Preparation of Freeze-Dried Clostridium Chauvoei Master Seed Bank from Local Isolate for Production of Safe, Effective and Acceptable Quality of Black Leg Vaccine
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Date
2021
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Addis Ababa University
Abstract
The research was accompanied from December 2020 to May 2021 amid to develop new preservation method spore form of Cl. Chauvoei from local isolate with the objective of mainly improving vaccine immunogenicity. Currently, in National Veterinary Institute (NVI) of Ethiopia viande-foie (V.F) medium is the sole medium used for preservation of the strain as a vaccine master seed bank. However, this method of preservation has its own drawbacks like low antigenicity maintenance, prone to contamination and difficulty in transportation. Intended for this purpose, samples were composed from Adea district from cattle following outbreak information of black leg disease. The samples were taken to NVI using cold chain system for isolation purpose. The blood and tissue samples were isolated as Cl. Chauvoei. The isolates were identified by cultural, morphological and biochemical features. With the aim of additional confirmation by means of pathogenicity study and acuity estimation, the isolates were inoculated on gunia pigs and up on postmortem examination it was confirmed as Cl. Chauvoei. The study of PCR also revealed the existence of Cl. Chauvoei. New method of preservation was then developed through lyophilization or freeze-drying of the isolated strain. Quality control tests like vacuum, moister content, viability, sterility, safety, lethal dose determination for potency test, and identity tests of freeze-dried spore form of Cl. Chauvoei seed bank were performed. Accordingly, the quality control tests of the freeze-dried product revealed; vacuum (1.5%), moister content (97.5%), LD50/ML (1 x 10 7.2), known to be viable, safe and free of contaminant. Regarding potency test 10 gunia pigs were challenged with LD50 dose of virulent challenge strain, and only one of them was dead, thus it is 90 % protective. It is believed that freeze-dried spore stored at -20 0 C retain viability for years, however in this specific study stability test at different storage temperatures for long period of time is essential to conclude this idea. Moreover, study reinforced by DNA sequencing is also indispensable to finalize these inferences.
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Keywords
Freeze-drying, Glyceration, Preservation, Vaccine