Study On Etiology, Pathology Of Tilapia Streptococcus Agalactiae Disease And On The Prokaryotic Expression Of CpsE Gene

Tilapia is one of the most important fish species for freshwater breeding as well as one of the species that is highly promoted to culture in southern China. Tilapia possesses favourable biological habitual behavior and is capable of surviving in water environment of poor quality. Infections caused by various pathogens including streptococcus, Flavobacterium cloumnare, Aeromonas hydrophila and Edwardsiellosis have brought obstacles to the Tilapia culture at the steady development. Among which, the infections of Streptococcus iniae and S. agalatiae are serious and have caused high economic losses in Tilapia culture. In2009summer, there was an outbreak of streptococcal infection in Tilapia in Hainan province. With high infectiosity, fast outbreak and high mortality, this infection has caused tremendous losses economically. In this study, infected tilapia cultured in several farms (Wenchang City, Hainan) were collected. The etiology and mechanism of pathological injury of infected Tilapia were studied using microbiology and histopathology technologies. The molecular characterization、 prokaryotic expression and further application on indirect in situ PCR and triple PCR of functional gene in pathogenic bacteria were investigated with molecular biology in order to provide scientific references to identification of pathogen to genus and species、analysis of biological characterization、pathogenic mechanism and establishment of fast diagnostic method and further to build theoretical foundation of new aquatic vaccine, with recombination protein from the functional gene of streptococcus as major antigen component.1. Isolation and identification of S. agalactiae from tilapiaSamples of seven batches were collected from infected tilapia cultured in ponds in Hainan province. The samples were subjected to isolation purification and identification of the pathogenic bacteria. Seven isolates were identified from the present study. Colonies of the isolated strains grown on nutrient agar medium were similar, producing zone of y-hemolysis with microscopic examination confirming as Gram-positive cocci.The regression test showed that the isolated strains could yield the same symptom by artificial infection over healthy tilapia with that of the spontaneous infection tilapia. Phylogenetic tree analysis between16S rDNA sequences of the isolated strains and streptococcus spp. showed that they all highly similar. Their similarity was over99.5%. The surface group-specific antigens of the isolated strains were identical to that of group-B streptococcus. Biochemical characteristic of API20Strep gave profile number3663414, which corresponded to excellent matches to S. agalactiae. Taken together, these seven isolated strains were S. agalactiae. Subsequent drug susceptibility test of the isolated strains was performed by employing slip agar diffusion method and the result showed that all the strains were well-sensitive to Cefoxitin, whereas were sensitive to other drugs to different extent. For instance, some strains showed complete drug resistance to drugs that are usually used to against streptococcal infection clinically such as penicillin and Gentamicin, while some strains were sensitive to. One strap was688bp molecular size and another one was272bp, which confirmed these isolates as la S. agalactiae as a result of its correspondence with that of standard strain of type la S. agalactiae. The LD50of liquids that contains and was lack of extracellular products were6.9×106CFU and2.4×107CFU, respectively. Results of content of total protein in extracellular products showed that concentration of total protein was1.6g/L. Enzymatic activity detection confirmed the activities of degradation of lipidase and urease, while inactivities of degradation of protease、 amylase and lecithinase.3. Establishment and application of triple PCR assay for fast diagnosis of S. agalactiae Genes of cpsE,sip and cpsLwere selected as templates to design three groups of specific primers, respectively. The triple PCR assay here described is well-specific and is available for distinctive identification of pathogenic bacteria between S. agalactiae and six other bacteria. The lowest concentration for detection of S. agalactiae DNA was0.32ng/μl, and the optimal concentration of Mg2+was37.5mM and total time the process taken was about100min. The present method was used to diagnose samples from artificially S. agalactiae infected and borderline case of clinically infected tilapia. The detection