The Developement Of Rapid Detection And Drug Susceptibility Testing For The Bovine Mastitis Pathogens

Mastitis remains the most economically important disease of dairy cattle throughout world which may decrease the quantitiy and quality of produced milk.It important to understand the pathogen's spacies of dairy cow mastitis and establish rapid identification methods to detect the pathogens caused mastitis and select sensitive drugs to treat bovine mastitis.In this dissertation,we conduct four tests to establish methods for these purpose.Trial one Identification of the pathogens from the bovine mastitis samplesIdentification of the pathogens from the bovine mastitis samples were carried out in three regions-Jinan.Taian,Linyi- in Shandong province.The highest infection rate of pathogens in three regions is fungl,fungl, Streptococcus or Bacillus in small farmer dairy respectively but Streptococcus and then Staphylococcus in big dairy in Jinan.Totally, Streptococcus is the most important pathogens caused clinical mastitis and then Staphylococcus, Fungi, Bacillus.The infectious rate of streptococcus and fungi from the clinical mastitis samples is higher than its from subclinical mastitis.However, The infectious rate of Staphylococcus, Enterobacteriaceae and Bacillus from subclinical mastitis is higher than the clinical mastitis.The results of this study indicate Staphylococcus, Enterobacteriaceae, Streptococcus are the major pathogens in shandong province.In some place in Shandong province Bacillus and Fungi have become the most important pathogens,especially the fungi which havethe highest infectious rate in small fanner dairy in Jinan and Taian areas.so we should regard them as a serious problem.Trial two The pathogenic yeast identification of mastitis and the establishment of fast detection methodThe method of identification of Candida albicans was studied and the infectious rate of yeast mastitis was investigated.The intermix infection rate and the infection rate of clinical mastitis and small dairy farm is higher than the subclinical mastitis and big bovine dairy farm.There is the fungi infection which the rate is high in different areas.A nested polymerase chain reaction(PCR) method based 18S rRNA gene was developed for specific , sensitive and rapid detection of fungi in raw milk. The direct sensitivity test indicate that the dilution of 1 CFU/mL Candida albicans in nutrition broth and lOCFU/mL in raw milk can be detected.Trial three a Nested PCR Method based 16S rRNA gene was developed to Detect Stretococcus agalactiae in Milka Nested PCR Method Based 16S rRNA gene can be detected Streptococcus and Stretococcus agalactiae in Milk. The direct sensitivity test indicate that the dilution of 101 CFU/mL Streptococcus agalactiae in nutrition broth, 102~103 CFU/mL in milk can be detected and 1 CFU/mL Streptococcus agalactiae in raw milk can be detected after culture over night. This method is more sensitive ,specific,and rapid than traditional methods. It can be used to detect Streptococcus agalactiae clinically and can be used for the early infectious investigation of Streptococcus agalactiae in the dairy herd and be effectively used in preventing and controlling of Streptococcus agalactiae to spread in herds.Trial four Drug susceptibility testing of pathogens from bovine mastitis. (one)The routine Drug Susceptibility TestingThe drug sensitive test of Keyanjing to 75 strains seperated from clinical bovine mastitis were operated with Kirby_Bauer agar.The result suggest that the total effective rate was 100% in which the high sensitive rate was 84% and the middle sensitive rate was 16%.In the research,many drugs ,used frequently in clinic ,were evaluated with 6 G+and 8 G'strains separated from bovine mastitis.The results indicate,single component drugs such as Enrofloxacin, Fluofenicol, Ceftriaxone and multi-component drugs Toubaoxujian,Keyanjing have good antibacterial effect to the G+ strains.But almost all of the strains have resistance to Penicillins (Oxacillin, Ampicillin)and Qunolones (Norfloxacin). Of all 49 drugs, all 8 G~strains was only sensitive to Amikacin, Florfenicol,some of the 2nd or 3rd generation of Cephalosporins,and all the multi-component drugs(except Trimeth/Sulfa). All 8 G~ strains were not sensitive to Streptomycin, Chlortetracycline and the Penicillins(except Piperacillin) and the Chloramphenicols(exceptFlorfenicol)and the Tetracyclines, Lincomycins, Macrolides, Nor-floxacin, NalidixicAcid and Cefalexin.AlI of the strains were completely not sensitive to Tetracyclines and Sulfonamides.(two) The study of DST used resaurin indicatorThe test ,used resazurin as an indicator ,was developed to detect the drug susceptibility by the Microdilution Resazurin Assay (MRA) method and mensurated fluorescence which estimated antibacterial effect.This test determine the Minimal Inhibitory Concentration(MIC) of six antibiotics for Staphylococcus aureus,Streptococcus, agalactiae, E. coli and antibacterial effect of three antiotics for Streptococcus, agalactiae in raw milk.To some extent,these methods simulation the drug environments of the mammary gland when treated bovine mastitis and it is impossble to select the susceptible drug quickly and then can be used to treat mastitis effectively .These methods avoid the shortages of agar diffuse method and test tube- phelometry which can not use raw milk to Drug Susceptibility Testing.