| The overall incidence of clinical mastitis in dairy cows is relatively high,causing serious harm to dairy cow health,milk production,and milk quality.The current mainstream antibiotic treatments are also facing problems such as increased bacterial resistance and drug residues,which restrict the development of the industry.In this study,milk samples of clinical mastitis from pastures in different areas of Jiangsu were collected in four seasons,to investigate the epidemiological of pathogens,and the dominant pathogens in the milk samples were isolated and identified.Based on the determination of staphylococcal drug resistance phenotype andβ-lactam resistance gene carrying status,molecular typing of staphylococcus was carried out.The aim is to clarify the correlation between the prevalence of clinical mastitis pathogens in dairy cows in Jiangsu area and the season and region,and investigate the drug resistance characteristics of mastitis-derived staphylococcus in dairy cows,so as to provide a theoretical basis for effective control of the occurrence of clinical mastitis in dairy cows and scientific prevention and treatment.This research is divided into two parts:1.Epidemiological investigation of pathogenic bacteria of cow clinical mastitis in Jiangsu areaFrom April 2020 to December 2020,taking clinical mastitis-affected dairy cows from seven large-scale ranches in northern Jiangsu,central Jiangsu,and southern Jiangsu as the research object,a total of clinical mastitis milk samples from three regions in each season were collected.In 316 samples,16-link Q-PCR was used for rapid diagnosis of pathogenic bacteria in milk samples.At the same time,bacterial isolation and culture combined with 16S rDNA sequencing were used to isolate dominant pathogens.The diagnostic results of 16-link Q-PCR are as follows:1.The overall detection rate of the sample is 90.82%,and all 14 pathogens except Serratia marcescens were detected;the detection rate of the sample was the highest in December,reaching 100%,5.The sample detection rate in June,June,and July was lower than other months;the sample detection rate in Central Jiangsu was the highest at 97.01%,followed by northern Jiangsu and southern Jiangsu.2.The number of samples with 2 and 3 pathogens was the largest,accounting for 33.10%and 28.92%of the total number of samples respectively;2 and 3 pathogens were mainly detected in each season,with the highest detection rate in summer,reaching the sample 74.11%of the total,the detection rate of 4 or more pathogenic bacteria samples in spring,autumn and winter is higher than that in summer.3.Klebsiella has the highest detection rate,reaching 55.05%,followed by coagulase-negative staphylococci,staphylococcus aureus,Escherichia coli and yeast;Klebsiella,coagulase-negative staphylococci throughout the year.The detection rate remains high.Staphylococcus aureus is prevalent in spring and summer,E.coli is prevalent in spring and winter,and streptococcus is prevalent in southern Jiangsu and central Jiangsu.The results of bacterial isolation and identification showed that the overall isolation rates of Staphylococcus,Klebsiella,and Escherichia coli were the highest,39.13%,17.66%,and 11.48%respectively;the identification results of dominant pathogens were basically consistent with the results of 16-link Q-PCR.2.Study on the molecular characteristics of Staphylococcus derived from cow mastitis.According to the guidelines of the 2013 version of CLSI,the KB disc diffusion method was used to determine the effects of 103 strains of staphylococci on 8 antibiotics(penicillin G,amoxicillin,oxacillin,gentamicin,tetracycline,lincomycin,and ciprofloxacin).The results showed that 103 strains of staphylococci were highly resistant to lincomycin,oxacillin,penicillin,and amoxicillin.The drug resistance rates reached 82.5%,67.6%,57.5%,43.2%.Sensitive to gentamicin,tetracycline,and chloramphenicol;moderately sensitive to ciprofloxacin.Multi-drug resistance is dominated by triple-drug resistance,accounting for 33.98%.Based on drug sensitivity results,103 strains of Staphylococcus were tested for β-lactamase resistance genes blaZ and mecA.The results showed that the overall detection rate of 103 strains of Staphylococcus blaZ gene was 45.57%,and the overall detection rate of mecA gene was 57.28%,and the change in the detection rate of the resistance genes of each staphylococcus was basically consistent with the resistance rate of the corresponding antibiotics.Fisher’s exact tests correlation analysis results show that there is a significant positive correlation between the severity of resistance of Staphylococcus to β-lactam antibiotics and the resistance genes it carries.MLST typing results show that ST188 is the dominant type of Staphylococcus aureus,ST51 is the dominant type of Staphylococcus chromogenes,and the sequence type of Staphylococcus haemolyticus is relatively dispersed,mainly ST77.SCCmec typing results showed that MRSA and methicillin-resistant staphylococcus saprophyticus were mainly SCCmec Ⅱ and Ⅲ,and the remaining MRCNS were mainly SCCmecⅣ. |
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