathogens for which DNA-based test methodologies are published or available commercially. However, in general, further research is required before standardised and validated DNA-based test protocols can be implemented for disease diagnosis and pathogen detection in the major aquaculture sectors in the Asia-Pacific region. Research needs vary for each pathogen depending on the existing knowledge base and state of the technology.
Recommendation Programs of international research cooperation should be developed and
coordinated by FAO/NACA. The research should be conducted by managed collaborative networks and provide the information and technology necessary for delivery of suitably specific and validated tests for pathogens of fish, shrimp and molluscs in the Asia-Pacific region. Two research programs are proposed:
Program A: Identification and characterisation of potential pathogens of molluscs, shrimp and fish in
the Asia-Pacific region.
This program should focus on improving the knowledge base by identification of new and emerging pathogens (through health screening, epidemiological investigation and subsequent molecular characterisation), relating pathogens in the region to those described elsewhere, and defining the extent of genetic variation between related pathogens in the region. The program should include the following priority projects:
x Health screening and pathogen identification in molluscs;
x Characterization of WSSV and YHV strain and pathotype variation in prawns;
x Characterization of Haplosporidium, Marteilia and Perkinsus spp. infecting molluscs in Asia; x Characterization of VNNV strain variation in grouper and other fish of economic importance; x Characterization of emerging fish diseases including red spot and streptococcal infections.
Program B: Development and validation of DNA-based diagnostic and detection methods for diseases
of aquaculture in the Asian region.
This program should draw on information currently available or obtained from Program A to develop suitably specific DNA-based diagnostic methods and to evaluate and validate the methods for disease diagnosis and pathogen screening programs. The research program should include the following priority projects:
x Standardisation and validation of group and strain-specific DNA-based detection tests for WSSV and YHV-complex viruses;
x Development and validation of species and strain-specific DNA-based detection tests for mycobacteriosis, viral nervous necrosis and epizootic ulcerative syndrome in Asia-Pacific;
x Development and validation of DNA-based detection tests for Haplosporidium, Marteilia and Perkinsus spp. in Asia-Pacific.
5.3 Training
needs
The implementation of effective DNA-based diagnosis is severely constrained by the availability of scientists and technicians with skills in pathology and molecular diagnostic technologies.
Recommendation. FAO/NACA should develop training programs for staff from key laboratories in the
region. Training is required in the following priority areas:
x The use of standard histopathological methods for health screening of fish and molluscs.
x The use of standard DNA-based methods for pathogen detection including sample collection, application of test protocols and the analysis and interpretation of test results. Initially, training should focus on detection of shrimp pathogens.
5.4 Communication
needs
There is a need to improve communication links between practitioners and scientists with recognised expertise in disease diagnosis and pathogen detection.
Recommendation. FAO/NACA should establish and maintain sector-based (fish, molluscs, shrimp)
communication networks of diagnostic practitioners and internationally recognised experts in aquatic animal health. Activities of the networks should include:
x Exchange on information pathogen distribution in the Asia-Pacific region and the availability of diagnostic tests and reagents;
x Development of cooperative research projects and training programs;
5.5 International
standardisation
Lack of standardisation of tests and test protocols is a major impediment to effective implementation of DNA-based methods in the Asia-Pacific region. Standardisation requires international agreement and cooperation in test selection, practitioner training and laboratory accreditation. Improvements in the reproducibility, validity and comparability of data resulting from accreditation will also assist OIE in assessing the suitability of DNA-based methods for detection of listed pathogens.
Recommendation. FAO/NACA should develop a program of accreditation of standard DNA-based
tests and laboratories with the required standards of operation and expertise to conduct the tests effectively. The program should be administered by NACA through pathogen-specific reference laboratories with the following functions:
x Maintain accredited tests and reagents including reference standards; x Monitor standards and provide technical advise to accredited laboratories; x Provide definitive diagnosis in difficult or unusual cases;
x Archive pathogens for future reference.
6 References
ADB/NACA. (1991). Fish Health Management in Asia-Pacific. Report on a regional study and workshop on fish disease and fish health management. ADB Agriculture Department Report Series No. 1. Network of Aquaculture Centres in the Asia Pacific, Bangkok, Thailand. ADB/NACA. Final Report on the Regional Study and Workshop on Aquaculture Sustainability and
the Environment (RETA 5534). Asian Development Bank and Network of Aquaculture Centres in Asia-Pacific. NACA, Bangkok, Thailand. (in press).
