SavedAdd your journal

Journals / Etlik Veteriner Mikrobiyoloji Dergisi / 2026 / Cilt: 37 Sayı: 1

Determinatin of Potency in Clostridium septicum Vaccines by In Vitro Toxin Neutralization Test Using Vero Cells

Clostridium septicum Aşılarında Toksin Nötralizasyon Testine Alternatif Vero Hücreleri Kullanılarak Etkinliğin Belirlenmesi

Oğuzhan Ayar(İZMİR BORNOVA VETERİNER KONTROL ENSTİTÜ MÜDÜRLÜĞÜ)0000-0001-6230-9781
Göksel Erbaş(ADNAN MENDERES ÜNİVERSİTESİ)0000-0002-1839-754X
Pages
17–24
Publication Date
2026-07-13
🏛️Bibliographic metadata and full text access for this article are provided via DergiPark.
DergiPark Record ↗
✨

AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

In conclusion, the Vero cell neutralization assay provided results comparable to TNT while requiring less time and cost, and offering more stable and reliable outcomes.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:This study aimed to evaluate the applicability of a Vero cell–based neutralization assay as an alternative to the toxin neutralization test (TNT), which does not comply with the 4R principles, for the control of clostridial vaccines.
📊
Key Findings & Data:In the study, 20 vaccines containing Clostridium septicum alpha toxoid and 10 vaccines lacking this component (clostridial or non-clostridial), submitted to the Bornova Veterinary Control Institute, Veterinary Biological Products Control Laboratory for routine marketing authorization, were used.
💡
Conclusion & Impact:In conclusion, the Vero cell neutralization assay provided results comparable to TNT while requiring less time and cost, and offering more stable and reliable outcomes.
🔬Design:Araştırma Makalesi
📍Scope:Comprehensive Sample
⚙️Variables:Aşı · Septicum toxin · Toksin nötralizasyon test

Abstract

This study aimed to evaluate the applicability of a Vero cell–based neutralization assay as an alternative to the toxin neutralization test (TNT), which does not comply with the 4R principles, for the control of clostridial vaccines. In the study, 20 vaccines containing Clostridium septicum alpha toxoid and 10 vaccines lacking this component (clostridial or non-clostridial), submitted to the Bornova Veterinary Control Institute, Veterinary Biological Products Control Laboratory for routine marketing authorization, were used. For each vaccine, ten New Zealand White rabbits with no history of disease or vaccination were immunized, and sera were collected. The sera were equally divided and subjected to neutralization analysis in Vero cell lines and to TNT using 15 mice per serum sample. According to TNT results, 16 of the 20 C. septicum–containing vaccines were found to be above the acceptance limit, while 4 were below the limit. In sera obtained from rabbits vaccinated with vaccines lacking C. septicum, no antitoxin formation was detected and all mice died in TNT. The Vero cell–based assay yielded results parallel to TNT, with the sensitivity and specificity of the new method calculated as 100%. In conclusion, the Vero cell neutralization assay provided results comparable to TNT while requiring less time and cost, and offering more stable and reliable outcomes.

Özet

Bu çalışmada, Clostridial aşıların kontrolünde 4R prensipleri ile örtüşmeyen klasik toksin nötralizasyon testi (TNT) yerine, Vero hücre hatlarında nötralizasyon analizinin uygulanabilirliğinin değerlendirilmesi amaçlanmıştır. Çalışmada, Bornova Veteriner Kontrol Enstitüsü Veteriner Biyolojik Ürünler Kontrol Laboratuvarına rutin satış izni kapsamında gönderilen farklı üreticilere ait 20 adet Clostridium septicum alfa toksoid içeren aşı ile 10 adet bu komponentten yoksun (clostridial veya clostridial olmayan) aşı kullanılmıştır. Her bir aşı için daha önce hastalık ve aşılama öyküsü bulunmayan New Zealand White ırkı 10 adet tavşan immünize edilmiş ve elde edilen serumlar eşit olarak bölünerek Vero hücre hatlarında nötralizasyon testi ve her serum için 15 adet fare kullanılarak TNT uygulanmıştır. TNT sonuçlarına göre, septicum içeren 20 aşının 16’sı limit geçer, 4’ü ise limit altında bulunmuş; septicum içermeyen aşıların serumlarında antitoksin oluşmadığından tüm fareler ölmüştür. Vero hücre nötralizasyon analizinde de TNT ile paralel sonuçlar elde edilmiş ve yöntemin TNT’ye göre sensitivite ve spesifitesi %100 olarak hesaplanmıştır. Sonuç olarak, Vero hücre nötralizasyon analizinin TNT ile uyumlu, daha az zaman ve maliyet gerektiren, stabil ve güvenilir bir alternatif yöntem olduğu ortaya konmuştur.

