| An acute infectious disease was outbreak in a meat duck farm in Sichuan.The main symptoms of sick ducks were blepharitis,astasia,green excrement et al.The liver,spleen,and brain of the sick ducks were all swollen due to haemorrhage,mesenteric congestion,pericarditis,etc.In order to identify if the ducks were infected by bacteria,the following experiments were carried out in this study:1.Isolation and identification of pathogenic bacteriaThe livers of dead ducks were selected and cultured in 5%sterile defibrillated sheep blood plate.A gram-negative strain was isolated and identified by morphological observation,biochemical test and 16S r RNA sequencing.Biochemical tests showed that the strain was positive in glucose fermentation test,oxidase test and thiase test,but negative in other sugar alcohol fermentation tests.After sequencing the gene encoding 16S r RNA of the strain,it was found that the 16S r RNA gene of the strain was more than 98%identity to that of the iguana-derived Neisseria.According to the above results,this bacterium was defined as Neisseria of duck,preliminarily.2.The biological characteristics analysis of this NeisseriaIn order to explore the biological characteristics of this Neisseria,the growth characteristics,drug resistance,pathogenicity and evolutionary analysis were studied.The bacterium was cultured on 5%sterile defibrillated sheep blood plate through streaklines.After 24 h of culture at 37℃,gray,moist,smooth and round bacterial colonies could be observed,and no hemolysis occurred.The isolated Neisseria was cultured in TSB medium with shaking.The result was shown that it was grown well and the highest OD600was close to 3.5.MIC test experiment showed that Neisseria was sensitive to 24 kinds of antibiotics,such as cefoxitin and amtrinan,and resistant to amoxicillin,ampicillin and streptomycin.The 3-day-old ducklings were infected with the dose of 1011 CFU/duckling via eyes,and no symptoms,such as conjunctivitis and eye swelling,were observed within a week.The 3-day-old ducklings were infected with leg muscle at the dose of 1011 CFU.No death was found in the ducklings within a week,but the bacteria could be isolated from the lesions such as bleeding in the liver and brain.In conclusion,the Neisseria isolated in this study was an attenuated strain.It was not the main cause for the death of ducks.3.Whole genome sequencing and evolutionary analysis of NeisseriaIn order to further study this isolated bacterium,the second generation sequencing technology was used to complete the whole genome of the Neisseria.The results showed that the total length of the Neisseria gene was about 2 088 147 bp,the average length of the gene was about 824.38 bp,the average GC content was about 50.89%,and the total number of the predicted genes was about 2533.The predicted coding sequences of Neisseria were annotated in 11 COG、GO、KEGG databases.The results showed that the genes encoded by Neisseria were mainly involved in cellular process and metabolic process in GO database.It was noted in COG database that the coding genes of this bacterium were mainly involved in the cellular and signal transduction（Information）categories.It was noted in the KEGG database that these coding genes are related to the processes of cell metabolism.In summary,the predicted biological processes of the genes are mainly involved in metabolism and enzyme synthesis and transport.A total of 27 secreted systematic-effector proteins of typeⅢwere annotated in the VFDB database,which were analyzed as potential virulence factors of Neisseria.The 16S r RNA sequence obtained by sequencing was compared with the Neisseria from different sources for evolutionary tree analysis.The Neisseria was closely related to other species of Neisseria,and the Neisseria from lizards was the most similar.In conclusion,Neisseria is not the main pathogen threatening the development of duck industry,but it is necessary to prevent the secondary infection of Neisseria. |