Alday-Sanz, V. and Flegel, T.W. (1997). The risk of introducing yellow-head and white-spot viral infections from Asia to the Americas. CD ROM Paper No. 1, IV Congreso Ecuatoriano de Acicultura, 22-27 October, 1997, Guayaquil, Ecuador, 9 p.
Ambrosia, R.E. and De Wall, D.T. (1990). Diagnosis of parasitic disease. Reviews of Sci Techn.,
Office Intern. Epizool. 9, 759-778.
Chamberlain, G.W. Sustainability of world shrimp farming. In: E.K. Pikitch, D.D. Huppert, and
M.P. Sissenwine (eds.) In: Global Trends: Fisheries Management. American Fisheries
Society Symposium 20, Bethesda, Maryland . (in press).
Chanratchakool, P., Turnbull, J.F., Funge-Smith, S.J., MacRae, I.H. and Limsuwan, C. (1998). Health Management in Shrimp Ponds. Aquatic Animal Health Research Institute, Bangkok, Thailand, 152 p.
Knowles, D.P. and Gorham, J. R. (1990). Diagnosis of viral and bacterial diseases. Reviews of Sci.
Techn., Off. Intern. Epizool. 9, 733-757.
Lightner, D.V. and Redman, R.M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture 164, 201-220.
Lo C.-F., Ho, C.-H., Peng, S.-E., Chen, C.-H., Hsu, H.-C., Chiu, Y.-L., Chang, C.-F., Liu, K.-F., Su, M.-F., Wang, C.-H. and Kou, G.-H. (1996a). White spot syndrome (WSBV) detected in
cultured and captured shrimp, crabs and other arthropods. Diseases of Aquatic Organisms
27, 215-225.
Lo C.-F., Leu, J.-H., Ho, C.-H., Chen, C.-H., Peng, S.-E., Chen, Y.-T. Chou, C.-M., Yeh, P.-Y., Huang, C.-J., Chou, H.-Y., Wang, C.-H. and Kou, G.-H. (1996b). Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain
reaction.Diseases of Aquatic Organisms 25, 133-141.
Lo C.-F., Hsu, H.-C., Tsai, M.-F., Ho, C.-H., Peng, S.-E., Kou, G.-H. and Lightner, D.V. (1999). Specific genomic DNA fragment analysis of different geographical clinical samples of
Lotz, J.M. (1997). Special topic review: Viruses, biosecurity and specific pathogen-free stocks in
shrimp aquaculture. World Journal of Microbiology and Biotechnology 13, 405-413.
Muroga, K. (1997). Recent advances in infectious diseases of marine fish with particular reference to
the case in Japan. p. 21-31. In: T.W. Flegel and I.H. MacRae (eds.) In: Diseases in Asian
Aquaculture III. Fish Health Section, Asian Fish. Soc., Manila.
Plumb, J.A. (1997). Trends in freshwater fish disease research. p. 35-47. In: T.W. Flegel and I.H.
MacRae (eds.) In: Diseases in Asian Aquaculture III. Fish Health Section, Asian Fish.
Soc., Manila.
Rana, K.J. (1997). Recent trends in global aquaculture production: 1984-1995. 14-19p. FAO Aquaculture Newsletter. No. 16. August 1997, FAO, Rome. 28pp.
Technological constraints to disease prevention and
control in aquatic animals, with special rference
to pathogen detection
Sharon M
cGladdery
Fisheries and Oceans Canada, Gulf Fisheries Centre, PO Box 5030 (343 Ave. Université Ave.), Moncton, NB E1C 9B6 (E1C 5K4), Canada
Introduction
Effective disease management and risk analyses rely on accurate data and information. For aquatic organisms, much of this information has been derived from a relatively narrow array of diagnostic tools, most of which are either non-pathogen-specific or have undergone “the test of time” rather than methodical validation or standardization procedures. Recently, however, this range of diagnostic methods has expanded to encompass the molecular expertise pioneered by human health and agricultural food production needs. The complexity of many of these techniques, and their rapid adaptation to “field kits” for use by non-specialist personnel, has prompted a serious re-evaluation of what we use in aquatic animal health management and why (Hiney, 1997).
