Optimization Of Isolation Method Of Campylobacter From Milk And Epidemiological Investigation Of Campylobacter In Milk Production During Breeding Stage

Campylobacter is a foodbore pathogenic bacterium that has received widespread attention at home and abroad.It can cause human infection with Campylobacter,causing abdominal pain,diarrhea and other diseases.The infection of immunocompromised patients can produce related complications such as MillerFisher Syndrome(MFS)and Guillain-Barre Syndrome(GBS).Campylobacter-contaminated food comes from a wide range of sources,and drinking contaminated milk is a common cause of Campylobacteriosis outbreaks.Therefore,it is extremely important to monitor the prevalence of Campylobacter in dairy cow production at the breeding stage.Since the complex components in milk have a certain influence on the detection of Campylobacter,this study optimized the separation method of Campylobacter in milk and applied it to milk production at the breeding stage to explore the prevalence of Campylobacter and resistance level in the process of milk production.The molecular traceability analysis of Campylobacter isolates was carried out by using whole genome sequencing technology,and the transmission law of Campylobacter in milking process was analyzed,in order to provide reference for the feeding management of dairy cows and the prevention and control of Campylobacter in milking process.1.Optimization of isolation and detection method of Campylobacter in milkIn this study,the effects of centrifugal force and centrifugation time on the recovery rate of Campylobacter in milk were determined by single factor experiment.A two-factor and three-level experiment was designed by response surface methodology to optimize the centrifugal parameters and verify the optimal centrifugal parameters.The optimal centrifugal force was determined to be 8500×g and the centrifugation time to be 15 min.The culture medium and method were determined based on the optimal centrifugation conditions,that is,adding 5%fetal bovine serum to MH culture medium for 24 h had the best effect.The optimized method was compared with the FDA method by artificially contaminated milk with different fat contents and actual samples.The results showed that the detection rate of the optimized method was higher than that of the FDA method on skim milk,low-fat milk and whole milk.In the actual samples,the detection rate of the optimized method was also higher than that of the FDA method.Only the detection rate of thick milk was lower than that of FDA method,indicating that the optimized method in this study is superior to FDA method on the whole and has the advantage of wide application range.2.Epidemiological investigation of Campylobacter in milk productionIn this study,A total of 1186 samples were collected from a large-scale dairy farm in Jiangsu Province,including 180 raw milk samples,288 anal swab samples,250 fresh feces samples,and 468 environmental samples,with an average positive rate of 4.72%.A total of 56 Campylobacter positive samples were detected,including 56 strains of Campylobacter jejuni and 5 strains of Campylobacter coli.The average positive rate of Campylobacter jejuni was significantly higher than that of Campylobacter coli(P<0.05),indicating that Campylobacter jejuni was the dominant species in this large-scale dairy farm.Among them,the investigation and analysis of Campylobacter during milking showed that the average positive rate of campylobacter after milking(4.93%)was higher than that before milking(2.25%),and the positive rate of Campylobacter from milk abandoned source was 3.33%before milking.The positive rates of Campylobacter from fresh feces and fresh milk were 11.00%and 3.33%,respectively.In order to evaluate the drug resistance of Campylobacter isolates,the drug sensitivity test was carried out.The results showed that Campylobacter isolates were generally resistant to ciprofloxacin,tetracycline and ampicillin,and other tested strains were all sensitive.In addition,the analysis of drug resistance spectrum of Campylobacter showed that 52 strains of 59 Campylobacter isolates had multiple drug resistance,only 7 strains were resistant to one antibiotic,and the multiple drug resistance spectrum was mainly 2 resistance(45.76%)and 3 resistance(42.37%).The drug resistance spectrum was CIP-TET and AMP-CIP-TET.Compared with the drug resistance spectrum of Campylobacter coli,the multiple drug resistance of Campylobacter jejuni was more serious.Through the whole genome sequencing technology,the Campylobacter isolates from milk were traced by molecular analysis.Three evolutionary branches showed that the Campylobacter isolates from milk and stool belonged to the same evolutionary branch,indicating that Campylobacter isolates from milk and stool had a certain genetic relationship.The drug-resistant genes blaOXA-61 and TET(O)were mainly concentrated in ST21,ST38,ST61 and ST604.Virulence gene detection results of sequenced strains showed that all tested strains carried six virulence genes,including iamA,ciaB,cdtB,cdtC,cheY and cadF.Among them,two ST7470 isolates also carried virulence genes cdtA and flaA,indicating that virulence gene distribution of different ST type strains was different.