Identification And Bological Characteristics Of Pasteurella Multocida Double Gene Delelation Mutants(PmCQ2Δ893Δ894)

Pasteurella multocida(P.multocida),a Gram-negative facultative anaerobic pathogenic bacteria,the capsule form the basis for classification into five serotypes(A,B,D,E,and F).The P.multocida serotypes A,B,and F infect many wild and domestic animal species and cause acute and chronic infectious diseases.Diseases caused by P.multocida include hemorrhagic septicemia in cattle,atrophic rhinitis in pigs,fowl cholera in birds,snuffles in the rabbit.P.multocida of serotypes A is the major pathogens causing respiratory disease,such as pneumonia in cattle,which has caused serious economic losses to the husbandry of livestock all over the world.At present,the prevention of P.multocida is mainly based on vaccine prevention,while for the animals that have developed the disease,antibiotics are mainly used to reduce the mortality rate.Antibiotics have the risk of drug resistance,which not only brings difficulties to the control and treatment of diseases but also aggravates the residue of antibiotics in cattle and poses certain risks to animal food safety.In the early stage,a mutant strain with obvious morphological changes was obtained during the construction of the gene-deleted strain.Transcriptomics sequencing of the mutant strain showed that 6 genes were not expressed.According to the transcriptomics results,some genes in the mutant were verified,and it was finally found that the mutant may be a PmCQ2Δ893Δ894double-gene deletion mutant.This paper will identify the double-gene deletion mutants through related experiments.In addition,in vivo and in vitro experiments were conducted to explore the biological characteristics of the deleted strains and wild strains in strain morphology,growth rate,biofilm and capsule formation,pathogenicity to mice and their immune protection.Finally,transcriptomics sequencing was used to explore the mechanism of PmCQ2Δ893Δ894 virulence reduction.1.Identification and bological characteristics of Pasteurella multocida double gene delelation mutants(PmCQ2Δ893Δ894)1.1 Identification and growth characteristics of double gene delelation mutants(PmCQ2Δ893Δ894)Firstly,the double-gene deletion strain was identified,and secondly,the biological characteristics of the mutant strain and PmCQ2 were compared in this paper.Under the same culture conditions,the mutant strain was found to grow slowly.According to the in vitro growth curve,the overall growth rate of the mutant strain was delayed by at least 2h compared with the PmCQ2 strain.The capsular content of PmCQ2Δ893Δ894 was significantly reduced.In addition,according to the iron utilization test of the mutant and the wild strain,it was found that the double-gene deletion strain could not obtain a single iron source heme.1.2 The virulence of PmCQ2Δ893Δ894 strain was significantly reducedIn this study,a mouse infection model with a double-gene deletion strain was successfully constructed,and the LD50 was calculated.The results showed that the virulence of the double-gene deletion mutant strain was significantly reduced.Compared with the wild strain,the LD50 of the mutant strain was increased by approximately 1.35×10~9times;the double-gene deletion strain had bacterial loads in mouse lung,liver and spleen tissues.It is lower than that of the wild strain;and the amount of bacterial colonization in the organs of mice 8h after infection with the double-gene deletion strain is lower than that of the bacteria colonizing 4h.1.3PmCQ2Δ893Δ894Strain has Good Cross-immunity ProtectionThis project evaluated the cross-protective efficacy of PmCQ2Δ893Δ894 as an attenuated vaccine.Muscle-immunized mice with PmCQ2Δ893Δ894 strain can effectively resist P.multocida type A and type B infections,and the immune protection rate is 100%.In addition,the immune protection rate of PmCQ2Δ893Δ894 against F type P.multocida is 40%.In addition,mice with the immune-inactivated PmCQ2Δ893Δ894 strain can effectively resist infections of P.multocida serotype A and P.multocida serotype B,with immune protection rates of 100%and 87.5%,respectively.The inactivated PmCQ2 strain has an immune protection rate of 87.5%and 37.5%for type A and type B Pm,respectively.The survival rate of the mice in the immune-inactivated PmCQ2Δ893Δ894 group was higher than that of the mice in the immune-inactivated PmCQ2 group,Which implies the possibility that it can be used as a vaccine candidate strain.2.PmCQ2Δ893Δ894 and PmCQ2 Transcriptomics Sequencing in Vitro and AnalysisTranscriptomics was used to analyze the expression of related virulence genes and immunoprotective antigens in PmCQ2Δ893Δ894.First,the analysis of DEGs related to virulence factors revealed that a total of 58 virulence factors were significantly down-regulated.Among them,capsule synthesis/transport,LPS and outer membrane protein related virulence genes were significantly down-regulated.Secondly,the expression of Ton B,Exb B,Hmu T and Hmu U genes in the PmCQ2Δ893Δ894 mutant strain was significantly reduced,and these genes are involved in bacterial heme transport.Finally,analysis of immunoprotective antigen-related genes DEGs showed that the expression of 110 genes was significantly up-regulated,and the proteins expressed by 12antigen-related genes have good immunoprotective effects in animals.3.Bio-informatics Analysis and Cloning Expression of Bovine Serotype A P.multocida DUF2623.1 DUF262 protein bio-informatics analysisUsing Signal P-5.0 online analysis software to predict the signal peptide of DUF262 protein,it is concluded that the protein has no signal peptide.Using TMHMM Server v2.0 software to analyze the transmembrane domain of DUF262,the results show that DUF262 has no transmembrane domain,and the probability of cells being located on the outer membrane is the greatest.The NCBI was used to analyze that the protein exists in a variety of common strains,and they are all proteins with unknown functions.3.2 Immune Protection of Recombinant Protein r DUF262The recombinant protein r DUF262 was constructed with PET-32a(+)as a carrier,and the immunoprotection of the protein was explored.Mice immunized with r DUF262 protein resist infections of the same serotype(Pmtype A)and non-serotypes(Pmtype B),and their immune protection rates are 30%,50%,and 40%,respectively.It shows that the immune PmCQ2Δ893Δ894strain can play a certain immune protection against Pmof different serotypes.