Isolation And Identification Of Aeromonas Hydrophila And Histopathological Observation In Rice Field Eel, Monopterus Albus

Rice field eel,Monopterus albus is one of the high economical value fish in China; it wins customers high favor for its delicious and high nutritive value.From May to June in 2007,we examined a case of high mortality hemorrhagic disease in a commercial rice field eel farm in Sichuan province,China.Its typical clinical syndrome and pathology were that: ecchymosed on body surface obviously,red swelling of the anus,enlarged and pale liver, empty intestine filled by yellowish fluid.The disease has caused large economical loss.In order to understand the pathogen and pathogennesy of the disease,theaetiology,pathology and virulence factors were studied.A pathogenic bacterium(jyl strain) was isolated from the liver,kidney and spleen of the diseased eel.Atifical infection proved that the bacterium was the pathogen of the disease.The carps and loaches had died in 72h after intraperitoneal injection.The strain grew well in nutrient broth,which had flocculent precipitate in the bottom and mycoderm on the surface.On the nutrient agar,bacterium colonies were white to gray,round and convex,and entire margins.The strain was that:facultative anaerobe,mobility, gram-negative rod-shaped bacteria,no spore stage and capsule.The results of biological characteristics were as follows:oxidase-positive,potassium-cyanine-positive, V-P-test-positive,arginine-diphydrolase-positive,H₂S-positive,altose-positive,and so on. It produced gas and acid by means of glucose and arabinose,respectively.The 1419bp 16S rRNA partial sequence of the isolated strain was checked by amplificated using bacteria general PCR primer.Compared to the sequences available in GenBank,the amplified nucleotide sequence(GenBank accession number EU678635) had high homology with the others 16S rRNA sequences of A.hydrophila.A phylogenetic tree was constructed by comparing the16S rRNA sequences of the isolated strain(GenBank) with another relative bacteria species in the GenBank databases.The homology between the isolated strain and another A.hydrophila was 99%.According to the morphological,physiological and biological characteristics and the resultes of phylogenetic analysis,the isolated strain(jyl) was identified as A.hydrophila.Based on antibiotic susceptibility studies,the strain was susceptible to eposerin, spectinomycin,nalidixie acid,efotaxime sodium,lomefloxacin,ciprofloxacin,norfloxain, rocephin and sulfafurazole;but it had an intrinsically high resistance to cefradine,cefalexin, novobiocin,trimethoprim,and ampicillin.Pathological changes of rice field eel infected A.hydrophila were observed in this study.Infection outbreak was an impatient process.The clinical symptoms after atifical infection were similar with the natural disease.The peak of death was between 15h to 33h after post infectionem.9h after atifical infection,there had visible symptoms,such as red swelling of the anus and so on.In 15h after infection,the eel began to death.In 18h after infection,the body surface and abdomen had ecchymosis;the oral of dead eel had red mucous.Pathological changes were characterized by general hemolytic anemia,extensive cellular degeneration and necrosis.The first pathological changes were observed in kidney, swelling in nephric tubule epithelium,and bleeding in interstitial in 3h after infection. Hepatocytes were vacuolar degeneration and pyknotic nuclei in 12h to 15h after infection. In 21h to 27h,kidney epithelial were disintegration,nephric tubules disappeared, catholicity hemorrhage and vacuolar degeneration in liver.In 33h to 39h,glomcrulus and hepatoeyte were degeneration necrosis.