| Currently there is no yet satisfactory commercial bovine mastitis vaccine against Staphylococcus aureus(S.aureus).As recombinant protein derived from S.aureus adhesins(Fibronectin-binding proteins B and clumping factors A,FnBPB-ClfA)induced more robust systemic and mammary gland localized humoral response after its fusion with Fc fragment of bovine IgG(Fc-FnBPB-ClfA).So present research aimed to testify whether improved efficiency of antigen transport would happen with FcRn,and to evaluate the immune effects of Fc-FnBPB-ClfA based vaccine on cows.The parallel aim was to develop a method based on differential chromogenic media for monitoring the immune effectiveness of vaccination,which would be simple with convenient operation compared with traditional methods.1.Test for antigen transport efficiency for S.aureus subunit vaccine:in vitro transcytosis assay used polarized primary bovine mammary epithelial cells(pbMECs)that were cultivated on porous membrane inserts to form natural epithelial barrier with integrity.Recombinant protein was added into the upper chamber with or without whether inhibitor of transcytosis pathway or staphylococal Protein A.Alteration of intracellular localization and transcellular transport of FnBPB-ClfA or Fc-FnBPB-ClfA caused by different tempretures,interfering Fc-FnBPB-ClfA’s interaction with the FcRn using staphylococal Protein A and inhibitor of common transcytosis pathway were assessed by Western blot analyses with samples from the lower chamber.The results showed that the uptake and transcellular transport of both two recombinant proteins were associated with FcRn-mediated transcytosis,and FcRn enhanced transcellular transport efficiency of Fc-FnBPB-ClfA.2.The development of differential chromogenic media:First,pathogenic bacteria in mastitis milk were collected including 13 genera attributing to 43 species.They were divided into three groups for identification,and bioinformatical database IMG together with metabolic pathways and Pathcomp in KEGG were used to screen intragroup conserved and specific enzymes and metabolizable carbon sources of different species or genus of bacteria within one group.Second,synthesized chromogenic substrate of specific enzyme,carbon source,acid-base indicator and other basic compositions provided by KOMODO database were sterilized to prepare differential chromogenic media,and standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media,and color changes of colonies and medium matrix were evaluated.Third,Plackets-Burman tests were used to find out which composition has significant effect on growth of bacteria,and BOX-Behnken Designs of Surface Response were used to optimize compositions of media.Fourth,milk samples from mastitic quarters were cultured aerobically at 37“C to identify bacteria using differential chromogenic media and traditional methods.The colony color and color change of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media were morphologically distinct and visible to the naked eyes,showing highly consistent results referring to traditional identifications.3.Test for enhancement of immune effects on cows by S.aureus subunit vaccine:5 healthy Holstein cows without mastitis were marked as control group treated with equavilent physiological saline,and another 10 healthy Holstein cows were divided into two equivalent groups that were respectively immunized with recombinant protein of Fc-FnBPB-ClfA or FnBPB-ClfA without adjuvant.There is only one time of vaccination conducted via teat canal perfusion and milking was conducted 6 h after vaccination.Sera samples and milk samples at 0 d and 14 d were collected to test specific antibody titer via indirect Enzyme-Linked Immuno Sorbent Assay(ELISA).The results showed that the specific antibody level in sera or whey from Fc-FnBPB-ClfA-vaccinated group was significantly higher than that from FnBPB-ClfA-vaccinated group(P<0.001).4.Evaluation for immune effectiveness of S.aureus subunit vaccine:60 Holstein cows were randomized sampled and divided into two groups,vaccinated group were immunized with vaccine of Fc-FnBPB-ClfA with levamisole hydrochloride as adjuvant,the primary vaccination was conducted via muscle injection and the booster vaccination was conducted via teat canal perfusion.And control group was treated with equavilent physiological saline.Sera samples and milk samples at 0 d,14 d,28 d,60 d were collected,and specific antibody titer was tested via indirect ELISA,and somatic cell count(SCC)in milk was tested using somatic cell counter,and bacteria isolation and identification was conducted using differential chromogenic media.Results showed that specific antibody levels in sera and whey were higher than that of control group(P<0.001)since 14 d after primary vaccination,and peaked at 28 d separately for 1:8192 and 1:1024.Compared with control group,vaccinated group showed no additional staphylococcal infection and mean SCC level in milk did not decrease.The validity,reliability and practicability of differential chromogenic media for identifying pathogenic bacteria in mastitis milk after vaccination were quite well.In conclusion,FcRn in pbMECs is involved in enhancing transcytosis efficiency of Fc fusion antigen protein of S.aureus subunit vaccine,which furtherly induces more significantly robust systemic humoral response and mammary gland localized humoral response.Fc-FnBPB-ClfA emulsified with levamisole hydrochloride shows great potential in exerting great immune effectivenss with significantly more robust systemic humoral response and mammary gland localized humoral response of cows.Differential chromogenic media,being simple and direct-viewing,shows pretty well validity,reliability and practicability as a bacteriological evaluation method for immune effectiveness before and after vaccination. |
PDF Full Text Request