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

Fereshteh Yavari,Seyed Ali Pourbakhsh,Ramezan Ali Khavarinejad,Hossein Goudarzi

doi:10.5539/jmbr.v8n1p61

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.

Highlights

Mycoplasma agalactiae is the cause of small ruminant syndrome that infects he main target organs of animals, including mammary glands, joints, respiratory system and eyes (Bergonier and Berthelo,1997)
To assess the species specificity of P40 protein, the results were confirmed by two primers which amplified the 920 bp fragment of p40 gene from the genome of Mycoplasma agalactiae strains
The p40 gene of Mycoplasma agalactiae was chosen for work because based on recent reports, its expression level increases in inflammation and immune defense and it is present in all Mycoplasma agalactiae strains, implying that p40 expression is specific to Mycoplasma agalactiae species. 19 samples from 11 provinces of Iran were positive for contagious agalactia infection

Summary

Introduction

Mycoplasma agalactiae is the cause of small ruminant syndrome that infects he main target organs of animals, including mammary glands, joints, respiratory system and eyes (Bergonier and Berthelo,1997). Agalactia, which is induced by other mastitis-causing Mollicutes like Mycoplasma putrefaciens, Mycoplasma mycoides and Mycoplasma capricolum (Nouvel, 2010), has been reported in the Mediterranean countries of Asia Minor like Iran, Iraq, the United Arab Emirates, Afghanistan and Pakistan with serious economic losses in shepherding due to the reduction in milk, cheese production and mortality of lambs. Contagious agalactia has been observed in different continents, the major foci of stable and long-standing infection in goats has been found in Iran, Spain, Switzerland, Iraq, France and Italy (Bergonier and Berthelo, 1997). Many surface lipoproteins including P30, P40, P80, P55, Vpma, P48, etc. Genomic and proteomic analyses have provided powerful immunological insights into a few proteins of this pathogen (including P40, Vpma and P48) as suitable agents for determination kits or detection reports.

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Conclusion