Scientists Develop Rapid, User-friendly Test Kit for Marine Toxins

Linda Benedict  |  4/27/2006 9:13:02 PM

Douglas L. Park and Carlos E. Ayala

Ciguatera fish poisoning is a type of food poisoning caused by ingestion of certain tropical and subtropical marine fish that harbor natural toxins originating from microscopic algae (dinoflagellates). The illness is widespread in the tropical Caribbean, subtropical North Atlantic and the Pacific regions. More than 400  different fish species, including amberjack, moray eel, barracuda, Spanish mackerel, triggerfish, snapper, parrot fish, surgeon fish and grouper have been associated with ciguatera outbreaks.

Ciguatera exhibits itself in a variety of ways with many symptoms ranging from gastrointestinal to neurological to cardiovascular disorders. The symptoms last from an initial duration of 14 to 21 days, to months or even years. The onset of symptoms usually occurs within 3 to 5 hours after eating a toxic fish. General symptoms are flu-like. Prolonged cases may also exhibit depression and phobia development. Low blood pressure, reduced blood volume, coma or death may occur. Susceptibility to the toxins and severity of the symptoms vary greatly among individuals because of the possible presence of several different toxins. Immunity does not develop. Evidence suggests that individuals who have been previously exposed are more susceptible and react to lower levels of the toxin. Additionally, the severity of the symptoms increases with subsequent ingestions of ciguatoxic fish.

The major source of the toxins is a group of dinoflagellates, which are planktonic unicellular aquatic microorganisms. Most, like plants, contain chlorophyll for photosynthesis and are primary producers of energy in the ocean food chain. Dinoflagellates show traits of both animals and plants. Zoologists classify them as protozoans and botanists as algae. They can sometimes reproduce in enormous numbers, called a bloom. Certain species produce a strong nerve toxin and are responsible for the blooms called red tides that have killed large numbers of fish and have contaminated clams and mussels, which may then be lethal to humans who eat them.

Ciguatera toxins are odorless, tasteless and difficult to detect by any simple chemical test. They are lipidsoluble, heat-resistant and acid-stable. This means the toxins cannot be eliminated by boiling, salting, drying, freezing, marinating or cooking the fish.

Detecting the toxin
There has been increasing interest in development of a simple, rapid and inexpensive way to detect ciguatoxin and related toxins. Since the early 1990s, HawaiiChemtect International (Pasadena, California), in association with LSU Agricultural Center researchers, has been working on development of a commercial kit named Ciguatect, which involves an innovative rapid solid-phase immunobead assay. Before the development of the Ciguatect kit, there was no test available which could be conducted outside of a laboratory. This test will allow fishers, processors and individuals at various stages in the food chain to detect the presence of ciguatoxins.

The Ciguatect kit is a qualitative method for detecting the presence of specific antigens, such as toxins, on a special membrane attached to a plastic strip for support. The suspected sample (tissue or its extract containing the toxin) is immobilized on the membrane and exposed to an immunobead solution. This solution is prepared by combining an antibody specific to the toxin with microscopic colored latex beads. This process allows the antibody to be bound to the beads’ surface. The resulting immunobeads are then capable of binding to the toxin whenever present on the membrane. In running the test, the specific immunobeads get bound to the immobilized antigen within a few minutes, resulting in a color change on the membrane that indicates the presence of the antigen. The assay can be considered semi-quantitative, since the intensity of the color reflects the antigen magnitude in the sample.

Test procedure
The Ciguatect test kit procedure for ciguatera fish poisoning toxins in whole fish is simple and rapid. The user normally makes a deep incision about 1 inch behind the head of the sample fish and inserts the membrane end of the test strip. The strip is placed on a flat surface until the membrane is dry (about 5 minutes). The membrane end of the test strip is immersed in methanol solution and then allowed to dry for about 5 minutes. This step helps the toxin migrate from the tissue to the membrane structure where it is immobilized. The membrane end of the test strip is immersed in the immunobead solution and left undisturbed for 10 minutes. No color on the membrane is indicative of negative toxicity, and it is given a score of zero. The presence of color on the membrane denotes the presence of ciguatoxin in the fish. A faint color indicates borderline toxicity. The intensity of color is compared to a set of positive results ranging from 1 to 5. The average value for the scores from duplicate or triplicate sample strips is calculated and recorded for the corresponding laboratory report.

Applications
The Ciguatect kit can be used for the detection of toxins associated with ciguatera fish poisoning and in rapid screening programs of toxic fish and shellfish in harvesting areas and the marketplace. In a kit configuration, this type of marine toxin detection assay can be used routinely for high-volume screening of suspect toxic fish on board ships, in rudimentary dockside laboratories and at aquaculture facilities, as well as in regulatory agency laboratories.

Ciguatect can be adapted to test mussel samples for the presence of the main toxin responsible for diarrheic shellfish poisoning in some parts of the world. In addition to helping to monitor shellfish beds for shellfish poisoning toxins, the kit can be applied in shellfish depuration operations for elimination of the toxins, and to screen for toxic mussels, scallops and oysters in the marketplace.

Technologies such as this are useful to governments around the world concerned with rapid, inexpensive commercial testing procedures to identify unsafe fishing locations. They also help in random testing of suspect commercial catches to ensure the safest seafood products for the public.

This test kit is not yet available commercially. The LSU AgCenter continues to be involved in its development.

Acknowledgment
Ciguatect is a registered trademark of HawaiiChemtect International, which helped to fund this research.

(This article was published in the spring 2000 issue of Louisiana Agriculture.)
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