| Crucian carp is the most common freshwater fish in China.In 2012,the production of crucian carp in China was nearly 2 million 450 thousand tons.Allogynogenetic silver crucian carp(C.auratus gibelio ?×Cyprinus carpio var.Singuonensis ?)is the most extensive variety for promotion in the past twenty years.But since 2012,a serious novel disease(hemorrhagic disease of gill)has occurred in the Allogynogenetic Crucian Carp and caused huge economic losses.Different from the other diseases of allogynogenetic silver crucian carp,the disease has the characteristics that occur at low water temperature,strong infectious,rapid onset,high mortality,and difficult to be prevented and controled.Before this,there is no report of this disease in China.The cause of the disease is unknown and the regulartiy of epidemic is not clear.Therefore,it is urgent to carry out the epidemiological investigation of the novel epidemic disease,to identify the pathogens and the cause of the disease in order to establish an effective detection method.On this basis,the possible pathogenicity mechanism of the pathogen was further clarified by using the methods of transcriptomics and proteomics as well as metabonomics.By using the method of metabonomics,research on pathogenic effects of allogynogenetic crucian carp metabolism was carried out to identify the differential metabolites,which laid the foundation for the prevention and treatment of the new disease in the future.1.Epidemiological investigationAccording to "Standards of Aquaculture Disease Monitoring and Reporting in Jiangsu province",epidemic pathogenic investigation was carried out and showed follwing:(1)The gill hemorrhage of allogynogenetic silver crucian carp appeared in spring and autumn when the water temperature at 15?25 ?.(2)The symptoms of the diseased fish included gill hemorrhage,caudal and dorsal fin was pale,hemorrhagic ecchymosis on symptoms of bladder.(3)This disease had obvious infectivity,mainly infected juvenile and adult crucian carp in the pond.(4)The pathogen had strong host specificity and did not infect other species of freshwater fishes in the same culture environment.(5)In ponds with centralized feeding,the disease has a higher rate of infection.2.Histopathology,ultrastructural pathology and pathogen determination of gill hemorrhage disease of allogynogenetic silver crucian carpThe pathological results showed that lamellibranch of diseased fish appeared blood cell infiltration,fusion of gill lamellae,accompanied by necrosis and shedding of epithelial cells.Lamellibranch in clusters of phagocytic cells with the size of the cytoplasmic vacuoles,with weak basophilic display.The glomeruli of the kidney had focal necrosis and the capillary wall was dilated.The nucleuses of the glomerular hypertrophy were constriction,fragmentation,and severe vacuolization.The blood cells and lymphoid cells infiltrated in the spleen,and there was a serious vacuolization.Many splenic cells had marginal chromatin and some appeared to be fragmented.The nucleus of the liver enlarges,had the inclusion body in the nucleus,the nucleuses hypertrophy was accompanied by nuclear pyknosis,and there was a serious vacuolization.The intrahepatic bile duct shows inflammation,and the tube wall cells swollen with each other,resulting in a rough and abnormal tube wall.The intestinal epithelial cells fall off,the intestinal mucosa was damaged,and the connective tissue was proliferated.No pathological changes showed in muscle tissue.The semi quantitative pathological evaluation of the corresponding tissues shown the most significant pathological changes were the kidneys,followed by gills,spleen and liver.Ultrastructural pathology observation revealed virus infection in the gills,spleen and kidneys.The viruses were duplicated and assembled in renal hematopoietic cell.In the nucleus,the virus nucleocapsid naked size is 95-110 nm,enveloped virus particle size is 170-200 nm,more than one together in the cytoplasm.During the maturation process of virus in the cytoplasm,virus particles passed through Golgi complex and entered a vesicle with glycoprotein,forming envelope virus in cell vesicles.Through the two package of cytoplasmic capsid,eventually mature virus particles were formed in the cytoplasm.The ultrastructural characteristics of the observed virus particles are similar to those reported herpes virus previously.The PCR method was used to identify the pathogen in