Ciguatera poisoning in French Polynesia: A review of the distribution and toxicity of Gambierdiscus spp., and related impacts on food web components and human health.

Ciguatera Poisoning (CP) is a seafood poisoning highly prevalent in French Polynesia. This illness results from the consumption of seafood contaminated with ciguatoxins (CTXs) produced by Gambierdiscus, a benthic dinoflagellate. Ciguatera significantly degrades the health and economic well-being of local communities largely dependent on reef fisheries for their subsistence. French Polynesia has been the site of rich and active CP research since the 1960's. The environmental, toxicological, and epidemiological data obtained in the frame of large-scale field surveys and a country-wide CP case reporting program conducted over the past three decades in the five island groups of French Polynesia are reviewed. Results show toxin production in Gambierdiscus in the natural environment may vary considerably at a temporal and spatial scale, and that several locales clearly represent Gambierdiscus spp. "biodiversity hotspots". Current data also suggest the "hot" species G. polynesiensis could be the primary source of CTXs in local ciguateric biotopes, pending formal confirmation. The prevalence of ciguatoxic fish and the CTX levels observed in several locales were remarkably high, with herbivores and omnivores often as toxic as carnivores. Results also confirm the strong local influence of Gambierdiscus spp. on the CTX toxin profiles characterized across multiple food web components including in CP-prone marine invertebrates. The statistics, obtained in the frame of a long-term epidemiological surveillance program established in 2007, point towards an apparent decline in the number of CP cases in French Polynesia as a whole; however, incidence rates remain dangerously high in some islands. Several of the challenges and opportunities, most notably those linked to the strong cultural ramifications of CP among local communities, that need to be considered to define effective risk management strategies are addressed.

Ciguatera poisonings: A global review of occurrences and trends.

Ciguatera Poisoning (CP) is the most prevalent, phycotoxin related seafood poisoning across the globe, affecting between 10,000 and 50,000 people annually. This illness results from the consumption of seafood contaminated with lipid soluble toxins known as ciguatoxins (CTXs) that are produced by benthic dinoflagellates in the genera Gambierdiscus and Fukuyoa. The present work reviews the global occurrence of CP events and outbreaks, based on both scientific and gray literature. Ciguatera prevalence is significantly underestimated due to a lack of recognition of ciguatera symptoms, limited collection of epidemiological data on a global level, and reticence to report ciguatera in CP-endemic regions. Analysis of the time-series data available for a limited number of countries indicates the highest incidence rates are consistently reported from two historical CP-endemic areas i.e., the Pacific and Caribbean regions, a situation due in part to the strong reliance of local communities on marine resources. Ciguatera-related fatalities are rare (<0.1% of reported cases). The vast majority of outbreaks involve carnivorous fish including snappers, groupers, wrasses, and barracudas. Since 2000, an expansion of the geographical range of CP has been observed in several areas like Macaronesia and east and southeast Asia. In some of these locales, random surveys confirmed the presence of CTXs in locally sourced fish, consistent with the concurrent report of novel CP incidents (e.g., Canary Islands, Madeira, Selvagens Islands, New South Wales). One characteristic of outbreaks occurring in Asia is that they often present as large disease clusters due to group consumption of a single contaminated fish. Similar observations are reported from the Indian Ocean in the form of shark poisoning outbreaks which often lead to singular types of CP characterized by a high fatality rate. Other atypical forms of CP linked to the consumption of marine invertebrates also have been documented recently. Owing to the significant health, socioeconomic and socio-cultural impacts of ciguatera, there is an urgent need for increased, standardized, coordinated efforts in ciguatera education, monitoring and research programs. Several regional and international initiatives have emerged recently, that may help improve patients' care, data collection at a global scale, and risk monitoring and management capabilities in countries most vulnerable to CP's toxic threat.

Ciguatera poisoning in French Polynesia: insights into the novel trends of an ancient disease.

Ciguatera is a non-bacterial seafood poisoning highly prevalent in French Polynesia where it constitutes a major health issue and a major threat to food sustainability and food security for local populations. Ciguatera results from the bioaccumulation in marine food webs of toxins known as ciguatoxins, originating from benthic dinoflagellates in the genera Gambierdiscus and Fukuyoa. Ciguatera is characterized by a complex array of gastrointestinal, neurological and cardiovascular symptoms. The effective management of patients is significantly hampered by the occurrence of atypical forms and/or chronic sequelae in some patients, and the lack of both a confirmatory diagnosis test and a specific antidote. In addition, recent findings have outlined the implication of novel species of the causative organisms as well as new vectors, namely marine invertebrates, in ciguatera outbreaks. Another novel trend relates to the geographical expansion of this disease to previously unaffected areas, not only in certain island groups of French Polynesia but also in temperate regions worldwide, as a likely consequence of the effects of climate change.