🔖

Cite This Article

Ayar, O., Erbaş, G. (2026). Determinatin of Potency in Clostridium septicum Vaccines by In Vitro Toxin Neutralization Test Using Vero Cells. Etlik Veteriner Mikrobiyoloji Dergisi, Cilt: 37 Sayı: 1, 17–24. https://doi.org/10.35864/evmd.1861616

📄 Full Text PDF Viewer

📄

PDF görüntüleyici hazırlanıyor...

References (27)

  1. Alves, M. L. F., Ferreira, M. R. A., Donassolo, R. A., Rodrigues, R. R., Conceição, F. R. (2021). Clostridium septicum: A review in the light of alpha-toxin and development of vaccines. Vaccine, 39(35), 4949–4956. doi:10.1016/j.vaccine.2021.07.019
  2. Arslan, A., Erbaş, G. (2023). Determining the Potency of Vaccines Containing Clostridium perfringens Epsilon Toxoid via Toxicity Analysis in MDCK Cell Lines. Etlik Veteriner Mikrobiyoloji Dergisi, 34(1), 59–66. https://doi.org/10.35864/evmd.1192288
  3. Borrmann, E., Schulze, F. (1999). Detection of Clostridium novyi type B α toxin by cell culture systems. FEMS Immunology and Medical Microbiology, 24(3), 275–280. https://doi.org/10.1111/j.1574-695x.1999.tb01294.x
  4. Borrmann, E., Schulze, F., Cussler, K., Hänel, I., Diller, R. (2006). Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin. Veterinary Microbiology, 114(1–2), 41–50. https://doi.org/10.1016/j.vetmic.2005.10.043
  5. Ebert, E., Oppling, V., Werner, E., Cussler, K. (1999). Development and prevalidation of two differentsystems fort he potency testing of Clostridium perfringens β and Ꜫ- toxoid containing veterinary vaccines, FEMS Immunology & Medical Microbiology. 24, 299-311. doi: 10.1111/j.1574-695X.1999.tb01298.x.
  6. European Pharmacopoeia (EP). (2022). Clostridium septicum vaccine for veterinary use (Monograpf 0364), Version 10.8, 6834-6836.
  7. Feoktistova, M., Geserick, P., & Leverkus, M. (2016). Crystal violet assay for determining viability of cultured cells. Cold Spring Harbor Protocols, 2016(4), 343–346. https://doi.org/10.1101/pdb.prot087379
  8. Gazioglu, A., Karagülle, B., Yüksel, H., Nuri Açlk, M., Keçeci, H., Dörtbudak, M. B., Çetinkaya, B. (2018). Sudden death due to gas gangrene caused by Clostridium septicum in goats. BMC Veterinary Research, 14(1), 1–6. https://doi.org/10.1186/s12917-018-1747-y
  9. Hartung, T. (2010). Comparative analysis of the revised directive 2010/63/EU for the protection of laboratory animals with its predecessor 86/609/EEC - A t4 report. Altex, 27(4), 285–303. https://doi.org/10.14573/altex.2010.4.285
  10. Huang, J., Zhao, B., Liu, T., Kang, L., Li, J., Guo, Z., Chen, M., Gao, S., Wang, J., Li, Y., Wang, J., Xin, W. (2023). Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases. International Journal of Molecular Sciences, 24(6). https://doi.org/10.3390/ijms24065414
  11. Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K., Okhura Y., Ueno, K., (1996). A Combined Assay of Cell Viability and in Vitro Cytotoxicity with a Highly Water- Soluble Tetrazolium Salt, Neutral Red and Crystal Violet. Biological and Pharmaceutical Bulletin 19(11) 1518-1520 Vol. 19, No. 11 doi: 10.1248/bpb.19.1518
  12. Jeong, C. G., Seo, B. J., Nazki, S., Jung, B. K., Khatun, A., Yang, M. S., Kim, S. C., Noh, S. H., Shin, J. H., Kim, B., and Kim, W. Il. (2020). Characterization of Clostridium novyi isolated from a sow in a sudden death case in Korea. BMC Veterinary Research, 16(1), 1–9. https://doi.org/10.1186/s12917-020-02349-9
  13. Kang, M., Long, T., Chang, C., Meng, T., Ma, H., Li, Z., Li, P., Chen, Y. (2022). A Review of the Ethical Use of Animals in Functional Experimental Research in China Based on the “four R” Principles of Reduction, Replacement, Refinement, and Responsibility. Medical Science Monitor, 28, 1–6. https://doi.org/10.12659/MSM.938807
  14. Kaushik, H., Deshmukh, S. K., Solanki, A. K., Bhatia, B., Tiwari, A., Garg, L. C. (2019). Immunization with recombinant fusion of LTB and linear epitope (40–62) of epsilon toxin elicits protective immune response against the epsilon toxin of Clostridium perfringens type D. AMB Express, 9(1). https://doi.org/10.1186/s13568-019-0824-3 Kocaçalışkan İ., Akanıl Bingöl, N. (2021). Biyoistatistik. N. Argun (Ed.) Korelasyon ve Regresyon Testi (4. bs., ss. 14- 21)
