Combating food poisoning from seafood
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|Sensitive polymerase chain reaction (PCR)-based detection of aquatic Vibrios|
A rapid and highly sensitive DNA test is now available to screen seafood for bacteria. Infected seafood, particularly shellfish, can cause food poisoning. Outbreaks damage consumer confidence and producers suffer, especially the poorest. Previously, screening for bacteria was slow, taking up to 7 days, and was not always accurate. These diagnostic techniques have been extensively tested on coastal and estuarine seafood in India, Bangladesh, China, Malaysia and Japan. They’ve been used not only for detecting bacteria in seafood but also for food safety tests and for monitoring bacteria in coastal areas popular for water sports. Hundreds of laboratories have adopted these methods and they are widely accepted by international food safety authorities. They will probably become routine for ensuring that fish exports meet EU, US and Japanese import standards.
Project Ref: PHF10:
Post Harvest Fisheries Research Programme
Relevant Research Projects:
Pathogenic Vibrio bacteria are commonly found along inshore coastal and estuarine environments in Asia and can be isolated from water, sediment, plankton, fish and particularly shellfish. Shellfish concentrate a range of Vibrio spp. through their filter feeding and as a result seafood (e.g. clams, cockles, oysters, lobsters, scallops, sardines, shrimp) is commonly contaminated with vibrios. Human consumption of pathogenic vibrios usually gives symptoms ranging from mild gastroenteritis to severe debilitating dysentery, although for some vibrios such as Vibrio vulnificus, life-threatening septicaemia and eye, ear and wound infections can also occur especially in immuno-compromised individuals.
Gastro-intestinal infections are usually due to consumption of raw or semi-cooked shellfish, which is popular for several types of local Asian foods. Traditionally Vibrio spp. were detected using culture-based methods, employing enrichment media followed by isolation on selective culture media. Such culture-based methods suffer the disadvantages of being time-consuming (7 days for a positive identification) as well as cells of many Vibrio spp. entering a viable but non-culturable (VBNC) state. As a result in the mid 1990s, the Post Harvest Fisheries Programme funded research to develop much more rapid polymerase chain reaction (PCR)-based diagnostic techniques for pathogenic marine vibrios. PCR is a technique which relies on the amplification of a nucleic-acid fragment from short DNA sequences that have been identified as pathogen-specific. PCR techniques were developed for V. parahaemolyticus, V. hollisae (reclassified as Grimontia hollisae) and V. cholerae.
These PCR-based techniques were shown in 1994-1996 under R5793 to have great potential for the quick and sensitive detection and diagnosis of pathogenic Vibrios in seafood in India. Since then, PCR-based techniques for detection and typing of Vibrio spp have been extensively validated not only within India, but also in many other countries (e.g. Bangladesh, China, Malaysia, Japan). PCR has been found to be useful for not only typing Vibrio infections, but also real-time PCR and alkaline-phosphatase DNA probes have been found to be rapid enumeration methods for evaluation of postharvest treatments to reduce Vibrio levels in seafood. PCR tools are also being used in selected laboratories worldwide for food safety testing, as well as monitoring the levels of pathogenic Vibrio spp. in coastal waters used for both recreational (e.g. water sports) and occupational purposes, such as fishing.
Seafood such as oysters, shrimp, eel, cockles, crab, clams, lobsters, scallops, sardines, and squid. The diagnostic tool outputs could be applied to other commodities that could have come into contact with contaminated seafood to assess these for the presence of pathogenic Vibrio spp.
