Evaluation of room temperature (30°C to 35°C) lyophilized vaccine with radio inactivated Mannheimia haemolytica whole cells isolated from infected sheep

Abstract

Background and Aim: Vaccines are one of the important tools for fighting diseases and limiting their spread. The development of vaccines with high efficacy against diseases is essential. Ionizing radiation is the method used for the preparation of the irradiated gamma Mannheimia haemolytica vaccine. The study aimed to measure the metabolic activity and electron microscopic examination of the irradiated bacterial cells and immunological efficiency of different preparations of the irradiated M. haemolytica vaccine. Materials and Methods: The irradiated vaccines were prepared in three forms at a dose of 2×109 colony-forming unit (CFU) (irradiated M. haemolytica, trehalose irradiated M. haemolytica, and trehalose lyophilized irradiated M. haemolytica). The formalin-killed vaccine was prepared at a dose of 2×109 CFU. Scanning electron microscopy was used to determine the difference between the non-irradiated bacterial cells and the bacterial cells exposed to gamma radiation. The metabolic activity of the irradiated bacterial cells was measured using the Alamar blue technique. Rabbits were divided into five groups (control, vaccinated groups with the formalin-killed vaccine, irradiated bacterial cells without trehalose, trehalose irradiated bacteria, and trehalose lyophilized irradiated bacterial cells). The rabbits were subcutaneously inoculated twice in 2-week intervals. Enzyme-linked immunosorbent assay, interferon-gamma (IFNγ), and interleukin 4 (IL4) assays were used to evaluate the vaccines' immunological efficiency in rabbits. Results: The metabolic activity tests showed that the bacterial cells exposed to gamma radiation at the lowest lethal dose have metabolic activity. The difference in the metabolic activity between preparations of the irradiated bacterial cells varied according to the cell concentration and incubation time. The highest level of metabolic activity was 8 h after incubation in the nutrient broth medium compared with 4 and 18 h. The scanning electron microscopy of irradiated bacterial cells showed a cavity at the bacterial cell center without rupture of the surrounding cell membrane compared to the non-irradiated bacterial cells. The antibody level in the groups vaccinated with the different preparations of the irradiated bacterial cells was high compared with the control and formalin-killed vaccine groups. The level of the IFNγ showed an increase after the second dose in the group vaccinated with irradiated bacterial cells without trehalose compared with the other groups. The IL4 level in the vaccinated groups with the irradiated bacterial cells without trehalose, irradiated bacterial cells with trehalose, and trehalose lyophilized irradiated bacterial cells were at a high level when compared with the formalin-killed vaccinated group and control group after the second inoculation. Conclusion: The irradiated M. haemolytica vaccine provides a wide range of humoral and cellular immunity. This study showed high immunological efficiency in rabbits inoculated with the irradiated M. haemolytica vaccine that was shown in the high levels of antibodies (IFNγ and IL4) compared with the group treated with the formalin-killed vaccine. The second dose of irradiated M. haemolytica vaccine is an immune booster that gives the irradiated vaccine a long-acting immunological efficiency.