After my last posting about the Monk Seals, several of you asked about the nature of the "antidote" mentioned in the text. This is an antibody against saxitoxin provided by Mark Poli of the Toxinlogy Division, US Army MRIID, Fort Detrick. It was give to the Marine Mammal Commission, but their job was only to deliver it to those working on the seals. Several studies have shown that anti-saxitoxin or anti-tetrodotoxin antisera can protect animals, so there is some possibility that this may work. There are, however, numerous problems. First, a great deal of antibody will be needed for a single 100kg seal - perhaps more antiserum than is available. Second, the antibody is specific for STX with little cross-reactivity with other PSP toxins. No one has provided HPLC toxin composition data for the algae in the area of the seal mortality. Third, it will be exceedingly difficult to get the antibody into a seal, as this will require an IV, somehow administered in a cave when the tide is low enough to permit entry. And somehow the seal will have to be prevented from going back to the toxin source if it recovers.
Anyway - those are but a few of the problems. It will be interesting to see what develops. What follows is the latest information on the seals, clarifying some of what was in the last posting.
Don Anderson
Since May 17, a unusually high Monk seal (Monachus monachus) mortality was observed by the permanent monitoring field team of the Spanish Monk Seal LIFE/CE Project, which has been studying the population since 1992 in collaboration with the national mauritanian authority in nature conservation (Parc National du Banc d=B4Arguin).
Previous analysis (MARMAM 23/May/1997) of water samples revealed the presence of toxic dinoflagellates, mostly Alexandrium minutum all over the coast. Two liver samples from Monk Seals dead on May 21 tested by mice bioassays following the methodology of Falconer (In: Falconer IR. (ed.) 1993. Algal Toxins in Seafood and Drinking Water. Academic Press, London. pp:165-167) yielded positive results and a dose of 0.917 plus/minus 0.12 mouse units over 6 mice assayed was detected.
An inmunofluorescence technique was employed to detect saxitoxins in brain cryosections from 4 Monk seals. These included 2 adult males (Nos 59 and 366), one sub adult female (No 365) and one pregnant, adult female (No 368). All these were found dead between June 3rd and June 12th in beaches of the Western Sahara. Brain samples were kept frozen at minus 20C until analysis.
Frozen sections 4 microns thick were made from these brain samples using a cryotome Leica CM 1500 and mounted Poly-L-Lysine coated microscope slides. Brain sections from unexposed mice and stranded Striped dolphin (Stenella coeruleoalba) were also analysed as negative controls.=20
Sections were incubated and results obtained following a sandwich technique described by Costas et al (1995. Journal Appl. Phycology 7:407-411) and Costas & Lopez-Rodas (1997. Journal of Phycology 33(3): in press). Two monoclonal mouse antibodies were used as primary antibodies: Industries UVE Inc., Japan and Industries Rouger Inc., Canada. A commercially available FITC-conjugated goat anti-mouse antibody from Sigma, Spain, was used as secondary antibody. Sections were examined using a Zeiss Epifluorescence microscope with a blue filter set (BP 450-490 nm) and a emission cut off for FITC. Fluorescence was measured by microscope photometry.
Results of the presence of saxitoxins in brain sections from all four seals were positive. Control sections using unexposed mouse and dolphin brain tissue yielded negative quantitative and quantitative results. Qualitative and quantitative results from the four seals correlate well with those obtained in mice given experimental doses of saxitoxin (Dr. E. Costas, unpubl. data).
Positive mouse bioassays indicate that phycotoxins were present in both liver assayed. Most Reference Laboratories use the Association of Official Analytical Chemists (AOAC) mouse bioassays for detection of PSP toxin in seafood (Hollinworth T & Wekell M 1990.In: K. Hellrich (ed). Official Methods of Analysis of the AOAC, 15th Edition, Arlington, USA, p881-882). The Falconer method differs from AOAC mouse bioassay in pH adjustement and the obtention of 4 extracts with different pHs, thus limiting the influence of pH during extraction on results. Mouse bioassays in a non selective method but, is currently the only bioassay reliable for detecting all PSP toxins (Fernandez et al, 1996. In: Yasumoto, T. et al (eds) Harmful and Toxic Algal Blooms. Intergov. Oceanographic Commision of UNESCO, pp: 11-14).
Inmunological techniques are being successfuly used in the detection of biotoxins in water and foodstuff samples (Costas et al 1995; Usleber et al 1995., Food Addit. Contam. 12:405-413). Results of the inmunological technique indicated that saxitoxins were present in the brain sections of all four seals. Although antibodies assayed have high specificity and sensivity for saxitoxins, they do not distinghish between different saxitoxins. Both antibodies cross-react with all toxins from the saxitoxin family including, STX, NeoSTX, dcSTX, GTX1-4, B1, C1-4 etc.
No brains from unexposed monk seals were available to be included as negative control but, brains from unexposed mice and stranded dolphins were used. No inmunostaning was obtained in neither of the negative controls. Thus, cross-reaction with some of the tissue components is unlikely. Fluorescence results are a strong suggestion that saxitoxin poisoning have played an important role in the monk seal mortalities.
Results in this study demosntrate that (1) saxitoxins were present in the four Monk seal brains analysed, (2) fluorescence acitivity correlates well with those obtained in mice given experimental doses of saxitoxin and, (3) the technique can be successfully applied to detect these and other biotoxins in target organs of marine mammals.
Prof. Dr Eduardo Costas