Isolation And Identification Of Pseudomonas Aeruginosa From Mink And The Construction Of ?Hsp33 Deletion Strain

Mink hemorrhagic pneumonia is an acute infectious disease caused by Pseudomonas aeruginosa?PA?characterized by hemorrhagic pneumonia.Our team confirmed that the mainly prevalent serotypes of PA were type G and type D,and developed a bivalent inactivated vaccine of mink hemorrhagic pneumonia Successfully.Previous studies of our team confirmed for the first time that the main prevalent serotypes of mink-derived PA were G-type and B-type,and developed a bivalent inactivated vaccine for mink hemorrhagic pneumonia..In this study,the biological characteristics and immunogenicity of serotypes C and D of PA strains,the gene characteristics of virulent strain HY-1 and laboratory-preserved attenuated strain PA1060 and the pathogenicity of virulence factor Hsp33 were studied.The main results are as follows:The biological characteristics and pathogenic characteristics were elucidated.Serotype,exercise ability,biofilm,pyocyanin,growth curve,drug resistance and pathogenicity of PA strains were studied.The results showed that eighteen serotypes of mink-derived PA were G-12 strains,B-1 strain,C-1 strain,D-3 strains and E-1strain.The virulence of C-type and D-type strains was strong,and their immune protection against G-type and B-type standard strains was determined.The swimming ability of 18 PA ranged from 1.8 cm to 9.2 cm,the swarming ability ranged from 1.00cm to 3.15 cm and the twitching ability areas were between 0.008 cm2 to 12.567 cm²..There was no obvious correlation between them.5 PA strains had strong biofilm formation ability,10 strains a medium ability and only three strains are weak in biofilm formation.All 18 strains of PA could produce Pseudomonas aeruginosa and their growth trend was the same.They entered the logarithmic growth phase at 4 h and the stationary phase at 18 h.18 PA strains were 100%sensitive to IPM?AK?CN?CIP?CFP?FEP?LEV?PRL?CAZ?ATM.HY-1 was the most virulent strain with LD₅₀ 1.5 x 10⁶ CFU in mice.The results showed that the LD₅₀ of C type Virulent strain PK13 to mice was 7.5 x 10⁶ CFU and to mink was 7 x 10⁵ CFU and the D type Virulent strain to mice was 2.05 x 10⁷ CFU and to mink was 2.2 x 10⁶ CFU.The immune protection rates of PK13 and H2F were 100%for this serotype strain and80%to 90%for the heterogeneous serotype strainThe genomic differences between attenuated strain PA1060 and virulent strain HY-1 were elucidated.Through comparative analysis of genome sequencing and genome composition of attenuated strain PA60 and virulent strain HY-1,it was found that the number of coding genes scattered in repetitive sequence,non-coding RNA and gene island of attenuated strain was more than that of virulent strain HY-1,but PA60 lacked a gene encoding T3SS secretory system compared with HY-1.Therefore,it was speculated that the main causes of PA60 virulence weakening may include deletion of T3SS secretory system,insertion sequence and gene rearrangement.HY-1 gene-deleted?Hsp33 gene-deleted strain was constructed and its function was verified.The LD₅₀0 of Hsp33 was?6.83+0.38?*10⁵CFU,and the pathogenicity of Hsp33 was lower than wild strain HY-1.The growth curves of deleted strains,complementary strains and wild strains showed that Hsp33 had the weakest growth ability;The biofilm formation ability,swimming and twitching ability were unchanged,the swarming ability of?Hsp33 was?13.76±0.32?cm,which was higher than HY-1.The colour of pyocyanin secreted by?Hsp33 was yellow-green and HY-1 was light green.The adherence rate of Hsp33 to mink lung epithelial cells was 12.6%+2.4%,which was 6.53%lower than that of HY-1.Drug susceptibility test showed that?Hsp33 could induce tetracycline and gentamicin resistance from moderate to sensitive.In summary,type C and type D of PA strains isolated in this study have good immunogenicity,which provides a basis for the development of mink hemorrhagic pneumonia quadruple inactivated vaccine?G+B+C+D?;The whole genome sequence of PA1060 and HY-1 provided data support for analysis the differences between attenuated strain and virulent strain;The function of Hsp33 gene was preliminarily analyzing.This study provides a basis for further prevention and control of mink hemorrhagic pneumonia.