Construction And Biological Characteristics Of Pasteurella Multocida L31 And SpeF Gene Deletion Strain From Bovine Type A

Bovine encapsulated Pasteurella multocida(Pm)is one of the important pathogens causing bovine respiratory disease(BRD).With the continuous development of ruminant animal husbandry,large-scale breeding has become the main body,and the harm caused by the cattle-derived capsular type A Pm has become increasingly prominent.At present,there is no other better treatment method than the administration of antibiotics,but this method is bound to make the bacteria highly resistant.Therefore,there is an urgent need to develop new safe and effective vaccines or drug resistance inhibitors for the prevention and treatment of bovine-derived capsular type A Pm.This study took the virulent Pasteurella multocida strain Pm3 isolated from cattle farms as the research object.Based on the transcriptome sequencing results of Pm3 and Pasteurella multocida attenuated strain Pm64 in the previous laboratory,two differentially expressed genes(DEGs)that may be related to virulence are 50 S ribosomal protein encoding gene L31 and ornithine decarboxylase encoding gene speF,respectively.Compared with Pm64,the expression of L31 gene was up-regulated by 6.8 times and speF gene was down-regulated by 7 times in Pm3.Therefore,L31 and speF genes were selected as the research focus to explore the regulatory roles of L31 gene and speF gene in Pm.In this study,L31 and speF gene deletion strains and complemented strains were constructed,and the phenotypes of growth,drug resistance and virulence were compared and analyzed.The biological characteristics of the deletion strain ΔL31 are as follows: The morphological characteristics of the deletion strain ΔL31 and the wild strain Pm3 are basically the same as observed by Gram staining,and the growth curve measurement results show that there is no significant difference in the growth rate between the deletion strain and the wild strain;Star as the representative drug,the minimum inhibitory concentration of enrofloxacin,resistance formation time and tolerance were determined.The results showed that the tolerance of the deletion strain to enrofloxacin was reduced,and the resistance formation time was shorter than The wild strain is prolonged;in terms of virulence,by comparing the LD50 of mice and the survival ability of macrophages,compared with the wild strain,the LD50 of the deletion strain ΔL31 is increased by 10 times,and the pathological changes caused by the deletion strain to the lungs of mice Compared with the wild strain,its survival ability in mouse mononuclear macrophage RAW264.7 was significantly lower than that of the wild strain.The results of the biological characteristics of the deletion strain ΔspeF are as follows: Observed by Gram staining,the morphology of the deletion strain ΔspeF and the wild strain Pm3 has not changed significantly,but the growth curve measurement results show that the growth rate of ΔspeF in the logarithmic growth phase is faster.ΔspeF entered the plateau phase earlier than the wild strain Pm3;in terms of drug resistance,enrofloxacin was also used as the representative drug,and its minimum inhibitory concentration,drug resistance formation time and tolerance to enrofloxacin were determined,and the results showed that the drug was missing.The minimum inhibitory concentration of the strain did not change,but the formation time of resistance to enrofloxacin was faster than that of the wild strain and the tolerance was improved;in terms of virulence,the LD50 of ΔspeF was 2 times lower than that of the deletion strain Pm3.The pulmonary hemorrhage of the latter mice was severe,and there were a large number of inflammatory cells infiltrated.The survival ability of the mouse mononuclear macrophage RAW264.7 was not significantly different from that of the wild strain Pm3.The above results indicate that L31 gene may have an important relationship with the virulence of bovine capsular type A Pm,and it may even be an important target gene when it exerts virulence,which can be used as a potential virulence factor of bovine capsular type A Pm.As a regulatory gene that affects the production of putrescine,the deletion of speF not only affects the virulence of the strain,but also has a certain impact on the growth of the strain,the formation time of drug resistance and the tolerance of drugs.The above test results are of great significance for the exploration of the pathogenic mechanism of bovine capsular type A Pm or the development of new vaccines in the future.