Identification And Pathogenicity Of Aeromonas Strains Associated With Skin Ulceration Disease Of Cynoglossus Semilaevis Günther

Cynoglossus semilaevis Günther is popular with both Chinese consumers and farmers,for it has a high nutritional value and economic value.However,with the continuous development of the factory farming model and deterioration of aquaculture environment,diseases become a key factor restricting the healthy development of C.semilaevis Günther aquaculture.The most common diseases of tongue sole are skin ulceration disease and tail-rot disease.C.semilaevis Günther cultured in a factory in Jiangsu province suffered from severe skin ulceration disease.On the body surface and head,there were ulcers with different sizes and different degrees.The wounds of severe diseased fish were big and bleeding,and the fish was dying.Dominant bacterial strains were isolated from depths of these ulcers before identification of bacteria,analysis of biochemical characteristics,drug susceptibility test and study on pathogenicity.1.Isolation and molecular identification of bacterial strains associated with skin ulceration disease of C.semilaevis GüntherTwo dominant bacterial strains(HX0416 and HX1316)were isolated from depths of ulcers of C.semilaevis Günther suffering from severe skin ulceration disease using the sheep blood agar,and there were more bacterial strain HX0416 than HX1316.Moreover,the two strains are both gram negative,and bacterial strain HX0416 is beta-haemolytic,different from the bacterial strain HX1316.Based on the PCR amplification for 16 S rRNA gene,bacterial strain HX1316 was identified asAeromonas media.Meanwhile,based on 16 S rRNA gene and gyrB gene as well,the bacterial strain HX0416 was finally identified as Aeromonas salmonicida.2.Biochemical characteristics and antimicrobial susceptibility tests of Aeromonas strains associated with skin ulceration diseaseThe strain HX0416 was positive for these characteristics such as oxidase,O/129,indole,arginine dihydrolase,fermentation of glucose,sucrose,maltose,arabinose and esculin hydrolysis.These characters are inaccordance to those of Aeromonas salmonicida.Besides,in all the items detected by API 20 NE,the strain HX0416 was only negative to five items such as urea,capric acid,adipic acid,citric acid and Phenylacetic acid.According to the manual “Bacterial fish pathogens: disease of farmed and wild fish”,HX0416 was closest to Aeromonas salmonicida subsp.salmonicida.Most of the biochemical characters of Aeromonas media HX1316 are same as those of strain HX0416 except for the several items including arginine dihydrolase,Ala-Phe-Pro-Arylamidase,?-glucosidase,L-proline arylamidase,palatinose,D-sorbitol,courmarate and Glu-Gly-Arg-arylamidase.The results of the antimicrobial susceptibility test showed that the isolate HX0416 was susceptible to Chloramphenicol,ciprofloxacin,meropenem,ceftriaxone,gentamicin,cefepime,enrofloxacin and norfloxacin,while resistant to neomycin sulfate,rifampin,florfenicol,oxytetracycline,and seriously resistant to ampicillin and sulfamethoxazole.Compared to the strain HX0416,strain HX1316 was susceptible to chloramphenicol,rifampicin,meropenem,ceftriaxone,gentamicin,cefepime,while resistant to neomycin sulfate,enrofloxacin,erythromycin,doxycycline,and seriously resistant to ciprofloxacin,oxytetracycline,ampicillin,norfloxacin and sulfamethoxazole.3.Study on pathogenicity of Aeromonas strains associated with skin ulceration diseaseThe artificial infection tests showed that the two Aeromonas strains both arepathogenic to C.semilaevis Günther.However,the virulence of Aeromonas media was significantly lower than that of Aeromonas salmonicida,suggesting that the ulcer symptoms of C.semilaevis Günther should be mainly caused by Aeromonas salmonicida.In order to investigate the mechanism of the skin ulceration disease,extracellular proteins of the two strains were collected and gel separated by electrophoresis,respectively.Analysis of the SDS-PAGE spectrum of the two strains indicated that a strong stained protein band appeared with a molecular weight of around 56 KD,which appeared only in protein profiles of Aeromonas salmonicida.The protein band was then analyzed by mass spectrometry,and the data demonstrated that the band mainly consisted of aerolysin.Obviously,the exotoxin aerolysin was responsible to the high virulence of Aeromonas salmonicida.Based on the above analysis and the common pathogenic characteristics of Aeromonas salmonicida,the Aeromonas salmonicida HX0416 was considered to be the causative pathogen of the ulceration disease of C.semilaevis Günther.