Isolation,Identification and pathogenicity of Streptococcus dysgalactiae from Siberian Sturgeon,Acipenser baerii

Siberian sturgeon,Acipenser baerii is one of the major cultured sturgeons in China in recent years.In this study,ultra-thin section and transmission electron microscope observation,bacteria isolation and identification,artificial infection tests were used for analysis the cause of the severe disease.Bacterial cocci were found in kidney,spleen,liver and heart tissue sections under electron microscope.And bacteria were isolated from the four tissues using 5% sheep blood agar and BHI agar.Two isolates,one named AeBF070904 isolated from the liver of sturgeon cultured in flowing water pond in Beijing and the other named AbHT070912 isolated from the heart of sturgeon cultured in cage of reservoir in Hebei Province were obtained.The biochemical and physiological characteristics of the two isolates were learned by using conventional method,API System(20 STREP)and ATP Expression System(ID32 STREP). The tests showed that the isolates were Gram-positive cocci arranging in chains andα-haemolytic on 5%sheep blood agar.They grew at 20℃,28℃and 37℃,but not at 10℃and 45℃.They grew in 1%NaCl,2%NaCl and 3%NaCl,but not in 6.5%NaCl.CAMP test and Voges-Proskauer test were negative.They producedβ-glucuronidase,leucine arylamidase and alanine-phenylalanine-proline arylamid ase but not urease,arginine dihydrolysis,hippurate hydrolysis,β-glucosidase,β-galactosidase,α-galactosidase,pyroglutamic acid arylamidase,glycyl-tryptophane arylamidase and acetyl-β-glucosaminidase.They utilized trehalose,ribose,amygdaline,sucrose,pullulan and Maltose.They didn’t utilize 12 kinds of tested sugar such as lactose,salicin,raffinose.Both the isolates were identified by conventional method,API System(20 STREP)and ATP Expression System(ID32 STREP)as Streptococcus dysgalactiae,59.1%S.dysgalactiae subsp.dysgalactiae and 97.9%S.dysgalactiae subsp.equisimilis,respectively.The 16S rRNA gene sequence of the two isolates were amplified and compared with those of other Streptococcus retrieved from GenBank.The 16S rRNA gene sequence analysis showed that the two isolates were 100%identity,both most homology with that of S.dysgalactiae in GenBank.The phylogenetic tree of Streptococcus showed that the isolates were clusted together with S.dysgalactiae. 16S rRNA gene sequence analysis further proved that the isolates belonged to S.dysgalactiae.The virulent tests were carried out by using the pure culture of the bacteria artificially infected Acipenser baerii,Hybrid sturgeon(Acipenser baerii♀×Acipenser schrencki♂)and aquatic laboratory model animal,Xiphophorus helleri.The morphological and biochemical characteristics of the re-isolated bacteria from the artificially infected diseased fish were the same as those of the original infected bacteria.Both AeBF070904 and AbHT070912 were pathogenic to the three species of experimental fish.The virulence of the bacteria cultured at 37℃was stronger than that of the bacteria cultured at 28℃.The test of sensitivity of two strains to 18 kinds of chemotherapeutant showed that both strains were sensitive to penicillin,ampicillin,amoxicillin,piperacillin,norfloxacin,enrofloxacin,levofloxacin and mid-sentive to erythromycin but resistant to cefazolin and gentamicin.There were some differences in sensitivity to other 8 kinds of medicaments between two isolates.For example,the susceptibility to kanamycin and amikacin,strain AeBF070904 was sensitive but AbHT070912 was resistant.While to spectinomycine,strain AeBF070904 was resistant but AbHT070912 was sensitive.It showed that there could be some differences in chemotherapeutant sensitivity among strains of bacteria belonging to the same species from different place.