| For the characteristics of big individual,good meat quality,great taste and rich in unsaturated fatty acid etc,Atlantic salmon(Salmo salar L)was popular in the world.Now superior varieties have been introduced in China,Atlantic salmon are cultured by using Recirculating Aquaculture System.However,disease caused by Aeromonas salmonicida has seriously restricted the development of the aquaculture industry of Atlantic salmon.A.salmonicida is opportunistic pathogen and only cause disease when in appropriate environment.Therefore,it is very important to explore the impact of ecological factors on the pathogenicity of A.salmonicida,but it is difficult to find reports on this.This study combined with the production data and aseries of experiments,screened interaction among bacteria and water temperature as two important ecological factors,and verified the regulatory function of these factors on the bacterial virulence.Main conclusions are as follows:In production,water temperature is related to the mortality of Atlantic salmon with the infection of A.salmonicidaDuring the period of survey,water quality was controlled well in the workshop generally.In addition to the concentration of nitrite fluctuating over 1 mg/L,other water parameters satisfied with Atlantic salmon culture practices.The variation trend of water parameters correlate with each other,especially,water temperature is negative correlated with the change of nitrite concentration(Pearson correlation coefficient-0.505,P<0.01),and ammonia is significantly positive correlated nitrite concentration(Pearson correlation coefficient 0.587,P<0.01).There is significant correlation between water temperature/nitrite concentration and mortality of Atlantic salmon,especially water temperature is negatively correlated with mortality(P<0.01).By regression analysis,mortality of Atlantic salmon M=19.573-1.042T+0.155 NO2-,P<0.01,adjusted R2 0.494,indicating this quation can be used to predict the mortality of Atlantic salmon with water parameters when A.salmonicida outbreaking.AI-2 quorum sensing exists in A.salmonicidaAI-2 activity could be detected from 6 different A.salmonicida strains.In C4,AI-2 activity changes in different growth phase,and is influenced of different culture condition.Gene cloning and sequencing demonstrate AI-2 quorum sensing related lux S and pfs genes integrally exist in A.salmonicida.Bioinformation shows that Lux S in A.salmonicida has key amino acid sites His54,His 58,Cys128 and Gly93 for AI-2 production,and functional protein secondary structure.The result shows that LuxS has function to synthesis AI-2 signals.Phylogenetic tree indicates that LuxS of A.salmonicida mostly homology with other Aeromonas.Bacteria,then Edwardsiella tarda.AI-2 quorum sensing affects pathogenicity of A.salmonicidaThis study constructed a A.salmonicida luxS deficiency strain by homologous recombination,and verified the right recombination by both genetic and phenotype.In same culture condition,C4ΔluxS mutant grows better than wild type,and expression level of most virulence genes do not decrease.However,expression of A-layer protein coding gene vapA decreases significantly in mutant strain(P<0.01),cell hydrophobicity decreases 3.59-fold and auto-aggregation disappears.Results above are verified by SEM observation,and cell size,morphology also change with the lost of Lux S.In challenge experiment,Atlantic salmon infected with C4ΔluxS express innate immune factors TLR5S、IFNα、SOD and POD earlier.Study data indicates that A-layer protein is regulated by LuxS,so LuxS could assist A.salmonicida self-protect,and prolong the survival time in host.Temperature affects virulence of A.salmonicidaECPs of A.salmonicida which cultured under 8℃,10℃,13℃,15℃,18℃ and 28℃ are different with each other,in which bacteria from 28℃ could secret more proteins than other from temperature,but protein of 40 kDa and 100 kDa are lost art 28℃.Growth rate,AI-2 activity and morphology are different for bacteria cultured under 10℃,18℃,28℃.Among these three groups,843,1212 and 521 genes differentialy express,which concentrate on the reaction such as catalize and bingding.Differential expressed genes distribute in 114 KEGG pathways,for example,expression of type three secretion system related coding genes are negatively regulated at 28℃.Results demonstrate that temperature could affect the virulence of A.salmonicida. |
