Study On The Immunologic Characteristics Of F17A-MF59Vaccine Against Escherichia Coli Bovine Mastitis

Escherichia coli (E. coli) is the most important environmental pathogen of bovine mastitis, which caused great economic losses on dairy industry. In this study, molecular epidemiology of E. coli from mastitis was investigated by the method of bacteria isolation and identification, and the results showed that70E. coli isolates (10.6%) were isolated from663mastitis samples which were collected from6dairy farms located in Beijing, Hebei and Tianjin province during the period of2012September to2013Octomber. E. coli can induce mastitis by colonizing on the mammary gland epithelial cells by its fimbria, multiplying in the mammary gland and releasing variety of toxins. For further explore the pathogenic factors of mastitis associated is. coli, phylogenetic group, virulence factors and antimicrobial resistance were detected by PCR method based on the previous studies. The results showed that the E. coli isolates from mastitis mainly belonged to group B1(58.6%) and group A (35.7%), and the most prevalent virulence genes were f17A (15.7%), irp2(14.3%), astA (10.0%), iucD (7.1%) and colV (5.7%), as well as virulence factors of the isolates in group B1distributed more widely than in group A. Additionally, the majority of isolates (87.1%) were resistant to at least one antimicrobial compound, especially to streptomycin, kanamycin and ampicillin, and those from group B1were more resistant to streptomycin than isolates from group A. This latter feature was supported by the distribution of streptomycin resistance genes observed in group B1compared to group A.F17fimbria is the most predominant fimbria in mastitis associated E. coli isolates, which was verified in the previous studies and account for preparation of F17fimbria subunit vaccine to prevent bovine mastits in our study. Firstly, f17A gene was amplified from E. coli strain labeled as E-BJ-1and inserted into pET28a vetor. Then the recombinant f17A-pET28a vetor was sequenced and transformed into E. coli BL21(DE3) competent cells. After induction with1mM IPTG, expression of F17A protein were significantly enhanced in the recombinant strains and the production was increased to a high level at4h with the induction time extension. SDS-PAGE showed that F17A exsited in the form of inclusion body and can be dissolved in the inclusions solution after lysing by ultrasonication. Finally, F17A protein of high quality was obtained after purification and renaturation.F17A-MF59subunit vaccine was prepared with F17A as antigen and MF59as adjuvant, and immunological traits of which was assessed. Safety experiments showed that the mice after inoculation behaved normally in clinical signs and pathological histology, which indicated that this vaccine was safe. Immunogenicity assay showed that the special antibody level of F17A in the F17A-MF59-vaccinated group was significantly higher than the single F17A-vaccinated group, which functioned with longer duration and mainly as IgG1and IgG2a. This declared that MF59adjuvant can not only enhance the humoral immune response effectively but also the cellular immune mediated by Th1cells. Survival test showed that survival rate of the mice in the vaccinated group was significantly higher than the non-vaccinated group, and the mice in the vaccinated group recovered more quickly. The result of bacteria clearance test and histopathology assessment showed that E.coli eradication rate of liver, spleen and kidney of the mice in the vaccinated group were also significantly higher than the non-vaccinated group regardless of the time or the account, especially in liver and spleen; as well as the histopathology changes displayed more slight in the vaccinated group.Classification of T lymphocytes showed that both of CD4+and CD8+T lymphocytes proportion was decreased in the non-vaccinated group, whereas CD4+T lymphocytes proportion was increased and CD8+decreased in the vaccinated group, comparing with the normal group. This may resulted from that immune system of the mice was damaged by the challenge with E. coli or the cellular immune function was suppressed of that E. coli mainly was extracellular pathogen. In general, F17A-MF59vaccine can effectively enhance the immune response against E. coli, especially humoral immunity, and this study confirmed the potential application values of F17A-MF59vaccine, which provide the theoretical and practical basis for preparation of mastitis vaccine in the future.