Isolation,genome Sequencing And Development Of Propolis Adjuvant Inactivated Vaccine Of Enterococcus Casseliflavus FML1805 From Moschus Berezovskii

Enterococcus casseliflavus is a common opportunistic pathogen of zoonoses,which can lead to pneumonia and death in artificially raised musk deer.An infectious pneumonia with high mortality occurred in an artificial musk deer farm in Hanyuan,Ya'an in April2018.In this study,a strain of Enterococcus casseliflavus was isolated from the lungs of forest musk deer,which died of infectious pneumonia.It was named FML1805.After physiological and biochemical identification as well as 16S r RNA sequence analysis of FML1805 strain,the mouse bacteria attack test,genome sequencing analysis and ANI calculation analysis were also carried out,and then the propolis inactivated vaccine was trial produced.After animal safety tests,the vaccine was injected into rabbits and musk deer respectively,and the titer of serum antibody of rabbits and musk deer within 84 days after three immunizations was detected by indirect ELISA.The main test results are as follows:1.Isolation and pathogenicity test of Enterococcus casseliflavus from lung of musk deerCombined with the biochemical test of the isolated strains and the comparison of 16S r RNA determination,the isolated strain from the lungs of dead musk deer is Enterococcus,and has the highest homology with 16S r RNA of Enterococcus casseliflavus.The isolated strain could cause mouse disease,the autopsy showed that the lung lesions of mice were more obvious,and other organs also had different degrees of lesions.After the preparation of pathological sections,it was found that the heart,liver,spleen,lung and kidney of mice had histopathological changes.The half lethal dose of the isolated strains to mice is 5.9×10⁸CFU/m L.The results of drug sensitivity test showed that the isolated strains were resistant to vancomycin,aminoglycosides and cephalosporins.2.Sequencing and analysis results of the whole genomeThe genome size of the isolate was 5321641 bp,including 6521 open reading frames(ORF).The results showed that the average sequence of the whole genome of it was99.54%consistent with that of Enterococcus casseliflavus EC20.The whole genome data was successfully uploaded to NCBI,named FML1805,login number:WEKZ0000000.The analysis of the whole genome showed that there were 19 genes related to drug resistance and 16 genes related to virulence.3.Trial production of Propolis adjuvant inactivated vaccineIn this study,natural propolis was used as adjuvant to develop inactivated vaccine,which passed the animal safety test after passing the test.The serum antibody level of rabbits and musk deer was measured by indirect ELISA within 84 days after three immunizations When the OD₆₀₀nm value(N)is greater than or equal to 2.1,the minimum serum dilution multiple is used to express the serum antibody level.The results showed that the serum antibody titer of rabbits is stable at about 3100 times within three months after completing the immune program,and that of forest musk deer is stable at about 1500times within three months after completing the immune program.In conclusion,a Gram-positivecocci named FML1805 was isolated and identified from the lung of musk deer.The whole genome of FML1805 was determined to be a vancomycin resistant variant of Enterococcus casseliflavus,and the pathogenicity and resistance mechanism of the secondary strain were studied.Finally,Propolis adjuvant inactivated vaccine was trial produced,which laid the foundation for the clinical treatment and prevention of Enterococcus casseliflavus.