The RNRRS output of project R5793 was to develop PCR techniques for Vibrio parahaemolyticus, V. hollisae and V. cholerae. Since the completion of these studies there have been 100s of reports from other labs using these and other PCR-based tools for detection of pathogenic Vibrio spp. Nucleic acid diagnostic techniques were in their infancy during R5793, and recently a range of highly sophisticated PCR-based techniques have been developed with which it would make sense to cluster outputs. These include:
With regard to RNRRS funded outputs, the Vibrio PCR tests developed would have potential value for a range of other outputs dealing with the food safety and market quality of seafood products, in particular
How the outputs were validated:
The research outputs are sensitive nucleic-acid based diagnostic tests for the detection of a range of pathogenic Vibrio species. Scientific research under R5793 carried out by the Mangalore College of Fisheries (India) was used to validate these diagnostic tests and this was sufficiently rigorous to result in the following high impact peer reviewed publications.Karunasagar, I; Sugumar, G; Karunasagar, I; Reilly, PJA (1993). Rapid detection of pathogenic marine vibrios by polymerase chain reaction (PCR). Int J Syst Evol Microbiol 53 (1993), 1615-1617
Karunasagar I, Sugumar G, Karunasagar I, Reilly A. (1995) Rapid detection of Vibrio choleraecontamination of seafood by polymerase chain reaction. Mol Mar Biol Biotechnol. 4:365-8
Karunasagar I, Sugumar G, Karunasagar I, Reilly PJ (1996) Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods. Int J Food Microbiol. 31: 317-23
The scientific validation of the tests was done by adopting an approach of determining the sensitivity and specificity of the tests, together with ensuring they would work on seafood samples. Detection of a mere 10 cells of the highly pathogenic Kanagawa positive strains of V. parahaemolyticus was achieved using extracts prepared directly from fish homogenates and enrichment culture in alkaline peptone water. Similarly, V. cholerae contamination of seafood could be detected at 1000 V. cholerae/ml fish homogenate without enrichment and as few as 1-2 cells with prior enrichment in alkaline peptone water.
Since the completion of project R5793, there have been many research reports from the above, as well as other, laboratories using modifications of these and other PCR-based tools for detection of pathogenic vibrios.
Although validation was done on laboratory samples and did not result in any increases in productivity, the validation results would be most applicable to extreme vulnerable fisher communities as it is this income category that suffers most when consumer confidence in seafood safety is damaged.
Where the Outputs were Validated:
The scientific research and validation under funding from the Post Harvest Fisheries Research Programme was carried out in 1993-1996 by Prof Iddya Karunasagar’s research team at Mangalore College of Fisheries. However, nucleic acid diagnostic techniques were in their infancy during this project (R5793), and as a result the validations that were done were limited. In the past decade, DNA-based techniques have evolved and as a result a range of highly sophisticated similar PCR-based techniques have been developed for pathogenic vibrios not only in the above laboratory but also in institutes in the following RIUP target countries. The institutes (with active research contact names in brackets where available) include:
All validations target the coastal artisanal farming systems.
Who are the Users?
The diagnostic tests developed through project R 5793 have been superseded and no evidence has been found of their significant use. However, tests resulting from the improvement of R5793 tests, or of similar technologies, are being used by many research laboratories interested in food safety as well as clinical diagnostics.
International regulatory authorities, such as the United States Food and Drug Administration (USFDA) have constructed guidelines that state seafood should contain less than 10 000 cells of V. parahaemolyticus per gram. Sanitary and Phytosanitary Measures (SPS) established in 1994 under the Uruguay Round of GATT encourage member countries to use international standards, guidelines and recommendations where they exist.
Since the completion of R5793, laboratories in the Australia, Japan and the USA have been most active in assisting the RIUP target countries to improve their molecular diagnostic capability for pathogenic vibrio species.
PCR techniques for pathogenic vibrios are thus used to determine the presence of particular Vibriogenes in clinical samples, to monitor the spread of particular pandemics and to test the safety of retail and trade seafood samples. The diagnostic technologies also allow the effectiveness of appropriate hygiene measures to be measured and hence assist the development of measures to avoid infected seafood contaminating other foods through contact in retail outlets.
Recently the tests have also been proposed as valuable to assist the monitoring of the safety of particular estuarine waters for recreational as well as fishing purposes.
Where the outputs have been used:
Modifications of the diagnostic tests developed are still being used actively by the Mangalore College of Fisheries (India). Detailed investigations on the prevalence and presence of particular genotypes of both V. parahaemolyticus and V. vulnificus in oysters from the southern Indian coast have been carried out using these tests (Environmental Microbiology (2005) 7, 995-1002). Recently, alkaline phosphatase probes have also been developed (research accepted for publication in Environmental Microbiology, 2006) to detect pathogenic vibrios in seafood. These probes have the advantage over PCR techniques in not being affected by the presence of PCR-inhibitory compounds in seafood which can give rise to false-negative results.
