Epidemiological Investigation And Expression Analysis Of Toll-Like Receptors(TLRs) In Responding To Aeromonas Challenge Asian Swamp Eel

In view of the frequent occurrence of Asian swamp eel breeding diseases in summer and autumn,a bacterial epidemiological investigation on Asian swamp eel breeding in Jingzhou city in late summer and early autumn was carried out.A total of 4 kinds of bacteria were identified in the collected objects,namely Aeromonas hydrophila,Aeromonas veronii,Plesiomonas shigelloides and Vibrio mimicus.Among them,8 strains of A.hydrophila accounted for 50% of the total bacteria detected.On the whole,the main pathogenic bacteria of Asian swamp eel in summer and autumn are still A.hydrophila and A.veronii.Toll-like receptors(TLRs)are an important class of protein molecules involved in non-specific immunity and also an important class of pattern recognition receptors.It can recognize and bind pathogen-associated molecular patterns of various pathogenic microorganisms,such as lipopolysaccharide,lipopeptide and ds RNA.TLRs are characterized by extracellular leucine-rich repeats domains(LRRs),transmembrane domains and intracellular Toll/interleukin-1 receptor(TIR)domain.TLR specificity is determined by sequence variation and number of LRR motifs which form horseshoe-like shaped solenoid and involve in recognition of PAMPs.In the TIR domain,there are three conserved functional areas Box1,Box2 and Box3.These conserved functional regions activate the My D88 signaling pathway or TRIF signaling pathway through signal protein transduction,inducing the release of pro-inflammatory cytokines,type I interferons and chemokines,thereby generating an immune response to eliminate the invasion of pathogens.It can be said that the research on TLR and its ligands is the basis for us to understand the mechanism of fish disease resistance.In this study,the sequence characteristics and homology of 12 Asian swamp eel TLR genes(TLR1,TLR3,TLR5 s,TLR5m,TLR7,TLR8,TLR9,TLR13,TLR14,TLR21,TLR22 and TLR23)were identified and analyzed.Sequence similarity analysis,functional domain prediction,protein 3-level structure and phylogenetic analysis supported their annotation and homology.Then the relative m RNA expression levels were detected in the skin,muscle,intestine,liver,spleen,gonad,head kidney,middle kidney and blood tissue of healthy Asian swamp eel.The results showed that TLR3 was highly expressed in the intestine and skin,TLR5 s and TLR22 were highly expressed in the kidney and spleen,and TLR14 was highly expressed in the spleen and muscle.TLR1,TLR5 m,TLR7,TLR8,TLR9,TLR13,TLR21,and TLR23 are all highly expressed in the spleen,which are related to host immunity.The differential expression of TLR gene was detected after artificial infection with A.veronii.The results showed that all TLR genes were induced and expressed in various patterns.Intestinal TLR5 s were induced at 1 h,4 h,and 12 h in the early stage of infection,and peaked at 1 h.TLR8 was significantly down-regulated after1 h,and TLR9 was significantly down-regulated at all time points.Except TLR13 was slightly up-regulated at 4 h,other TLR genes were suppressed to the lowest fold.TLR5 s and TLR5 m were induced in spleen at 1 h,4 h,12 h and 4 h,24 h,respectively.TLR1,TLR3,TLR7,TLR8,TLR9,TLR13 were inhibited at all time points,and the expression of TLR23 was significantly decreased at three time points at 4 h,12 h,and 72 h.TLR14,TLR21,TLR22 gene changes were not obvious.In kidney,except for TLR5 s,which was up-regulated at 1 h and 4 h at the early stage of infection,all other TLR genes were down-regulated to the lowest level at 4 h,and then returned to the basal level at 24 h.This suggests that TLRs play an important role in the innate immune response of Asian swamp eel to Gram-negative bacteria.Finally,the spleen and intestinal samples of the diseased Asian swamp eel were collected to detect the expression of TLR gene relative to the healthy Asian swamp eel when they were infected by A.hydrophila.The results showed that the expression of TLR5 s and TLR7 was upregulated,and the expressions of TLR5 m,TLR8,TLR14 and TLR22 were slightly upregulated in the spleen.In the intestine,all except TLR13 tend to be up-regulated.Among them,the up-regulation of TLR3,TLR5 s and TLR8 was significantly different from that in the control group.