What is Ciguatera Fish Poisoning?

 

Ciguatera (or Ciguatera Fish Poisoning, CFP) is a foodborne illness following the consumption of fish and marine products from coral reef, in perfect freshness and usually safe to eat, made toxic by the presence of toxins which come from a micro-algae, the Gambierdiscus dinoflagellate.

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Ciguatera thoughout the centuries...

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Origin of the ciguatoxic phenomenon

The true origin of ciguatera fish poisoning is only known since the mid-1970s and involves a benthic unicellular dinoflagellate, Gambierdiscus spp., whose populations preferentially grow in algal "turf" covering damaged coral. The genus Gambierdiscus is widely spread around the globe. At least six species are endemic to French Polynesia: G. toxicus, G. australes, G. pacificus,G. carpenter, G. caribaeus and G. polynesiensis, the latter being the most toxic.

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Cells of the benthic dinoflagellate Gambierdiscus spp. © ILM; (A): Optical microscope view ; B): Scanning electron microscope view; (EP): Epitheca; (HYP): hypotheca.

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Example of Gambierdiscus sp. ‘support-algae’: A) Turbinaria ornata et B) Halimeda spp.© ILM

 Under the influence of still poorly identified factors, but generally related to natural (hurricanes, tsunami…) or anthropogenic (pollution, constructions...), environmental imbalance, populations of Gambierdiscus will begin to proliferate in a sporadic way.

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Examples of natural (cyclone) and anthropogenic (constructions,bank) factors that may lead to coral mortality and increase of Gambierdiscus blooms.

For reasons still poorly documented, only some microalgae strains will be able to produce extremely powerful (CTXs) toxins. In other terms, it is not the presence of high densities of Gambierdiscus, which causes ciguatera but also that of toxic strains.

Massive colonization of the coral reef ecosystem by Gambierdiscus toxic strains therefore constitutes the starting point of the reef’s food chain contamination. CTXs produced by the dinoflagellate are gradually accumulated in herbivorous fish when they graze on the “micro-algal lawn” covering dead corals. Transfer of theses toxins to carnivorous fish , however, occurs with predation, when carnivorous fish prey on toxic herbivorous fish. This bioaccumulation of algal CTXs doubled by a biotransformation, leads, over time, to the poisoning of consumers, which are at the top of the food chain, when a certain degree of tolerance or symptomatic threshold is reached. In total, an estimated of 400 lagoon fish species are potentially contaminated.

     
      
 
List of the main fish species identified as ciguatera fish poisoning carriers.
                                                                                  
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Did you say "Ciguatera Shellfish Poisoning"?

Although Gambierdiscus is considered as the main causative agent of ciguatera, recent studies have shown that some marine cyanobacteria could also contribute to a ciguatera-like syndrome.

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Examples of cyanobacteria. Macroscopic observations: A) Hydrocoleum cf. floccosum, B) Anabaena sp.; C) Aulosira schauinslandii; D) optical microscope view of Oscillatoria bonnemaisonii trichomes, © S. Golubic. 

Polymorphism of symptoms and complexity of ciguatera in a broad sense, would partly be related to the diversity of these toxin-producing organisms. Similarly, reef fish have long been regarded as the only vectors of ciguatoxins, however, some marine invertebrates such as clams, sea urchins and gastropods (shellfish) may also be involved in atypical forms of ciguatera.

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A)Tridacna maxima (giant clam) © M. Roué  and B) Tripneustes gratilla (sea urchin) © JJ. Eckert, other potential links in ciguatera’s trophic food chain. 
  

Symptoms of these marine invertebrates poisoning include characteristic symptoms of ciguatera (see chapter on symptoms) but also faster and more severe additional symptoms such as, almost immediate burning sensations of the mouth and throat, strong metallic taste, sometimes followed by severe paralysis. Hypothesis of the existence of a new way of contamination due to cyanobacterias (and not to dinoflagellates typically responsible for ciguatera), involving filtering bivalves was then proposed. This new phenomenon is called “Ciguatera Shellfish Poisoning” (CSP).

