| Due to misdiagnosis of pathogens, it is difficult to treat the cow’s mastitis caused by fungus, so this kind of mastitis often becomes "intractable" mastitis. In order to investigate the yeast infection condition and the main yeast pathogen of the cows mastitis in a dairy farm in Nanning, we collected 198 milk samples from this dairy cattle farm,ninety-two (46.46%) milk samples of clinical mastitis and seventeen(8.58%) milk samples of subclinical mastitis were detected by detecting their somatic cell counts, combined with the cows’milk yield, the clinical findings,the milk appearance et al. Isolated and identified the yeasts from these milk samples, there were fifty-one milk samples with yeasts. After identification of pseudomycelium and the test of biochemical identification tube of the yeasts, checked the TH 15-C yeasts biochemical identification code books, and the results were as follows:there were thirty-seven milk samples had candida albicans(72.54%), there were six milk samples had candida famata(11.76%), there were six milk samples had pichia ohmeri (11.76%) and there were two milk samples had rhodotorula mucilaginosa(3.94%). The antibiotic sensitivity was randomly measured to twenty-one strains from the clinical isolated yeasts strains, the results were as follows:they all have higher sensitivity to nystatin, mostly strains have medium sensitivity to ketoconazole and amphotericin B, and they were mostly all resistanted to fluconazole, amikacin, caftriaxone, gentamicin and streptomycin. We provided reference of the treatment for the yeast mastitis cows and the theoretical basis for the prevention of the yeasts mastitis.The yeast mastitis could cause different degree and irreversible pathological damage to the breast tissue, or even could caused the cows to be eliminated or death.Candida albicans is one of the most important pathogenitic bacteria of yeast mastitis in dairy cows. It could also infected humans. It was harmful to both human and animals. The clinical still used the traditional method to detect Candida albicans, which were time costly that may be delayed the time of treating the disease that might make things worse. Hence, it is necessary to establish a fast and accuracy method to detect candida albicans in mastitis cows. According to the ITS 2 conserved domain of the candida albicans, we designed one pair of the specific primers,and established a rapidly diagnostic method to detect the bovine mastitis Candida albicans by SYBR Green-I qPCR technique. The standard equation was:y=-3.448x+39.72, R2=1.00, its efficiency was 95%. Its minimal detectable concentration of the samples was 1.03×101copies/uL. The melting curve analysis indicated the Tm value of the candida albicans was 87.25 ℃-88.25 ℃. Use this method to detect S.aureus, E.coli, S.agalactiae and other pathogens which could also caused cows mastitis, no melting curves were produced, which indicated that this method had a perfect specificity. The repetitive tests showed that the within-batch CV% were 0.90%,0.39%, 0.34%,0.13%, 0.12% respectively and the between-batch CV% were 0.21%, 0.26%,0.37% ,0.13,0.19% respectively, they were all below 5% that indicated the method had a perfect repeatability. Use this method to detect 40 clinical samples, use the traditional method as the standard, its sensitivity, specificity, accuracy were 100%,90.91% ,96.77% respectively. Use PCR method as the standard, its sensitivity,specificity,accuracy were 93.75% ,100%,95.23% respectively. The above results showed that the SYBR Green-I qPCR technique achieved high specificity,sensitivity and repeatability. It provided scientific basis for detection of the cows mastitis candida albicans, and it is still need a large number of practical research to be used in clinical diagnostic. |
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