Artificial Infection, Preparation And Application Of Hyperimmune Serum With PRRSV In Pigs

Porcine reproductive and respiratory syndrome(PRRS)is a new kind of impetuous,hyper infectious and contiguous diseases caused by Porcine reproductive and respiratory syndrome virus (PRRSV) in pigs.It broke out in June,2006,which first happened in the south of China and swept most of the provinces in China,with new epidemic characteristics and complex pathogenetic condition,and threatened Chinese pig-raising industry,pork supply and food safety.The PRRS infection was artificially replicated in pigs by inoculation with variant PRRSV (NVDC-JXA1).Antigen localization and histopathologic features were observed on the cases of artificial infection,by direct immunohistochemistry and H.E.staining.The results showed that pulmonary edema,congestion,and hemorrhage was observed in the lung of the initial dead cases,and then consolation in foci,hepatization,and interstitial proliferation,detelectasis,rubber-liked geloses on the surface,found in lung of the finial dead cases,diffuse interstitial pneumonia were observed under the microscope.It presented a marble-liked bleeding in the lymph nodes,hemorrhagic infarctions on the edge or surface of the spleen,nonsuppurative encephalitis,needle-sized bleeding point in the kidney, petechia and necrosis foci in the liver,and hemorrhage in the skin,the heart and the instestine.Direct immunohistochemistry method was established to locate PRRSV antigen in the lung and brain.The antibody was prepared and purified with salt precipitation(Octenic acid-saturated ammonium sulfate precipitation)and DEAE52 cellulose anion exchange method.The concentration of the primary antibody was 2.03mg/ml.After labelling with the alkali phosphatase,the concentration was 1.02mg/ml.Sevaral conditions of direct immunohistochemistry test were optimized as following:The antigen recovery buffer was citrate buffer solution(0.01M pH6.0),the antigen was recoveried by the high temperature and pressure,the enzyme labelled antibody was diluted as 50 times in antibody diluent solution,and the best time of incubation was 90 min in 37℃.By immunohistochemcal analysis,it showed positive reaction for PRRSV antigen in the cytoplasm of phagocytes,monocytes and cerebullar nernon cells in the pigs at 9-day post-infection.By RT-PCR, they also showed positive for PRRSV gene in the tissue of the lung.Then the hyperimmune sera against PRRSV were prepared and applicated.At 21-day of post fundamental inoculations with twicely immunization with PRRSV inactivated vaccine,Antibody in five immunized pigs were still negative(S/P＜0.4),and Antibody in the other pigs became positive(S/P＞0.4) among 17 pigs.On the base of the value of antibody,the experimental pigs were divided into three group.The five positive and eight negative pigs were immuned with live PRRSV NVDC-JXA1.and the four pigs were immuned with PRRS inactivated vaccine(8ml/kg). Subsequently,at 28 days of post-final inoculation,the rate of antibody with high ELISA titer in experimental pigs were 7/8,4/5 and 2/5.It showed that the high titer serum can be produced by final inoculating live PRRSV.If the pigs injected intramuscularly with hyper immune serum(1ml/kg) within three days after challenged with PRRSV,their survival rate was 7/7;The detective result of antigen by RT-PCR revealed that three experimental pigs were not detected PRRSV in serum and five pigs were not detected PRRSV in lung among all pigs(seven)of therapeuticⅡgroup.And after fourteen days of injecting the hyperimmune sera,the rate of antibody with positive(S/P值≥0.4)in experimental pigs were 6/8.If the pigs injected intramuscularly with hyper immune serum(0.5ml/kg) within three days after challenged with PRRSV,their survival rate was 5/7:The detective result of antigen by RT-PCR reveal that six experimental pigs were not detected PRRSV in serum,four pigs were detected in lung and six pigs were not detected in brain among all pigs(seven)of preventive proup.Which present that the hyperimmune sera have some effect against PRRSV in prevention and therapy.But,It didn't present preventive and therapeutic efficacy in sera produced by pigs inoculating three times with PRRS inactivative vaccines.This experiment aimed to apply Data for diagnosing,preventing and treating of PRRS by artificial infection,preparing and application of high immune serum.