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With water pollution aggravated and intensive aquaculture spread,the occurrence of opportunistic infections has become a significant health problem in farmed fish.Citrobacter freundii is an enterobacterium commonly isolated from diseased and healthy animals,including human,mammals and fish,which is considered to be an opportunistic pathogen.Skin is an essential protective barrier for fish and functions as a first line of defense against invading pathogens,but its molecular mechanism remains unclear.The objective of this study is to screen the differential gene expression in response to pathogen infection in skin of fish,and provide a basis for understanding the fish skin immune mechanism to the pathogen at the molecular level.Here,a strain of bacteria was isolated from intestine in Ctenopharyngodon idellus,which was identified by using the morphological observation,physiological and biochemical characteristics,and 16S rDNA sequence analysis.The results showed that the isolated strain was identified species levels as Citrobacter freundii,and animal tests showed that the strain was lethal to zebrafish.Immune response in skin of fish analyzed by using Citrobacter freundii induced zebrafish as a model,which applied the Affymetrix zebrafish cDNA microarray hybridized to the skin tissues.Total RNAs were isolated from the skin tissues of the zebrafish and labeled with biotin,and hybridized to zebrafish cDNA gene chips.The expression profiles from the hybridization to 15617 genes in zebrafish cDNA array were analyzed by the GeneChip Operating Software(GCOS 1.4).Out of 15617 genes in zebrafish cDNA chips,total of 88 immune-related genes were identified to be significantly expressed in the skin tissues,of which 74 genes are up-regulated and 14 are down-regulated in the zebrafish skin tissues.Furthermore,the differential expression genes could be categorized into eight functional groups by using the Gene Ontology(GO)method,including complement activation,acute phase response,defense immune response,platelet activation,coagulation factors,response to stress and stimulus,apoptosis,antigen processing and presentation,cell adhesion and migration,etc.In addition,the analysis of the pathways showed that the MAPK(hsp70,daxx,nfkbiab),JAK/STAT(ifn1),and TGF-β(thbs1) signaling pathways may be involved in the immune responses of skin in fish.The Affymetrix zebrafish cDNA microarray is useful for identifying the functional genes involved in the skin immune defense of fish,and future functional analysis of these genes may contribute to understanding the mechanisms of skin immune responses and offer a scientific reference for the Citrobacter-zebrafish model of bacterial pathogenesis. |
