| In recent years, Streptococus agalactiae is a major bacterial pathogens that cause tilapia become sickly, and it cause a loss to the tilapia aquaculture. Other studies have shown that morbidity and mortality of tilapia streptococcus was higher, and the virulence related gene's expression of Streptococcus agalactiae was up-regulation under the condition of high temperature. But most of these studies based on single of clinical isolates in study, whether or not it is a universal phenomenon of Streptococc-us agalactiae that is unknown. Hemolysis and hyaluronidase are the key of Streptococcus agalactiae virulence factors. The aim of this research is to study the effect of culturing temperature on the hemolysis titer (HT) and hyaluronidase activity of Streptococus agalactiae isolates,which from different separation of the year? different regions?different hosts and different capsule polysaccharide serotype. With this support, to understand the temperature effect on virulence rules of Streptococcus agalactiae epidemic strains,and further regulation of farming conditions, to reduce the pathogen pathogenicity and immune prevention and control streptococcus development provide theoretical basis for disease control and prevention method.Eighteen of nineteen isolates used in this research are from Hainan Wenchang, Qionghai, Lingao and Haikou tilapia farms which have an outbreak of typical streptococcicosis and the rest are from trachinotus ovatus. And in 2009 and 2015, strains were isolated by 11 and 7 strains respectively. Peart river fisheries research institute provide three stains for our research are bighead carp, tiger frog and tilapia.There have 22 isolates in this study.Primarily, with bacterial morphological exam, physiology and biochemistry, gene of 16S rDNA and cfb to identify 22 strains.And combining the NCBI database streptococcus genetic sequences to build 16S phylogenetic tree and cfb gene system N-J tree respectively. By using PCR to amplify capsular polysaccharides (cps) gene strain analyses serotype. Results showed that the physiological and biochemical identification of 22 isolates are gram positive, physiological and biochemical indicators and Streptococcus agalactiae standard strains are highly consistent.16S rDNA and cfb gene identification that 22 strains are Streptococcus agalactiae.16S phylogenetic tree and cfb gene system N-J tree showed that 22 strains and Streptococcus agalactiae gathered, homology are as high as 99.9% above. The capsule polysaccharide serotype of 21 isolates were type I a and the rest was type III.Then Doubling dilution hemolysis test method was employed to determine the hemolysis titer of the isolates under 3 different temperatures (25??30? and 37?) culture conditions,and the hemolysis titer of the isolates under 37? static culture and 37? shaking culture were also studied. The results showed that the HTs of 18 isolates were significantly affected by temperature. Among the 22 isolates, the HTs of 18 isolates had the highest values under 37? culture, that of 2 isolates were under 25? culture, and that of 2 isolate was under 30? cultured. The HTs of the isolates under 37? static culture and 37? shaking culture showed that 18 isolates were significantly affected by shaking culture conditions. The highest HT of trachinotus ovatus origin strain of PBSA1401 was cultured under 30?, bighead carp origin strain of HN1404 and tilapia origin strain of PBSA0905 was cultured under 25 ?,and tiger frog origin strain of FWL1404 and the rest of 18 isloates was cultured under 37 ?. The HTs of the diversity of 21 type I a isolates. PBSA0901 and PBSA1505 was separated fromWenchang DaZhiPo, their HTs are much higher than other srains under the same culture conditions.Although, the HTs of 22 isolated had no association with their different regions?different hosts and different capsule polysaccharide serotype, All of 7 isolated at Year 2015 showed the same characteristics that HTs are increasing while the culturing temperature rising.All of isolates were systematically observed by agar-method and their hyaluronidase activity were quantitatively examinedunder 3 different temperatures (25?,30? and 37?) culture conditions. The results showed that the hyaluronidase-producing activity of 21 isolates were significantly affected by temperature. Among the 22 isolates, the hyaluronidase activity of 17 isolates had the highest values under 30? culture, that of 2 isolates were under 25 ? culture, and that of 3 isolate was under 37? cultured. The highest hyaluronidase activity of tilapia origin strain of PBSA0901 and WC1501 was cultured under 25 ?, tiger frog origin strain of FWL1404 and tilapia origin strain of PBS A 1505and PBSA1506 was cultured under 37 ?, and trachinotus ovatus origin strain of PBSA1401, bighead carp origin strain of HN1404 and the restof 17 isloates was cultured under 30 ?. The hyaluronidase activities of the diversity of 21 type I a isolates. PBSA0903 was separated fromWenchang DaZhiPo, too. The HTs is much higher than other srains under the same culture conditions. Although, the hyaluronidase activities of 22 isolated have no association with their different regions?different hosts and different capsule polysaccharide serotype.Although, the HTs of 22 isolates were significantly affected by temperature,they all have their own characters.But All of 7 isolated at Year 2015 showed the same characteristics that HTs are increasing while the culturing temperature rising, and the HTs of 81.8% isolates had the highest values under 37? culture. The hyaluronidase activity of 95.5% isolates were significantly affected by temperature, and the hyaluronidase activity of 77.3% isolates had the highest values under 30 ? culture.Type I a capsule polysaccharide serotype of 21 isolates exist diverse on HTs and hyaluronidase activities. |
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