Establishment And Application Of Dual-PCR Method To Rapid Detection Of Bacteria And Yeast From Bovine Mastitis

Mastitis is the most common infectious disease affecting dairy cows and remains the most economically important disease of dairy industries around the world. Because of the complex cause and no effective therapy, it has brought large economic loss to the cattle farms. But there are so far no effective ways to solve the problems completely. Therefore, exploring new methods of prevention and treatment of mastitis is the main tendency in the future including safety, economy and high performance. Generally, bovine mastitis is classified into clinical mastitis and subclinical mastitis. The incidence of mastitis due to bacteria is usually very often in dairy herds. In recent years, owing to the abuse of antibiotic, steroid and immune inhibitor, one type of obstinate mastitis which was immune to antibiotic treatment appeared. Mostly, this kind of mastitis was caused by fungi; above all, fungal infection of the mammary gland was predominantly caused by microzyme. The identifying and fast diagnosis of mastitis is essential; however, it is difficult to ascertain the etiological agent only depending on the clinical symptoms of the cow and at the same time optimal treatments differ for each type of etiological agent.Traditonal methods for detection and characterization of microorganism are time-consuming and less-sensitive. And blind antibiotics treatment not only has ill-effects, it also decreases the quality of milk and increase treatment difficulty, so it is necessary to find new methods to detect cow mastitis. Basing on backgrounds above, we carried out a series of studies about dairy cows mastitis. In this study, some results were reported as follows:Part One: Bacteria which caused udder infection were analysed systemicly.Two hundred and twenty strains of bacteria were isolated from the milk of dairy cows with mastitis from 3 dairy farms in Taian. By using the culture character, morphological observation and biochemical test, the result demonstrated that among the 220 strains, 66 of them are Staphylococcus (30%), 25 are Streptococcus (11.4%), 20 are Other Gram-positive coccus (9%), 40 are Gram-positive Bacillus (18.2%), 46 are Gram-negative Bacillus (20.9%), 23 are Yeast (10.5%). 50% of samples were infected by multiple pathogens.Part Two: Isolation, identification, medicine sensitivity test and Vancomycin sensitivity test of Staphylococcus of dairy cow mastitisMastitis is mainly caused by Staphylococcus. Eighty milk samples were collected from 5 cattle farms in Taian from 2006 to 2007, and 29 Staphylococcus strains were isolated from these samples, including S.aureus 14/29 (47.4%),S.chromogenes 3/29 (10.3%),S.xylosus,S.cpidermidis,S.saproghyticus and S.sciuri 2/29 (6.9%) respectively, S.caplitis and S.simulans 1/29 (3.3%) respectively. S.aureus was primary pathogenic bacteria of Staphylococcus. The results indicated that all of Staphylococcus was hypersensitive to cephradine and Amikacin and partially resistant to Clindamycin, Teracycline, Trythromycin and TMP/SMZ which used frequently in clinic. Average diameter of circle that controlled S.aureus and Coagulase-negative Staphylococcus for Vancomycin was 19.1mm and 20.3mm respectively. As a result, S.aureus which could resist Vancomycin was not found.Part Three: Establishment and application of Dual-PCR method to rapid detection of bacteria and yeast from bovine mastitisA Double polymerase chain reaction (PCR) was developed for simultaneous detection of germ group classification of the bovine mastitis. We develop a new method that retains both bacteria and yeast DNA form milk byβ-mercaptoethanol which can decompose cell wall, lysozyme and snailase which can assimilate cell wall and quartz sand which can further abrade the outside of cell. The target sequences of bacteria and yeast were conserved regions from 16S rRNA and 18S rRNA respectively. We evaluated the technique for the detection and differentiation of bacteria in artificially infected milk and milk from cows with moderate or severe clinical mastitis. The performances of the Dual-PCR and traditional culture method were evaluated by 84 mastitis milk samples. The results indicated that the sensitivity of Dual-PCR in detecting bacteria and yeast was 102CFU/mL and 103CFU/mL respectively. There was not different from the culture method in bacteria (P>0.05), but was more sensitive than culture in detecting yeasts (P<0.01).