Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines

Abstract

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

Keywords:

• Brucella abortus
• real-time PCR
• potency testing
• eryC gene

Ethical approval

Animal experiments were conducted in acquiescence with regulations set by Institutional Animal Ethics Committee.

Acknowledgments

The authors highly acknowledge essential facilities provided by ICAR-Indian Veterinary Research Institute, Izatnagar to carry out this research work. This work was a part of M.V. Sc. thesis of first author.

Disclosure statement

No potential conflict of interest was reported by the author(s).