This paper is aimed at determining which areas of aquatic animal health management are limited by diagnostic and pathogen detection technology, and which are adequately met by traditional methods. Specific disease examples are described elsewhere in these proceedings by the specialists working with them, thus, the points below are deliberately general and provided as “food for thought”.
The Issues
Aquatic animal health management needs arise from two separate situations:
1. The proliferation of opportunistic pathogens in physiologically stressed or immunologically compromised host populations, requiring sensitive, early, detection of potential pathogens.
2. The spread of a primary infectious organisms between infected and uninfected individuals, stocks or populations, requiring accurate identification of the pathogens responsible for disease outbreaks, sensitive detection of pathogens in sub-clinical carriers or abnormal hosts and accurate differentiation between benign and significant infectious organisms.
Disease diagnosis - identification of the cause of a disease outbreak.
Some diseases can be diagnosed in the field with minimal technology or the need to isolate the causative agent, e.g., bacterial gill disease in association with stressful rearing conditions (Thoesen, 1994). Others present clinical signs which defy rapid or conclusive diagnosis, e.g., Malpeque disease
of American oysters, Crassostrea virginica (McGladdery, 1993). Yet other diseases are caused by a
range of different infectious agents e.g., chitinolytic fungal and bacterial shell diseases of crustaceans (Brock and Lightner, 1990). These situations can lead to diagnostic confusion (misdiagnosis) and ineffective management. First time disease outbreaks may (and should) require sample referrals to laboratories or diagnosticians which have experience with the putative pathogen - experience often being as important as technology for rapid and accurate disease diagnosis.
What do we have?
The tools available for disease diagnosis differ between the types of aquatic organisms being examined. For finfish, there is a relatively broad range of diagnostic techniques, many of which can be used as cross checks for diagnosis of a single disease. For example, epizootic haematopoietic necrosis
of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) can be confirmed by: i) conventional isolation on BF-2 (bluefin gill 2) or FHM (fathead minnow) cell lines with serological identification of the iridovirus agent; ii) an indirect immunofluorescence antibody test (IFAT); iii) an enzyme-linked immunosorbent assay (ELISA); or iv) polymerase chain reaction (PCR) amplification and subsequent sequencing of the iridovirus DNA, using two published primers (OIE, 1997). Viral encephalopathy and retinopathy (viral nervous necrosis) virus and related nodaviruses are detectable using a range of specific and less specific techniques including: i) ultrastructural confirmation of virus-induced histopathology; ii) immunohistochemistry; iii) DFAT; iv) ELISA; and v) PCR amplification and sequence analysis (OIE, 1997). Some diseases with a more limited range of diagnostic options can be diagnosed accurately using the techniques available e.g. whirling disease of
salmonids, caused by Myxosoma cerebralis, is presumptively diagnosed by behaviour, with
confirmatory observation of the myxosporean spores in cartilage digests or histology preparations. Furthermore, few finfish diseases with a single aetiology, have defied conclusive diagnosis for long periods.
The role of multiple infectious agents in a disease can usually be resolved through experimental research and verification using Koch’s postulates. One example for finfish (which has an as yet unidentified aetiologic agent) is erythrocytic inclusion body syndrome (EIBS). Although the causative agent is believed to be viral in nature, secondary infections by bacteria and fungi can confound
diagnosis (Thoesen, 1994; Jarp et al., 1996). Once the primary infectious agent for such diseases is
identified, subsequent diagnosis is simplified and, generally, ignores the presence of the secondary pathogens. The classic example of one such multi-factorial disease is epizootic ulcerative syndrome (EUS) which is described in detail elsewhere in these proceedings.
The range of diagnostic techniques available for molluscan and crustacean diseases is narrower than that for finfish. Most significantly, aquatic invertebrates lack self-replicating cell lines for isolation and identification of intracellular pathogens. Finfish cell lines can be used, but the nature of the isolated viruses is often subject to question, since they could be vertebrate contaminants rather than
primary invertebrate pathogens (Hill et al., 1986). In addition, Koch’s postulates have rarely been
fulfilled or replicated for molluscan or crustacean isolates from fish cellm lines. Thus, most intracellular infections which cause overt disease in crustaceans and molluscs require histopathology for presumptive diagnosis, with ultrastructural confirmation of viral or bacterial aetiology. Histology,
although laborious, has the advantage that it provides a permanent record of the pathogen in situ and
can be used to assess focal or systemic histopathology. Conversely, it is limited in sensitivity to infectious agents which can be detected, and identified, at the light microscope level, eliminating most viruses, many bacteria, protists and even some metazoan parasites (which require whole mount or adult-stage identification).
