Investigation of the use of the ELISA procedure in determining the potency of Clostridium chauvoei vaccines

Yıl 2025, Cilt: 10 Sayı: 6, 883 - 888, 30.11.2025

Deha Ali Deniz , Şükrü Kırkan

https://doi.org/10.35229/jaes.1812152

Öz

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.

Anahtar Kelimeler

Clostridium , Blackleg , Elisa , Potency , Vaccine

Etik Beyan

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).

Proje Numarası

Aydın Adnan Menderes University Scientific Research Projects Coordination Unit (Project number: VTF-15066),

Teşekkür

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.

Clostridium chauvoei aşılarının potensinin Belirlenmesinde ELISA prosedürünün Kullanılmasının araştırılması

Öz

Clostridium chauvoei tarafından oluşturulan Yanıkara hastalığı, sığır ve küçük ruminantlarda hâlen ciddi kayıplara neden olan önemli bir enfeksiyondur. C. chauvoei aşılarının potentlik testleri günümüzde hâlâ kobaylarla yapılan canlı hayvan deneme testlerine dayanmaktadır. Bu çalışmada 350–450 g ağırlığında 300 Wistar Albino kobay kullanılmıştır. Referans yöntem olarak kabul edilmekle birlikte bu testler, yoğun iş gücü gerektiren, zaman alıcı ve hayvan refahı açısından sakıncalar içeren uygulamalardır. Bu çalışmanın amacı, inaktive edilmiş C. chauvoei aşılarının potentlik testlerinde, antijen temelli bir ELISA yönteminin hızlı, tekrarlanabilir ve hayvan dostu bir in vitro alternatif olarak kullanılıp kullanılamayacağını değerlendirmektir. Toplamda 20 ticari aşı (15’i C. chauvoei antijeni içeren, 5’i içermeyen) hem kobay deneme testi hem de flagellar antijene özgü ELISA ile değerlendirilmiştir. Her bir aşı için 10 kobay aşılanmış, 5 kobay kontrol grubunda tutulmuştur. Göreceli potens (RP) değeri standart referans aşıya göre hesaplanmıştır. Yöntemler arasındaki korelasyon, duyarlılık ve özgüllük istatistiksel olarak belirlenmiştir. Elde edilen sonuçlara göre geliştirilen ELISA, %93,3 duyarlılık, %100 özgüllük ve %95,0 göreceli doğruluk göstermiştir. Pozitif aşıların RP değerleri 1,02–5,44 arasında değişmiş ve in vivo sonuçlarla güçlü bir korelasyon (r = 0,882; p < 0,001) göstermiştir. İntra-plaka varyasyonu %10’un, inter-plaka varyasyonu %15’in altında bulunmuştur. Yanlış pozitif sonuçlara rastlanmamış; C. chauvoei antijeni içermeyen aşılar oldukça düşük RP değerleri vermiştir. Sonuç olarak geliştirilen ELISA yöntemi, hayvan denemelerine güvenilir bir alternatif sunmakta; test süresini ve hayvan kullanımını önemli ölçüde azaltırken doğruluk düzeyini korumaktadır. Bu yaklaşım, 3R ilkeleri (Replacement, Reduction, Refinement) ile uyumlu olup, aşı kalite kontrol süreçlerinin verimliliğini artırma potansiyeline sahiptir.

Anahtar Kelimeler

Aşı , Clostridium , ELİSA , Potens , Yanıkara.

Proje Numarası

Aydın Adnan Menderes University Scientific Research Projects Coordination Unit (Project number: VTF-15066),

Kaynakça

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