Studies on the origin of ciguatera

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Toxins '89 Symposium

CtrtT~[x species of plankton dinoflagellates produce potent neurological toxins which can find their way through shellfish to humans. When humans eat shellfish contaminated by these dinoflagellates, they may suffer a variety of gastro-intestinal and neurological illnesses. These include paralytic shellfish poisoning (PSP), which in extreme cages can lead to death through respiratory paralysis, and diarrhetic shellfish poisoning (DSP), which causes severe gastro-intestinal problems and can promote stomach tumors . PSP toxins in the Australian region are caused by the dinoflagellates Gymnodinium catertatum (Southern Tasmania), Alexandrium catenella (Port Phillip Bay, Melbourne) and Alexandrittm minutum (Adelaide) . The first two species produce mainly sulphamate saxitoxins (fractions C,, C2, C C~ while the latter species produces exclusively gonyautoxins l, 2, 3 and 4. These toxin fractions show widely different toxic potencies when injected i.p . into mice, ranging from 2045 MU/itmole (saxitoxin) to 16 MU/kmole (fraction C,), in which 1 MU (mouse unit) is the amount of toxin to kill a mouse weighing 20 g in 15 min . DSP toxins in the Australian region are caused by the dinoflagellates Dinophysis acurninata and Dinophysis fortü (Southern Tasmania, New Zealand). The fat-soluble polyether toxins produced by these species include okadaic acid and dinophysis-toxin l . Detection methods for these shellfish toxins (mouse bioassay, HPLC) and control measures for the shellfish industry will be discussed. Plant toxins and thefood chain. M. P. HZ:caxTSr (CSIRO, Division of Tropical Crops and Pastures, St Lucia, Queensland, Australia) . Tt~ puaL[c generally perceives that natural foods and feeds are safe, contrary to the available scientific evidence which indicates that natural substances in the diet are not necessarily safer than added artificial compounds, and could be significantly more hazardous. Foods may contain very stable exogenous chemicals, e.g . pesticide residues and pollutants, which can have adverse effects further along the food chain. Little is known about natural toxicants in this context, partly because of the very large number of potential toxins, and the limited distribution of some of them in plants . This paper summarizes information on natural toxins which are found in milk or tissues of animals which have eaten poisonous plants . The following classes of plant toxicants ate considered : hepatotoxic pyrrolizidine alkaloids from species of Crotalaria and Senecio, goitrogenic compounds from cruciferous pastures or weeds, and a sesquiterpene from Eupatorium rugosum which causes a disease known as "trembles" . Recently, a highly polar, hepatotoxic amino acid present in some species of Irrdigojera has been shown to accumulate in the tissues of horses eating these plants, and to cause severe liver damage in dogs which have eaten the meat from these horses. While milk can be a vehicle for transmission of plant toxins, in isolated instances, to man and other animals, it is doubtful if this poses any significant health hazard to the human population in general. Conotoxin clortirtg.~ constant and hypervariable regions in propeptide precursors . Dw[o R. HILLYARD and SCOTT WooOwexO (Department of Pathology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT, U.S .A.). Cor[oro~nrts are small cysteine-rich peptides found in the venom of the predatory cone snails (Comos) which have proven to be useful high aflùtity ligands For various receptors and ion channels . The cloning data for wnotoxins suggests how such small peptides may be folded into a specific disulfide configuration, and how conotoxins with diverse specificities may be generated. The King-Kong peptide is a conotoxin found in the venom of the Cloth-of-Gold cone, Comos textile. Analysis of cDNA clones of the King-Kong peptide revealed a family of related toxin transcripts. Three different propeptide cDNA sequences were obtained: when the predicted amino acid sequences are compared, welldefined conserved and hypervariable regions can be identified . The hypervariable regions comprise four regions between Cys residues in the final peptide toxins ; the remainder of the propeptide sequences, i.e . the excised Nterminal regions and the disulfide-bonded Cys residues are highly conserved. An analysis of all conotoxin sequences uncovered to date reveals that the organization of cysteine residues in conotoxins comprises a very restricted subset of the total possible. Together with the present cloning results, this suggests that specific cassettes of amino acids between cystcine residues may be systematically scrambled, generating sets of functionally diverse peptide ligands wnserved in their structural frameworks . Studies on the origin of ciguatera. M. J. Hot,~s, R. J. Lew[s and N. C. GILLESP~ (Southern Fisheries Research Centre, Queensland, DPI, P.O. Box 76, Deception Bay, 4508 Queensland, Australia) . CtiOUn~u is a form of food poisoning caused by eating otherwise edible tropical reef fishes . In Qucensland 527

people were reported poisoned between 1965 and 1984 (GILC.FSPIE et al., 1986). Flesh of toxic fish in Queensland contains ciguatoxin, which is a lipid-soluble polyether compound (LEw[s and Erroenx, 1983, 1984). Ciguatoxin is

