title: Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism.
abstract: Liver is a reliable matrix for laboratory confirmation of avian botulism using real-time PCR. Here, we developed, optimized, and validated the analytical steps preceding PCR to maximize the detection of Clostridium botulinum group III in avian liver. These pre-PCR steps included enrichment incubation of the whole liver (maximum 25 g) at 37°C for at least 24 h in an anaerobic chamber and DNA extraction using an enzymatic digestion step followed by a DNA purification step. Conditions of sample storage before analysis appear to have a strong effect on the detection of group III C. botulinum strains and our results recommend storage at temperatures below -18°C. Short-term storage at 5°C is possible for up to 24 h, but a decrease in sensitivity was observed at 48 h of storage at this temperature. Analysis of whole livers (maximum 25 g) is required and pooling samples before enrichment culturing must be avoided. Pooling is however possible before or after DNA extraction under certain conditions. Whole livers should be 10-fold diluted in enrichment medium and homogenized using a Pulsifier® blender (Microgen, Surrey, UK) instead of a conventional paddle blender. Spiked liver samples showed a limit of detection of 5 spores/g liver for types C and D and 250 spores/g for type E. Using the method developed here, the analysis of 268 samples from 73 suspected outbreaks showed 100% specificity and 95.35% sensitivity compared with other PCR-based methods considered as reference. The mosaic type C/D was the most common neurotoxin type found in examined samples, which included both wild and domestic birds.
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Le Maréchal
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Rouxel
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Valentine
authors.initials: V
authors.lastname: Ballan
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Houard
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Typhaine
authors.initials: T
authors.lastname: Poezevara
authors.affiliation: Labocea, Ploufragan, France.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Bayon-Auboyer
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité d'Epidémiologie et bien-être en aviculture et cuniculture, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Rozenn
authors.initials: R
authors.lastname: Souillard
authors.affiliation: Labocea, Ploufragan, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Morvan
authors.affiliation: Labocea, Ploufragan, France.
authors.forename: Marie-Agnès
authors.initials: MA
authors.lastname: Baudouard
authors.affiliation: ANSES, Laboratoire de sécurité des aliments, Maisons Alfort, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Woudstra
authors.affiliation: Institut Pasteur, Bactéries anaérobies et Toxines, Paris, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Mazuet
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité d'Epidémiologie et bien-être en aviculture et cuniculture, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Bouquin
authors.affiliation: ANSES, Laboratoire de sécurité des aliments, Maisons Alfort, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
authors.affiliation: Institut Pasteur, Bactéries anaérobies et Toxines, Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Popoff
authors.affiliation: ANSES, Laboratoire de Ploufragan - Plouzané, Unité Hygiène et qualité des produits avicoles et porcins, Université Bretagne-Loire, Ploufragan, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Chemaly
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000382:microbiology*; D001906:Botulism / Q000382:microbiology / Q000662:veterinary*; D003014:Clostridium botulinum / Q000235:genetics / Q000302:isolation & purification; D008099:Liver / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D011200:Poultry / Q000382:microbiology