Screening Of Main Pathogens Of Dairy Cow Mastitis In Part Of The Pastures In Jiangsu Province Using TaqMan Probe-fluorescence Quantitative PCR

Dairy cow mastitis was a common and frequently-occurring disease in dairy farms,which was the local inflammation caused mainly by microbial invasion or physical and chemical damage to breast tissue.In the process of dairy industry processing and production,dairy cow mastitis had seriously affected the development of dairy industry,aggravated the economic losses of pasture and dairy industry,and became one of the biggest diseases that caused the loss of dairy industry.With the development of dairy industry and concerns about the safety of dairy products,more attentions were paid to the occurrence and harm of cow mastitis,and the ways to judge,detect and treat dairy cow mastitis were constantly looked for.The purpose of this scientific research was to investigate the relationship between somatic cell number and dairy cow mastitis,and then to use Taq Man probe fluorescence quantitative PCR to quickly screen the pathogenic bacteria of dairy cow mastitis in Jiangsu Province,to research the pathogenic bacteria and extend PCR testing technology to production practice.Based on the investigation,sampling,experimental research and analysis of the results,a series of suggestions and measures for the detection and prevention of mastitis in dairy cows were put forward.The results were as follows:1.A total of 10 large-scale rangelands in Jiangsu Province(nine pastures in northern Jiangsu Province and one in central Jiangsu Province,a total of about 8000 lacting cows)were investigated,sampled and DHI determined for 6 straight months.And the results of investigation and measurement showed that the average number of somatic cells per month was 315800 per milliliter,the average monthly incidence of mastitis was 16.35%,and the average daily milk production was 29 kilogram per day.The daily yielding decreased with the increase of somatic cell number and the difference was significant(P<0.05).The incidence of mastitis increased with the increase of somatic cell number and the difference was significant(P<0.05).The daily yielding decreased with the increase of mastitis incidence and the difference was significant(P<0.05).Therefore,dairy cow SCC played a first warning role in the incidence of mastitis and the decline of DMY,and dairy cow mastitis seriously affected the dairy herd improvement of dairy cows.2.In this experiment,the clinical mastitis and subclinical mastitis cows from various large-scale dairy farms in Jiangsu Province(pastures are mainly distributed in northern Jiangsu Province and central Jiangsu Province,including twelve in northern Jiangsu Province and three in central Jiangsu Province)were sampled,Taqman probe real-time fluorescence quantitative method was used to quickly screen the pathogenic bacteria of dairy cow mastitis and received the result that the main pathogenic bacteria of dairy cow mastitis in Jiangsu Province were Streptococcus agalactiae,Mycoplasma bovis,Escherichia coli and Klebsiella spp.Through bacterial isolation,culture and sequencing identification,a total of seven pathogenic bacteria were obtained.They were Escherichia coli,Streptococcus agalactiae,Streptococcus uberis,Enterococcus spp,Klebsiella spp,Serratia marcescens and Staphylococcus aureus.The drug sensitivity test showed that Gram-negative bacteria were sensitive to oxytetracycline and doxycycline.Gram-positive bacteria were strongly sensitive to oxytetracycline and also were sensitive to doxycycline.The mixed samples were sensitive to oxytetracycline and doxycycline.3.This experiment selected two kinds of bacteria:Streptococcus agalactiae(a kind of gram-positive bacteria)and Klebsiella pneumoniae(a kind of gram-negative bacteria)as the representative,the concentration selected for the scientific research were 10~5CFU/m L,10~6CFU/m L,10~8CFU/m L.They infected bovine mammary epithelial cells for 4 hours,8hours,12 hours and 24 hours.Compared with normal cells,the bacteria that at the concerntration of 10~5CFU/m L infected the cell for 8 hours made the most obvious changes in cell morphology.According to this concentration and time,the analysis of pyroptosis test showed that the number of pyroptosis infected by Streptococcus agalactiae accounted for4.4%of the total cells;the number of pyroptosis infected by Klebsiella pneumoniae accounted for 11.1%of the total cells.4.Through comparing the sequence of Serratia marcescens and Streptococcus agalactiae,primers were designed,PCR were used for amplification,electrophoretic were used for verification,sequencing were used for reverification and other operational approaches were used to design specific primers for the two bacteria,and a fluorescence quantitative PCR detection method for Serratia marcescens was established.Through the relationship between dairy cow mastitis and dairy herd improvement,it could guide pastures to judge whether cows had been ill by DHI data and to monitor the overall pasture management level and dairy cow production level in Jiangsu Province.Through the research of dairy cow mastitis pathogenic bacteria,the targeted drug sterilization scheme and the degree of harm of different pathogenic bacteria to dairy cow mammary gland could be explored,and the foundation for the further study of dairy cow mastitis pathogenic bacteria could also be established.