PARETIC SYNDROME IN GULLS FROM SOUTHERN PORTUGAL: SEARCHING FOR THE CAUSATIVE AGENT

the reason of the continuous morbidity and mortality episodes. Pasteur Institute (Paris) and the Spanish Oceanographic Institute (IEO Vigo) agreed to perform toxin analyses.

• Domoic acid and Paralytic Shellfish Toxins do not seem to be the cause of the gulls paretic syndrome.
• Samples tested point to Clostridium botulinum as the possible cause of the gulls paretic syndrome, or at least one of the causes.This bacterium is considered as a frequent cause of massive mortality events in seabirds.The symptoms caused in seabirds are very similar to the ones produced by PSTs.
• Further studies are needed to evaluate the source of C. botulinum contamination.
• Additional studies are planned in other Institutions to evaluate the potential implication of Cyanotoxins and Tetrodotoxins in the toxic episodes.

CONCLUSIONS METHODS & RESULTS
food offered •Water ad libitum •Subcutaneous and intravenous fluid therapy •Repeat fluid therapy for 24 or 48 hours until tube feeding is safeThe aim of this preliminary study was to evaluate if Amnesic Shellfish Toxins (Domoic acid, (DA)), Paralytic Shellfish Toxins (PSTs) or botulinum toxins could be the cause of the gulls paretic syndrome.This presentation compiles the results of the Clostridium botulinum and marine biotoxins analysis conducted at the French National Reference Centre for anaerobic bacteria and botulism, Pasteur Institute (Paris) and the Vigo Centre of the Spanish Oceanographic Institute, respectively.IEO analyses were funded by project DIANAS (CTM2017-86066-R, MICINN), GRC-GAIN (IN607A 2019/04), and are a contribution of Unidad Asociada IEO-CSIC Microalgas Nocivas.We thank Sanidad Exterior (Vigo) for providing DA standard and Wade Rourke (Canadian Food Inspection Agency) for support with chromatographic conditions.REFERENCES HPLC-FLD DETECTION•Samples: twenty three gull samples: ten livers, ten intestines and three cloacae contents.•Drawbacks: small sample size, difficult homogenization (specially for intestines, hard to cut by Ultraturrax, pre-cutting needed).•Single dispersive extraction: with MEOH 50% • Liquid Chromatography coupled to High Resolution Mass Spectrometry.DA was not detected in any of the samples tested • Samples: livers & intestines from 5 gulls.Pooled in two groups according to the tissue.• Enrichment cultures of tissues in fortified cooked meat medium at 37°C and 15°C under anaerobic conditions.C. botulinum was isolated on agar selective medium.• DNA extraction from samples.• C. botulinum was investigated by targeted Real Time Polymerase Chain Reaction (PCR) on neurotoxin genes.•Presence of botulinum toxins was confirmed by a lethality test on mice (mouse bioassay).Mice were intraperitoneally injected with filtered supernatant of the culture.Samples were positive for C. Botulimum types C/D Domoic acid analyses • Gulls samples: ten kidneys & contents from one cloacae.• Drawbacks: very small sample size, difficult homogenization, extraction had to be scaled down.• Double extraction with 0.1M HCl.Sample deproteination with trichloroacetic acid.• Analyses by Liquid Chromatography with Postcolumn Derivatization & Fluorescence Detection.• Samples re-run without oxidation: checking for naturally fluorescent compounds needed analyses • De La Iglesia, P.; Barber, E.; Giménez, G.; et al.High-throughput analysis of amnesic shellfish poisoning toxins in shellfish by ultra-performance rapid resolution LC-MS/MS.J. AOAC Int.2011, 94, 555-564.• Gibble, C.M.; Hoover, B.A. Interactions between Seabirds &Harmful Algal Blooms.In Harmful Algal Blooms: A Compendium Desk Reference; S.E.Shumway, J.M. Burkholder & S.L. Morton Eds; John Wiley & Sons Ltd. 2018; pp.223-242.• Mazuet C., Sautereau J., et al.An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham.J Clin Microbiol.2015, 53:722-726.• Rourke, W.A.; Murphy, C.J.; Pitcher, G.; Van De Riet, J.M.; et al.Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue.J. AOAC Int.2008, 91, 589-597.• Rodríguez, F.; Garrido, J.L.; Sobrino, C.; Johnsen, G.; Riobó, P.; et al.Divinyl chlorophyll a in the marine eukaryotic protist Alexandrium ostenfeldii (Dinophyceae).Environ.Microbiol.2016, 18, 627-643.• Sonne, C.; Alstrup, A.K.O.; Therkildsen, O.R. A review of the factors causing paralysis in wild birds: Implications for the paralytic syndrome observed in the Baltic Sea.Sci.Total Environ.2012, 416, 32-39.• Van Hemert, C.; Schoen, S.K.; Litaker, R.W.; Smith, M.M.; et al.Algal toxins in Alaskan seabirds: Evaluating the role of saxitoxin and domoic acid in a large-scale die-off of Common Murres.Harmful Algae 2020, 92, 101730.• Vanhomwegen J, Berthet, N. Mazuet, C.; et al.Application of high-density DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.PLoS One 2013, 8:e67510