The National Institute of Cholera and Enteric Diseases in Calcutta. India, is also using PCR tests to monitor the prevalence of the pandemic genotype of V. parahaemolyticus.
The International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh, has used PCR tests for V. parahaemolyticus to determine the presence of the particularly virulent pandemic genotype in hospitalised patients, as well as toxigenic Vibrio cholerae in the aquatic environment of Mathbaria (Alam et al., (2006) Appl Environ Microbiol. 72: 2849-2855).
Various agricultural universities in China have been using PCR tests to demonstrate that retail seafood samples are commonly contaminated with V. parahaemolyticus (FEMS Immunol Med Microbiol 46 (2006) 180-186)
In Malaysia, universities (see 11) have used PCR-based tests to highlight estuarine waters that may be unsafe for recreational purposes. PCR tests have also shown cockle samples to contain high levels of infection (62%) highlighting the risk of consumption of such seafood in an undercooked state (Bilung et al., 2005. FEMS Microbiology Letters 252: 85-88).
Scale of Current Use:
It has not been possible to obtain an accurate estimation of the current usage as the little data is available other than that published in scientific reports. The increased prevalence of scientific papers outlining the use of the techniques for clinical diagnoses and shellfish safety analysis in 2005-2006 suggests that PCR tests for pathogenic vibrios are becoming increasingly popular for detection, enumeration and typing of Vibrio spp. Of relevance here is that international regulatory authorities such as the USFDA now accept the application of nucleic acid tests such as PCR and probe hybridisation tests for food safety analysis.
Considering that these nucleic acid tests are rapid, highly sensitive and specific, it appears probable that they will become increasingly popular for routine analyses in food quality control laboratories particularly to ensure that fish products for exports meet EU, US and Japanese import regulations.
Regulations in the EU have tightened particularly in light of the first European incidence of gastroenteritis due to V. parahaemolyticus, derived from the consumption of live oysters in Spain, being reported in 2003. There will be an increasing requirement for the rapid and accurate detection of pathogenic vibrios against a background of numerically greater numbers of non-pathogenic vibrios and other heterotrophic bacteria to prevent the emergence of seafood-associated disease outbreaks. The non-radioactively labelled probes developed by the R5793 partner (post project funding) offer a very promising technique for such diagnostic purposes as they are less sensitive to the presence of a range of compounds present in shellfish than some of the PCR techniques.
Policy and Institutional Structures, and Key Components for Success:
Promotional activities for the diagnostic outputs outlined do not appear to be taking place. However, International regulatory authorities such as the USFDA have indirectly assisted with the promotion of the outputs by recommending the use of non-radioactively labelled DNA probes for the enumeration of pathogenic V. parahaemolyticus to test whether shellfish to be certified meets food safety requirements.
The new EU General Food Law which became effective in 2006 is also resulting in EU importing countries tightening their food safety legislation and demanding the adoption by exporting countries of agreed inspection, examination and certification procedures.
Direct and Indirect Environmental Benefits:
The adoption of DNA-based diagnostic technologies assist the improvement of food quality control laboratories and the ability to meet international trade requirements regarding food safety.
Adverse Environmental Impacts:
The DNA based diagnostic tests involve the use of carcinogenic and mutagenic compounds that need to be disposed of correctly to ensure that there are no adverse environmental impacts. Certified waste disposal systems are sometimes lacking in developing countries.
Coping with the Effects of Climate Change, or Risk from Natural Disasters:
The outputs can be used as monitoring tools to determine the changed distribution of marine Vibrios due to climate change. For example, V. parahaemolyticus is widespread along marine coastal waters globally, with higher numbers usually encountered during warmer summer months. A slight increase in the temperature of waters due to global warming is likely to increase the incidence of Vibrio pandemics.
Relevant Research Projects, with links to the