Livers provide a reliable matrix for real-time PCR confirmation of avian botulism

Maréchal, Caroline Le; Ballan, Valentine; Rouxel, Sandra; Bayon-Auboyer, Marie-Hélène; Baudouard, Marie-Agnès; Morvan, Hervé; Houard, Emmanuelle; Poëzévara, Typhaine; Souillard, Rozenn; Woudstra, Cédric; Bouquin, Sophie Le; Fach, Patrick; Chemaly, Marianne

doi:10.1016/j.anaerobe.2015.10.014

Cited by 13 publications

(26 citation statements)

References 27 publications

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“…The analysis of four livers is usually recommended for avian botulism confirmation using real-time PCR [3]. When considering our results on an outbreak basis (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…Laboratory confirmation for animal botulism can be done by different strategies including demonstration of the presence of BoNT or BoNT-producing clostridia in serum, gastrointestinal content, liver and wound [18]. We recently showed statistically that the analysis of four livers to confirm avian botulism using real-time PCR is a reliable method for avian botulism diagnosis [3]. However, the steps preceding the PCR had not been yet optimized to maximize the sensitivity of the method.…”

Section: Discussionmentioning

confidence: 99%

“…When considering the results at the outbreak level and not at the individual sample level, only two suspected outbreaks tested positive with Hill method and negative with our method. For one suspected outbreak, only two livers were analyzed and not four as recommended [3]. Regarding the second suspicion, the mouse bioassay was additionally performed with sera collected on birds was negative for BoNT C, D and E. Ionophore intoxication was highly suspected to explain the observed mortality.…”

Section: Discussionmentioning

confidence: 99%

“…Outbreaks due to types D, D/C or C have been also reported but at a lower rate [3, 4]. Clostridium botulinum , which produces BoNT is divided into four physiological groups.…”

Section: Introductionmentioning

confidence: 99%

“…botulinum types C, D, C/D, D/C and E using real-time PCR [3]. However, the steps preceding PCR have not yet been optimized.…”

Section: Introductionmentioning

confidence: 99%

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Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism

et al. 2017

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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.

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“…The analysis of four livers is usually recommended for avian botulism confirmation using real-time PCR [3]. When considering our results on an outbreak basis (i.e.…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Outbreaks due to types D, D/C or C have been also reported but at a lower rate [3, 4]. Clostridium botulinum , which produces BoNT is divided into four physiological groups.…”

Section: Introductionmentioning

confidence: 99%

“…botulinum types C, D, C/D, D/C and E using real-time PCR [3]. However, the steps preceding PCR have not yet been optimized.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations