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Cleavage Combinatorial Screening And Identification Based On Campylobacter Genotype And Phage Biological Characteristics And Its Application

Posted on:2024-06-16Degree:MasterType:Thesis
Country:ChinaCandidate:Z C SunFull Text:PDF
GTID:2544306914994889Subject:Master of Science in Biology and Medicine (Professional Degree)
Abstract/Summary:PDF Full Text Request
Campylobacter jejuni is a common zoonotic pathogen widely present in poultry and poultry products,which can cause intestinal diseases in humans.The widespread use of antibiotics in poultry farming has led to an increasingly serious problem of multiple drug resistance of Campylobacter jejuni,and there is an urgent need for new antibacterial agents.As a bacterial virus,phages can specifically recognize and cleave Campylobacter jejuni.Compared with antibiotics,phages have the advantages of simple use,strong specificity,fast proliferation speed,and strong antibacterial ability.However,due to the specificity of phages towards the host,their lysis spectrum is narrow,which is subject to many limitations in application.The biological characteristics of phages themselves are closely related to their living environment,invasion of host bacteria,etc.Therefore,selecting phages with different biological characteristics and combining them can meet practical application needs.This study used different genotypes of Campylobacter jejuni for initial and secondary screening to obtain 6 strains of Campylobacter jejuni phages with wide lysis spectrum and strong lysis ability.Their biological characteristics were identified,and on this basis,different combinations of phages were constructed for application in broiler breeding production.The optimal concentration and mode of phage inoculation were explored,providing new ideas for the application of phages and the prevention and control of Campylobacter jejuni.1 Screening and identification of bacteriophage combinations of Campylobacter jejuniScreen phages based on different genotypes of Campylobacterjejuni,determine their biological characteristics,and combine phages with different characteristics to meet the needs of subsequent applications.In the screening of phages,20 strains of Campylobacter jejuni isolated from different genotypes and hosts were used as test strains for preliminary screening of 58 phages.Based on the bacteriophage lysis spectrum and degree,14 phages were obtained.Afterwards,12 strains of Campylobacter jejuni isolated from broiler farms were used for re screening of the 14 phages initially screened.Finally,6 phages with strong lysis ability and wide lysis spectrum,13,35,P14,P53,P81,and P152,were selected for combination.In the combination of phages,the biological characteristics of phages were determined,including lysis spectrum,morphology,optimal infection complex,one-step growth curve,thermal stability,acid-base stability,and disinfectant tolerance.The six selected phages were able to extensively amplify and form clear and transparent plaques,all belonging to the family Muscoviridae.The optimal infection multiples for phages were 0.1 and 0.01.The incubation period of six phages is relatively short,ranging from 35 to 45 minutes,and the lysis period is relatively long,ranging from 60 to 110 minutes.Phages P53 and P152 exhibit high lysis activity,with a lysis amount of 300 PFU/cell.The optimal growth temperature for phages is below 50℃,and phages 35 and P152 have strong resistance to high temperatures.After being treated at 70℃ for 1 hour,they still exhibit 1 ×10~2 PFU/mL.Phages have strong pH tolerance and can still maintain a certain titer in pH 3-12 environments.Compared to the P14 and P81 strains,the acid resistance of phages is relatively poor.Several commonly used disinfectants in chicken farms(ethanol,isopropanol,formaldehyde,sodium hypochlorite)are used to treat phages.The inactivation of phages varies depending on the type of disinfectant used.Formaldehyde has the best effect,with a 10%concentration of formaldehyde being able to completely inactivate the bacteriophage,followed by sodium hypochlorite,which requires a concentration of 500 mg/L.However,isopropanol and ethanol have poor effects,and only when the concentration reaches 50%or more,can the bacteriophage be completely inactivated,Among them,phages 35 and P81 have a higher tolerance to different disinfectants.The reduction effect of mixed multi phages on Campylobacter jejuni is significantly better than that of single phages(p<0.001)and can effectively reduce the production of resistant strains.Moreover,the reduction effect is more stable when combined with three or more phages.Based on the biological characteristics of six bacteriophage strains determined,they were classified and combined,resulting in four bacteriophage combinations:combination 1(35,P53,P152),combination 2(35,P14,P81),combination 3(13,P81,P152),and combination 4(13,P14,P53).2 Application of Campylobacter jejuni phage combination in broiler breedingEvaluate the efficacy of bacteriophage combinations in reducing Campylobacter jejuni through in vivo and in vitro experiments,and further explore different application pathways of phages in broiler breeding.In vitro experiments have shown that all four bacteriophage combinations can achieve a reduction of more than 2 lg of Campylobacter jejuni,with a maximum of 4 lg,and effectively inhibit the growth of Campylobacter jejuni within 24 hours.Determine the lysis spectrum of bacteriophage combinations using chicken derived Campylobacter jejuni isolated from different regions.In vivo experiments,strain 85 isolated from the breeding farm was added to the drinking water of broilers,and after continuous addition for 1 week,the Campylobacter jejuni in their cloaca remained stable at 1×10~7 CFU/g。After inoculation with different combinations of phages,the bacterial load of Campylobacter jejuni in broiler feces showed a decreasing trend.The bacterial load began to increase at 12 days,and decreased by 0.94 lg1.78 lg at 16 days.Phage combination 1 had a better effect on the reduction of Campylobacter jejuni in broiler feces.The experimental results showed that different feeding modes(direct oral administration,daily drinking water,continuous drinking water)did not affect the maximum reduction of phages against Campylobacter jejuni.Direct oral administration of phages can quickly reduce the bacterial load of Campylobacter jejuni in feces,and continuous drinking water feeding is suitable for large-scale use.In addition,in order to effectively reduce Campylobacter jejuni,it is necessary to inoculate at least 105 PFU/mL of phages.There are differences in the colonization amount of Campylobacter jejuni in different intestines,with an average colonization amount of in the contents of the jejunum,cecum,and colon,respectively 2.6×10~2,7.4×10~7 and 3.9×10~6 CFU/g,phage combination 1 can persist in the intestine and reduce the bacterial load in the cecum by 2.5 lg and the colon by 1.9 lg.
Keywords/Search Tags:Campylobacter jejuni, Phage, Broiler farming, application
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