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Objective: To compared seed culture of hemorrhagic septicemia (HS) bacteria which was used to produce vaccine for its antibody induction efficiency before and after passaging in natural host (calf) using laboratory animals. Methods: Serial dilution of virulent bacteria was injected in to mice which were immunized with HS vaccine which was obtained from seed bacteria before and after back passaged in calf. Ratio of survived and dead was calculated by Reed-Meunch hypothesis and the LD50 value for each vaccine trial groups were calculated. Results: The immunological study revealed that vaccine prepared from back passaged seed culture showed greater improvement in its immunopotency than seed vaccine (before back passage). Around 200 mice were used to study the immuno efficiency of vaccine. Each mouse was from the same source, which were free from the Pastuerella infection previous to expose to trial infection. The same broth culture of HS was used to induce infection in mice in both trials (vaccine before back passage and vaccine after back passage). The 0.2 mL of broth dilution from 10−1 to 10−10 was used, as dilution increases, death rate decreases. It indicates the minimum load of bacterium is required to induced infection. Conclusions: Obtained results revealed that back passaged vaccine seed HS bacteria in its natural host had provided better immune efficiency to the culture than laboratory stock culture, and this findings recommended that regular annual back passage was mandatory for the vaccine seed culture of Pastuerella multocida bacteria for better establishment of immune potent vaccines.