Identification And Pathogenicity Study Of Hafnia Alvei From Rainbow Trout(Oncorhynchus Mykiss)

As a gram-negative and facultative anaerobic bacteria,Hafnia alvei is a kind of conditional pathogenic bacteria of Enterobacteriaceae family,which widely exists in the intestines of mammals,and can also be detected in natural environment and frozen food because of its putrefactive characteristics.Infection of Hafnia alvei could cause the infected rainbow trout（Oncorhynchus mykiss）surface bleeding and inflammatory reactions,and also acute death,all of these lead a greatly affecting to the rainbow trout breeding industry.Therefore,it is of great significance to isolate and systematically study the biological characteristics and pathogenicity of Hafnia alvei strains from rainbow trout,in order to further study the pathogenic mechanism and prevention and control of Hafnia honeycomb.A dominant bacterial strain was isolated in this study,named HA01,from the diseased fish lesion tissue of a certain rainbow trout farm in Liaoning.All the diseased rainbow trout had slow swimming and black bleeding on the body surface.The biological characteristics,drug resistance,and pathogenicity of the strain were studied using 16S r RNA sequence analysis,physiological and biochemical identification,drug sensitivity disk method,and q RT-PCR methods.The research results are as follows:1.The results of biological characteristics showed that HA01 strain was gray-white,round,with relatively neat edges on TSA plates,and could use glucose,water lactose,escin,V-P experiment,ONPG experiment,OF experiment,catalase experiment was positive,nitrate reduction,hydrogen sulfide,hydrolyzed gelatin,starch,etc.The 16S r RNA comparison results showed that the homology between HA01 strain and the reference strain ATCC 29926 of Haffni was 99.65%.The physicochemical and molecular characteristics of the strain were combined to determine that the HA01 strain was Haffni hive.2.The results of drug resistance showed that the HA01 strain was sensitive to enrofloxacin,gentamicin,streptomycin,chloramphenicol,and methoxypyrimidine,but resistance to amoxicillin,penicillin,erythromycin,rifampicin,and ampicillin.3.The pathogenicity results showed that the semi-lethal concentration(LD₅₀)of HA01 strain to rainbow trout juveniles was 4.80×10⁹ CFU/m L,and Hafnia alvei could be re-isolated from artificially infected experimental fish,which conformed to Koch’s rule.The observation results of histopathological sections showed that pathological changes were observed in the spleen,liver,and intestines of infected rainbow trout:spleen lymphocytes were reduced,red blood cells were greatly increased,and there was obvious blood cell infiltration.Hepatocytes swelling,mild watery degeneration,partial hepatocyte nucleus dissolution disappears,cytosol is loose and lightly stained;Intestinal cells vacuolize,inflammatory cells infiltrate,intestinal villi rupture.By using the plate counting method to detect the pathogen load in various tissues of experimental fish after infection with HA01 strain,the results showed that Hafnia alvei were detected in the liver,spleen,kidney and intestine 6 h after infection,and the highest pathogenic bacterial load in the liver was 6.7×10⁷CFU/g.At 24 h of infection,the pathogenic bacteria load of each tissue reached the maximum,and the liver pathogenic bacteria load was the highest,which was 8.3×107 CFU/g,and the pathogenic bacteria load of other tissues was slightly lower than that of the liver.From 24 h to 48 h,the pathogenic bacterial load of each tissue decreased significantly,and gradually decreased after 48 h until complete elimination.4.The immune response results of rainbow trout indicate that the content of red blood cells and hemoglobin in the blood of the test fish after infection showed a trend of first decreasing and then increasing,the content of 72 h was significantly lower than that of other time points（P<0.05）,the content of white blood cells showed a trend of first increasing and then decreasing,and the content of 48h was significantly higher than that of other time points（P<0.05）;liver lysozyme（LZM）,alanine aminotransferase（GPT）,alkaline phosphatase（AKP）,Superoxide dismutase（SOD）,catalase（CAT）and total antioxidant capacity（T-AOC）all showed a trend of first increasing and then decreasing,and peaked at 48 h.It was significantly higher than that of other time points（P<0.05）,and the expression of related genes that indicate immune levels and inflammatory Ig M,IL10,growth-related genes FGF and proinflammatory factor SAA in liver and spleen showed a trend of first increasing and then decreasing,and the change between each time point was significant（P<0.05）,and the expression of each detected gene peaked at 48 h.In summary,the HA01 strain isolated from the lesion tissue of diseased rainbow trout was identified as Hafnia alvei.After infecting rainbow trout,it can cause pathological changes in the liver,spleen,and intestinal tissues of diseased fish.Infected fish suppressed the content of pathogenic bacteria in the body by improving antioxidant capacity and immune function.And the pathogen is sensitive to drugs such as enrofloxacin and gentamicin,which provides a theoretical basis for the prevention and treatment of diseases caused by Hafnia alvei infection in aquaculture production.