Abstract
Clostridium chauvoei is the etiologic agent of blackleg in cattle, inducing fever, severe myonecrosis, oedemic lesions and ultimately death of infected animals. The pathogen often results in such rapid death that antibiotic therapy is futile and thus vaccination is the only efficient strategy in order to control the disease. The β-barrel pore forming leucocidin Clostridium chauvoei toxin A (CctA) is one of the best characterised toxins of C. chauvoei and has been shown to be an important virulence factor. It has been reported to induce protective immunity and is conserved across C. chauvoei strains collected from diverse geographical locations for more than 50 years. The aim of this study was to identify the location of the CctA toxin during liquid culture fermentation and to use CctA to develop an in vitro assay to replace the current guinea pig challenge assay for vaccine potency in standard batch release procedures. We report that CctA is fully secreted in C. chauvoei culture and show that it is found abundantly in the supernatant of liquid cultures. Sera from cattle vaccinated with a commercial blackleg vaccine revealed strong haemolysin-neutralizing activity against recombinant CctA which reached titres of 1000 times 28 days post-vaccination. Similarly, guinea pig sera from an official potency control test reached titres of 600 times 14 days post-vaccination. In contrast, ELISA was not able to specifically measure anti-CctA antibodies in cattle serum due to strong cross-reactions with antibodies against other proteins present pre-vaccination. We conclude that haemolysin-neutralizing antibodies are a valuable measurement for protective immunity against blackleg and have the potential to be a suitable replacement of the guinea pig challenge potency test, which would forego the unnecessary challenge of laboratory animals.
Highlights
Clostridium (C.) chauvoei is the causative agent of blackleg referred to black quarter and quarter evil, a severe infectiousAbbreviations: Clostridium chauvoei toxin A (CctA), Clostridium chauvoei leucocidin toxin A; rCctA-NusA, recombinant Clostridium chauvoei leucocidin toxin A fused to E. coli NusA protein; rCctA-cent, recombinant antigenic central fragment of CctA.Shared first authorship.P
Blackleg vaccines are based on a mixture of detoxified culture supernatant of C. chauvoei and inactivated bacteria but there is no conclusive information concerning the protective antigens and where they reside in this vaccine
To demonstrate the specificity of our anti-CctA antibody [13] which provides the basis of our experiments, we loaded the recombinantly generated CctA produced in this study, encompassing the central antigenic domain of CctA-cent (rCctA-cent comprising amino acids (AA) 177e271), with a predicted molecular mass of 18.7 kDa and a commercially purchased recombinant mature CctA (AA29-317) with a stated molecular mass of 59.53 kDa on to a 15% SDS-polyacrylamide gel followed by Western blotting
Summary
Clostridium (C.) chauvoei is the causative agent of blackleg referred to black quarter and quarter evil, a severe infectious. P. Nicholson et al / Anaerobe 56 (2019) 78e87 factor is Clostridium chauvoei toxin A (CctA) belonging to the bbarrel pore forming leucocidin family [12]. Nicholson et al / Anaerobe 56 (2019) 78e87 factor is Clostridium chauvoei toxin A (CctA) belonging to the bbarrel pore forming leucocidin family [12] This factor has been shown to be the main cytotoxic and haemolytic agent of C. chauvoei [13]. Along with a highly active DNAse, hyaluronidase and sialidase, they seem to constitute the central virulence arsenal of C. chauvoei [1,3,7,13e15]. Flagella have been reported to be involved in the pathogenesis of C. chauvoei infections [11,16e23]
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