Development of rp48d -ELISA and evaluation of its performance for detection of specific mycoplasma agalactiae antibody in vaccinated sheep

Abstract Contagious agalactiae is a disease affecting small ruminants, caused by the bacterium Mycoplasma agalactiae. It is characterized by mastitis, polyarthritis, and keratoconjunctivitis. In regions heavily reliant on animal husbandry, this disease can result in substantial economic losses. Effective prevention methods include vaccination and accurate disease diagnosis in sheep and goats. The Surface lipoprotein P48 of M. agalactiae is constantly expressed and considered an immunodominant antigen present in all isolates. Antigenic variation is the primary cause of sensitivity differences in the p48-based serum assay. Recent studies have revealed fundamental differences in the p48 protein of Iranian isolates compared to previous ones. Therefore, rp48d protein was expressed in Escherichia coli BL21 (DE3) cells and purified using Ni-resin through an affinity batch formation method. Experimental sheep were divided into four groups for immunization: rp48d, RVSRI agalactiae vaccine, and two control groups (adjuvant and non-vaccinated control). Additionally, one hundred blood serum samples taken from vaccinated and non-vaccinated sheep (Fashandi breed) from various herds in the Taleghan region were analyzed using indirect ELISA. The results demonstrated that 97.50% and 100% sensitivity and specificity, respectively in rp48d-ELISA. Based on our findings, we can conclude that the in-house rp48d-ELISA is highly effective in detecting specific antibodies in vaccinated or infected sheep. This is due to the significant protein similarity between the structure of the rp48d protein utilized in the design of the ELISA test and mycoplasma agalactiae circulating in the region and used in vaccine production.