Aquafin CRC - Atlantic Salmon Aquaculture Subprogram: development of selective enrichment culture-polymerase chain reaction (SEC-PCR) for the detection of bacterial pathogens in covertly infected farmed salmonid fish
University of Tasmania (UTAS)
The ability to detect infected animals is an essential requirement in animal health monitoring and surveillance. A major problem of testing farmed and wild fish is the absence of simple diagnostic tests for the detection of asymptomatic carrier fish. Where tests are available, they are resource intensive and time consuming such as the heat+corticosteroid stress test for furunculosis in salmonids. This test is used for disease control measures in eastern Canada and has been instrumental in limiting spread of furunculosis to sea cage farms (Olivier 1992). Active surveillance of animal populations is considered an important approach in animal health monitoring (Stark 1996) and is of particular relevance with publication by the Office International des Epizooties of its guidelines in the International Aquatic Animal Health Code (Anon 1997) for defining disease-free status. Demonstration of freedom from disease, both covert and overt, within a region or a country, can be an asset when selling live and uncooked product in markets overseas. As global trade develops, Australia will need to demonstrate freedom from disease not just as a marketing strategy but as an essential requirement of trade and as a means of protecting or limiting the spread of disease. This project aims to develop a hybrid technology derived from the food industry. It will require adaptation and refinement for use with fish pathogens and development of test protocols for screening adequate numbers of fish. The use of specialised enrichment culture media with the sensitive and specific techniques of PCR should provide a useful and sensitive tool in active surveillance of fish populations and fish products. This technology will also have application in screening ornamental fish entering Australia as well as uncooked fish products. Detection of bacterial pathogens using immunological markers or DNA are termed proxy tests since the presence of the pathogen is inferred. Proxy tests pose two major problems: firstly what is the relationship of the proxy measure to the intact target pathogen and secondly, what is the biological significance of the proxy test? Validation of proxy tests is a recognised problem that if unresolved can seriously restrict the use of such tests (Hiney 1997). The proposed project solves many of these issues of validation: the primary test requires amplification of the target pathogen by culture and hence is not a proxy test. Detection of the target pathogen after culture utilises a secondary proxy test but it can be internally validated by secondary culture as required. The test system will be evaluated against farmed populations of fish to determine the significance of findings, a pre-requisite for external validation. Correlations of this type have not been undertaken previously and the strategies proposed in this project represent a realistic attempt to convert bench tests into practical and robust diagnostic tools. References: Anon (1997) International Aquatic Animal Health Code. 2nd edit. Office International des Epizooties, Paris. Hiney M. (1997) How to test a test: methods of field validation for non-culture based detection techniques. Bull. Eur. Ass. Fish Path. 17:245-250 Olivier G (1992) Furunculosis in the Atlantic provinces: an overview. Bull. Aquacul. Assoc. Canada 92-1: 4-10. Stark K D C (1996) Animal health monitoring and surveillance in Switzerland. Aus. Vet. J. 73:96-97.
1. Develop a procedure for extracting bacterial DNA from the selective enrichment media that is suitable for the PCR process and is suitable for processing multiple samples.
2. Determine optimum conditions for the PCR test to maximise specificity and sensitivity of the procedure.
3. Develop a test procedure based on immuno-ELISA capture that will verify any positive PCR reactions using a secondary confirmatory gene probe and is suitable for testing multiple samples.
4. Optimise the PCR conditions to incorporate a PCR protection system to protect tests against false positive reactions arising from contamination.
5. Optimise the culture conditions and PCR detection process to ensure the minimum test time between sample collection and test result.
6. Test populations of salmonids with the optimised SEC-PCR system to verify test performance and obtain baseline data on carrier prevalence.
Bacterial disease is a major cause of stock loss in aquaculture. The severity of infection may range from acute to chronic through to benign. This latter condition, termed covert infection, is insidious, as fish may appear to be outwardly healthy but during periods of stress, these carriers may breakdown leading to spread of infection and development of a disease outbreak.
Several bacterial pathogens, known to exist in Australia and the cause of significant disease episodes in Atlantic salmon and rainbow trout, can cause covert infections including: atypical Aeromonas salmonicida, Lactococcus garvieae, Tenacibaculum maritimum and Yersinia ruckeri.
Early detection of covertly infected fish is considered desirable as it provides a means of determining a suitable disease control strategy such as imposing movement restrictions to prevent the spread of disease, changing management practices to avoid stress or determining the spread of disease in a population at risk of infection. The standard method for identifying carriers is to stress a cohort of fish using a combination of heat andi mmunosuppression to force covertly infected fish to breakdown with disease. This form of testing is undesirable for animal welfare considerations, is difficult to accomplish and takes over three weeks to generate results.
Keywords: Aeromonas salmonicida, Lactococcus garvieae, Tenacibaculum maritimum, Yersinia ruckeri, Atlantic salmon, rainbow trout, covert infection, diagnosis, PCR, RT-PCR amplification, enzyme hybridization, DNA extraction