Comparative Study of p40 Gene Expression of Mycoplasma Agalactiae Isolated from Iranian Provinces

Abstract

Mycoplasma agalactiae pathogen is the main cause of agalactia in small ruminants and as such, results in some economic losses in Iran. Contagious agalactia disease should be regarded as a syndrome, caused by various Mycoplasmas which infect several organs. In the present study, 30 suspected samples from 11 provinces of Iran were isolated from mammary gland, joint and eyes of sheep and goats and subjected to genus and species detection via PCR technique for 2 genes. These Mycoplasma strains and three Iranian vaccinal strains of Mycoplasma agalactiae were submitted to sequence analysis of a surface lipoprotein called p40 protein. Based on a comparative study between Iranian strains and PG2 Spanish strain of Mycoplasma agalactiae, most Iranian strains presented 97% homology, whereas some strains showed 80-88% and three vaccinal strains were associated with 99% homology. According to PCR and bioinformatics analysis outcomes, 6 provinces of Iran were recognized as infection areas with different expressions of this effecter protein, suggesting that finding the individual characteristics of a particular effecter may require empirical testing for vaccination. Finally, the selected sequence of p40 gene was cloned into pGEMB1cloning vector and subsequently expressed in Escherichia coli by pET-22b+ expression plasmid under the control of the T7 promoter. The expression of this fusion protein was absorbed and confirmed by SDS-PAGE. The recombinant P40 protein was expressed with a molecular mass of 37 kDa on SDS-PAGE. The sera taken from rabbits infected with Mycoplasma agalactiae produced polyclonal antibody which was then used for westernblotting. The rabbits were bled 10 days after the booster immunization using cardiac puncture. Significantly, the use of recombinant specific antigens instead of other tools for diagnosis of a disease could improve the discrimination and separation of positive and negative animals in the area under investigation and therefore, it can be applied to control infected animals and reduce economic losses.