Establishment And Application Of Screening Method Based On CRISPR Sequence Typing Of Riemerella Anatipestifer Vaccine Strain

Riemerella anatipestifer?RA?is a contagious infectious disease.It infects most poultry and causes symptoms such as cellulose pericarditis,perihepatitis,airbag inflammation,and meningitis.Infesting ducks aged 1-8 weeks poses a great threat to the duck industry.This study aims to establish an efficient screening method for vaccine strains.Compared with traditional screening methods for vaccine strains,it greatly reduces the workload and cost and provides a powerful technical means for vaccine development.Strains with strong pathogenicity usually have better immune protection effects.When the background knowledge of a certain bacterial research is ambiguous and the virulence factors are not yet clear,the screening method of vaccine strains is usually to sequentially determine the virulence of each isolate It is commonly used to infect animals with different doses of bacteria.By measuring the minimum lethal dose?MLD?or half lethal dose(LD₅₀),you can also use the minimum infection amount?MID?or half infection amount(LD₅₀)to evaluate.These methods all require one-by-one determination of clinically isolated bacteria,which requires a large amount of work,is time-consuming and laborious,and limits the number of comparative strains.Therefore,the establishment of a highly effective vaccine strain screening method has a decisive influence on vaccine development.In this research method,taking Riemerella anatipestifer as an example,using several RA strains stored in a group laboratory as samples,using CRISPR sequences of Riemerella anatipestifer as the identification tag,the primers RAC2-F/RAC2-R were designed It can successfully amplify CRISPR sequences of different serotype RA with an efficiency of 94.74%;on this basis,a challenge model of mixed strain infection has been established;In order to determine the fastest time for dominant strains to break through the blood-brain barrier of ducks,the gradient setting time of RA separation from duck brains after mixed infection was 3h,6h,9h,and 12h,respectively,to comprehensively evaluate the efficiency of RA separation and RA growth on the plate.The time to isolate RA after challenge was determined to be 6h;pre-tests were conducted with 4 RA strains of 3 different serotypes,and the screening efficiency of dominant strains reached 100%.Then,taking the type 1 RA strain as an example,A-D 4 parallel experiments were carried out.The most dominant strains in each group were FJ-HJH,FJ-LYP,GD-DCR,and R9.The proportion of the most dominant strains in each group was RA 83.3%,75%,100%,and 77.8%,respectively,and the screening efficiency is ideal.In order to investigate whether there is a corresponding relationship between the predominant strains screened and virulence,the LD₅₀0 of the four most predominant strains were determined.The FJ-HJH,FJ-LYP,GD-DCR,and R9 LD₅₀0 values were3.40×10⁵,1.15×10⁶,1.91×10⁴,1.43×10⁵ CFU/piece;the four most dominant strains were mixed again to infect ducks.The results showed that the sequence of the isolated RA strain was 77.8%identical to the GD-DCR sequence,and the sequence was consistent with R9 The ratio is 11.1%,and the sequence consistent with FY-HJH accounts for 11.1%.There is a corresponding relationship between the screening efficiency and the virulence of the strains,and it can be concluded that the dominant strains have strong virulence.Sensitivity test,the results show that there is no significant difference,the results from this study show that there may be no association between virulence and drug resistance.In summary,this study successfully established a screening method for the vaccine strains of Riemerella anatipestifer based on CRISPR sequence typing,the screening efficiency is ideal,and provides a powerful technical means for efficient screening of vaccine strains;The dominant strains selected in this experiment can be applied to the development and preparation of Riemerella anatipestifer vaccine;it is verified that the dominant strain is a highly virulent strain.In the immune response of ducks,Preferentially break through the blood-brain barrier;and explored the link between strain virulence and drug resistance.