Isolation And Screen Of Campylobacter Phage And Exploration Of Its Application In Poultry Production

Campylobacter is one of the most common food-borne pathogens in the world.It often causes human intestinal diseases.Poultry,as the most common host,play an important role in the prevalence and transmission of Campylobacter.A variety of antimicrobial agents have been used to control the contamination of Campylobacter in poultry production.As bacterial virus,phages can specifically kill the host strains and expected to become a new type of antibacterial agent.However,the host specificity of phages usually leads to narrow lysis spectrum,so we need to screen and combine phages to form phage cocktails to meet the application demand.In this study,we made a screen of the Campylobacter phage and combined a phage cocktail.Then,we explored the prevalence of Campylobacter in poultry during rearing and slaughtering phase from the perspective of qualitative research,quantitative research,and molecular epidemiological research.After the research,we analyzed the necessity of Campylobacter prevention in the rearing phase and used the phage cocktail for the prevention and control of Campylobacter.1 Isolation of Campylobacter phage and the screening of phage cocktailFrom September 2017 to June 2018,495 feces samples of chicken were collected in Jiangsu and Shandong.A total of 180 positive samples of Campylobacter phages were detected and 205 strains of Campylobacter phages were isolated.The results showed that the positive rate of phage in poultry market samples was 53.3 3%,which was significantly higher than that in farms(6.67%).In this study,lytic ability of 193 bacteriophages had been determined by using 29 Campylobacter strains with different ST type and PFGE type as test strains.According to the results,we screened out 24 phages,and their lytic ability to 229 C.jejuni isolates and 99 C.coli isolates had been tested.The results showed that 54.17%of the phages had a lytic rate between 20-40%and all phages had intensive lytic rate between 0-40%to C.jejuni isolates;62.50%of the phages had a lytic rate between 60-100%and 67.66%of phages had intensive lytic rate between 40-80%to C.coli isolates.With result of lysis spectrum,we combined 3 phages to form a phage cocktail.After combination,the lytic rate and intensive lytic rate against C jejuni isolates was increased to 41.05%and 86.46%respectively,and the lytic rate and intensive lytic rate against the C.coli isolates was increased to 77.78%and 93.94%respectively.2 Epidemiology of Campylobacter in poultry rearing and slaughtering phase and the application research of phagesFrom July 2018 to July 2019,samples from poultry slaughterhouses and farms were collected in Jiangsu and Shandong.A total of 2889 samples from 2 slaughterhouses and 7 farms were collected,1170 cloacal swab samples and 430 environment samples from farms,245 cloacal samples,911 carcass wiping samples and 133 environment samples from slaughterhouses.Qualitative research showed that a total of 558 positive samples of Campylobacter were detected in the rearing and slaughtering phase,and the average positive rate was 19.38%.349 C.jejuni and 288 C.coli strains were isolated.In rearing phase,the positive rate showed an upward trend,which gradually increased from 21 days old(0%)to 42 days old(34.69%);the positive rate of dehairing,evisceration,cooling and partition were 21.6%,44.16%,22.42%and 18.98%respectively.The positive rate of cage rear(8.45%)was significantly lower than free range(46.19%)in different rearing mode.The positive rate during slaughtering phase was significantly different according to the contaminated levels of chickens,and the positive rate in slaughtering phase from low contamination flocks(4.00%)was 1.2%,while high contamination flocks(46.00%)was 74.00%.The dominant species distribution of Campylobacter isolates was different in different production phase.C.jejuni was the dominant species in rearing phase(69.30%)and C.coli was the dominant species in slaughtering phase(59.04%).Quantitative research showed that enumeration of Campylobacter throughout the rearing phase showed an upward trend from 9.27×105 CFU/g in 28 days to 5.10×106 CFU/g in 42 days.During the slaughtering phase,the amount of Campylobacter loaded in evisceration was 1.17×106 CFU/100cm2 witch significantly higher than that in dehairing(1.66×105 CFU/g,p<0.05),and the Campylobacter counts in cooling(2.56×103 CFU/100cm2)and partition(3.98×103 CFU/100cm2)was significantly lower than evisceration(p<0.001).The Campylobacter counts of carcass in large slaughterhouse was lower than medium slaughterhouse(p<0.05).The cross-link tracking of some chicken flocks was carried out in rearing and slaughtering phases,and the isolates were sequenced for molecular epidemiological research.The results of MLST typing showed that C.jejuni isolates contained ST 8089 and three new ST types,of which ST 10242 were dominant type(65.00%),and C.coli had six ST types,of which,ST1586 was dominant type(70.00%).Strains with same ST type can be isolated from same chicken from rearing phase to slaughtering phase,and different ST type strains can be isolated from same chicken in slaughtering phase,which indicates that Campylobacter can transmitted along the production link,and cross-contamination occurred in the slaughtering phase.The above research showed that the Campylobacter in rearing phase can be transmitted to the slaughtering phase,resulting in serious contamination in the downstream.So,it is of great significance to prevent and control Campylobacter in the rearing phase.Therefore,wo explored the effect of phage cocktail on the reduction of Campylobacter in poultry.In vitro,phage cocktail can reduce the number of Campylobacter by 3 1g in 24h,and the effect of cocktail is stronger than single phage.In vivo,phages were inoculated by oral and drinking water.The qualitative results showed that the positive rate of chicken flocks reached 100%after 2 and a half weeks,and the positive rate of experimental group did not show a decrease trend after inoculation with phage.The quaitative results showed that both drinking and oral group had significant reduction of Campylobacter after 6-9 days in chicken feces by 2.1lg and gradually weakened within 28 days until no obvious reduction effect was found.The colonization of Campylobacter in intestinal contents decreased by 1.5lg,2.4lg and 2lg respectively,and he colonization of Campylobacter in intestinal contents decreased by 1.3lg,1.1lg and 1.7lg respectively.