Neuroprotective effects of rosmarinic acid on ciguatoxin in primary human neurons.

Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200 nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.

Use of folk tests to detect ciguateric fish: a scientific evaluation of their effectiveness in Raivavae Island (Australes, French Polynesia).

Ciguatera fish poisoning is a seafood intoxication commonly afflicting island communities in the Pacific. These populations, which are strongly dependent on fish resources, have developed over centuries various strategies to decrease the risk of intoxication, including the use of folk tests to detect ciguateric fish. This study aims to evaluate the effectiveness of two folk tests commonly used in Raivavae Island (Australes, French Polynesia): the rigor mortis test (RMT) and the bleeding test (BT). A total of 107 fish were collected in Raivavae Lagoon, among which 80 were tested by five testers using the RMT versus 107 tested by four testers using BT. First, the performance between testers was compared. Second, the efficiency of these tests was compared with toxicity data obtained via the receptor binding assay (RBA) by assessing various parameter's values such as sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). Comparisons of outcomes between folk tests and RBA analyses were considered: tests used separately or in a parallel versus the series approach by each tester. The overall efficiency of the RMT and BT tests was also evaluated when the judgments of all testers were "pooled". The results demonstrate that efficiencies varied between testers with one showing the best scores in detecting toxic fish: 55% with RMT and 69.2% with BT. BT gave the best results in detecting toxic fish as compared with RMT, giving also better agreement between testers. If high NPV and Se values were to be privileged, the data also suggest that the best way to limit cases of intoxication would be to use RMT and BT tests in a parallel approach. The use of traditional knowledge and a good knowledge of risky versus healthy fishing areas may help reduce the risk of intoxication among communities where ciguatera fish poisoning is highly prevalent.

Ciguatera risk assessment in two toxic sites of French Polynesia using the receptor-binding assay.

Ciguatera Fish Poisoning (CFP) is a tropical syndrome well known in remote archipelagos where the population is still dependent on fish resources. In order to assess the ciguatera risk in two islands of French Polynesia, Tubuai (Australes) and Nuku Hiva (Marquesas), a study was carried out on both Gambierdiscus populations as well as on various fish species using the receptor-binding assay (RBA) to detect and quantify ciguatoxins. Relationship between RBA data and size or weight of fish was evaluated, and when only few individuals for a particular species were available the trophic level was used to help comparisons between studied areas. According to epidemiological data, toxic versus safe areas were explored and compared in both islands. In Tubuai Island, Gambierdiscus cells were surprisingly absent in the north area, considered as a toxic area, but almost 94% of fishes were classified as RBA+. In contrast, the south area, supposed to be safe, was evolving to be a risky area because of the presence of Gambierdiscus cells and 74% of fishes being RBA+. In Nuku Hiva Island, Gambierdiscus cells were present in the toxic areas, Anaho, Taiohae and Taipivei, with two toxic blooms in Anaho Bay, but none in Terre Déserte, the fishing area of this island. With RBA data, fishes were analyzed to be RBA+ at a high percentage in Anaho and Taiohae, higher than in Taipivei and Terre Déserte areas. In general, our findings were congruent with epidemiological data and the knowledge of local people only for risky fish species.

Neurologic signs of ciguatera disease: evidence of their persistence.

Ciguatoxins exert their effect on the voltage-sensitive sodium channels of the cellular membranes of all excitable tissues. This effect confers to ciguatera disease (CD) its neurologic hallmarks. A prospective study among French Polynesian adults over a two-month period was conducted to characterize and determine the persistence of neurologic symptoms of CD. We compared 47 patients with CD with 125 controls. In the acute phase of the disease, patients had mainly sensory disturbances as detected by an hypoesthesia on the palm of the hand and poorer sway performance compared with controls. Follow-up two months showed improvement of sway performance that eventually reaching control levels. However, for light-touch threshold, even if we observed a decrease threshold towards normal values, more than 50% of patients did not reach normal values 60 days after disease onset. Our results support the existence of neurologic impairments of CD and suggest their persistence for at least two months after onset.

Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium.

