| The aim of the studies was to develop a Chinese medicinal formulae that can treat bovine mastitis caused by Candida albicans and find a method which can diagnose this disease quickly. The studies were devided into four parts. In part1, A pair of specific primers were designed according to the conserved sequence ITS1-ITS2 of the Candida albicans. PCR method to identify the Candida albicans in mammary gland from infections of cows was established; in part2, based on the Chinese veterinary medicine theories that determine the treatment according to different of symptoms and signs and antifungal effect of Chinese herb medicine, we formed the complex prescription with Chinese herb medicine, named as"Ruyanxiao"which was used to treat the bovine mastitis caused by Candida albicans; the acute toxicity and cytotoxicity of"Ruyanxiao"were tested in part3; in part4, the therapeutic effects of the Chinese herbal medicine was tested using milk goats as experimental model animal, and the clinical therapeutic effets was also assayed.PartI Diagnosis of Bovine Mastitis caused by Candida albicans by PCRRefered to GenBank, a pair of specific primers were designed according to the conserved sequences ITS1-ITS2 of the Candida albicans, 4 Candida albicans from clinical milk samples were amplified by the specific primers. The results indicated that all of them can be amplified by PCR, and the length of PCR products was 500bp. it only costs 2.5h to detect sampels by PCR. Furthermore, the contrast experiment showed that other species of usual mastitis pathogen including Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, could not be amplified. The sensitivity test indicated that the sensitivity of this method can reach 1.25×103 CFU/mL. 50 milk samples were cultivated and then tested respectively by biochemical test and PCR, as a result, 16% samples were identified to have Candida albicans by traditional method, 18% samples were detected by PCR method. The positive samples which were identified by traditional method were also positive by PCR. And the detection rate of PCR is higher than traditional method. So the PCR method is a more rapid, sensitive, and specific method that has a potential application in veterinary detection of bovine mastitis caused by Candida albicans.PartII The screening of Chinese herbal medicine formulae which was used to treat the bovine yeast mastitisBased on the Chinese veterinary medicine theories that determine the treatment according to different symptoms and signs, we chosed herbs which can promote blood circulation, disperse blood stasis and dredge collateral to form a prescription, and three Chinese medicine formulaes were formed. We selected one pure Chinese medicine formulae according to the antifungal effect of one single Chinese medicines and three Chinese medicine formulaes, named as"ruyanxiao". The minimal inhibitory concentration(MIC) of the formulae was detected, the results showed that"ruyanxiao"inhibited the main pathogenic bacteria effectively, and the average MIC is 0.116g/mL.PartIII The toxicity detection of"ruyanxiao"formulaesTo make sure the safety of clinical application, the acute toxicity and cytotcoxicity of"ruyanxiao"was detected in vivo and in vitro . The Half Lethal Dose (LD50) and the Maximally Tolerant Dose (MTD) were assayed in vivo. By intragastric administration of"ruyyanxiao"at the dose of 40g/kg?w . the mice were still live a week later, then they were given the"ruyanxiao"at the dose of 120g/kg?w everyday at the condition of the most tolerant dose. After 1 week, the mice were killed to observe the pathological change. In vitro,0% toxicity concentration (TC0) of"ruyanxiao"in L929 cells was determined by microscopy and MTT assay. Firstly, the acute toxicity of"ruyanxiao"was very low, and the LD50 and MTD was above 40g/kg?w and 120g/kg?w respectively. According to the toxicity level standard, the short period use of"ruyanxiao"was safe. Secondly, TC0 of"ruyanxiao"in the L929 cells was 1.56g/L.PartIV The study of the treatment effectiveness of"ruyanxiao"30 healthy milk goats were used as experimental animal models to study how to cure cow mastitis caused by Candida albicans. They were devided into three groups : morbility medication group, morbility no medication group and healthy control group,ten goats per group. Candida albicans were injected into milk vessel of the morbility medication group and morbility no medication group. The result showed that inflammatory reactions with different degree occured after 48h, the ill goats were depressed and their appetite was lower than healthy control group. Candida albicans was isolated from milk samples of the milk goats which were ill. Goats of morbility medication group were intragastricly administrated with chinese medicinal formulae"ruyanxiao", After five days, in the morbility medication group, the symptom of 8 goats disappeared and 2 goats relieved obviously and the spirit state was obviously improved. But the goats'symptom of morbility no medication group didn't increased or improved obviously. The blood samples were obtained from the jugular vein of all the 30 milk goats respectively before and after medication. The blood samples was used to analyze the blood rheology and blastisation of lymphocyte. Milk samples from the 30 milk goats were used to analyze the milk component. The results showed that all the health indicators of the morbility medication group approached or achieved the normal level after medication, but there were no obviously improved in the morbility no medication group. Taken together, our data suggested that the chinese herbal medicine formulae"ruyanxiao"was effective to cure mastitis caused by Candida albicans. The experiment is helpful to the study of cow mastitis caused by Candida albicans.Clinical therapeutic test indicated that the Chinese herbal medicine formulae can eliminate the clinical symptom and make the milk production return to almost the normal level. It indicated that"ruyanxiao"is effective to cure the bovine mastitis caused by Candida albicans.
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