| Spiroplasma eriocheiris is a first found and isolated spiroplasma pathogenic microorganisms from aquatic crustaceans. It is a novel aquatic crustacean pathogen. Taxonomically, S. eriocheiris belong to the domain Bacteria, phylum Firmicutes, class Mollicutes, order Entomoplasmatales, family Spiroplasmataceae and genus Spiroplasma. It belongs to a new Spiroplasma group XLIII. S. eriocheiris possess wall-less, helical morphology and motility, it also be able to pass through the 0.22μm millipore filter and grow in MID or R2 liquid media at temperatures between 20℃and 40℃(optimum growth occurred at 30℃) in vitro. S. eriocheiris has the extensive host range, which could infect Eriocheir sinensis, Procambarus clarku, Macrobrachium rosenbergii and Penaeus vannamei, and caused serious diseases and catastrophic economic losses in aquaculture. Furthermore, S. eriocheiris also can infect suckling mice and cause cataract. The prevenient studies on the physiological and biochemical properties, evolutionary state, pathogen detection, screening of the effective medications, pharmacokinetic in E. sinensis and the whole genome sequencing and annotation were the base of this work. There is a limited known about the molecular biology of S. eriocheiris, especially for what function genes in the pathogen infection process. In the present study, we establish a primary cell culture system for in vitro culture of the hemocytes of E. sinensis with high viability. Selection and study of the important function genes from the infected host (on individual level and cellular level) by S. eriocheiris was performed. Meanwhile, the cytopathic effects and the direct immune relationship between S. eriocheiris and host’s hemocytes were presented. Based on the result of SCOTS, Spiralin-like protein SLP31 from S. eriocheiris was cloned and expressed which could be as a potential antigen for immunodiagnostics of tremor disease in E. sinensis. We also establish a multiplex PCR method for simultaneous and rapid detection of S. eriocheiris and white spot syndrome virus (WSSV) in P. clarkii. Main results of this dissertation included the following four parts:1. Establishing a primary cell culture system for in vitro culture of the hemocytes of E. sinensisA primary culture system for in vitro culture of the hemocytes of E. sinensis with high viability was developed. Sterile anticoagulant citrate dextrose solution B (ACD-B) and phosphate buffered saline (PBS) were employed, the results showed ACD-B had the better anticoagulant effect than PBS. We chose two kinds of common culture mediums (Modified L-15 medium and M199 medium) to culture the hemocytes. Our results demonstrated that the Modified L-15 medium were more efficiency for promoting growth and cell survival of hemocytes of E. sinensis than M199 medium. In disagreement with other crustacean cell lines culture work, the cell cultures were achieved at a temperature of 25-28℃rather than 16-20℃which has been favored for other freshwater invertebrates. In brief, the modified Leibovitz’s L-15 medium consisting of 15% fetal bovine serum,100 U ml-1 penicillin, and 100 U ml-1 streptomycin was suitable for hemocytes culture from the E. sinensis adjusting pH 7.2-7.4, and incubated at 28℃with 5% carbondioxide. The cells survival lasted over 35 days in Modified L-15 medium in the optimization condition.2. Variations in the number of hemocytes in E. sinensis and the expression levels of ALF, Pox and cSP genes in primary culture of hemocytes from E. sinensis after S. eriocheiris challengedIn the former study, we have established the tremor diseased model of E. sinensis. In the study, we used the model to research the variations in the number of hemocytes in E. sinensis after challenged by S. eriocheiris. From the symptoms, there were no significant changes at the early infection stage; the vitality of E. sinensis in experiment groups was declined obviously at the middle infection stage; most of E. sinensis in the experiment groups showed signs of weakness and began to tremor. Some experiment groups began to appear the concentrated death at the late stage. For the variations in the number of hemocytes, the numbers retained stable (1.13×107 ml-1) in the negative group. The hemocytes numbers appear declined (3.65×106 ml-1) after infected two days; the hemocytes numbers appear significant increase (10.8×106 ml-1) after infected four days; the hemocytes numbers appear declined (8.75×106 ml-1 and 6.25×105 ml-1) again after infected eight and ten days; the hemocytes numbers continued to decline (5.0×105 ml-1) after infected twelve days, and the experiment groups appear the concentrated death.The effects of S. eriocheiris stimulation on the expression levels of anti-lipopolysaccharide factor (ALF), Peroxinectin (Pox) and clip domain serine protease (cSP) genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced. After S. eriocheiris stimulation, the relative abundance of the ALF mRNA was up-regulated and peaked at 12 h (10-fold, P<0.05). The expression of Pox dropped gradually, after receiving S. eriocheiris. As time progressed, Pox transcripts significantly increased and peaked at 24 h (36-fold, P<0.05), and then after 24 h, the Pox mRNA was down-regulated. In contrast, cSP transcripts were first down-regulated in 2 h, whereafter the relative abundance of cSP was up-regulated and peaked at 48 h (2.4-fold, P <0.05), and then significant reduced at 72 h. The Pox transcripts greatly increased and peaked at 24 h (36-fold, P< 0.05); the fold was outweighing the other two immune genes’ greatly (ALF and cSP) which indicated that the Pox gene played more important role after S. eriocheiris challenge.3. Selection and study of the important function genes from the infected host by S. eriocheiris based on SCOTS and prokaryotic expression of Spiralin-like protein SLP31 from S. eriocheirisFifty-seven effective sequences were captured from the different infected samples (on individual level and cellular level) by SCOTS. There were 27 effective sequences with the annotation information in the S. eriocheiris genome. Some of these proteins were related with metabolic of S. eriocheiris, such as lipid metabolism, sugar metabolism and nucleic acid metabolism. Some proteins were related with S. eriocheiris pathogenicity. Adhesin-like protein and EF-Tu were related with adhere and infection. Furthermore, the putative spiralin was also be selected in the study. However, NAD-dependent glycerol-3-phosphate dehydrogenase, thiol peroxidase, ferritin, glycerol uptake facilitator protein, ferrous iron transport protein B, endonuclease I, serine-threonine protein phosphatase and N-acetylglucosaminidase were related with the S. eriocheiris metabolism, which control and realize the virulence effect through relevant metabolic. Based on the results of SCOTS, we described here the identification of a spiralin-like protein (SLP31) from S. eriocheiris and expression in Escherichia coli. Analysis of the nucleotide sequence revealed that the clone has an open reading frame of 837 bp encoding a protein of 279 amino acids. Theoretical isoelectric point and molar mass for SLP31 are 7.72 and 31 kDa, respectively. The similarity of SLP31 deduced amino acid sequence shared with the spiralin from other species indicated that the gene may be a member of spiralin family. However, spiralin-like protein gene from S. mirum could not be cloned which confirmed that this is a special gene which can be used to distinguish S. mirum and S. eriocheiris. After cloning the SLP31, the gene was site-mutated from TGA to TGG and transcribed in E. coli to full expression of SLP31. The purified recombinant protein was used to determine the immune reactivity by Western blotting which suggests that SLP31 could be a good antigen for immunodiagnostic of tremor disease in E. sinensis. 4. Simultaneous and rapid detection of S. eriocheir and white spot syndrome virus (WSSV) by multiplex polymerase chain reaction in P. clarkiiS. eriocheiris and WSSV are the important pathogenic microorganisms from aquatic crustaceans; both of them could infect crayfish and cause serious diseases and catastrophic economic losses in recent years. The aim of the study is to establish a multiplex PCR method for simultaneous and rapid detection of S. eriocheiris and white spot syndrome virus (WSSV) in P. clarkii. Three primer sets were mixed at a ratio of 1:3:1 to amplify specific fragments of the S. eriocheiris, WSSV, and P. clarkii genomes, respectively. S. eriocheiris and WSSV were used to challenge the different groups. Total DNA of the samples were purified and detected by multiplex PCR. The PCR amplified products produced four cases. One fragment of 1195 bp showing no pathogen amplified by primer set ITS-crayfish/28S-crayfish as an internal control. In samples only challenged by S. eriocheiris or WSSV, two fragments could be seen:either from S. eriocheiris (271 bp) plus the internal control or WSSV (530 bp) plus the internal control, respectively. In cases of double challenged, all three amplified products were detected simultaneously. Simultaneous and rapid detection of two pathogens in P. clarkii is important to crayfish aquaculture. The direct detection of S. eriocheiris and WSSV from P. clarkii is possible with multiplex PCR. The new method provides a novel and highly practical detection assay for S. eriocheirs and WSSV infections, and has the potential to be widely used in the aquaculture industry, entry-exit inspections and quarantines. |
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