As with finfish, multiple diagnostic techniques are available for a number of shrimp diseases (Lightner, 1996) but most clinical cases can be presumptively diagnosed using non-specific techniques (gross observation, histology and tissue smears). Confirmatory diagnosis is then achieved using culture e.g. crayfish plague (Alderman and Polglase, 1986) or electron microscopic examination of ultrastructural features (Lightner, 1996). Pathogen culture is rarely used for diagnosis of molluscan
diseases with the exception of two groups of significant disease agents: i) Perkinsus spp. (Ray, 1966;
Gauthier and Vasta, 1993; LaPeyre et al., 1993); and ii) Labyrinthuloides-like protists (Bower, 1987;
Kleinschuster et al., 1998). In clinical cases, however, these are also readily diagnosed using standard
histology. Most other significant disease agents of molluscs are difficult to culture, but can be isolated
under certain conditions (Hervio et al., 1993; 1995) i.e. acute infections. However, pathogen isolation
is normally reserved for development of more specific detection and identification techniques rather than clinical diagnoses.
What are the limitations?
Speed of diagnosis is always a concern, especially with acute losses relying on histopathology, ultrastructural confirmation or long periods of tissue/media culture. The time span required for confirmatory diagnosis is frequently overcome by remedial action being based on presumptive diagnoses such as tissue smears, gross pathology or behavioural changes. This is most effective in areas with a well defined history of the disease e.g. Denman Island disease which is caused by Mikrocytos mackini in Pacific oysters (Crassostrea gigas) on the west coast of Canada (Bower, 1988). First time disease outbreaks in new species to culture, or appearing at a location for the first time, can undergo protracted periods of non-diagnosis or, worse, misdiagnosis. Examples include a serious
disease of hard shell clams, Mercenaria mercenaria, caused by an unidentified Labyrinthuloides-like
organism, “QPX”. This may have been causing mortalities in pre-culture history (Drinnan and Henderson, 1963) but its significance was not fully realised until hatchery broodstock and cultured
stock on grow-out beds started to die (Whyte et al., 1994; Ragone Calvo et al., 1997; Smolowitz et al.,
1996). Similarly, infectious salmon anaemia of Atlantic salmon (Salmo salar) was described as
haemorrhagic kidney syndrome (HKS) when first detected in Atlantic Canada (Getchell, 1997). It took over a year before the causative agent was recognised as a virus and identified as ISAV, an agent previously known only from Norway (Hastein, 1997).
Where can molecular techniques enhance disease diagnosis?
Significant pathogens that require long, complex culture or histology-based confirmatory diagnosis are prime candidates for rapid, pathogen-specific diagnostic kits. This applies predominantly to microbial pathogens, but may be equally appropriate for protists which are difficult to distinguish morphologically at the light microscope level or which have a diverse host-range. Rapid, pathogen-specific diagnostics would be particularly appropriate for disease management and control when diseases emerge in new geographic locations or host species, as described under limitations. An additional application for molecular techniques is for research into the pathogenesis of a disease via non-lethal sampling e.g. of haemolymph, fin- or gill-clips. This would provide useful information on pathogen proliferation, haemolymph profiles etc. but negates examination of the physical host-pathogen interface.
Pathogen screening – detecting infectious agents in sub-clinical or healthy organisms.
Screening for infectious agents in healthy hosts is probably the most controversial area of aquatic animal health management. This is due to: i) inconclusive negative results; ii) the potential disease risks; and iii) the difficulty of controlling a disease outbreak in naïve and/or open-water populations. Since pathogen screening is frequently a pivotal part of disease risk assessments prior to transboundary transfers, the techniques used can also be “politically sensitive”. More recently, pathogen and/or disease screening is being used to define aquatic zones (intra-national and, more rarely, international) based on health profiles. This is usually limited to specific pathogens of commercially important host species (OIE, 1977). These zones can then be used for management purposes, to allow movement of pathogen carriers between non-confluent waters where the pathogen is known to occur (“like-to-like” transfers). Pathogen detection in healthy (carrier) hosts never assures 100% confidence, statistically, therefore negative samples all have a level of error which can be directly related to the sensitivity of the screening technique(s) applied.Since disease risk analyses have been, and continue to be, well-debated (DeVoe, 1992), more effort has been focussed on epidemiological principals in an effort to quantify and standardise the broad range of qualitative-based risk evaluations (Hiney, 1997; Thorburn, 1999). This has revealed a broad gap between the probability of detection of a single pathogen in a given sample and the statistical confidence in that detection. This reflects non-survey-based assumptions for pathogen prevalence in wild or open-water populations, as well as detection sensitivity, since prevalence is one of the critical factors determining confidence of detection of a single pathogen in any given sample size (Ossiander and Wedemeyer, 1973).