Minute amount of Brevetoxin PbTx-3 (400 microg; 0.446 micromol) was converted into an hemisuccinate derivative (PbTx-3 HS) then covalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) in a reversed micellar medium. According to the efficient cyclic synthetic procedure described, the epitope density of the conjugates was around 10 and 20 for OVA and BSA carriers, respectively. The kinetics of antibody production in sequential sera harvested from a single BALB/c mouse immunised by multiple intraperitoneal (i.p.) injections of PbTx-3-BSA conjugate was performed by enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies (MAbs) against PbTx-3 were selected from fusion of the mouse immune splenocytes with the P3-X63-Ag 8.653 myeloma cells. In competitive inhibition ELISA experiments, both polyclonal antibodies and MAbs exhibited strong cross-reactivity (> or = 100%) to other PbTx-2-type toxins (PbTx-2 and -9) but low or moderate cross-reactivity (6-15%) to a PbTx-1-type toxin (PbTx-1). Moreover, using these two MAbs, a low cross-reactivity with okadaic acid (3%) was noticed but no significant cross-reactivity was observed with two ciguatoxins (CTX-1B and CTX-3C) over the concentration range studied. The apparent dissociation constant (K(D)) for the interaction of these MAbs with free PbTx-2-type toxins was in the 10(-6)-10(-7)M range. The performance of this MAb-based assay (limit of detection approximately 5ng/well; working range=8-150ng/well) coupled with adequate extraction methods would provide an alternative assay to the mouse i.p. bioassay for routine shellfish monitoring. This production and characterisation of MAbs using small amount of polyether toxins in a reversed micellar medium appear most valuable for the development of immunoassays to other highly potent but poorly available marine polyether toxins like ciguatoxins (CTXs).

Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins.

As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of JKLM ring fragment, a carboxylic derivative of the right-hand tetracyclic terminus portion of CTX-1B (the most potent CTX) with two carrier proteins have been synthesized. Two procedures using different amount of hapten were evaluated: (i) a bulk technique (3-5 mg) via the N-hydroxysuccinimide ester of the carboxylic fragment in the presence of a water-soluble carbodiimide according to the standard method in aqueous buffer, or (ii) a micro-scale technique (300 microg) via the mixed anhydride method performed in a reversed micellar medium. In both cases, bovine serum albumin and ovalbumin were respectively used for immunization of BALB/c mice and antibody screening by a solid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedule appeared to be more efficient to specifically trigger the mice immune system. These antisera titers determined in an end-point titration standard ELISA format were found around 1/128,000 as compared to 1/16,000 obtained in the short-term protocol (immunogen prepared via the bulk procedure). In competitive inhibition ELISA experiments, both types of antisera did not significantly cross-react with a brevetoxin congener (PbTx-3), okadaic acid (OA), monensin or other polyether compounds, but only sera from the short-term protocol did show high cross-reactivity to CTX-1B (133%). With sera from the long-term protocol, a lower detection limit for JKLM (1.23 x 10(-9) M) was achieved by implementation of a biotin-avidin amplification system rather than by miniaturization of the assay in Terasaki plates. This study confirms the feasibility of the immunological approach for CTXs assay in fish tissues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen, (ii) the immunization schedule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs.

Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay.

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.

An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens.

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate.

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (ABA) and two other closely related small chromophoric haptens. The amide bond-generating mixed anhydride method of Erlanger was modified to promote conjugation to various proteins (bovine serum albumin, ovalbumin, casein and hemocyanin) or to a synthetic homopolymer (Poly-DL-lysine). The key process in this synthesis is the use of a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement at characteristic hapten absorption peaks. This coupling procedure is applicable to as little hapten material as 0.2 micromol and is disclosed to be most valuable for other rare lipid haptens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple intraperitoneal injections of (ABA)23-BSA conjugate as revealed by indirect and competitive ELISA. Calculated KD for the interaction of the antibodies with free ABA was found to be 5 X 10(-5)M.

Are Streptomyces bacteria involved in the ciguatoxicity of the surgeon fish Ctenochaetus striatus?

Several Streptomyces strains have been isolated from the digestive tract of the herbivorous fish C. striatus, a preeminent ciguateric fish of Polynesian waters. In order to study the possible role played by these bacteria in the toxicity of this fish, the quantitative and qualitative distributions of these isolates within toxic and non toxic fish are compared. The preliminary results are discussed.

Toxicity of French Polynesian strains of Gambierdiscus toxicus in cultures.

Seven clonal strains of Gambierdiscus toxicus isolated from three ciguateric areas around Tahiti island were mass cultured and extracted for ciguatoxins and maitotoxin. CTX analogs were detected only in one clone (GTP1), suggesting that CTX production may be strain-dependent. However, this in vitro production of CTXs, which remains fairly poor with regards to the toxicity levels encountered in wild G. toxicus, is not a stable temporal characteristic. On the other hand, maitotoxic compounds were detected in all 7 strains in copious amount, especially in clone GTH2.