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Illustration of ciguatera toxins transfer across the food chain. Main vectors of "Ciguatera Fish Poisoning" are marine vertebrates (herbivorous and carnivorous lagoon fish). "Ciguatera Shellfish Poisoning" preferentially involves marine invertebrates such as clams and sea urchins; © F. Rossi.

                                           
     
   

Toxins: nature and modes of action


The genus Gambierdiscus can synthesize at least two families of toxins: one lipid-soluble, Ciguatoxins (CTXs) and the other water-soluble, Maitotoxins (MTXs). It is generally agreed that only CTXs are responsible for ciguatera fish poisoning, MTXs are usually not involved in human poisonings.

In humans, the average dose at which 50% of humans fall ill, is estimated to be as low as 2 ng/kg of body weight (only 2/100,000th of a gram!), making CTXs one of the most potent natural substances known.


Other molecules such as gambieric acids, which exhibit antifungal activity, and gambierol have also been isolated from cultures of this micro-algae, but their role in ciguatera fish poisoning is still to be confirmed.

One might wonder about the ecological benefit for Gambierdiscus to produce these toxins. One hypothesis is that these metabolites provide an environmental benefit (e.g. defense mechanism) over potential competitors or predators.

  Ciguatoxins

Ciguatoxins (CTXs) are polycyclic polyether compounds, lipid-soluble, with a molecular weight between 1.023 and 1.159 Da. There are 3 main groups of CTXs throughout the 3 main areas affected by ciguatera: Pacific ciguatoxins or P-CTXs, Caribbean ciguatoxins or C-CTXs and Indian Ocean ciguatoxins or I-CTXs. P-CTXs, composed of 13 ether rings, are described in two types 1 and 2, the difference resides mainly in the E cycle. C-CTXs have, on the other hand, 14 cyclic ethers. To date, the structure of I-CTXs is yet to discover. In total, there are more than 40 different CTXs, which have been isolated primarily from Gambierdiscus cells and toxic fish.

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Chemical structures of Pacific ciguatoxins (P-CTXs) differentiated by two different types (type-1 and -2), and a Caribbean ciguatoxin (C-CTX -1), (modified from Caillaud et al.)

 The panel of CTXs in contaminated fish can also vary significantly from one fish species to another. One single species may host several types of CTXs. So, one may talk about a “toxic profile” for a given trophic level.

Algal CTXs produced by Gambierdiscus undergo transformations from their accumulation in herbivorous fish until they pass through carnivorous fish. As a result, CTXs polarity as well as their toxicity increase. Thus, P-CTX-1B only found in carnivorous fish is, for example, 30 times more toxic than P-CTX-4B present in Gambierdiscus. This biotransformation phenomenon is what makes the wide variability of the toxic profiles with respect to the fish and trophic level considered.

These understandings partly illustrate the complex mechanisms underlying the significant variation of ciguatera outbreaks severity, depending on an area to another.

   CTXs' mode of action

CTXs’ main molecular targets are voltage sensitive sodium channels (VSSC), which are responsible for action potentials initiation and propagation. CTXs binding to electrically excitable cells, prolong the opening of VSSC even at resting membrane potential, which results in a continuous inflow of Na+ ions in the cells. To counter this intracellular increase of sodium level, cellular mechanisms are put into place allowing an outward flow of Na+ against an inward flow of Ca2+, leading to an increase of intracellular calcium levels. This quasi-irreversible binding of CTXs on VSSC results in both, nerve conduction and nervous cells morphology impairments. More recently, studies have shown that L-type calcium channels is another potential target of CTXs. CTXs binding to these calcium channels would lead to a cascade of enzymatic events resulting in an overproduction of NO radicals. 

