| During August and September of 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhage and nodules showing on epicardium has been occurred in the Acipenser baerii in Hanyuan lake, Ya'an, SichuanProvince. To elucidate the aetiology, the bacteria smeared and isolated from the livers, spleens and kidneys, and artificial infection, physiological and biochemical analysis and molecular analysis of the isolate were conducted. The results showed that numerous sphere bacteria were exhibited in spleens and livers and a Gram-positive, chain-forming coccus (Ab 130920) was isolated and also was the same in transmission electron microscope. Artificial infection showed that the strain was lethal to A. baerii, physiological and biochemical characteristics were consistent with Sreptococcus iniae (ATCC29178), besides the reactions to arginine phosphokinase and ribose;the 16S rRNA gene sequencing analysis by Blast in GenBank indicated that the isolate (KJ162337) showed high levels of similarity to S.iniae (99%); A phylogenetic tree was constructed based on the 16S rDNA sequences of the isolate and the homologous sequences of other strains of Streptococcus, and the strain and other S.iniae strains formed a tight cluster; Meanwhile, a expectedsegment of 870 bp was yielded in the specific PCR detection of S.iniae based on S.iniaelctO gene. And combined phenotypic and molecular characteristics detection and identificatedthe isolate Ab 130920 as S. iniae. The drug sensitivity results showed the isolate was sensitive to amoxicillin, doxycycline and florfenicol et al, but resistance to novobiocin, ofloxacin, rifampicin.To investigate the virulence of the Ab 130920, the serum bactericidal test, the serotyping, the adhesion and invasion to EPC, the detection of virulence and the pathological study were conducted. The result of serum bactericidal test indicated the healthy serum can kill bacteria in percent of 43.8, and the surivial may enter the bloodstream to result in a systemic infection; and some of bacteria could adhere (3.28%) and invade (0.08%) to the EPC, indicated that they could colonize inner the host cells; the strain was serotyp ? proved by the the reactions to arginine phosphokinase and ribose and the RAPD analysis, simultaneously. As expected, the strain Ab 130920 was positive by multiplex PCR for all five virulence genes (cpsD?simA?sagA?pdi and scp1) test, showing amplification products of the expected sizes, which was further identical the isolate to S.iniae and confirmed the pathogenicity of it, and consistent with the result of artificial infection.Histopathologically, edema, hemorrhage, degeneration, necrosis and the inflammatory response occurred in many organs, especially in liver, spleen, kidney, heart and muscle. Further, necrotichepatitis, blood vesselinflammation of liver, glomerularnephritis, interstitialnephritis, acute splenitis, carditis, Sang Renxin and necrotic myositis were evident. Besides, lamellae inflammation, gastritis, enteritidis, meningitis and were also observed.To study the evolution of capsular polysaccharide genes, the capsular polysaccharide genes of the Ab 130920 and the standard strain ATCC29178 were amplificated by PCR and sequenced, and downloaded and searched squences from GenBank and related documents to conduct multile sequence alignment aimed to clear the variability of these genes. Chosed variable genescpsD, downloaded and searched squences from GenBank and related documents to Bayesian phylogenetic analysis. The variation was found in the genescpsY, cpsC, cpsD, cpsE, cpsG and cpsY, especially the cpsD and cpsE which owned a high degree of variability. As expected, the cpsD and cpsE gene of the Ab 130920 were various too. The mutation genes in corresponding points insertion, deletion and frameshift resulted in the changes of amino acid. Chosing the cpsD gene for Bayesian analysisshowed a great deal of variability of the Ab 130920. |
PDF Full Text Request