DNA - based molecular
diagnostic technics:

Research needs for standardization and
validation of the detection of aquatic
animal pathogens and diseases

During 7-9 February 1999, FAO in collaboration with NACA, ACIAR, CSIRO, and DFID conducted an Expert Workshop on DNA-based Molecular Diagnostic Techniques: Research Needs for Standardization and Validation of the Detection of Aquatic Animal Pathogens and Diseases. Twenty-two experts, most of them who are currently working on the development of DNA-based rapid diagnostic techniques for the detection of aquatic animal pathogens, and representatives from other concerned agencies participated in this workshop which was held at NACA Headquarters (Bangkok, Thailand). 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. The report of the workshop together with the review papers are being published as a FAO Fisheries Technical Report. This article outlines the background and the major findings and recommendations from the workshop.

Impact of disease on aquaculture

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. Disease is now recognized as the primary limiting factor for shrimp farming today, and the impact of aquatic animal diseases on sustainable rural livelihoods also been duly recognized among the communities where aquaculture plays a role in rural development. 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.
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 for detection and diagnosis. However, these methods are time-consuming and costly, and, for shrimp and other crustaceans, cell lines suitable for virus culture have not been available.

Role of DNA-based technologies in diagnosis
and pathogen detection

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 diagnostic 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 programmes in aquaculture, in field epidemiology and in efforts to prevent the international spread of pathogens.
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 haematopoietic 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). Striped jack (Pseudocaranx dentex) broodstock have been screened with the aid of PRC 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 haematopoietic 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 parasite 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 reliable detection 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 Epizooties (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.


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). 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. 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. Large differences in sensitivity are related to the PCR method used. PCR methodologies are highly susceptible to contamination by foreign DNA during processing that may result in false positives. PCR tests must be conducted in very well managed, clean laboratories. "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.

Figure 1. In situ hybridization

Figure 2. Fluorescense Antibody Test

The expert workshop

The main objectives of the workshop was to (a) 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, (b) evaluate needs for rapid diagnostic techniques for the principal diseases of cultured fish and shellfish, (c) review the status of research towards meeting these needs, (d) identify problems and key constraints related to establishing international standards for protocols and procedures for such tests and make recommendations towards their solution, and (e) make recommendations for research programmes to be developed by 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.

Key outcomes and recommendations

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 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 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 programmes recognize 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 standardized 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, specificity, and relatively low cost (to other detection procedures) of these tests, and the economic returns experienced through accurate detection of serious pathogens 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 programmes to achieve improved performance and international standardization should be developed. It is also essential that these programmes should assist and complement the activities of OIE in obtaining internationally agreed test standards.

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 standardized 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 Programmes 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 programmes are proposed:

Programme A: Identification and characterization of potential pathogens of molluscs, shrimp and fish in the Asia-Pacific region.

This programme 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 programme should include the following priority projects:

• Health screening and pathogen identification in molluscs;

• Characterization of WSSV and YHV strain and pathotype variation in prawns;

• Characterization of Haplosporidium, Marteilia and Perkinsus spp. infecting molluscs in Asia;

• Characterization of VNNV strain variation in grouper and other fish of economic importance;

• Characterization of emerging fish diseases including red spot and streptococcal infections.

Programme B: Development and validation of DNA-based diagnostic and detection methods for diseases of aquaculture in the Asian region.

This programme should draw on information currently available or obtained from Programme A to develop suitably specific DNA-based diagnostic methods and to evaluate and validate the methods for disease diagnosis and pathogen screening programmes. The research programme should include the following priority projects:

• Standardization and validation of group and strain-specific DNA-based detection tests for WSSV and YHV-complex viruses;

• Development and validation of species and strain-specific DNA-based detection tests for

mycobacteriosis, viral nervous necrosis and epizootic ulcerative syndrome in Asia-Pacific;

• Development and validation of DNA-based detection tests for Haplosporidium, Marteilia and Perkinsus spp. in Asia-Pacific.

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 programmes for staff from key laboratories in the region.
Training is required in the following priority areas:

• The use of standard histopathological methods for health screening of fish and molluscs.

• 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.

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 species-based (fish, molluscs, shrimp) communication networks of diagnostic practitioners and internationally recognized experts in aquatic animal health. Activities of the networks should include:

• Exchange on information pathogen distribution in the Asia-Pacific region and the availability of diagnostic tests and reagents;

• Development of cooperative research projects and training programmes;

• Development of cooperative programmes for test validation and laboratory accreditation.
  
  

International standardization

Lack of standardization 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 programme of accreditation of standard DNA-based tests and laboratories with the required standards of operation and expertise to conduct the tests effectively. The programme should be administered by NACA through pathogen-specific reference laboratories with the following functions:

• Maintain accredited tests and reagents including reference standards.

• Monitor standards and provide technical advise to accredited laboratories.

• Provide definitive diagnosis in difficult or unusual cases.

• Archive pathogens for future reference.

References

Ambrosia, R.E. and De Wall, D.T. (1990). Diagnosis of parasitic disease. Reviews of Sci. Techn., Office Intern. Epizool. 9, 759-778.

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 pp.

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. 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. Diseases in Asian Aquaculture III. Fish Health Section, Asian Fish. Soc., Manila.

Figure 3. In situ hybridization

Photos courtesy of Drs Gene Burreson (USA) and Sharon McGladdery (Canada)