| Campylobacter is one kind of zoonotic bacterium that is prevalent in the world.Among them,Campylobacter jejuni(C.jejuni)and Campylobacter coli(C.coli)are mostly responsible for infection and transmission.Both of them can cause food poisoning or gastrointestinal inflammation in consumers.Symptoms of infection mainly include diarrhea,abdominal pain,fever,nausea,and vomiting.Generally,it is a self-limiting disease,but it may also cause serious complications such as gastritis and Guillain-Barre syndrome.The production and sales of poultry is the easiest way of Campylobacter spread.At present,additional antibiotics are added to inhibit the growth of miscellaneous bacteria when isolating and purifying Campylobacter,but differences exist in the selection of antibiotics in different regions.Based on the GB4789.9-2014 national standard,this research established the optimal antibiotic selections for the isolation and purification of Campylobacter from samples with different cleanliness;investigated on the prevalence of Campylobacter in a large broiler slaughtering enterprise in North China;established the transmission model of the poultry slaughtering and processing chain and the preliminary exploration of the spread and contamination range of Campylobacter.This study aims to provide better combinations of antibiotics for the isolation of Campylobacter,provide new ideas for epidemiological research and prevention of Campylobacter within poultry slaughtering process.1.Combination optimization of antibiotics used for Campylobacter isolationBased on the Microbiological examination of food hygiene-Examination of Campylobacter jejuni(GB4789.9-2014),six antibiotics,including cefoperazone,polymyxin B,trimethoprim,rifampicin,cycloheximide,and amphotericin B were classified and combined into three combinations of three antibiotics(cefoperazone,polymyxin B,trimethoprim),four antibiotics(cefoperazone,polymyxin B,trimethoprim,rifampicin),and the same six antibiotics.Samples of anal swab,carcass swab and disinfectant were used,and the effectiveness of using different antibiotic combinations was investigated based on the criteria of prevalence and bacterial count.The results showed that the biofilm-forming ability of Campylobacter decreased with increasing antibiotic use.Antibiotic selection also varied for samples with different degrees of cleanliness.For anal swab samples,the optimal antibiotic combination was the six-antibiotic combination,which showed higher qualitative(17.3%)and quantitative(7.64×104 CFU/g)detection rates than the four-antibiotic and threeantibiotic combinations(P<0.05).For swab samples,the four-antibiotic culture medium had the highest positive detection rate(35.6%)(P<0.05)and bacterial load(2.33×102 CFU/100cm2)(P<0.05).For liquid samples,the four-antibiotic culture medium had the highest positive detection rate(38.9%)(P<0.05),while the threeantibiotic combination had the highest bacterial load(1.05×102 CFU/mL)(P<0.001).Therefore,different combinations of antibiotics should be used to isolate Campylobacter for different species and clean samples within slaughtering process.2.Epidemiological investigation and transmission characteristics analysis of Campylobacter in poultry processing chainA total of 3050 samples were collected from a large poultry slaughtering enterprise in North China,including anal swabs,carcass swabs,and environmental samples of conveyors,hooks,tools and others.Campylobacter was isolated using the optimized antibiotic combination of A6,A4 and A3 CCDA culture medium and molecular epidemiology techniques were employed to investigate the prevalence and contamination of bacteria in processing chain.MATLAB was used to perform polynomial fitting on the sampled data,establishing transmission and contamination models of Campylobacter that describes and predicts the transmission characteristics and contamination scope of Campylobacter in the slaughtering chain and partition link.Results showed that 22.3%(146/650)of the broiler samples were positive for Campylobacter,with the positivity rate gradually increasing along the processing chain.At partition,50.8%(66/130)of the samples were contaminated with Campylobacter(P<0.001).The quantitative results showed an initial increase and subsequent decrease.At partition,the average bacterial load remained low at only 2.73×102 CFU/100cm2(P<0.001).The contamination and spread of Campylobacter in product samples were found to be closely related to the length of the processing chain in the enterprise,as shown by the transmission and contamination model.The model predicted that the spread of Campylobacter contamination was not limited to the processing chain but also showed the possibility of positive detection in areas surrounding the enterprise.For environmental samples,19.4%(466/2400)of all were positive for Campylobacter,with positive samples mainly concentrated in partition.The positivity rate was 73.3%(33/45)for chicken leg storage frames(P<0.0001),86.7%(39/45)for chicken leg conveyor belts(P<0.0001),and 33.3%(10/30)for chopping boards(P<0.05),all significantly higher than average.The bacterial load of environmental samples was similar to that of broiler samples and decreased sharply after cooling.Regarding the concentrated sampling of high positive rate sites in the partition link and the results of the transmission contamination model,it was found that the spread of Campylobacter contamination in the working area around the conveyor belts within partition was relatively extensive,while the ability of Campylobacter to spread and contaminate surrounding areas was significantly reduced in the product storage frames and chopping board(P<0.05).The MLST results of the isolated strains around conveyor belts confirmed that 84.8%(28/33)of the strains belong to the same single ST type,indicating a severe aggregation contamination phenomenon of Campylobacter around conveyor belts in partition link.The positivity rate significantly decreased after 10 days in frozen storage(P<0.05)and no Campylobacter was detected after 30 days.The positivity rate and bacterial load of Campylobacter remained stable across spring,autumn,and winter,with no significant seasonal variations.To sum up,efforts should be made to strengthen the prevention and control of Campylobacter contamination in the surrounding areas of the enterprise and around the conveyor belts within the partition link. |
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