Research On Analyzation Of Main Pathogenic Bacteria And Detection Of Virulence Genes In Staphylococcus Aureus From Bovine Subclinical Mastitis Cases In Urumqi

Milk samples of subclinical mastitis were collected from three dairy farms in XinJiang, 91strains of bacteria which contain 21 species were isolated and identified from milk specimens. There were 28(30.78%) strains of Staphylococci, 21(23.07%) strains of Streptococcus ,25(27.48%) strains of bacillus and 17 (18.67%)strains of other bacteria. 49 Staphylococcus aureus were typed by Enterobacterial Repetitive Intergenic Consens–PCR(ERIC-PCR). ERIC-PCR could be amplified out 3~10 straps,revealeing 4 genotype. Isolates from the same cow farm belonged to the different gene types, and isolates from different farms belonged to the same gene type.This suggested that the different genotypes of pathogenic bacteria can exist in different environments .Milk samples of Xinjiang Brown Cattle and Chinese Holstein Cattle from Urumqi Breeding Cattle Farm were collected and isolated. The effects of subclinical mastitis on different varieties were compared, and information of Somatic Cell Counts (SCC) and colony forming units(CFU),SCC and different ages from different varieties were analyzed. The result showed that there was difference in category of pathogenic bacteria and Xinjiang Brown Cattle was prior to Chinese Holstein Cattle on the infected rate of staphylococci; the former has a stronger resistance to disease than that of the latter; there was a extreme1y significant association two varieties between CFU of SCC﹤200000/mL and CFU of SCC﹥500000/mL (P<0.05),while there was no significant correlation between CFU of 200000/mL﹤SCC﹤500000/mL and CFU of SCC﹤200000/mL,SCC﹥500000/mL(P>0.05),Statistical analysis on total SCC and culture data showed that there was no association between SCC and bacterial isolation and there was effected by other factors; there was the trend of escalation on SCS of Xinjiang Brown Cattle and Chinese Holstein Cattle and there was significant correlation on SCS of different varieties.To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the major bacterial causes of bovine mastitis for Staphycococcus ,Streptococcus and E.coli.The target sequences of Staphycococcus, Streptococcus and E.coli were the 16S rRNA regions. The results showed that the assay was specific.The sensitivity of mutiplex PCR in detecting Staphycococcus , Streptococcus and E.coli was 103 CFU/ mL ,103 CFU/ mL , 104 CFU/ mL respectively. To detect clinical samples, 25 milk samples from herdswere directly detected. 15samples were positive for Staphycococcus, 5 samples were positive for Slagalactiae and 10 samples were positive for E.coli by dectecting of this multiplex PCR.Molecular investigations on the genes encoding virulence determinants entA,tsst-1,nuc,clfA,fnbA,fnbB,hla,hlb were conducted within 49 strains of Staphylococcus aureus isolated from subclinical mastitis by means of PCR.The results revealed that the positive rate of tsst-1,nuc,clfA,fnbA,fnbB,hla,hlb,entA were 4%,59.2%,63.3%,69.4%,22.5%,46.9%,53.1%,0%, respectively.ClfA and fnbA are predominant virulence determinants.The results of 30 drugs sensitivity test showed pathogenic bacteria almost were ineffective to penicillin and tetracycline frequently used, while Levofloxacin,furadantin,cefotaxim,amikacin had a fairly good bacteriostasis.