Isolation And Identification Of Photobacterium Damselae In Litopenaeus Vannamei And Preliminary Study On The Pathogenic Mechanism

The Litopenaeus vannamei which is cultured in small sheds in Rudong County(Jiangsu Province)died sporadically and regionally for unknown reasons in March 2022,caused economic crisis,and the farmers called it"Dixing Disease".In order to explore the cause of the disease,this study conducted the pathogen isolation,identification and pathogenicity study of the diseased Litopenaeus vannamei.This can provide scientific basis for the prevention of the disease.A dominant strain MRY0520 was isolated from the hepatopancreas of dying diseased shrimp,and its pathogenicity was confirmed by artificial challenge.The strain MRY0520 was observed morphologically,identified physiologically and biochemically,and identified by molecular biology using 16S rRNA gene.Through phenotypic biological observation and molecular biological analysis,the strain MRY0520 was identified as Photobacterium damselae subsp.damselae.The artificially infected Litopenaeus vannamei could cause the same symptoms and death.Pathogens were reisolated from infected individuals,and it was found that the dominant strain isolated from the hepatopancreas of endangered shrimp was consistent with the morphological characteristics,physicochemical properties,and 16S rRNA sequence of MRY0520,confirming its pathogenicity.The results of artificial challenge showed that the LD50 of the bacteria to Litopenaeus vannamei was 2.15×10~5 CFU/g,the drug sensitivity test showed that strain MRY0520 was sensitive to neomycin(μg/disc),doxycycline(μg/disc)and other drugs.The results showed that the pathogen causing the death of Litopenaeus vannamei was Photobacterium damselae subsp.damselae,which was pathogenic to Litopenaeus vannamei and could cause the death of it.In order to understand the whole genome sequence information of Photobacterium damselae MRY0520 and further analyze its pathogenic mechanism.Through the second generation of Illumina NovaSeq and the third generation of PacBio Sequel sequencing platform,the whole genome of MRY0520 was sequenced.The sequencing data were corrected for genome assembly,component prediction and functional annotation,meanwhile the differences between MRY0520 and other strains were analyzed by comparative genomics.The genome size of strain MRY0520 is 4,451,849 bp,and the GC content is 40.87%;A total of 3 663 genes were encoded with a total length of 3,691,779bp,accounting for 83%of the genome.It contains 208 tRNA genes,22 5S rRNA genes,20 16S rRNA genes,20 23S rRNA genes,2 CRISPR sequences and 2 gene islands.3 655,2 399,3 121 and 2 780 genes were annotated in Nr,KEGG,GO and COG databases respectively.Comparative genomic analysis showed that strain MRY0520 had high homology with strains Phdp-Wu-1.The sequence information of strain MRY0520 was revealed by whole-genome sequencing technology,which provided a theoretical basis for further understanding the pathogenesis of MRY0520.For further study the molecular mechanism of Litopenaeus vannamei response to the infection of Photobacterium damselae,reveal its immune and metabolism response effect,and provide a valuable genetic data source for scientific prevention and control of vibrio infection,this paper used the hepatopancreas tissues of L.vannamei infected group(injected with P.damselae suspension)and control group(injected with the same amount of sterile normal saline)as experimental materials,and used the Illumina HiSeq 2500 to sequence.The original sequencing data were assembled,annotated,and the differentially expressed genes under the infection conditions were screened for analysis.A total of222,611,760 clean reads were obtained through splicing and assembly,and the Q30 of each sample clean reads reached about 90%.The comparison efficiency between the sequenced clean reads and the reference genome was 86.24%～88.46%,indicating that the transcriptome data was reliable.Use Stringtie to splice Mapped Reads(Reads that are compared to the reference genome).A total of 1693 new genes were discovered,and these new genes were put into the Nr,KEGG,and GO databases for functional annotation.Finally,a total of 1196 new genes were annotated.Among them,595 new genes were annotated in GO database,accounting for 49.8%;The proportion of new genes annotated in KEGG database was the least,17.9%.Based on the screening conditions of FDR<0.05and|log2FC|>log2(2),1521 differentially expressed genes(DEGs)were obtained,of which 763 genes were significantly up-regulated and 758 genes were significantly down-regulated.Functional enrichment analysis showed that DEGs were mainly enriched in biological processes and metabolic pathways.Eight DEGs were selected,and transcriptome data were verified by Real time RT-PCR.The results showed that it was reliable to screen DEGs under infection conditions based on transcriptome sequencing.Among them,metabolism related genes such as betaine homocysteine methyltransferase gene was significantly down regulated,and immune related genes such as heat shock protein was significantly up regulated.The experiment shows that infection of shrimp with P.damselae can affect the host’s metabolic process and stimulate the immune response.