One of the reasons for failures of mastitis therapy and conventional vaccines is the ability of many mastitis causing microorganisms to grow in biofilms (BF) in mammary gland, thus developing an innate resistance to almost all therapeutic agents. Presently no true biofilm based mastitis vaccine is available. Our invention is a Biofilm Based Mastitis Vaccine against S.aureus and E.coli comprising of biofilm based inactivated bacterial culture along with adjuvant S.aureus and E.coli are the the major mastitis causing pathogens, we have grown them under the biofilm mode for the first time. We used Biofilm grown bacteria as vaccine candidate against mastitis causing organisms in bovines. The prevalence of inter cellular adhesion A (ica A) and Biofilm associated protein (Bap) genes among S. aureus isolates by PCR studies, suggested it"s role in biofilm production. Further, ERIC and Multiplex PCR differentiated E.coli strains. In vitro growth conditions were standardized for optimum production of bentonite clay based biofilms by S. aureus and E. coli. SDS-PAGE of Outer Membrane Proteins showed expression and repression of several polypeptides. Immunoblotting of BF and Free cell (FC) OMPs confirmed the cross reactivity among strains / serotypes. Vaccination trials in rabbits indicated the low values of mastitis indicators in both homologous and heterologous challenged groups among S.aureus and E.coli BF vaccinates. Significantly elevated levels of serum IgG and milk IgG & IgA levels in indirect ELISA were detected in BF vaccinated rabbits. Vaccination trials using bentonite clay based S.aureus and E.coli BF and FC vaccines in cattle in early lactation showed that in S.aureus and E.coli BF vaccinated cattle, significant enhancement in CD4 and CD8 cells by Flow cytometry and enhanced serum IgG levels by indirect ELISA were detected when compared to FC and control groups. The studies indicated the supremacy of the BF vaccine developed in the present study over FC vaccine.