Toxins '89 Symposium

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the only toxin (apart from scaritoxin found only in French Polynesia) that has been found in the flesh of ciguateric ûshes in the Pacific . The presumed origin of ciguatoxin is the benthic dinoflagellate Gambierdiscus toxicus, which is a common but minor component of the reef benthos along the tZttcensland coast (GILLESPrE et al., 19ß5a). However, we have found no ciguatoxin in either wild or cultured G . toxicas populations in tZrrcensland (GILI.ES1':, N . C., LEwrs, R ., BURKE, J . and HOLAffS, M . (19ß5b) In : Proceedings of the Fijth International Coral Reef Congress Tabiti, pp. 43741, (GAete>E, C . and SALVAT, B ., Eds .) 4. GILLE3P~, N . C ., LEwts, R . J., PEARN, J . H ., BouxI~E, A . T . C ., HOLDS, M . J ., BoutslcE, J . B. and StüELDS, W . J. (1986) Med. !. Aust . 145, (11/I2), 584-590 . HOLbiF3, M . J ., GILI .ESPIE, N. C. and Lewis, R . J . (l9ß86) In : Proceedings ojrhe Sixth International Coral Reef Symposium, Townsville. (In press.) HoLe~s, M . J., LEwts, R . J. and GtLLE4P~, N . C . (19ß8a) Third International Phycological Congress, Melbourne, 40 . HOr.xms, M . J., LEwts, R . J . and GILLFSI'IE, N . C . (1989) In : Proceedings of the Australian Biochemical Society, Gold Coast. LEwts, R . J . and ENDEAN, R . (1983) Toxicon 21, 19-24 . LEwts, R. J. and ENDEAN, R . (1984) Toxicon 22, 805-810. LEwrs, R . J., GILI.ESPIE, N . C., HOLt~s, M . J., BuntcE, J. B ., KEYS, A. B., FtFOOT, A . T. and SrteEE-r, R . (l9ß8) Proceedings ojthe Sixth International Coral Reef Symposium, Townsville . (In press .) Antibody to Pseudomonas pseudomallei exotoxin in sheep.' evidence jor in vivo toxin production during infection . GHAZALLY ISMAIL, RAtIMAH MOHAMED, SITI ROHANA Iutzts and Noox Ester (Department of Biochemistry, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia) . Pseadomonas pseadomallei has been the cause of infrequent outbreaks of melioidosis among animals (SenTH et al., 1986) . Our previous studies on the virulence determinants of melioidosis point to the importance of exotoxin in the pathogenicity of P. pseudomallei (NYONYA RAZAK et al., 1986) . The exotoxin is a heat-labile protein with a mol. wt of approximately 31,000 and possesses lethal, necrotic, hemolytic and cytotoxic activities (I3MAIL et al., 1987). At the cellular level, it inhibits protein and DNA synthesis in cultured macrophages (MoHAt~n et al., 1989) . Despite evidence of the exotoxins activity and its detection among the majority of P. pseudamallei isolates (Noox Esmt et a/., 1988), there has been no demonstration that the exotoxin is produced during infections. In this study, we present evidence that P. pseadomallei exotoxin is released in quantities that elicit antibodies in diseased sheep as well as those in the same flock passively exposed to the organism . Antitoxin antibody was determined in 288 sera obtained from 3 groups of sheep presumed to have been passively exposed to P. pseadomallei via acutely infected animals that occurred within the flocks, Additionally, for control group, 60 serum samples were obtained from another sheep farm known to have been free of melioidosis outbreak for some years . An enzyme-linked immunosorbent assay (ELISA) detected antibody in 80.9% of shcep sera obtained from passively exposed flocks. We however failed to show the prevalence of specific antibody to exotoxin in control sera . Serum antitoxin titers of 10,000 were observed in five sheep; and 49 .3% of animals from passively exposed flocks exhibited serum titers of 1000. All sera giving titers of more than 100 were considered positive for antitoxin antibody. The ELISA reactivity of all positive sera could be completely absorbed with purified P. pseudonrallei exotoxin . Similarly, preincubation of the exotoxin~oated wells with antisera of exotoxin-immunized rabbits completely inhibited the ELISA reactivity of sheep's sera ; indicating that the exotoxin produced during infection is similar to the exotoxin produced in laboratory cultures of P. pseudomallei. The development of serum antitoxin antibody suggests that exotoxin is produced during P. psettdomallei