Study On Major Pathogen And Diagnosis In The Acute Epidemic Diseases In Chinese Soft-Shelled Turtles, Pelodiscus Sinensis, In Yuhang Region, Zhejiang Province

Culture of Chinese soft-shelled turtles is an important part of aquaculture and high economic benefit.With the development of the culture industry,there were more and more diseases and outbreak-infective disease was one of the most serious.Mass death-off in pond cultivated turtles was recorded in Yuhang district during May to July,2006,with the symptoms of swelled neck,pale the tortoise-plastron, branchial gland congestion.Ulcer and congestion were also observed in gastrointestinal tract congestion,with purple black spleen and swelled and flower-streak liver in some serious turtles.Aerornonas T0608 was isolated and used for challenging with injection of healthy turtles i.m by cultivated aerornonas at the dose of 1×10~8CFU/ind.No death was recorded in 7 days after the aeromonas injection.Death was recorded in healthy turtles after which were injected with the bacteria-free tissue homogenates supematants collected from typical symptom of diseased turtles,indicating the possible virus pathogen.The death turtles were found 5 days after injection,with the similar signs with the natural diseased turtles.The symptom was repeated for 5 generations,with the similar injection.Spherical viruses particles sized with 127nm were observed in the intestinal tissue by TEM.Two cell lines of Chinese soft-shelled turtles from renal tissues and embryo have been successfully established,which are named PsK-0612 and PsE-0612 respectively.The primary cell cultured with tissue culture method by DMEM medium containing 15%calf serum and incubated at temperature about 28-30℃.The subculture are by DMEM medium containing 12%newborn bovine serum.Now PsK-0612 and PsE-0612 are through more than 80 and 70 subculture passages respectively.They look like epithelioid cells and the chromosome number is 66(2n=66).The rejuvenation of the subculture passage cells was tested to be well.Also cell growth curve and mitosis index are measured.PsK-0612 and PsE-0612 were applied to preliminary study on viral pathogen.The bacteria-free tissue homogenates supernatants is collected from typical symptom of diseased turtles.The cell pathological effect(CPE) of PsK-0612 were observed 4-6 days after viral supernatants inoculation,with increased granulations,voids and cell layer desquamates,compared with the control cell inoculated with the heat inactived viral supernatants.The death turtles were found 6 days after injection with 0.5mL/ind of the 4 generation's cell suspension,with the similar signs with the natural diseased turtles.The CPE of PsE-0612 didn't be observed.The classification of virus is still to be clarified.Aeromonas hydrophila T0608,isolated from diseased Chinese soft-shelled turtles, Pelodiscus sinensis,was used for rabbit anti-serum preparation by injection the formalin-inactivated bacteria.The agglutination titer of rabbit serum was 1:1280.The rabbit immunoglobulin G was purified by Protein G column and labeled with biotin.A biotin-avidin-amplified enzyme-linked immunosorbent assay(BA-ELISA) was developed for detection of T0608.The sensitivity of ABC-ELISA,indirect ELISA and BA-ELISA was compared.The detection limit of BA-ELISA was approximately 2.5×10~3 CFU,or 0.5ng bacterial protein,16 times more sensitive than ABC-ELISA and indirect ELISA.No cross-reaction was observed when fish A.hydrophila,BSK-10,Vibrio mimicus,Streptococcus iniae and E.coli were applied instead of T0608.BA-ELISA can be directly used to detect aeromonads in water,intestine and liver samples of Chinese soft-shelled turtles and red-eared slider turtles.BA-ELISA can be considered as a useful method for early diagnosis of Chinese soft-shelled turtles infected with aeromonads,as well as aeromonads detection in Chinese soft-shelled turtles farms.