Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content content-type="genus-species">Clostridium botulinum</jats:named-content>types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the<jats:named-content content-type="genus-species">C. botulinum</jats:named-content>subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including<jats:named-content content-type="genus-species">C. botulinum</jats:named-content>types C (<jats:italic>n</jats:italic>= 12), C-D (<jats:italic>n</jats:italic>= 29), D (<jats:italic>n</jats:italic>= 5), and D-C (<jats:italic>n</jats:italic>= 10), other botulinum neurotoxin (BoNT)-producing<jats:named-content content-type="genus-species">Clostridium</jats:named-content>strains (<jats:italic>n</jats:italic>= 20), non-BoNT-producing clostridia (<jats:italic>n</jats:italic>= 20), and other bacterial species (<jats:italic>n</jats:italic>= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of<jats:named-content content-type="genus-species">C. botulinum</jats:named-content>in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (<jats:italic>n</jats:italic>= 108), poultry (<jats:italic>n</jats:italic>= 60), and bovines (<jats:italic>n</jats:italic>= 56). Among the 292 samples, 144 were positive for either the<jats:italic>bont/</jats:italic>C-D or the<jats:italic>bont/</jats:italic>D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.</jats:p>

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

“…Before DNA extraction, which was performed as described elsewhere, 26 each aliquot of enrichment broth was supplemented with an amount of approximately 100 cells of process control (PC). PC consisted of a 4-day-old culture of a C. botulinum type B-like strain (a C. botulinum strain that had naturally lost the gene encoding the toxin) or, alternatively, a 4-day-old culture of Clostridium sporogenes strain ATCC 19404.…”

“…DNA extraction was performed using 1 ml of each cultured strain, as reported elsewhere. 26 Positive realtime PCR controls were generated by cloning each PCR product into 4 plasmids. Cloning procedures were carried out by MWG Eurofins (Ebersber, Germany).…”

“…The criteria for laboratory confirmation have been extensively reported elsewhere. 4,9,25,26 Among BoNT-producing clostridia strains, C. botulinum strains that produce types C, D, C/D, and D/C toxins (group III organisms) are the organisms mainly responsible for animal botulism outbreaks. 6,7 Rapid detection and typing of these organisms and their toxins are a prerequisite for a prompt diagnosis, for treatment of sick animals, and to prevent or minimize further cases.…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…In particular, assays able to detect bont/a, /b, /e, and /f, which are known to be pathogenic to humans, in a single reaction have been developed (Lindström et al 2001;Shin et al 2007;Kirchner et al 2010;Satterfield et al 2010;Fach et al 2011). Assays covering bont/c, /d, and their mosaic forms have been developed to meet the needs of veterinary medicine Woudstra et al 2012).…”

The Workshop on Animal Botulism in Europe

“…Cedric Woudstra presented the GeneDisc technology and the 2 prototypes for detection of botulinum neurotoxin gene of type C, D, and mosaic types C/D and D/C toxin. 21 The Gegenees program and GeneDisc technology were also demonstrated for participants to get a hands-on experience from using the different methods. It was agreed that gene detection methods are suitable for detection of C. botulinum in research settings and for screening of samples in order to minimize the use of the mouse bioassay.…”