Development Of Detection Methods For Common Bacterials Causing Cow Mastitis

Dairy cow mastitis is one of the most common and severediseases in dairy farms,extremely impedingthe development of dairy industry.Pathogens causing mastitis are numerous.Streptococcus agalactiae,Staphylococcus aureus,Streptococcus dysgalactiae,Streptococcus uberis are the most common pathogens among them.It has been reportedthat these bacteria are not only found in the milk of mastitis cows,but also in the milk of cows without clinical signs.In China,however,the conditions of these bacteria within milk is not clear.One of the reasons is that there are still lacking of detection methods with rapid,sensitive and effective ways.Therefore,in this study,common bacteria（S.agalactiae,S.dysgalactiae,S.uberis,S.aureus）are isolated and identifiedin milk from dairy cows with no clinical mastitis in Guangdong province.Additionly,a multiplex PCR assay for rapid diagnosis of these 4bacteria wasdeveloped,as well as an insulated isothermal PC R assay for rapid diagnosis of S.aureus.The aim of this stud y is to provide reference and technical support for the epidemiological investigation of S.agalactiae,S.dysgalactiae,S.uberis,S.aureusin milk.A total of 460 milk samples were collected from 10 dairy farms in Guangdong province.The above bacteria were isolatedand identifiedbybacterial culture and 16srRNA Sequencing.A total of 47 isolates were identified.They were 11 strains of S.agalactiae（2.40%）,onestrains of S.dysgalactiae（0.22%）,13 strains of S.uberis（2.82%）and22 strains of S.aureus（4.78%）.The strain ofS.aureusisolates rank top among them.Secondly,four pairs of primers were designed for multiplex PCR,based on gene sequences of Surface Immunogenic ProteinofS.agalactiae,immunogenic secreted proteinfoS.dysgalactia,plasminogen activator A ofS.uberis,thermonuclease ofS.aureus.The working conditions of the multiplex PCR assay were optimized and its sentitivity and specificity were evaluated.The detection limits of the PC R for S.agalactiae,S.dysgalactiae,S.uberis,S.aureus were 10⁵ CFU/mL,10⁴ CFU/mL,10⁵ CFU/mL and 10⁵ CFU/mL,respectively.Addition,an insulated isothermal PC R assay were developed viaprimers designedfor thermonuclease gene of S.aureus.The reaction conditions of the PCR were optimized and its specificity and sensitivity were assessed.The insulated isothermal PCR had good specificity and sensitivity of 1 CFU/mL,which was higher than that of common PCR（10¹ CFU/mL）.The insulated isothermal PCR exhibited higher sensitivity,simpler operation and less time consuming,compared with common PCR.In summary,a sensitive and specific multiplex PCR were developed for detection of S.agalactiae,S.dysgalactiae,S.uberis andS.aureusin milk,as well as an insulated isothermal PCR assay for rapid diagnosis of S.aureus.These couldprovide methodsto support for detection of dairy cow mastitis and bacterial check of milk samples in China.In this study,bacterial pathogens were detected in the milk from dairy cows with no clinical sign of mastitis in Guangdong Province.The detection rates were close to those of the foreign epidemiological investigation.This phenomenon should be taken attention by government and society regarding dairy cow mastitis prevention and co ntrol and dairy food safetyin Guangdong province.