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Study on Major Pathogen and Diagnosis in the Acute Epidemic Diseases in Chinese Soft-Shelled Turtles, Pelodiscus Sinensis, in Yuhang Region, Zhejiang Province

Author: HuDaYan
Tutor: ChenChangFu；QianDong
School: Huazhong Agricultural University
Course: Aquaculture
Keywords: Soft-shelled Turtle Virus Cell lines Biotin - avidin double-antibody sandwich ELISA Aeromonas hydrophila Detect
CLC: S947.1
Type: Master's thesis
Year: 2008
Downloads: 129
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Abstract

The soft-shelled turtle breeding industry is an important part of China's aquaculture industry, aquaculture cultured species of high economic returns. But with the soft-shelled turtle breeding expanding variety of diseases growing number of outbreaks of sexually transmitted diseases is a cause for concern. May-July 2006, Yuhang Area Chinese turtle farms ecological turtle breeding pond soft-shelled turtle acute onset and the emergence of a large number of deaths, the death of soft-shelled turtle neck swelling, backplane white, throat gills shaped tissue group hair protruding, congestive heart erosion the gastrointestinal mucosa serious congestive spleen purple-black, enlargement of the liver was spent porphyritic. Incidence of soft-shelled turtle separation bacterial and viral pathogens: Aeromonas monocytogenes bacteria isolated from the incidence of soft-shelled turtle liver, spleen, kidney, infection test normal soft-shelled turtle, 1 × 10 ~ 8cfu/ind challenged not the incidence of soft-shelled turtle; take the typical symptoms of the onset of the soft-shelled turtle visceral homogenate supernatant sterilization filter to 1:50-1:5000 diluted to 0.5mL/ind intramuscular healthy soft-shelled turtle, 5d succession the incidence of death, death soft-shelled turtle neck swelling, throat and gastrointestinal mucosa severe congestive spleen purple-black, enlargement of the liver was spent porphyritic natural incidence of soft-shelled turtle Similar symptoms; take artificial infection incidence of soft-shelled turtle tissue homogenates sterilization continue to infect healthy soft-shelled turtle has been successfully repeated infections in healthy soft-shelled turtle generation, can replicate the same symptoms, not isolated to the bacteria in artificially infected the incidence turtle visceral; take the incidence of soft-shelled turtle tissue fixed with 2.5% glutaraldehyde, phosphotungstic acid staining super thin section electron microscopy, spherical virus, the virus particle size of 127 ± 4 nm from the intestinal tissue. DMEM medium were used for further research on the virus characteristics, soft-shelled turtle kidney tissue, embryonic material primary kidney cells and embryonic cell culture, tissue culture method, with 15% fetal bovine serum and 12% fetal bovine serum in primary cultured and passaged in cell culture, soft-shelled turtle kidney cell line PsK-0612 the embryonic cell the strains PsE-0612. The two built strains cells were passaged on behalf of more than 80 and more than 70 generations, are epithelioid cell growth and stable passage; passage cryopreservation recovery in good condition, the optimal incubation temperature of 28-30 ° C. The growth of cell lines of the two built index, mitotic index determined to draw a growth curve and mitotic index curve results PsK-0612 PsE-0612 growth was slow - fast - stable trend; mitotic index, respectively inoculated 4 days and 2 days peaked; analysis showed that the 50 49th-generation the metaphase PsK-0612 cells and the 37th generation of metaphase PsE-0612 cells, respectively, the PsK-0612 with the PsE-0612 chromosome number of 2n = 66 ± 2. Typical symptoms incidence of soft-shelled turtle tissue homogenates the filtrate vaccination of soft-shelled turtle kidney cell line established PsK-0612 and the embryonic cell lines PsE-0612;, after inoculation PsK-0612 cultured at 30 ℃ 4-6d cytopathic performance for the cell granules increased significantly, a large number of empty, followed by a large number of off control cell cultures no obvious lesions in the same time; diseased cells in suspension with the 4th generation 0.5mL/ind injection of healthy soft-shelled turtle, control for the first The 4 generation diseased cells suspension was inactivated at 56 ℃ for 30 min, 6d, soft-shelled turtle Incidence and Mortality control Soft-shelled Turtle not disease: PsE-0612 does not appear to significantly lesions. Taxonomy of Viruses work is still in progress. Hydrophila isolated from soft-shelled turtle Aeromonas pathogenic strain T0608 virulent of the strains, is an important secondary pathogens. T0608 formalin inactivated bacterial injection immunization of New Zealand rabbits was prepared High serum, serum bacterial agglutination titer of up to 1/1280; by protein G affinity chromatography, purified rabbit IgG, an IgG with biological labeled biotin - avidin double-antibody sandwich enzyme-linked immunosorbent assay (BA-ELISA); minimum detection limit of the method is 2.5 × 10 ~ 3 cfu, equivalent to 0.5 ng bacterial protein, its detection sensitivity of ABC-ELISA (4.0 × 10 4 cfu) and indirect ELISA (4.0 × 10 4 cfu) 16 times; Aeromonas Act fish gas mimicus, no dolphins streptococcus, E. coli, etc. cross-reactions, with good specificity. Terrapin farm in Huzhou City, watery BA-ELISA, turtles and red-eared liver, intestinal contents, testing, results show that the method can be used to directly detect the onset of soft-shelled turtle liver, intestine Aeromonas hydrophila Aeromonas can be better applied to the detection of the pathogen incidence of soft-shelled turtle early and rapid diagnosis and breeding soft-shelled turtle pathogen.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Other aquatic pests and diseases and their prevention > Turtle
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