What do we have now?
Pathogen screening of aquatic animals involves the same techniques described above for disease diagnosis. In addition to culture-based techniques, immunoassays (fluorescent antibody tests, agglutination tests, ELISA) and nucleic acid probes have been available for finfish pathogens for
years, and some form the basis of kits now used for pathogen screening (e.g. Aeromonas salmonicida
which causes furunculosis, and Renibacterium salmoninarum which causes bacterial kidney disease).
Pathogen-sensitive techniques for molluscs pathogens have been developed more recently e.g.
immunoassays for Perkinsus marinus (Dungan and Roberson, 1993), Bonamia ostreae (Mialhe et al.,
1988),Vibrio tapetis (causative agent of brown ring disease of Manila and Portuguese carpet clams,
Ruditapes philippinarum and R. descussatus, respectively) (Castro et al., 1995) and giant rickettisia of
the sea scallop Pecten maximus (Le Gall et al., 1992). However, none of these techniques has yet
been transformed from research to routine diagnostic application and histology remains the most broadly used detection/diagnostic method applied to molluscs. Detection of sub-clinical infections in shrimp is limited to infectious hypodermal and hematopoietic necrosis virus (IHHNV), using
bioassays as well as in situ and dot blot hybridisation or PCR of viral product in haemolymph or tissue
homogenates, and baculoviral midgut gland necrosis virus (BMNV) using bioassays in susceptible Penaeus japonicus and a fluorescent antibody test (Lightner, 1996). Stress-induced enhancement of infections is another procedure used to enhance some sub-clinical viral infections in shrimp (and other aquatic species) which cannot be detected by histology (Lightner, 1996).
What are the limitations?
For cryptic infectious agents (mainly microbial) routine diagnostic procedures on healthy animals, especially non-culture-based techniques, are particularly weak in detection sensitivity. Thoesen (1994) lists several diseases caused by primary pathogens for which there are no procedures
documented for detecting sub-clinical infections (e.g. Vibrio salmonicida, cold-water vibriosis or
“Hitra disease”; channel catfish virus, CCV; Haplosporidium nelsoni, MSX; and H. costale, SSO, of
American oysters, Crassostrea virginica). Lightner (1996) lists very few pathogens of shrimp for
which there no methods to detect sub-clinical infections (hepatopancreas parvovirus, HPV). However, some pathogens can only be detected following stress-testing (e.g. monodon baculoviruses, MBV). For most disease agents in sub-clinical or abnormal “carrier” hosts, this means that sample size or sampling frequency has to be increased to enhance the level of confidence in detection. For techniques such as histology and ultrastructure, this frequently involves compromise between sample size (confidence level) and resource capability (time and manpower). For other more sensitive techniques (tissue culture, immunoassays and nucleic acid probes), the compromise may involve time, expense and specialist resource factors.
Where can molecular techniques enhance zonation establishment and surveillance or transfer disease risk analysis?
As described above, most agents of significant infectious diseases are difficult to detect using routine diagnostic techniques in healthy, sub-clinical hosts. This means that establishing an area which is designated free of a specific pathogen, inherently, includes a degree of error. Molecular screening techniques for specific pathogens could reduce this error margin by increasing confidence of detection. This would be especially important for areas that export live aquatic animals on a regular basis (“uninfected” zone to “uninfected” zone transfers). However, the pathogen specificity of these screening techniques negates detection of any other pathogenic or potentially significant organisms in the same specimens. Additional non-specific, but less sensitive, screening techniques may, therefore be required to give a true health “profile”. In addition, full-scale molecular-based testing of populations for a given pathogen, especially where there has been no history of the disease, could meet with varying degrees of resistance on both a practical and political level. Interpretation of low positive results from such an area would be especially problematic and difficult to resolve. In conclusion, molecular techniques might best serve as confirmatory screening to reinforce/refute results from