gills,kidney and spleen using the polymerase specific primers of cyprinid herpes virus(cyprinid herpesvirus).The products of 362 bp were amplified and 99%similar to the herpes virus(CyHV-2)of the hemopoietic organ necrosis of goldfish.Based on partial sequences of CyHV polymerase in NCBI,we constructed the phylogenetic tree.The virus and goldfish hemopoietic necrosis herpesvirus(CyHV-2)were strictly clustered into one branch(self guide value 76%).The results show the virus are CyHV-2.After artificial infection,the fish began to die on the third day with the typical naturally infected symptoms such as gill hemorrhage,caudal and dorsal fin was pale and hemorrhagic ecchymosis of bladder.Until the fifth day,all the fish in the infected group died.But no death occurred in the control group until fifteenth days.By molecular detection,the results of the infected fish were all CyHV-2 positive.With electron microscopy,we observed that the herpesvirus in the diseased fish.Consequently,we confirmed that the cyprinid herpesvirus ? is the pathogen of gill hemorrhage of gibel carp.3.Establishment of a detection method of fluorescence in situ hybridization for CyHV-2A CyHV-2 fluorescence in situ hybridization method was established.This method was used to detect the histopathological changes of Allogynogenetic Crucian Carp infected with CyHV-2.The results showed that the probe did not bind to healthy fish tissues with high specificity.The fluorescence intensity of the localized necrotic lesions in the kidney,spleen,liver and gill,which was in accordance with the results of HE staining of histopathology above.For the specific probe test,the maximum fluorescence intensity was 43 ? for CyHV-2 detection.It was also found that when the probe concentration was 5ng/?L,a specific and sufficient fluorescence could be obtained.The high concentration of the probe(50 ng/?L)resulted in the increase of fluorescence intensity,but the specificity decreased.4.The pathogenicity of CyHV-2 based on the study of transcriptomeics and proteomicsUsing transcriptome sequencing,TMT labeling,HPLC and mass spectrometry based quantitative proteomics technology,we detected the differentially expressed genes and proteins in the kidneys of crucian carp after three days infection with CyHV-2.After transcriptional sequence analysis,there were 3090 up-regulated genes in the head kidney and 3995 down-regulated genes.And then,197 proteins expressions were significantly up-regulated and the expressions of 53 proteins were significantly down-regulated by TMT protein quantitative technology.The crosstalk of transcriptomic and proteomic analysis showed that 86 of the transcripts and proteins were up-regulated,and 42 of them were down-regulated simultaneously.KEGG signaling pathway found that up-regulated proteins or genes are mainly concentrated in herpes virus infection signal pathway,RIG-?like signaling pathway,necrotic apoptosis and other immune related signaling pathways.QPCR results further showed that the transcription levels of LGP2,RIG-?,MDA5,FAS,PKZ and PKR genes in these three signaling pathways were significantly up-regulated after CyHV-2 infection,which is consistent with the trend of transcriptome group and proteome.5.Effect of CyHV-2 on metabolism of crucian carp based on metabonomicsPrevious studies showed that CyHV-2 infection had great effects on the metabolism of the kidneys of Allogynogenetic Crucian Carp,especially the two signal pathways including pyrimidine metabolism and arginine proline metabolism,but how the various metabolites changed,so far,it is not clear.In order to further explore the effect of CyHV-2 infection on the metabolism of Allogynogenetic Crucian Carp,LC-Q/TOF-MS analysis platform based on gibel carp CyHV-2 infection serum were investigated.Through the metabolomics data,we identified a total of 79 metabolites,including several carbohydrates(glucose,mannose,ribose),fatty acids(fifteen acid,four arachidonic acid,palmitic acid and twenty hydroxy twenty carbon carbon five acid,four acid,linoleic acid and its derivatives),most of the amino acids and their derivatives were significantly increased.At the same time,the substances such as guanine,cytosine,uridine,VB2 and other substances were significantly reduced.Metabolic pathway analysis showed that the ability of sugar and amino acid transport,tRNA and amino acid synthesis were enhanced. |
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