Isolation,identification Of The Pathogen Of Micropterus Salmoides Nocardiosis And Preliminary Study On The Toxic Function Of Mce4E Protein

In fish,nocardiosis is a kind of disease with a lot of white nodules in whole body.The disease has In fish,nocardiosis is a kind of disease with a lot of white nodules in whole body.The disease has a high mortality rate,spread rapidly,occurs year after year,and has poor drug use.It poses a serious threat to fish.In this study a gram-positive bacillus was isolated from skin ulcers,effusions from fish swimming bladder's cavity and liver of the diseased Micropterus salmoides in Qionglai,Sichuan Province.The pathogenicity of the bacterium was confirmed by regression tests.Physiological and biochemical characteristics,16 s rDNA gene construction phylogenetic tree and other traditional methods,the author believes that it is Nocardia seriolea,called HSY-NS02,which grows slowly.Scanning electron microscopy observations of this bacterium showed that the cells are branching.The average size of the bacteria was 10.1±2.3 ?m×0.7±0.2 ?m.The cells were clearly visible under transmission electron microscope.Two floors.Tthe results showed that the strain was sensitive to gentamicin,neomycin and nystatin.Histopathological observation and analysis of diseased largemouth bass showed that there were different degrees of chronic granulomatous lesions in skin ulcers,heart,liver,spleen,kidney and gill,of which spleen lesions were the most serious.To further study the pathogenicity logarithmic phase,this study performed genome-wide sequencing and analysis of the strain of Nocardia seriolea isolate HSY-NS02.The completed genome of HSY-NS02 comprises 776,219 bp(GenBank accession no.RCNK00000000).The ARDB,VFDB were used for pathogen analysis,showing that there were 17 drug resistance gene and 5 potential virulence factor.Further,the HSY-NS02 genomes were compared in silico to other N.seriolea by using the softwares BRIG.We found a particular gene sequence in HSY-NS02,called Mce4 which is the member of Mammalian cell entry Mce family proteins.Based on this,the mce4 E gene in the Mce4 operon was cloned and expressed,and the mce4 E protein was stimulated to express EPC cells and injected intraperitoneally into healthy Micropterus salmoides to explore the toxic function of the protein..The strain HSY-NS0 did the same operation.The results are as follows: 1 Effect of mce4 E protein and strain HSY-NS02 on EPC cellsThe mce4 E protein and the strain HSY-NS02 were separately incubated with EPCin 24 h.EPC was detected by flow cytometry,showing mce4 E protein casuing EPC a high early apoptosis rate with 5.15±0.13%,compared with the strain HSY-NS02.2 Effect of mce4 E protein and strain HSY-NS02 on Micropterus salmoidesMicropterus salmoides were died after intraperitoneal injected in mce4 E protein with 35% mortality rate.Histopathological observation and analysis of these largemouth bass showed that the mce4 E protein cause ballooming degeneration of liver cell without granulomatous lesions in any organization of largemouth bass.Non-specific immune indexes(including serum total protein,superoxide dismutase,lysozyme)were detected after injected to understand the effect of mce4 E protein on tilapia immune system.The detection of non-specific immune indicators showed that strain HSY-NS02 could cause the serum superoxide dismutase activity of largemouth bass to decrease and maintain at a low level,while serum total protein and lysozyme content increase and maintaine at a high level;mce4E protein can cause serum superoxide dismutase activity of largemouth bass to decrease at the first and increase later.And the difference was not significant with the control group at the final.But,mce4 E protein can cause serum total protein and lysozyme content increase at the first and decrease later.And the difference was not significant with the control group at the final.