Transcriptome Analysis Of Spleen Of The Monopterus Albus Infected With Aeromonas Hydrophila And Evaluating The Disinfetion Effect Of Povidone Lodine

Monopterus albus（swamp eel）is an important freshwater aquaculture fish in China.With the rapid development of intensive aquaculture,the outbreak of hemorrhagic disease has become more serious.At present,there is a lack of effective prevention and treatment measures against hemorrhagic diseases.In this study,on the one hand,the transcriptome information of swamp eel was obtained by high-throughput sequencing.On the other hand,the bactericidal effect of povidone-iodine was studied.This is all about reduce the negative influence of hemorrhagic disease on the aquaculture industry in theory and practice.The contents of this study are as follows:1.Transcriptome analysisof swamp eel spleenIn the present study,we performed RNA-Seq sequencing of spleen tissue from swamp eel,which were given intra-peritoneal injections containing PBS with or without a dose of Aeromonas hydrophila.The fish were sampled from the control group at 0 h,and from the experimental group at 4 h,8 h,16 h,24 h and 48 h.Totally,340,562,976 raw reads and 69,705 unigenes were obtained,17,346 unigenes were annotated in public database.After fliter,7571 differentially expressed genes were identified,which involved with 360 signal pathways.KEGG pathway enrichment analysis reveals that the differentially expressed genes were functionally enriched in Hematopoietic cell lineage,Complement and coagulation cascades,Antigen processing and presentation,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway and NOD-like receptor signaling pathway.2.Complement system mediates immune responses to A.hydrophila infectionComparing the relative expression of spleen reference genes（GAPDH,EF1α,RPL17,β-actin,and 18S rRNA）in different infection periods,it suggested that the 18S rRNA was the most stable reference gene,and it can be used as an internal reference gene.Quantitative analysis of the 15 complement associated genes revealed that alternative pathway was continuously activated during the period of infection,classical pathway and lectin pathway might be activated at the late period,it also indicated the complement system play important role in the immune mechanism of swamp eel.3.Disinfection effect of povidone iodineThe bactericidal efficacy of povidone iodine（PVPI）on A.hydrophila were compared with two different methods.The results showed that when treated with tube double dilution method,the minimum inhibitory concentrations and minimum bactericidal concentrations of PVPI were all in the range of 2-4 g/L.By contrast,using direct acting method,10 mg/L of PVPI could kill 100%A.hydrophila in 10 min.This study reveals the bactericidal efficacy of PVPI was susceptible to organic matter,so it is recommended to use the direct effect method to determine the effective bactericidal concentration in aquaculture.The direct acting method was used to determine the bactericidal effect of PVPI in different aquaculture waters.The results showed that the effective germicidal concentration of povidone iodine in outdoor aquaculture water was 25 mg/L.In indoor aquaculture water with 10⁵CFU/mL bacteria,10 mg/L povidone iodine could kill 99%of the bacteria.The minimal germicidal concentration of povidone iodine in deionized distilled water and Luria-Bertani broth was 1.25 mg/L and 4000 mg/L,respectively.The median lethal concentration test showed that the 24h-LC₅₀of povidone iodine to swamp eel was 173.91 mg/L,which was much lower than the germicidal concentrations in outdoor and indoor aquaculture water,indicating its safety and effectively in swamp eel aquaculture to against the A.hydrophila,and without any obvious harm to the individuals.