Studies On Fulminant Septicaemia Of Soft-shelled Turtle (trionyx Sinensis)

Fulminant septicaemia of soft-shelled turtle (Trionyx sinensis) originally occurred in Zhejiang Eco-Farm in 2005, breaked out frequently these years. This disease causes mass mortality and makes a severe hazard. The main symptoms were hemorrhagic septicemia. Epidemiology, pathology and ultrastructural studies were carried out. The pathogen was determined and studied.Epidemiological survey showed that the fulminant septicaemia of soft-shelled turtles mainly outbreaked at eco-farms in Zhejiang province. Both larvae and adult turtles can be infected and die, while sick turtles mainly weight 150-200 g. Epidemic season was from June to October, especially from June to September. Mortality within 10 days of the pond can reach 90-100%, main symptoms were hemorrhagic septicemia. Conventional drugs were not effectual. The contagion has caused great economic losses.Parasites were not found under microscopic observation. But under TEM, a large number of spherical enveloped viruses, with diameter of 90-100 nm, were observed in the tissues of sick turtles. Virus particles were isolated and purified from the sick turtle tissues. The tissue fluid and tissue homogenate filtrate from sick turtles were injected to health turtles, and caused classical hemorrhagic septicemia symptom and mortality to the turtles. Mortality rate of the tissue fluid attacking group was 15% higher than the tissue homogenate filtrate group. The above results showed that the disease was mainly caused by virus, and aggravated by bacteria.Histopathological observation found obvious pathological changes in the liver, intestine, stomach, kidney and spleen of sick turtles. Cells of liver, intestine, stomach were tumid and congestive, they were loosely and disorderly arranged. Destroyed blood cells were observed in tissues. Tissue cells of stomach and heart appeared vacuolization, blood cells in the heart decreased. Glomerular was atrophic, disintegrated and disappeared. Renal tubular and partial blood cells in spleen were ruptured. Ultrastructural results showed that liver had most severe lesions. There were spherical viruses in most tissue cells. The main ultrastructural changes were the reduction, rupture of mitochondrial cristae; vacuolization of mitochondrial, increasation of lysosomes; putrescence and apoptosis of cells.The virus cultivation in SPF chicken embryos was studied, using yolk sac and amniotic inoculation methods. Variance analysis of the survival time of inoculated chick embryos showed that experimental groups and control group, which were injected with PBS, had a significant difference (p <0.01). No significant difference appeared between the two experimental groups using different inoculation methods (p>0.05); a large number of virus, which were similar with the virus found in tissues in sick turtles,were observed in chick embryo yolk sac membrane of the experimental group under TEM; tissue homogenate filtrate of infected chicken embryo were injected to health turtles and caused infection, hemorrhagic septicemia symptom and death, similar with natural case. Virus was cultured two passages in chick embryo, and the culture time of experimental and control group had a significantly difference (p<0.01). The above results showed that culturing spherical virus of soft-shelled turtles was succeeded. The two inoculation methods were equivalent. Virus can be stable passaged in chick embryo.Physicochemical properties of the spherical virus were studied. The result showed that the virus was instability to heat (56℃), pH 3.0 and pH 11.0; the virus was sensitive to chloroform; the majority of virus could be inactivated by ultraviolet irradiation for 30 min.DNA of crudely purified virus was extracted. Six random primers were used to the virus nucleic acid amplification and preliminary sequencing. Four fragment sequences were acquired. Blastn results showed that four sequences had a low homology or coverage with the sequences of Genebank. Only one amino acid sequence could be read in the four sequences. Blastp results showed that the sequence was homologous to methylase family genes. Sequencing results associated with electron microscopy results indicated that the partially sequenced virus should be a new virus.Eight bacteria were isolated from sick soft-shelled turtles (Trionyx sinensis) associated with fulminant septicaemia. Morphology of the bacteria was characterized by LM, SEM and TEM. 16S rDNA of the bacteria were amplified and sequenced, Results showed that they were Lactococcus garvieae, Pseudomonas sp., Enterococcus hirae, Enterococcus faecium, Ochrobactrum anthropii, Haemophilus segnis, Citrobacter freundii and Edwardsiella tarda. Antibiotic sensitivity patterns of L. garvieae,C. freundii and E. tarda were evaluated against 20 antibiotics. L. garvieae was sensitive to 4 antibiotics including Penicillin. C. freundii was sensitive to 13 antibiotics including Cefazolin. E. tarda was sensitive to 3 antibiotics including Neomycin. Antibiotic sensitivity results could help curing this disease.Diversity of external and internal bacterium associated with the Soft-shelled turtle (Trionyx sinensis) was determined by 16S rDNA gene sequences. Results showed cloned partial sequences of external and internal bacterium clone library were belonged to 15 and 11 OUT respectively. The phylogenetic trees showed that, they mainly related to Bacteroidetes and Proteobacteria respectively. Flavobacterium sp. were detected in both cloned librarys, they were dominate species. Also several beneficial bacteria (Lactococcus lactis and Pseudomonas sp.) and conditional pathogens (E. tarda, C. freundii) were detected. Results demonstrated prolific diversity of external and internal bacterium associated with the soft-shelled turtle.