The Changes Of Immune Performance In Aeromonas Hydrophila Infection And Genome-wide Methylation Analysis Of Ctenopharyngodon Idella

Grass carp is the most important aquaculture fish species in China.Bacterial septicemia is a important disease caused by the infection of Aeromonas hydrophila is always restrict the development of the grass carp aquaculture.In addition,we measured the spleen genome DNA methylation of grass carp infected with Aeromonas hydrophila.Combine with immune index to explore the immune mechanism of grass carp infected with Aeromonas hydrophila.We aim to provide the theoretical basis for grass carp breeding for disease resistance.The results follows:1.Evaluate the healthy status of grass carp.We adopted hematological analysis,serologic assays and Histopathological assessment method to evaluated the grass carp health status.hematological analysis included the RBC,WBC,HCT,MCV and DLC.The DLC consist of the percentage of monocytes,neutrophils,thrombocytes,and lymphocytes.The serologic assays contain the level of ALT,AST,LYZ,POD,C3,C4 and IgM content.We use gill,liver,intestine and kidney to histopathological assessment,and found that the value of WBC was 10 times higher than reference value.The percentage of neutrophils was lower than the reference,and thrombocytes was higher than it.The result of serologic assays shows the low ALT and AST but high IgM content compare to the reference.The organ index of gill and liver was higher than other tissue,histological index was 21-40(range Class III-Class IV).The organ index of kidney and intestine was a little bit lower than other organs(on more than Class II).The result shows the grass carp may suffer from the injure from water quality or disorder of nutrition metabolism,the gill and liver became function impairment.We also not found the infection by pathogen.2.Immune index for grass carp infected with Aeromonas hydrophila.We took fishes to infection experiment,in case of the healthy status of grass carp were good.The medial lethal concentration was 107cfu/ml.Treatment group was injected bacteria solution via re-activation,control group was injected equivalent PBS.Three fish were sampled at 0,1,3,7,14,and 21 days post injection(DPI)from each group.The RBC,WBC,HCT,MCV and DLC of hematological analysis and TP,LYZ,POD,IgM,and ACH50 were measured.Every index was measured for three times.The result shows: the WBC of treatment group was significant higher than control group.The percentage of monocytes and neutrophils was significant higher than the control on 1 DPI.The percentage of thrombocytes in treatment was significant increase first then go down on 7-21 DPI,but the percentage of lymphocytes on the contrary.At the early stage of infection,the total protein content of treatment was significant increase in 1-3 DPI.LYZ activity was increase first and then decline,there was significant difference compare to the control.The POD was significant decrease compare to the control in 1 DPI.The content of IgM and total complement was significant increase in 7,21 DPI,respectively.Hence,we infer grass carp infected bacteria may produce intensive immune response.The increase of IgM stands for the joining of acquired immunity.The key time point may 3-7 DPI.3.Genome DNA mathylation for grass carp infected with Aeromonas hydrophila.We use medial lethal concentration of Aeromonas hydrophila infected grass carp.Spleen was obtain from 0,12,24,48,72 hours post injection(HPI).Genome DNA was extracted and the quality was detected with 1% agarose gelelectrophoresis.Then we use the DNA sample to 5-mc% detection.Found that the genome DNA mathylation was highest in 72 HPI.Rather than 0 HPI.So,we use two time points(0h and 72h)spleen sample to high-throughput sequencing,found CG,CHG and CHH sites had no difference between 0 and 72 HPI.The percentage of CG sites mathylation were increase with the cover degree rising up.But CHG and CHH sites were gradually decreased.We put four spleen samples into pairwise comparison,found there were 974 and 1010 differential expression genes in genebody.And there were 1812 and 2011 differential expression genes in TSS.693 differential expression genes were assigned to 1122 GO functional.And 236 differential expression genes were marched to 127 pathways.It was included the same gene can join in the different GO or pathways at the same time.In addition,we also discovering the significant increasing in methylation such as ABC transporters pathway,Ubiquitin mediated proteolysis pathway,the Fanconi anemia pathway and p53 pathway.We also found a markedly decreasing about Lysine degradation pathway,and Cell adhesion molecules(CAMs)pathway.These processes may play a important role in disease resistant of grass carp directly or indirectly.