PCR-based molecular diagnosis of botulism (types C and D) outbreaks in aquatic birds

Clostridium botulinum is a strictly anaerobic spore forming bacterium found in soil and sediments, producing botulinum neurotoxins. Group III strains of this organism are only able to produce neurotoxin types C, mosaic C/D, D/C, and D, which are associated with bird botulism. The threats and outbreak cycle of bird botulism are enhanced in the aquatic environment via a food web-environment-avian interface in response to global climatic changes. The aim of this review was to describe and discuss the development of PCR-based markers, diagnostic assays, and applications with special emphasis to botulism detection in aquatic birds. We employed a text-mining approach for collection of current diagnostic information to bird botulism in aquatic environments. Using the PubMed search engine, we have comprehensively collected relevant information from 124 articles and then summated for the purpose of this review. Genes coding for botulinum neurotoxin (bont/C, bont/C/D, bont/D/C, and bont/D), nontoxic components (ha70, ha17, ha33, and ntnh), and flagellin (fliC) were the molecular markers most commonly found for the genotyping of group III strains from environmental samples. “GeneDisc” real-time PCR system was a robust and reliable diagnostic technique for discrimination of neurotoxin types in the large sampled areas. PCR-based assays were the most perceptive and widely established for detection of bird botulism, even if several biochemical and molecular diagnostic techniques have been available. Thus, timely and accurate identification of its mortality is needed to provide a biosecurity, disease control management, and conservation of aquatic birds. PCR-based diagnosis is a promising alternative to the mouse bioassay. Hence, the present paper makes an attempt to review the PCR-based detection assays including primers, specificity, sensitivity, and detection limit and explore their potential applications in wildlife microbiology.