Construction Of The Research Model Of Bacterial Enteritis And Effects Of Bacterial Pathogen On Intestinal Microflora In Lined Seahorses,Hippocampus Erectus

In recent years,seahorse aquaculture is a growing industry due to increasing market demands and scarce wild resources.However,seahorses are extremely prone to stress,especially under farmed conditions,which makes them more vulnerable to infection and disease.Bacterial diseases,especially bacterial enteritis,were commonly found in farmed seahorse and caused heavy losses.Little is known about pathogenesis and treatment of bacterial enteritis,primarily because of the paucity of reproducible experimental models of intestinal inflammation in fish,although it is a widespread inflammatory disease in most farmed fish,In present study,lined seahorse(Hippocampus erectus),which is predominantly cultured in China,was selected as the research subject.The potential pathogens were isolated and identified from the diseased seahorses by traditional,molecular and biochemical methods,and the pathogenicities of these pathogens were confirmed by challenge experiments.For construction of reserch model,proper dose of chanllenge and sensitive indictors were evaluated.Finally,the effects of pathogen infection on both construction and function of intestinal microflora were investigated.And the results showed that:1.Four candidate pathogens were isolated and preliminarily identified as Edwardsiella tarda,Halomonas meridiana,Vibrio alginolyticus and Pseudomonas sp.by 16S r DNA sequencing.After intraperitoneal injection,both E.tarda and P.sp.could induce typical symptoms of bacterial enteritis,such as anorexia,little activity,anal inflammation,and even host death,while H.meridiana and V.alginolyticus could not.E.tarda was chosen for subsequent experiments,because P.sp has been reported previously.2.The pathogenic of E.tarda was precisely classfied by Gram staining,scanning electron microscope(SEM)analysis and biochemical test,and named as E.tarda YT1.3.E.tarda YT1 was diluted into three concentrations(1×10~5,1×10~7 and 1×10~9colony forming units[cfu]/ml)with sterile physiological saline solution(PSS),and were used as experimental groups for a 21-day chanllenge experiment with PSS as control.And the results showed that,contrasting with the PSS and lower doses injection groups(1×10~5 and 1×10~7 cfu/ml),seahorses began to die on day3 in the high dose group(1×10~9 cfu/ml)with the final mortality rate of 67.5%.Considering these observations,the challenge doses of 1×10~5 and 1×10~7 cfu/ml,which could effectively induce inflammation without causing death,were considered appropriate for constructing experimental enteritis models in seahorses.4.To monitor the inflammation process and to evaluate the severity of intestinal inflammation in fish,44 external,histological and molecular indicators were evaluated.The the results showed that:(1)the infection inhibited body length increase,significantly decreased body weight(P<0.05);(2)19 external(weight,height,anal inflammation,feeding status,and intestinal fluid retention),histological(goblet and inflammatory cell numbers and thickening of lamina propria and muscularis mucosae),and molecular(hepcidin,liver-expressed antimicrobial peptide[LEAP],lysozyme[LYZ],piscidin,interleukin[IL]-1?,IL-1?receptor,IL-2,IL-10,interferon1[IFN1],tumor necrosis factor[TNF]-?,and toll-like receptor 5[TLR5])indicators were suitable for model evaluation,as they could sensitively respond and varied similarly throughout the experiment;(3)the evaluating system could reflect the pattern and intensity of disease progression,and(4)TLR5 may play an essential role in triggering host immune responses during E.tarda-induced chronic enteritis.5.The results of 16S r DNA sequencing analyzing of intestinal microflora by using the above model showed that E.tarda YT1 infection could:(1)change the structure and richness of intestinal microflora,(2)significantly reduce the abundance of basic intestinal bacterial flora of genera Psychrobacter,Macrococcus and Rothia,and simultaneously increase the abundance of pathogenic genera Edwardsiella and Vibrio(P<0.05);(3)up-regulate the carbohydrate transport and metabolism and cell motility,and down-regulate lipid transport and metabolism,and biosynthesis,transport and catabolism of secondary metabolites of intestinal bacterial functions percentage.Therefore,our research may provide a valuable context in which to better understand the mechanism underlying the pathogenesis of fish enteritis and identify potential therapeutic drugs for intensive aquaculture,as well as search for markers suitable for genetic improvement of disease resistance in seahorses.