Study Of Detection Methods And Vaccine Of A Virus Isolated From Chickens With Stunt And Immunos Uppression Syndrome

Recently,Stunt and Immunosuppression Syndrome(tentative name.acronym for SIS)of Chickens was a importance infectious disease which caused severely jeopardize in chickens,It not only can cause serious damage to the industry to their own culture,but can i(?)crease susceptibility to other pathogeny on immunosuppression,lead to multiple mixed infection or secondary infection,and it is very difficulty to prevention and treatment the SIS. At present pathogeny of the disease has been isolated,and studies proved to be a new virus as the etiologic.But there is a lack of detection of the disease and control methods.This study because of this,with a view to achieving the virus detection and vaccine development to provide experimental evidence,to lay experimental basis for controlling the disease.1.Three methods on immune detection which were Dot enzyme-linked immuno-sorbent assay(Dot- ELISA),Dot immunogold staining(Dot-IGS)and Indirect enzyme-linked immunosorbent assay(Indirect ELISA)were established using the rabbit antiserum in the experimental lab.The optimal conditions of Dot- ELISA included the optimal coating antigen dilution was 1:200,the optimal working dilution of HRP labeled sheep anti-rabbit antibody was 1:1000,the optimal reaction time of substrate was 10 minutes,the total time of the test after coating antigen only 3 hours.The optimal conditions of Dot-IGS included the optimal coating antigen dilution was 1:200,the optimal working dilution of HRP labeled sheep anti(?)bbit antibody was 1:1000,the total time of test after coating antigen was 3hours.And the optimal conditions of Indirect ELISA included the optimal coating antigen dilution was 1:100,the optimal working dilution of HRP labeled sheep anti-rabbit antibody was 1:1000, the optimal reaction time of substrate was 30 minutes,the total time of test after coating antigen required 5 hours.The test results of specificity and repeated showed Dot- indirect ELISA and indirect ELISA all have good specificity and reproducibility.And the results of compareing of three methods showed Dot-ELISA has other advantages,such as simple,save antigen,highsensitivity,detect a short time,the use of simple instruments,detection of lowcost and the results can be preserved for long term,etc..2.Preparation of the Inactivated oil-emulsion vaccines of chickens with stunt and immunosuppression Syndrome After allantoid liquid which contains virus were inactivated by formaldehyde,add 4% axenic Tween-80,shake it up,and then the aqueous phase of vaccine were prepared,preemergency. Medical light quality vash oil,Span 80 and Aluminium stearate are compounded according to 94 mL:6 mL:2 g proportion,and then the oil phase(mixture)of vaccine were made,through autoclave sterilization,pre-emergency.According to 1:1proportion,aqueous phase is slowly added into oil phase which were whisking in 8000-10000r/min,emulsifies 3 minutes,added the Merthiolate solution to final concentration be 0.01%before the whisk were ended,then becomes milky oil emulsion vaccine.After emulsification,the vaccine have been taken 10mL to centrifuge with 3,000r/min for 10min.lf stratification,the vaccine should be whisked repeat.The vaccine of emulsifying were parted charging and seal-packing,labeled preserves at 4～8℃.The appearance of the vaccine is the milky oil shape and uniformity.The results of cold water surface test showed inactivated oil-emulsion vaccines is the water-in-oil,because it is a round droplet and indiffusion on the cold water surface.The stability tests that inactivated oil-emulsion vaccines have not seen stratification after the 3000r/min centrifugal for 30min. It showed that the stability is good.The viscosity meauresement test indicates that 1mL standard suction tubes,the perpendicularity disengages 0.4 ml vaccine for 8s at room temperature, suitable for injection.The aseptic test indicated that there were no other bacteria growth in common nutrition agar flat medium in 37℃for 48h,.Storege test showed that inactivated oil-emulsion vaccines have not the broken emulsion and no delamination phenomenon at 37℃for 21 days,and the vaccine remains uniform milky white oil and the absence of stratification which was preserved at 4-10℃(9 months),.The result of the ELLSA tests showed the antigen titer of the vaccine has not reduced basically.That acute toxicity tests showed that the chicken group normally until 24 days old by inoculated the inactivated oil-emulsion vaccines at 1-day-old,indicated the vaccine is safe.The optimal dose of the inactivated oil-emulsion vaccines of chickens with stunt and immunosuppression syndrome is 0.3mL,immune approach is neck hypodermic,immune time is 1 or 2 days.Antibody began to be produced 7 days after immunization,and the attacked chickens had been fully protected 21 days after immunization.The attack critical value of 100%protection is 1:128.Immunity after the 21 days to 14 weeks,The value of antibody titer is above the critical value of attack 100%protectionIt has the ability to fully protect attacked chickens.The protection term of the vaccine is three months.