Clostridium perfringens Epsilon Toksoid İçeren Aşılarda MDCK Hücre Hatlarında Toksisite Analizi ile Potensin Belirlenmesi

Yıl 2023, Cilt: 34 Sayı: 1, 59 - 66, 20.06.2023

Ahmet Arslan , Göksel Erbaş

https://doi.org/10.35864/evmd.1192288

Öz

Epsilon toksoid içeren klostridium aşılarının potens kontrolü Toksin Nötralizasyon Testi (TNT) ile yapılmaktadır. TNT standart bir metot olmakla birlikte hayvan refahı ve metot doğrulama çalışmaları açısından dezavantajları olan bir testtir. Bu nedenle alternatif metotların geliştirilmesi ve uygulanması teşvik edilmektedir. Ancak serolojik tabanlı in vitro metotlarda antijen, antikor ve referans standartların saflaştırılması ve standardizasyonu halen önemli bir sorun olarak durmaktadır. Bu bakımdan benzer reaktifler ile gerçekleştirilen hücre kültürü analizleri klostridial aşıların potens testlerinde olası alternatifler içinde görülmektedir. Bu yaklaşımla aşılanan tavşan serumlarında epsilon antitoksin düzeyi fare TNT ve Madin-Darby Canine Kidney (MDCK) hücre kültürü sitotoksisite analizi ile belirlenmiştir. Hücre kültüründeki toksisite yanıtı farelerdeki yanıta benzerdir. İki metodun sonuçları arasında yüksek bir korelasyon ve iyi bir lineer ilişki gözlenmiştir. Non-spesifik reaksiyon olmamıştır. Bu sonuçlar Clostridium perfringens epsilon toksoid içeren aşıların potens testlerinde, in vivo fare TNT yerine, in vitro MDCK hücre hattı seronötralizasyon analizinin başarıyla kullanılabileceğini göstermektedir.

Anahtar Kelimeler

Klostrial aşı, Epsilon toksin, MDCK, Potens, TNT

Destekleyen Kurum

Aydın Adnan Menderes Üniversitesi Bilimsel Araştırma Projeleri Birimi

Proje Numarası

VTF - 17050

Teşekkür

Araştırmanın yürütülmesinde katkı sağlayan Bornova Veteriner Kontrol Enstitüsü Müdürlüğü ve Aydın Adnan Menderes Üniversitesi Veteriner Fakültesi Mikrobiyoloji ABD’na teşekkür ederiz.

Determining the Potency of Vaccines Containing Clostridium perfringens Epsilon Toxoid via Toxicity Analysis in MDCK Cell Lines

Öz

The potency control of the clostridium vaccines containing epsilon toxoid is performed with the Toxin Neutralization Test (TNT). Although TNT is a standard method, it has some disadvantages in terms of animal welfare and method validation studies. For this reason, the development and application of alternative methods are encouraged. However, purification and standardization of antigen, antibody and reference standards in serological-based in vitro methods still remain a significant problem. In this respect, cell culture analyses performed using similar reagents are considered possible alternatives in potency tests of the clostridial vaccines. Based on this approach, epsilon antitoxin levels in the sera of vaccinated rabbits were determined by cytotoxicity assay in mouse TNT and Madin-Darby Canine Kidney (MDCK) cell cultures. The toxicity response in cell culture is similar to the response in mice. A high correlation and a good linear relationship were observed in the results of the two methods. There were no non-specific reactions. These results show that, in potency tests of vaccines containing Clostridium perfringens epsilon toxoid, in vitro MDCK cell line seroneutralization assay can be successfully used instead of in vivo mouse TNT.

