Multiplex Real-Time PCR for Detecting and Typing <i>Clostridium botulinum</i> Group III Organisms and Their Mosaic Variants

Anniballi, Fabrizio; Auricchio, Bruna; Woudstra, Cédric; Fach, Patrick; Fiore, Alfonsina; Skarin, Hanna; Bano, Luca; Segerman, Bo; Knutsson, Rickard; Medici, Dario De

doi:10.1089/bsp.2012.0084

Cited by 16 publications

(12 citation statements)

References 24 publications

(18 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples positive for all primers had also been reported in some other avian outbreaks [38]. It appears that type D is uncommon in avian botulism outbreaks and has not been reported in other available studies [16,21,22,38]. No type C BoNTs were detected either in the samples analyzed during our study or in previously published studies that distinguished type C/D from type C BoNTs [14,16].…”

Section: Discussionsupporting

confidence: 74%

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

Gratiet

Poëzévara

Rouxel

et al. 2020

Toxins

View full text Add to dashboard Cite

Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridium novyi sensu lato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.

show abstract

Section: Discussionsupporting

confidence: 74%

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

Gratiet

Poëzévara

Rouxel

et al. 2020

Toxins

View full text Add to dashboard Cite

show abstract

“…Incubation of at least 24 h appeared to be sufficient. Similarly, previous reports also propose an incubation time of 24 h [19, 20]. Therefore, diagnosis confirmation time can be shortened, especially in comparison with the mouse bioassay.…”

Section: Discussionmentioning

confidence: 89%

“…The temperature used for enrichment of C . botulinum group III varies among studies in the literature: 30°C [19, 20, 22], 37°C [2, 15, 35] and 40°C [21, 29]. Here, we compared three different incubation temperatures: 30°C, 37°C, and 41.5°C (Fig 6).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Alternative methods that do not require laboratory animals are for example polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and the endopeptidase mass spectrometric method called Endopep-MS. Prior to PCR analysis, the bacteria in the sample need to be anaerobically cultivated, and then the toxin gene can be detected [13][14][15][16]. It is a fast and cheap method, and it works well for detection as long as spores and/or vegetative cells of the bacteria are present in the sample, i.e., the test will be negative if only preformed BoNT is present.…”

Section: Introductionmentioning

confidence: 99%

Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks

2020

View full text Add to dashboard Cite

Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden.

show abstract

Multiplex Real-Time PCR for Detecting and Typing Clostridium botulinum Group III Organisms and Their Mosaic Variants

Cited by 16 publications

References 24 publications

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

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

Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks

Contact Info

Product

Resources

About