Ponlerd Chanratchakool, Dan Fegan, Tim Flegel, Indrani Karunsagar, Don Lightner, Gary Nash, Mohamed Shariff, Peter Walker.
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suggest some variability in the detection specificity of PCR tests (Park et al., 1998; R.A.J. Hodgson and P.J. Walker, unpublished data).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.
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:
maintaining an agreed test as the primary reference standard against which other testing protocols should be assessed;
maintaining standard PCR reagents including primers and suitable positive and negative controls;
monitoring standards and providing technical advise to accredited laboratories;
providing definitive diagnosis in difficult or unusual cases;
retaining and archiving virus isolates for reference;
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.
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.
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.
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.
Alexandra Adams, Supranee Chinabut, Barry Hill, Maura Hiney, James Lilley, Kamonporn Tonguthai.
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.
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.
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.
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.
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.