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Bacteria of the species Clostridium botulinum are sporeforming, gram-positive, anaerobic rods which are able to produce the most potent toxins in nature. Botulinum toxins are the etiologic factor of botulism in humans and animals. A lethal dose of botulinum toxin for a mouse is about 0.3 ng/kg, whilst a lethal dose for a human ranges from 0.2 µg/kg to 2 µg/kg. Historically, the differentiation of Clostridium botulinum strains is based on their ability to produce one of the seven botulinum toxins marked with letters from A to G. Nowadays, the classification based on the ability to produce botulinum toxins is not the only taxonomic criterion. C. botulinum strains are also divided into four groups which have different metabolic and culture features. Although, botulism is a rare disease, the outbreaks of botulism are difficult to control and cause economic losses in livestock. The species most susceptible to botulism are cattle and birds. The aim of this article is the characteristic of Clostridium botulinum, botulinum toxins, their action and botulism as a disease in some species of animals.
For validation purposes, characteristic parameters for quantitative detection were estimated according to the PN-EN ISO 16140. Additionally, a comparison between validated real-time PCR method and traditional methods based on the isolation of this pathogen on differential agar media was conducted. The validated method has shown the possibility of detection of 58 copies of ntnh gene (genome equivalents) for DNA obtained from dilution of pure Clostridium botulinum NCTC 887 cells. For DNA obtained from the contaminated food and feed samples inhibitory effect was observed. The tested method has shown high specificity proved by the examination of DNA obtained from C. botulinum reference strains and other strains of Clostridium sp. The specificity has also proved the obtained concordance between results from analyses using test on laboratory mice with those from analyses using the tested real-time PCR method. The obtained results have shown that the described method gives the possibility to detect the pathogen without isolation and to shorten time of analysis in comparison to the traditional methods, based on isolation of this pathogen on differential agar media.
The application of molecular-biology methods based on PCR and Real-time PCR in the diagnosis of botulism in mallard ducks was described. The participation of Clostridium botulinum C₁ toxin in the causing of the disease was demonstrated. The results were confirmed by the sequencing of PCR product. The application of molecular-biology methods provides fast and specific analysis of the occurrence and types of Clostridium botulinum toxins.
The aim of this work was to present selected data regarding traditional and modern methods for C. botulinum and its toxins detection. In this article, methods based on culturing techniques, mouse bioassay, immunological techniques, chromatography and PCR, PFGE, RFLP, AFLP are described. The mentioned techniques were evaluated considering their usefulness in the samples examination, genotyping of strains and the diagnostics of botulism.
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