Impaired nerve conduction

A well-known consequence of the CTXs binding to VSSC is the appearance of spontaneous and/or repetitive discharge of action potentials. This sharp increase of nervous excitability then leads to a sustained release of neurotransmitters (until exhaustion) at nerve endings, resulting in a modification of synaptic efficiency or a nerve transmission deficiency. In the symptomatic treatment of ciguatera, we try to counterbalance or eliminate these “disarranged” action potentials, by giving local anesthetics such as lidocaine or with intravenous (IV) D-mannitol or sucrose infusions.

Modification of cells morphology

Swelling of the nodes of Ranvier, due to water inflow in the cells, is another consequence of the constant inflow of Na+ ions. This wide set of CTXs-induced modifications ( depolarization and hyper-excitability, increased intracellular levels of Na+ and Ca2+, anarchic release of neuromediators, swelling associated to water inflow…) is what causes such a diversity of the clinical signs observed in CFP cases. Neurological: symptoms such as motor, sensation, cerebellar or psychiatric disorders, are direct consequences of the alteration of the peripheral, central and autonomic nervous system fibers. Gastrointestinal: high intracellular levels of Ca2+, would lead to profuse diarrhea. Cardiovascular: CTXs action occurs through the autonomic nervous system, bradycardia and hypotension being related to a parasympathetic hyperstimulation (hence, the efficiency of atropine in symptomatic treatment of ciguatera) and a low sympathetic tone. Muscular: increased intracellular calcium level results in an increased frequency and intensity of muscular contractions, while spontaneous and repetitive action potential discharges induce uncoordinated muscle contractions. These effects are more significant when they concern the heart muscle, as both nerve supply and heart muscle are affected.

 

… after that, what happens to these toxins?

After an episode of ciguatera, CTXs will circulate freely into the blood stream for a few days, however, some will be directly excreted in urine and feces.


Due to their high lipophilic properties, the un-excreted CTXs will then diffuse and strongly anchor themselves in different organs and tissues, such as the liver, muscles, fat and brain.

 

Even if ways and time for “detoxification” in humans remain unknown, toxicokinetic experiences conducted on eels have shown that complete elimination of toxins was long but possible (several months or years).

 

To date, western treatments enable to specifically act on toxins elimination are still to discover.

   

 

   
   

How to detect toxins?

 At present, CTXs detection is still a significant technical challenge, due to the toxins nature, the multiplicity of congeners to detect and the low levels of toxins present in fish flesh. However, even if several detection tests are now available, currently there is no standard test duly validated by the scientific community, that could enable public authorities to establish a marine products food security regulation at an European and international level. At present, the only reliable method to detect CTXs are laboratory tests based on toxins mode of action (functional tests), or on CTXs physicochemical properties.

  In vivo tests

 Biological test on mouse or “Mouse bio-assay” (MBA) was the first CTXs detection test used. It is based on the symptoms and mice survival time observed after a 24-48h period, following an intraperitoneal (ip) or intravenous (iv) injection of fish extract. Several animal species other than mice have been used: cats, young chicken or mongooses that have a better CTXs sensitivity but require large amount of extracts or, conversely, methods using invertebrates, such as mosquitoes, crayfish, fly larvae or shrimps, which require lesser amounts of the extract. With their lack of sensitivity and specificity these test are gradually replaced by other methods following the 3R rule: “Reduce, Refine, Replace”. These methods are based on CTXs chemical, pharmacological and immunological properties.

  Functional tests

 The radioligand-receptor test or “Receptor binding-assay” (RBA) is a neuropharmacological test based on the specific affinity of CTXs and brevetoxins (PbTxs) to the site 5 of VSSC’s alpha subunits found on excitables cells membranes. Concerning CTXs detection, RBA measures the binding of a radiolabelled toxin, tritiated PbTx ([3H]PbTx-3), to this receptor, which compete with unradiolabled CTXs contained within the extract to analyse. Well adapted to CTXs detection in complex and varied biological matrices, RBA offers a high sensitivity (10-10M detection limit) and allows the use of untreated or partially purified extracts. It seems to be economically more viable than the mouse bioassay, as it is easily automated to allow maximum processing capacity, making it an ideal tool for large-scale ciguatera risk monitoring programs. However, due to the regulatory constraints imposed by radioelements storing and handling, it appears difficult to generalize this test. But, recent labeling of brevetoxin with a fluorescent element gives hope for a greater applicability of this test.