rate in artificially infected samples of40tilapia was100%from liver and kidney tissues’DNA. And for samples of22clinically infected tilapia, it was72.7%. Moreover, the detection results using triple PCR method completely matched that using traditional bacteria identification method.4. Pathological damage caused by S. agalactiae to tilapia The pathogenicity of tilapia infected naturally or by S. agalactiae were evaluated through clinical symptom, histopathology and ultrastructure pathology. Signs of disease due to experimental infection were similar to those of naturally infected tilapia.Both naturally infected tilapia and experimentally infected tilapia showed some common clinical symptoms including haemorrhaging of the submaxilla、inside and on the edge of the operculum, congestion of fin ray and meninges; yellow-transparent mucus in the enteric cavity; red swelling of anus; uneven color and a dilation of the liver tissue with fragile texture and swelling gallbladder. However, exophthalmia was only observed in naturally infected fish. Histopathological changes in S. agalactiae infected tilapia were observed including acute inflammation all over the body and focal necrosis; Both liver and kidney exhibited degeneration and necrosis, together with cerebral edema, myocarditis and gastroenteritis. Ultrastructure pathological changes showed ultrastructure destroy in cells of liver, kidney and spleen. Secondary lysosome in cells were found increasing and further forming bacterial pinocytotic vesicles.6. Distribution and location of S. agalactiae in artificially infected Tilapia tissue Tilapia were artificially infected by intraperitoneal injection with viable bacteria fluid of S. agalactiae isolates, viable bacteria fluid of standard S. agalactiae and sodium chloride respectively to prepare specimens of positive control,negative control and specimen for detection. cpsE gene of S. agalactiae was used as a template to design a pair of specific primers and an oligonucleotide probe.IS-PCR was developed for application to specimens of positive and negative controls and subsequently detecting the bacteria of S. agalactiae. isolates aiming to study distribution pattern of S. agalactiae isolates in tilapia. The positive signals sporadically appeared in the spleen of tilapia after two hours since artificial injection; After four hours, the liver of tilapia was detected positive signals as well; Twelve hours post artificail injection, positive signals were found in kidney, cardiac muscle and eyeball, too. Twenty-four hours post artificail injection, positive signals were found in brain. And lots of positive spots were detected in many tissues of tilapia including liver, spleen,kidney, cardiac muscle, brain and eyeball in thirty-six hours post artificail injection6. Cloning、identification and molecular characterization of cpsE gene of S. agalactiae Cloning and identification of cpsE gene of S. agalactiae isolates were performed followed by molecular characterization analysis of amplified sequence of cpsE gene. Results indicated that amino acid sequence deduced by cpsE was highly conserved and has revealed a surprising degree(100%) of homology among strains isolated from human and other mammals. The highest content of amino acid in deduced sequence were Lys and Val, which were10.7%and10.1%, respectively; Polypeptide analyzed in this study contained a glycosyltransferases superfamily conserved domain functioned as enzyme that catalyze the transfer of sugar moieties. Moreover,the polypeptide possesed3phosphorylation sites which is related to post-translational modification. The hydrophilic regions were larger than hydrophobic regions. Bioinformatic software also confirmed the presence of transmembrane domain and absence of signal peptide in deduced sequence. Proportions of various structures in secondary structure were as follows: α-helice40.94%, β-strand25.50%, β-corner5.37%and loops structure28.19%.7. Prokaryotic expression of cpsE gene of S. agalactiae Recombinant expression plasmid pET-32a(+)-cpsE was constructed and then was expressed in Escherichia coli BL21(DE3) competent cells. The immunogenicity of the recombinant protein was studied by using SDS-PAGE、Western-blot and agar diffusion reaction test. Results showed protein with molecular size of36KDa which was correspondence with anticipate size of cpsE expression product under inducing condition induction at30℃、34℃and37℃with0.4mM IPTG for3h. The results presented in Western-blot analysis confirm favorable antigenicity of CpsE recombinant protein. The immunopotency of polyclonal antibody of rabbit anti-CpsE was1:32.