| Cyprinus carpio var.Koi are currently listed among the most important ornamental species in China,however,various infectious diseases in koi carp have emerged with great economic losses.Seven gram-negative strains were isolated from diseased koi carp and tentatively named strain KCL-1 to KCL-4,KCS-1 to KCS-3.Based on physiological and biochemical characteristics,and 16S r DNA gene and gyr B gene to confirmed the KCL-1 to KCL-4 were Aeromonas veronii（A.veronii）and KCS-1 to KCS-3 were Vibrio cholerae（V.cholerae）.Subsequently,study the pathogenicity of single-and dual-infection through experimental challenges.The infection experiment showed that the median lethal dosage(LD50)for KCL-1（A.veronii）was calculated as 6.93×10~4 CFU/g weight,average mortality is 82.5%;5.48×10~5 CFU/g weight for KCS-1（V.cholerae）and average mortality is 65%.Co-infection of four different doses caused markedly high mortality of 50-100%.Through the fluorescence in situ hybridization,showed both pathogens（V.cholerae and A.veronii）can actively together in the liver and spleen on co-infected koi carp.Then pathological observation by the H＆E staining,that the liver was hyperaemia and vacuolated with focal necrosis cell;the spleen has arise lots of focal necrosis,melano-macrophage center and hemosiderin deposition;the head kidney has haemorrhage and the mid-kidney showed renal tubular disappears;intestine showed inflammatory cell infiltrating,villi shedding and connective tissues became collapsed;on the gill showed secondary lamellar epithelial hyperplasia,lamellar fusion and epithelial detachment.And the AB-PAS staining showed the number of mucus cells of infection groups were more than the control group（P<0.05）.The healthy koi carp were infected with A.veronii,to study the changed of immune index and the effect on intestinal flora structure after infection.By the results,the acid phosphatase（ACP）and alkaline phosphatase（AKP）were significant reduction on head kidney（P<0.05）;peroxidase（POD）was significantly increased on liver（P<0.05）;malonic dialdehyde（MDA）in the liver,spleen,head-kidney and intestine was significantly increased（P<0.05）.The results of intestinal flora structure,showed the OTUs number is 323 for infection group,158 OTUs for control group,and 138 OTUs were shared by the two groups,there a significant difference for the OTUs number between the two groups（P<0.05）.Shannon,ACE and Chao index for the infection group were more than the control group,and the Simpson index was less than control group.There were 7 predominant phyla shared in the top 10 by the two groups,that were Proteobacteria,Fusobacteria,Bacteroidetes,Spirochaetes,Firmicutes,Verrucomicrobia,Cyanobacteria,respectively.The specific predominant phyla by the infection group were Lentisphaerae,Thermi and GN02;by the control were Tenericutes,Actinobacteria and TM7.In functional prediction of gut microbiota at the level 2 of KEGG,there were significant differences in 32 functions（P<0.05）.In conclusion,we have confirmed the strain A.veronii and strain V.cholerae were the pathogeny to koi carp,and the toxicity for A.veronii is higher than V.cholerae.When the koi carp has been infected,the tissue structure will have pathological changes.There was unable to distinguish between single-and dual-infection which through the pathological observation.And when the strain A.veronii infected koi carp,which will damage the internal organs,changes the structure of intestinal flora,improve the diversity and richness of intestinal flora,lead to the imbalance of intestinal flora and to die of koi carp. |