 Cell toxicity test or “Cell based-assay” (CBA) is another functional test allowing to measure out the “overall toxicity” of a sample, by measuring the viability of a cultured cell line. This test is commonly used for the detection of a wide range of marine toxins: e.g. those active on VSSCs (saxitoxins, tetrodotoxins, brevetoxins and ciguatoxins), those active on Na+/K+ ATPases pumps (palytoxins), maitotoxins acting on voltage sensitive calcium channels or VSCC, okadaic acid which inhibits serine / threonine protein phosphatases, or pectenotoxins and dinophysistoxins… Besides its capacity to detect a wide range of marine biotoxins, CBA is very also very sensitive (10-12M) and replicable, making it an excellent CTXs standard detection test candidate.

 Various immunological assays have been developed for CTXs screening: the radioimmunoassay (RIA) or the “sandwichtest or enzyme-linked immunosorbent assay (ELISA). These tests are based on the principle of a highly specific recognition between an antibody (CTXs anti-antibody) and its antigen (CTXs). Theoretically, this approach seems to be the most promising one for the implementation of a fast, reliable, sensitive (up to 5×10-12M) and cheaper screening test. Its operating principle could also enable high-throughput screening of marine samples, and, most of all, its direct use on the field by individuals. At present, two trials of developing such a test have been tried: the CIGUATECT ™ and Cigua-Check ® (ToxiTec Inc. / Oceanit). But, these test kits have been withdrawn from the market, partly due to high false positives and false negatives reactions.

 CTXs’ complexity and chemical diversity, their low natural immunogenicity related to their polycyclic polyether nature, as well as the limited availability of pure standards, partially, explains the apparent difficulties to develop a reliable test.

 

 

Physicochemical tests

Physicochemical tests (e.g. HPLC, LC-MS / MS) are based on high performance liquid chromatography techniques coupled with detection of each toxins families using Ultra-violet (UV), fluorescence, or tandem mass spectrometry. With a great sensitivity, these tests allow distinction and quantization of the different CTXs congeners within the same toxic family, but they require, as a prerequisite, to have the corresponding pure standards. Therefore, the main limitations of this technique are that it does not detect new toxic families and, unlike the so-called functional tests, it doesn’t provide indication on the fish sample “whole toxicity”. Also, this type of methodology appears difficult to adapt to a CTXs high-throughput screening due to the several preliminary purification steps of the biological matrices. Therefore, these tests are most of the time used as confirmatory testing.

 

Traditional tests

 As CTXs in toxic fish cannot be identified by appearance, odor, color or taste, island populations (which are exposed daily to CFP risks), have gradually developed a wide range of traditional tests in attempt to detect ciguatoxic fish. Across south pacific islands , depending on the archipelago or island, several detection methods coming form popular beliefs or long ancestral practices are used. These traditional tests consist of giving a piece of flesh or liver of the suspicious fish to an animal or insect; or by using a silver coin or some matches; or based on the appearance of the whole fish or some of its organs.

 A recent study has verified the effectiveness of two traditional detection tests (the rigor mortis method and hemorrhagic test), their results were compared to the Receptor Binding Assay (RBA). Despite a predictability rate not exceeding 70%, the use of these tests combined with the population knowledge on suspicious toxic species and fishing areas, may help to significantly reduce the risk of CFP within fish dependent communities, at the condition that the test users are accustomed to these tests. The opportunity for island populations of remote archipelagos to use on site and cost-effective validated traditional tests may therefore represent a day to day valuable asset in ciguatera risk management.

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Examples of traditional tests used by French Polynesia’s fishermen to differentiate toxic fish over healthy fish.© ILM

   
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