| Mastitis is a common frequently-occurring disease causing great economic losses on cow cultivation. At present, the antibiotic is mainly treatment of mastitis. The antibiotic residues in milk because of much use of the antibiotics to cause serious affected on physical and mental health of people. The dairy cattle produce the resistance to pathogenic bacteria and brought new challenges for prevention and treatment of mastitis. Therefore, the research of mastitis vaccine has become hots of scholars at home and abroad and show favorable development prospects. Mastitis vaccine is often less than ideal because of complex pathogens causing mastitis, weak antigen of main pathogens, many serotypes of a pathogen, "blood-milk" barriers and so on. The selection of effective adjuvant to enhance the effects of vaccine is an effective way in addition to screen strong virulence and cross-protective serotype strains.63 samples of mastitis milk were collected from three large-scale dairies of Lanzhou in order to find out the flora distribution and resistance of main pathogens arising from dairy mastitis of Lanzhou. Pathogens were isolated and identified from mastitis milk. Drug resistance was studied on main pathogens. The results of isolation and identification show that 9 kinds of bacterias and 103 strains were isolated and identified from 63 samples of mastitis milk.6 samples were bioclean.Plating efficiency is 90%. The pathogens of mastitis in 103 strains were 34 strains of Streptococcus agalactiae(33.16%),21strains of Streptococcus dysgalactiae(21.27%), 18 strains of Staphylococcus flavus(17.63%),12strains of Escherichia coli(11.75%),2strains of Staphylococcus epidermidis (0.02%),2 strains of Streptococcus uberis(0.02%),2 strains of Pseudomonas aeruginosa(0.02%),1 strains of Bacillus proteus(0.01%),and 1 strains of Klebsiella pneumoniae(0.01%). 5 strains of Streptococcus agalactiae,Streptococcus dysgalactiae, Staphylococcus flavus, Escherichia coli were used to be sensitization test. The strains were very sensitive to Azithromycin, CephalosporinV, Gentamycin, Amikacinsulphate,Ofloxacin,Norfloxacin,Rocephin, midrange sensitive to Penicillin, Ciprofloxacin, mild sensitive to Novobiocin, Erythromycin, Streptomycin and drug resistance to Acetylspiramycin.The purpose of the research is the development of efficient multi-vaccine and enhances of antigen content in per unit volume .The culture characteristics and influential factor in process of growth of Streptococcus agalactiae and Streptococcus dysgalactiae were studied. The results showed that Streptococcus agalactiae and Streptococcus dysgalactiae were cultured respectively in improved gravy soup, optimize medium and THB.The growth velocity and quantities of the bacterium in the optimize medium was more than two other nutrient mediums; Exponential phase of growth was decurtated obviously. The respective growth crest-time was about 10 and 12 hours. The result of influencing factor show that the optimum concentration of glucose is 10g/L in the optimize medium; Adding NaOH in the growth period of Streptococcus agalactiae and Streptococcus dysgalactiae can prolong the exponential growth phase and increase the production. In order to enhance the immune effects of multi-vaccine, three adjuvants of Aluminum,France206,FIA and three antigens (agalactiae, dysgalactiae and Staphylococcus flavus) were prepared for multi-vaccine against mastitis. 265 mice were randomly divided into four groups (Aluminum, oil, FIA and control group). Experimental mice were immuned on 0d and 21d respectively with three kinds of vaccines. The weight of thymus, spleen, phagocytic capacity and antibody titer were measured in different time after immunization. The result of the weight of thymus, spleen, phagocytic capacity shows that thymus index of three experimental groups compared with control group was not significant difference (P> 0.05) in all experimental time. The spleen index, phagocytic rate and phagocytic index of Aluminum compared with the control group was significant difference (P <0.05) on 35d and 65d.The result of serum antibody titer show that average antibody titer of three antigen (agalactiae, dysgalactiae and Staphylococcus flavus) compared with control group was significant difference (P<0.01), but antibody level among the three antigens were not significant differences (P>0.05) in different time (21d-65d) after immunization. Three experiment groups compared with the control group were significant differences (P <0.01).The time of highest serum antibody titer of FIA and Aluminum was on 35d, significantly higher than the oil group (P <0.01). FIA group has maintained a high level of antibodies after 30 days. The time of highest serum antibody titer of oil was on 50d, significantly higher than the oil group and the continuing high level of antibody can be longer.
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