Anahtar Kelimeler

Clostridial vaccine, Epsilon toxin, MDCK, Potency, TNT

Proje Numarası

VTF - 17050

Kaynakça

• Arslan A, Dilik Z, Özyer M, Oktay N, Yılmaz Ş. Klostridial aşıların potensinin belirlenmesinde toksin nötralizasyon testlerine alternatif olarak ELISA’nın kullanımı, TAGEM Program Değerlendirme Toplantısı, 15-19 Şubat 2016, Antalya.
• Bonistalli KN (2013). Monoclonal antibody production: A comparision of in vitro and in vivo methods and their use in clostridial vaccine manufacture, MSc, Institute of Veterinary, Animal and Biomedical Sciences, Veterinary Medicine at Massey University, Palmerston North, New Zealand.
• Borrmann E, Günther H, Köhler H (2001). Effect of Clostridium perfringens epsilon toxin on MDCK cells. FEMS Immunology and Medical Microbiology, 31, 85-92.
• Borrmann E, Schulze F, Cussler K, Hanel I, Diller R (2006). Development of a cell culture assay fort he quantitative determination of vaccination induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin. Veterinary Microbiology, 114, 41-50.
• Code of Federal Regulation (CFR) (2019). Clostridium perfringens type D toxoid and bacterin-toxoid, Section 113.112, Title 9:Animal and Animal Products (1-1-21 edition), U.S. Government Publishing Office, p 751-752.
• Council of Europe (EDQM) (2022a) Clostridium perfringens vaccine for veterinary use, Monograph 0363, European Pharmacopoeia Version 11, EDQM Publication, France p 1153-1155.
• Council of Europe (EDQM) (2022b). Vaccines for veterinary use, Monograph 0062, European Pharmacopoeia Version 11, EDQM Publication, France 955-959.
• Council of Europe (EDQM) (2022c). Substitution of in vivo methods by in vitro methods for the quality control of vaccines, Monograph 5.2.14, European Pharmacopoeia Version 11, EDQM Publication, France, 716-718.
• Crowthwer JR (2009). The ELISA Guidebook, Second Edition, Humana Press, New York, 2009, 566.
• Erbaş G (2011). Veteriner aşı ve biyolojik maddelerin kontrollerinde alternatif metotlar, Fırat Üniversitesi Sağlık Bilimleri Veteriner Dergisi, 25(3), 141-146.
• Hill RE (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 in Vaccinology 5, 141-145.
• Kulpa-Eddy J, Srinivas G, Halder M, Hill R, Brown K, Roth J, Draayer H, Galvin J, Claassen I, Gilford G, Woodland R, Doelling V, Jones B, Stokes WS (2011). Non-animal replacement methods for veterinary vaccine potency testing: state of the science and future directions, Procedia in Vaccinology 5, 60-83.
• Lang C, Kolaj-Robin O, CireficeG, Toconet L, Pel E, Jouette S, Buda M, Milne C, Charton E (2018). Replacement, reduction, refiment. animal welfare progress in European Pharmacopoeia Commission from 2007 to 2017, Pharmeuropa Bio&SN, 12-36
• Navarro MA, McClane BA, Uzal FA (2018). Mechanisms of action and cell death associated with Clostridium perfringens toxins. Toxins 10, 212, 1-21.
• Petit L, Gibert M, Gillet D, Laurent Winter C, Boquet P, Popoff MR (1997). Clostridium perfringens epsilon toxin acts on MDCK cells by forming a large membran complex. Journal of Bacteriology, 6480-6487.
• Prescott JF (2013). Clostridium. McVey DS, Kennedy M, Chengappa MM eds. Veterinary Microbiology (3rd edition), Wiley Blackwell, Danvers, p 245-262.
• Redhead K, Wood K, Jackson K (2011). Testing of veterinary clostridial vaccines: From mouse to microtitre plate, Jungback C. eds, Potency testing of veterinary vaccines for animals: The way from in vivo to in vitro, Vol 134, Karger AG, Basel, Switzerland, p 45-50.
• 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 in vivo to in vitro, Biologicals 40, 100-106.
• Salvarini FM, Lobato ZIP, Assis RA, Silva ROS, Pires PS, Lobato FCF (2010). In vitro evaluation of Clostridium septicum alpha toxoids, Arquivo Brasileiro de Medicina Veterinaria e Zootecnia 62(4), 778-783.
• Salvarini FM, Lobato ZIP, Pires PS, Silva ROS, Alves GG, Pareira PLL, Lobato FCF (2013). In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid. Arquivos do Instituto Biológico, 80 (4), 450-452.
• Sinitskaya N, Redhead K, Daas A, Bruckner I, Behr-Gross E (2015). Validation of alternative/3Rs methods for the in-process quality control of Clostridium septicum vaccines, BSP130 participants workshop report, Egmond aan Zee, Netherlands, 107.
• Soler Jover A. Blasi J, Aranda IG, Navarro P, Gibert M, Popoff MR, Martin Satue M (2004). Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo. Journal of Histochemistry and Cytochemistry, 52 (7), 931-942.
• Souza Junior MF, Lobato ZIP, Pires PS, Silva ROS, Salvarini FM, Assis RA, Lobato FCF (2010). Standardization of the titration of the epsilon toxin of Clostridiumj perfringens type D in cell line as an alternative to animal bioassay, Ciencia Rural, 40 (3), 600-603.
• Stiles BG, Barth G, Barth H, Popoff MR (2013). Clostridium perfringens epsilon toxin: A malevolent molecule for animals and man. Toxins 5, 2138-2160.
• Stokes WS, Brown K, Kulpa Eddy J, Srinivas G, Halder M, Draayer H, Galvin J, Claassen I, Gifford G, Woodland R, Doelling V, Jones B (2011). Improving animal welfare and reducing animal use for veterinary vaccine potency testing: state of the science and future directions, Procedia in Vaccinology 5, 84 – 105.
• United States Department of Agriculture (USDA) (2016): Potency test for Clostridium perfringens type D epsilon antitoxin using a cell assay testing protocol, BBPRO1008.03, Center for Veterinary Biologics, USDA Animal and Plant Health Inspection Service, Ames, IA, 1-8
• Uzal FA, Vidal JE, McClane B.A, Gurjar AA (2014). Clostridium perfringens toxin involved in mammalian veterinary disease. The Open Toxicology Journal 2, 24-42.
• Waritani T, Chang J, McKinney B, Terato K (2017). An ELISA protocol to improve the accuracy and reliability of serological antibody assays, MethodsX 2017, 4, 153–165.