Isolation And Identification Of Bovine Mastitis Pathogenic Bacteria And The Study On The Antimicrobial Activity Of BNBD4 And BNBD5

Bovine mammitis is one of the most cost diseases in dairy industry,which could be caused by physical factors,chemical factors or microorganisms.The pathogens include Streptococcus agalaciae,Staphylococcus aurous and Escherichia coli etc.So far,no effective methods have been found to prevent and cure bovine mammitis.?-defensins are antimicrobial peptides with positive charge.They have antibacterial activity not only by regulating the immunity system but also by disrupting the bacterial membrane directly.Moreover,drug resistance can not occur during the process.Mature bovine neutrophil ?-defensins 4 and 5(mBNBD4 and 5)are the mature peptides involved in the inflamation reactions in animals.Therefore,in this research,we isolated and identified the pathogenic bacterium from 159 milk samples obtained from the cows with bovine mammitis in Hebei Province,and expressed and purified mBNBD4 and 5 peptides by utilizing the pichia pastoris strain with recombinant plasmids mBNBD4-pPIC9 K,mBNBD5-pPIC9 K constructed in previous work.We used the purified ?-defensins to incubate with the characterized pathogenic bacterial and detect the antibacterial activity in vitro.The goals of the research are to do the epidemiological investigation for the pathogens of bovine mammitis in Hebei province and to further explore the application potential of the ?-defensins mBNBD4 and 5.Isolation and identification of bovine mastitis pathogenic bacterium.In total,159 milk samples were obtained from the the caws with bovine mammitis in Hebei Province.Different microbiological and molecular biological methods were used to grow,isolate and identify the bacterium including morphological test,biochemical analysis and PCR detection.As result,96 pathogenic bacterial strain among the three main classes Streptococcus agalaciae,Staphylococcus aurous and Escherichia coli were identified.Among them,56 Streptococcus agalaciae strains were detected.50 of them were ?-hemolytic as pathogenic type accounting for 89.3%.6 of them were ?-hemolytic type according for 10.7%.Besides that,23 strains were Staphylococcus aurous,and 17 strains were Escherichia coli.The percentage of Streptococcus agalaciae,Staphylococcus aurous and Escherichia coli was 58.3%,24.0%,17.7%,respectively.Anti-bacterial activity test of ?-defensins.Recombinant mBNBD4 and 5 peptides were induced,expressed and purified from the pichia pastoris strain with plasmids mBNBD4-pPIC9 K,mBNBD5-pPIC9 K.The antibacterial activity of the purified mBNBD4 and 5 were detected.In this work,the isolated Staphylococcus aurous and Escherichia coli were co-inculated with the purified peptides with 3 different concentrations on the agar plate.The concentrations were used as 5 ?g /mL,10 ?g /mL and 20 ?g /mL.The colony-forming unit(CFU)was calculated and analyzed.The result implied that both mBNBD4 and mBNBD5 with 20 ?g /mL showed significant antibacterial activity to either Staphylococcus aurous or Escherichia coli.Moreover,the antibacterial activity was positive correlated with the peptides concentration in the certain range.The activity to inhibit the Staphylococcus aurous was higher than that to the Escherichia coli.This research provides theoretical base to prevent and treat bovine mastitis.