Development of inactivated mycoplasma gallisepticum vaccine in chickens

Mycoplasma gallisepticum (MG) is the respiratory pathogen causing chronic respiratory disease (CRD) in poultry. More importantly, MG infection affects the economic losses of poultry industry due to decrease egg production and carcass quality. There are several procedures for MG monitoring and several commercial vaccines to prevent and control the MG infection. In Thailand, MG infection has been the one of major problems of poultry industry. The objectives of this study were to investigate the different molecular techniques for MG monitoring in the poultry farms and to prepare the inactivated vaccine or bacterin with Thai MG strain. There were 3 experiments to accomplish in this study. The first experiment; the investigation and comparison of the virulent mgc2 gene of MG outbreak in Thailand and in various countries were carried out. Targeted partial mgc2 gene of 17 Thai MG strains were analyzed with 2 molecular techniques including random amplified polymorphic DNA (RAPD) and gene-targeted sequencing (GTS) assay. The results showed that RAPD and GTS assay could classify Thai MG strains into 3 and 4 groups, respectively. In addition, the phylogenetic tree which conducted from partial mgc2 gene sequence showed that 11 Thai MG strains did not distinguish from Indian MG strains and Israel MG strain. The other studies; the bacterin preparing from Thai MG strain AHRL 20/52 and chitosan served as an adjuvant was determined against Thai MG strain AHRL 58/46. This study consisted of 2 experiments (the second and third experiment) to determine the safety and efficacy of chitosan-adjuvanted MG bacterin comparing with those of commercial MG bacterin. The second experiment was to investigate the local reaction at injection site, antibody responses, the histopathological tracheal lesion score and gross thoracic air sac lesion score. Chitosan-adjuvanted MG bacterin were prepared with different concentration of chitosan (0.25, 0.5 and 1%) and administered via intramuscular injection. The third experiment; chitosan-adjuvanted MG bacterin was determined based on the routes of vaccine administration including intraocular and/or intramuscular routes, and vaccine program. The serology, quantitative real-time PCR assay, and air sac and tracheal lesion scores were used to evaluate this experiment. The results showed that chitosan-adjuvanted MG bacterin caused milder tissue reaction at injection site than the commercial MG bacterin and provided the significantly effective protection on tracheal lesion (P< 0.05). In addition, birds vaccinated with either Chitosan-adjuvanted vaccine (IM) at 6 and 10 weeks of age or bird vaccinated with Chitosan-adjuvanted vaccine (IO) at 6 weeks of age and 10 weeks of age had the significantly lower mean tracheal lesion score than positive control group (P< 0.05). In addition, the commercial bacterin administered by intramuscular route followed by the chitosan adjuvant bacterin administered by intraocular route showed the best protection against the MG challenge. These results provided the interesting molecular technique and the potential adjuvant for preparation of MG bacterin. These data provided the useful knowledge to improve the monitoring, surveillance and protection program of MG in poultry industry in Thailand.