Investigation of the use of the ELISA procedure in determining the potency of Clostridium chauvoei vaccines
Abstract
Blackleg, caused by Clostridium chauvoei, remains a serious disease of cattle and small ruminants. Potency testing of C. chauvoei vaccines is still based on guinea pig challenge assays, which in this study involved 300 Wistar Albino guinea pigs (350–450 g). Although regarded as the reference method, it is labor-intensive, time-consuming, and raises animal welfare concerns. This study aimed to evaluate whether an antigen-based ELISA could serve as a rapid, reproducible, and animal-friendly in vitro alternative for potency testing of inactivated C. chauvoei vaccines. Twenty commercial vaccines (15 with C. chauvoei antigen, 5 without) were evaluated by both the guinea pig challenge test and a flagellar antigen–specific ELISA. For each vaccine, 10 guinea pigs were vaccinated and 5 remained as controls. Relative potency (RP) was calculated against a standardized reference vaccine. Correlation between methods, sensitivity, and specificity were determined. The ELISA achieved 93.3% sensitivity, 100% specificity, and 95.0% relative accuracy. RP values of positive vaccines ranged from 1.02 to 5.44, showing a strong correlation with in vivo results (r = 0.882, p < 0.001). Intra-plate (CV < 10%) and inter-plate (CV < 15%) variation were within acceptable limits. No false positives occurred, and vaccines lacking C. chauvoei antigen produced very low RP values. The developed ELISA represents a reliable alternative to animal testing, significantly reducing both animal use and testing duration while maintaining accuracy. Implementation would align with 3R principles (Replacement, Reduction, Refinement) and enhance the efficiency of vaccine quality control protocols.
Keywords
Clostridium, Blackleg, Elisa, Potency, Vaccine
Project Number
Aydın Adnan Menderes University Scientific Research Projects Coordination Unit (Project number: VTF-15066),
Ethical Statement
This research was supported by the Aydın Adnan Menderes University Scientific Research Projects Coordination Unit (Project number: VTF-15066), the Ministry of Food, Agriculture and Livestock – General Directorate of Agricultural Research and Policies (TAGEM), and İzmir/Bornova Veterinary Control Institute (Project number: TAGEM/HSGYAD/17/A05/P01/91).
Thanks
The authors would like to express their sincere gratitude to Zahide Dilik and Ahmet Arslan for their valuable contributions to the laboratory studies conducted during this research.
References
- Amitkumar, M., Prajapati, A., Bindu, S., Sairam, S., Namrutha, M.R., Anandamurthy, R., & Shivachandra, S.B. (2025). Comparative evaluation of indirect-ELISAs based on native antigens of Clostridium chauvoei for the detection of blackleg-specific antibodies in cattle. Anaerobe, 94, 102975. DOI: 10.1016/j.anaerobe.2025.102975
- Arslan A, Dilik Z, Özyer M, Oktay N, Yılmaz Ş. (2016) Klostridial aşıların potensinin belirlenmesinde toksin nötralizasyon testlerine alternatif olarak ELISA'nın kullanımı. TAGEM/ HSGYAD, 13/ A07 /P02 / 32, Araştırma - Geliştirme Destekleri Proje Sonuç Raporu 2016. 1-36.
- Crichton, R., Harriss, D.A., & McKay, D.J. (1986). Standards for Clostridium chauvoei vaccine: the relationship between the response of guinea pigs and sheep following vaccination and challenge with virulent C. chauvoei. Aust. Vet. J., 63, 68-70. DOI: 10.1111/j.1751-0813.1986.tb02820.x
- DeHaven, W.R. (2003). In vitro serial release potency test for complete product containing Clostridium chauvoei. USDA Vet. Serv. Mem., 800.104. 1-4.
- Dinse, G.E., & Umbach, D.M. (2013). Quantifying relative potency in dose–response studies. In: Lee, MLT; Gail, M; Pfeiffer, R and Satten, GA (Eds.), Risk assessment and evaluation of predictions. New York, Springer. 315-331. DOI:10.1007/978- 1-4614-8981-8_15.
- EDQM. (2022). Clostridium chauvoei vaccine for veterinary use. In: European Pharmacopoeia. 11th Edn., Strasbourg, EDQM. 1151-1152.
- Gohari, I.M., & Prescott, J.F. (2022). Clostridium. In: McVey, DS; Kennedy, M; Chengappa, MM and Wilkes, R (Eds.), Veterinary Microbiology. 4th Edn., USA, Wiley Blackwell. 309-334.
- Hamzavipour, R., Moazeni, M., Firoozeh, F., & Aghamiri, S.M. (2024). Protection efficacy and immunogenicity of Clostridium chauvoei cellular and extracellular proteins. Toxicon, 238, 108124. DOI: 10.1016/j.toxicon.2024.108124
- Hendriksen, C.F.M. (2009). Replacement, reduction and refinement alternatives to animal use in vaccine potency measurement. Expert Rev. Vaccines, 8, 313-322. DOI: 10.1586/14760584.8.3.313
- Hill, R.E. (2011). Alternative methods to reduce, refine, and replace the use of animals in the development and testing of veterinary biologics in the United States: a strategic priority. Procedia Vaccinol., 5, 141-145. DOI: 10.1016/j.provac.2011.10.019