  15. Lima, C. G. R., Lobato, Z. I. P., Pires, P., Silva, R. O., Salvarani, F. M., Assis, R. A., and Lobato, F. C. F. (2011). Padronização de teste de Potência in vitro Para vacinas que contenham toxoide alfa de Clostridium novyi tipo B. Arquivos Do Instituto Biológico, 78(4), 507–512. https://doi.org/10.1590/1808-1657v78p5072011
  16. Redhead, K., Wood, K., Jackson, K. (2012). Testing of veterinary clostridial vaccines: From Mouse to microtitre plate, In: Potency testing of veterinary vaccines for animals: The way from in vivo to in vitro, Jungback C. (Ed.), Vol 134, KArger AG, Basel, Switzerland, 45-50
  17. Romberg, J., Lang, S., Balks, E., Kamphuis, E., Duchow, K., Loos, D., Rau, H., Motitschke, A., Jungbäck, C. (2012). Potency testing of veterinary vaccines: The way from invivo to invitro. Biologicals, 40(1), 100–106. https://doi.org/10.1016/j.biologicals.2011.10.004
  18. Salvarani, F. M., Lobato, Z. I. P., Assis, R. A., Lima, C. G. R. D., Silva, R. O. S., Pires, P. S., Lobato, F. C. F. (2010). In vitro evaluation of Clostridium septicum alpha toxoid. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 62(4), 778–783. https://doi.org/10.1590/s0102-093520100004000022
  19. Salvarini, F. M., Lobato, Z. I. P., Pires, P. S., Silva, R. O. S., Alves, G. G., Pereira, P. L. L., and Lobato, F. C. F. (2013). In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid. Arquivos Do Instituto Biológico, 80(4), 450–452. https://doi.org/10.1590/s1808-16572013000400013
  20. Sinitskaya, N., Behr-Gross, M.-E. Validation of alternative/3Rs methods for the in-process quality control of Clostridium septicum vaccines (BSP130 participants workshop report) Blood transfusion at Council of Europe View project Bıologıcal Standardısatıon Programme View project. 2015, Egmond aan Zee, Netherlands
  21. Souza Júnior, M. F., Lobato, Z. I. P., Pires, P. S., Silva, R. O. S., Salvarani, F. M., Assis, R. A. de, Lobato, F. C. F. (2010). Padronização da titulação da toxina épsilon de Clostridium perfringens tipo D em linhagem contínua de células como alternativa ao bioensaio animal. Ciência Rural, 40(3), 600–603. https://doi.org/10.1590/s0103-84782010005000024
  22. Sümbüloğlu, K., Sümbüloğlu, V. (2021). Biyoistatistik. M.Tahir Hatipoğlu (Ed.) Korelasyon ve Regresyon Analizi: Korelasyon Analizi içinde Ankara: Hatipoğlu Yayıncılık, (20. bs.,ss. 186-196).
  23. USDA-CVB. (2011). Potency testing Clostridium septicum antigen, BBPRO1301.03, Center for Veterinary Biologics, USDA Animal and Plant Health Inspection Services, Ames, 1-10 https://www.aphis.usda.gov/animal_health/vet_biologics/publications/BBPRO1301.pdf
  24. USDA-CVB. (2022). Potency Test for Clostridium septicum Alpha Antitoxin Using a Cell Assay, BBPRO1009.04, Center for Veterinary Biologics, USDA Animal and Plant Health Inspection Services, Ames, 1-8 https://www.aphis.usda.gov/animal_health/vet_biologics/publications/cvb-pro-0009.pdf
  25. Zaragoza, N. E., Orellana, C. A., Moonen, G. A., Moutafis, G., and Marcellin, E. (2019). Vaccine production to protect animals against pathogenic clostridia. Toxins, 11(9), 1–29. https://doi.org/10.3390/toxins11090525 WEB 1. (2023).: Deneysel Ve Diğer Bilimsel Amaçlarla Kullanılan Hayvanların Refah Ve Korunmasına Dair Kılavuz https://www.tarimorman.gov.tr/Belgeler/Mevzuat/Talimatlar/gkgm/deneysel_hayvan_koruma_kilavuz.pdff (08.06.2023)
  26. Borrmann, E., Schulze, F. (1999). Detection of Clostridium novyi type B α toxin by cell culture systems. FEMS Immunology and Medical Microbiology, 24(3), 275–280. https://doi.org/10.1111/j.1574-695x.1999.tb01294.x
  27. Zaragoza, N. E., Orellana, C. A., Moonen, G. A., Moutafis, G., and Marcellin, E. (2019). Vaccine production to protect animals against pathogenic clostridia. Toxins, 11(9), 1–29. https://doi.org/10.3390/toxins11090525 WEB 1. (2023).: Deneysel Ve Diğer Bilimsel Amaçlarla Kullanılan Hayvanların Refah Ve Korunmasına Dair Kılavuz https://www.tarimorman.gov.tr/Belgeler/Mevzuat/Talimatlar/gkgm/deneysel_hayvan_koruma_kilavuz.pdff (08.06.2023)