Bacteria Investigation And Minimal Inhibition Concentration To Lysozyme On Bovine Mastitis In Three Conties In Chongqing

A total of 22,000 cows feed at Chongqing in 2006,which produced 9,000-10,000 tons of milk. Bovine mastitis is a serious disease in the dairy farm,caused the economic loss over 10 million RMB each year in Chongqing.Bacterium was mainly pathogen of Bovine mastitis.Penicillin and others antibiotics were widely used to treat the disease and residue in milk,which made the milk to be discarded.So,it is very important work to control bovine mastitis that study new drug without residues in the milk.The cows feededin three counties at Chongqing were investigated and the bacterium isolated from the milk sample of the cow tested was identified and detected by arbitrary primers PCR(AR-PCR)and mimal inhibition concentration test to lysozyme in the thesis.1.A total of 108 milk samples were collected in Hechuan,Rongchang and Yubei since September 2006 to June 2007 and detected using the Beijing mastitis test(BMT)for somatic cell in samples.The positive rate was 70%(21/30),65%(31/48)and 40%(13/30)respectively in the three counties.2.48 strains of bacteria were isolated from the 65 positive milk samples,and classified to Staphylococci 40 strains(83%),E.coli,6 strains(12.5%)and Streptococcus,2 strains(4.2%)by battery of biochemical reactions.All the bacteria isolated from the milk were sensitive to cephalosporins antibiotics,hoever,resisitance to Penicillinum and Ampicilli in vitro.3.40 strains of Staphylococci isolated from the milk coμL be divided three groups in morphology,five groups by dimater of the drug inbibition test and four types by AP-PCR.According to colonial morphology and microscopic appearance,40 strains Staphylococci were divided into three groups.The 1st group,26 srains,showed inaurate,with 2mm of the diameter and thyrsiform bolus.The 2nd group has 9 strains with stramineous colony,1mm of diameter,which were similar to thyrsiform and anomalism bolus and were bigger than the 1st group.The 3rd group contained 5 strains ivory petite colony with 0.2mm-0.3mm of diameter.40 strains of the Staphylococci may be classifie into five drug resistance groups,such as A,B,C,D and E.only 1 strain belongs to Group A, whichi resistnce all the 14 kinds of antibiotics tested.There was 4 strains in the group B with resisitant of Ofloxacin.7 strains to be sensitive to streptomycin belonged to group C.25 strains, more than 60%of the Staphylococci from the milk,group D,showed lowed sensitive to Ampicillin. The last group E,3 strains were sensitive to Gentamicin.According to the bands of the agarose gel eletrophoresis of the AP-PCR products,40 strains of the Staphylococci were divided into 6 genotypes-Ⅰ,Ⅱ,Ⅲ,Ⅳ,ⅤandⅥwhich contained 13,2,6,1,10 and 8 strains respectively by the genetic resemblance coefficient matrix and between-groups linkage.23 strains of Staphylococci isolate from the samples collected at Rongchang belonged to the 1st group by shape,group A,Band D by drag susceptible.,9,3,7,4strains of genotypeⅠ,Ⅲ,ⅤandⅥ.There were 10 strains Staphylococci from Hechuan,which contained 1,2 and 3 group by shape,group D and E by drug susceptible.There were 3,1,2,1,2,1 strains of genotypeⅠ,Ⅱ,Ⅲ,Ⅳ,ⅥandⅥ.In Yubei region,we got 7 strains Staphylococci,which belonged to group 2 by shape and group C by drag fast,genotypeⅠ(1),Ⅱ(1),Ⅲ(1),Ⅴ(1)andⅥ(3).4.The Minimum Inhibitory Concentration(MIC)of the four lysozyme samples to the bacterium isolated from the cow milk samples.The results showed that 4 lysozyme had no inhibit affection to the E.coli,LY,no inhibit effection both to Staphylococci and Streptococcus.The MIC of SK,GK and ZD to Staphylococci were 125mg/mL,0.312mg/mL,0.312mg/mL;SK and GK to Streptococcus were 0.125mg/mL and 0.312mg/mL.Our experiment results indicated that the lysozyme samples,Lysozyme samples include SK,GK,ZD and LY were be detected using Micrococcus turbidimetry,Coomassie brilliant blue,PAGE,the enzymatic activity each samples were 2100 U/mg,7320 U/mg,1880 U/mg and 80 U/mg.The content of Lysozyme in SK,GK and LY were 280.lug/mg,944ug/mg,and 976ug/mg.The content of antalzyme in ZD,however,couldn't be detected because of the carrier materials.SK showed best inhibition effect ion to the bacterium causing bovine mastitis,so it maybe the first candidate to develop new drug without antibiotic residue for bovine mastitis therapy.