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

Abstract

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.

Comparative Genomics of Clostridium baratii Reveals Strain-Level Diversity in Toxin Abundance

“…Clostridium species could be found in soil and residing in several animals' intestines, showing their widespread distribution [2]. This taxonomic group includes important animal and human pathogens that cause dangerous and potentially deadly diseases such as tetanus [3] (produced by tetanus toxin from C. tetani), gastroenteritis [4] (C. perfringens), Clostridioides difficile infection, and botulism [5,6] (produced by botulism neurotoxin from C. botulinum), among several others [7]. In addition to their medical importance, Clostridium species are well studied for their industrial properties in producing important bioproducts such as ethanol, organic acids, and therapeutic proteins [8].…”

Asymptomatic Carriage of C. botulinum Type D/C in Broiler Flocks as the Source of Contamination of a Massive Botulism Outbreak on a Dairy Cattle Farm

“…However such approaches require isolation of pure strains, thus limiting the number of samples that can be analyzed. Despite improvements regarding C. botulinum group III isolation ( Le Gratiet et al, 2020 ), it is still time-consuming and phages encoding BoNT are easily lost. MLVA has been available for C. botulinum group I and II for many years ( Fillo et al, 2011 ; Umeda et al, 2013 ; Anniballi et al, 2016 ).…”

“…Botulism is more common in animals than in humans and results in high mortality rate, raising significant animal welfare and economic concerns ( Anniballi et al, 2013 ; Relun et al, 2017 ; Rasetti-Escargueil et al, 2019 ). Avian botulism is generally associated with BoNT C/D, whereas bovine botulism is more frequently associated with BoNT D/C, and to a lesser extent, BoNT C ( Woudstra et al, 2012 ; Bano et al, 2017 ; Le Gratiet et al, 2020 ). Considering the serious consequences of botulism on bovine and avian species, a better understanding of this disease — particularly in terms of potential mechanisms of transmission — is crucial to improve prevention and management of animal botulism outbreaks in an efficient manner.…”

Bird-Area Water-Bodies Dataset (BAWD) and Predictive AI Model for Avian Botulism Outbreak (AVI-BoT)

“…Some of the recent outbreaks in England (2018), Wales (2018) 3 , Klamath National Wildlife Refuge (2020) 5 resulted in bird casualty as high as 40,000. Some of the widely known techniques to confirm the outbreak of Avian botulism are culture techniques 6 , assay test for presence of toxin 7 , PCR test 8 (See Table 1). Most of these techniques rely on actual ground level sample collection which is challenging due to the requirement of physical access.…”