| Campylobacter is one of the most common zoonotic food-borne pathogens in the world,which could cause human gastroenteritis such as diarrhea and abdominal pain.Poultry plays an important role in the spread of Campylobacter.Phage lysis of bacteria is of specificity and is a strategy of Campylobacter prevention and control.Besides,the isolation of phage with lytic activity is the prerequisite for the practical application of phage,the interaction between phage and bacteria,and the reformation of phage.However,Phages are especially difficult to isolate from samples with low numbers of virus particle.Therefore,optimizing the phage isolation method is beneficial to improve the phage isolation efficiency.This study optimized the enrichment and isolation method of Campylobacter phage,investigated the distribution of phage in broiler production stages to explore the factors affecting the distribution of phage,and we identified the characteristics of phage isolates,providing reference for the study of Campylobacter phage.1.Optimization of the enrichment and isolation method for Campylobacter jejuni phageIn order to improve the efficiency of phage isolation,the conditions of sample storage and phage enrichment were optimized.The variables in the sample storage process are type and dosage of buffer,time and temperature.The single-factor test results show that the SM buffer maintains the number and activity of phage with in 48h under the condition at 4-25℃.There are culture condition variables of using host bacteria to enrich phage,including medium,temperature,speed,and ionic additives.The recovery rate is used as an index to evaluate the enrichment effect,and test results show that temperature and speed are the key factors for enrichment of phage.Enrichment of phage with host bacteria is the key for improving the efficiency of phage isolation,and the culture conditions directly affect the efficiency of phage enrichment.The factors include culture medium,temperature,rotating speed,additive and the number of host bacteria in the process of culture.Based on the single factor test results,the response surface method was used to analyze the key factors on the enrichment effect.Taking 37℃ and 45 rpm as the basic parameters and the phage recovery rate as the dependent variable,a two factor three-level response surface test was carried out.The best predicted value showed that the temperature and speed are 38.95℃ and 9.46 rpm,with a phage recovery of 361.06%.Combined with the convenience of practical operation,the culture conditions were adjusted to 37℃ and 0 rpm,with a phage recovery of 3 54.12%.Therefore,enrichment culture conditions for Campylobacter jejuni phage were determined,that the host bacteria and phage were cocultured with Bolton broth at 37℃ and 0 rpm.Using this method to isolate Campylobacter jejuni phage from chicken feces,prepare sample filtrate and isolate phage after enrichment.Compared with the traditional single spot method,the detection rate of phage is increased by 260.00%,and the effect is good,and the method is feasible.Using optimized method to isolate phages from chicken feces,compared with the traditional single-spot method,the phage isolation rate is increased by 260.00%.2.Isolation and characteristics of Campylobacter jejuni phage from broiler production stagesIn the period December 2020 to October 2021,sampling were obtained from 12 farms and 4 agricultural products markets in Tengzhou,Shandong.A total of 3,310 samples were collected from chicken flocks,including 820 cloaca swabs,530 environmental samples and 1360 fresh feces from chicken farms,600 fresh feces from broiler markets.Of these samples,168 samples(8.57%,168/1960)were positive,the details are as follows,5.15%(70/1360)of breeding stage samples and 16.33%(98/600)of live poultry market samples.A total of 86 bacteriophages were isolated,including 8 strains originated from fresh feces of the farm,77 strains originated from fresh feces of the broiler market,and 1 strain originated from the cloaca swabs of broiler.Phage survey data of our laboratory were summarized.A total of 1,775 samples were collected in Yangzhou,Jiangsu Province during the 2017-2020 years,including 680 fresh originated from flocks,520 environmental samples from broiler market.and 575 fresh feces from live poultry wholesale markets.Based on the above data,the influencing factors of phage distribution were analyzed,the results showed that Campylobacter phages were widely distributed in chicken flocks,the characteristics of a flock include indoor poultry,high population density,long fecal cleaning cycle and presence of host bacteria.The positive rate was of no significant difference between the phage and host in the live poultry market,the percentages respectively were 16.33%(98/600)and 14%(84/600).The phages positive rate of indoor chickens was significantly higher than that of outdoor chickens,the rates were respectively 11.75%(229/1980)and 5.88%(20/340).In terms of feeding mode,phages could be isolated from approximately 28.53%(214/750)of ground breeding flocks,and 28.75%(23/80)of free range flocks in market,while the positive rate of phages in caged and freerange chickens in farms was lower than 6%.In terms of fecal cleaning interval period,56 of 392 samples were positive,the fecal cleaning cycle of this kind of chicken herd is about 42 days,with only 2.02%(44/2178)positive samples from chickens whose feces were cleaned up every day.Therefore,the longer fecal cleaning interval period,the higher positive rate of phages in the flocks(P<0.0001).The phage positive rates of fecal samples in small and medium-scale chicken farms and large-scale chicken farms were 18.75%(111/592)and 0.75%(17/2278).The positive rate of phage in small and medium scale chicken farms was significantly higher than that in large-scale chicken farms(P<0.0001).The density of chickens in large wholesale markets is relatively higher,and the phage-positive rate is 48.87%(281/575).In terms of sample types,the phage positive rates of fresh feces and cloaca swabs were respectively 7.25%(148/2040)and 1.22%(10/820),the phage positive rate of the former was significantly higher than that of the latter(P<0.0001).Phage isolates were characterized for lytic activity,specificity,receptor type,and morphology.80%of the C.jejuni isolates showed sensitivity to at least one bacteriophage.The sensitivity to individual bacteriophages ranged mainly below 50%.They were assigned to Myoviridae families,and there was no significant correlation between specificity and host origin.The identification of receptor types showed that 63.95%(55/86)of phages could lysis C.jejuni NCTC12662,of which 45.5%of the phage receptors were related to capsular polysaccharides,and the rest were related to flagella. |
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