Studies On Indirect Immunohistochemistry Method To Detect PRRSV And Distribution Of The Antigen In Artificially Infected Piglets

The anti-PRRSV IgG was obtained in rabbits after being inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) SC-1 strain which was purified by sucrose density gradient centrifugation. Indirect Immnohistochemical Method (â…¡M) was successfully developed and standardized to detect the PRRSV antigen in artificially infected piglets. Using this method, we can localize and find out the main target-cell of PRRSV in piglets. It proved that the Indirect Immunoperoxidase Staining Method (â…¡SM) has high sensitivity and specificity by absorption assay, replacement assay and positive and negative control assay. Through optimization, IISM could get excellent results under the following conditions: formalin-fixed tissues, treating clean slices by APES, keeping slices in 37℃ overnight, the dilution ratio of rabbit anti-PRRSV antibody is 1:20, the dilution ratio of enzyme labelling goat anti-rabbit antibody(HRP) is 1:30, repairing antigens by microwave for 10 min, treating slices in 3% H₂O₂-carbinol for 20min to eliminate the effect of endogenetic peroxidase, incubating the slices in 4℃ with the first antibody overnight, HRP for 30min, blocking serum for 30min, and staining slices with DAB for 5min.Only special positive signals emerged in the tissues of piglets infected by PRRSV, but not emerged in the liver tissues infected by Hog cholera virus, Porcine parvovirus, Porcine pseudorabies virus, Porcine Japanese encephalitis virus. Some infected piglets showed sneezing, eyelid edema, diarrhea after five days. Body temperature change was not evidence. Fourteen days post infection, pathological studies showed that submandibular lymph node, jugular lymph node, hilar lymph node, medial iliac lymph node . Groin lymph node underwent hemorrhage. At early stage of infection, the capillary vessel showed hyperaemia and endothelial cell edema, at later stage, infused interval pneumonia was found in the infected tissue of lung. Specimen of artificiallyinfected piglet was detected by IIM. And the results was as follows: PRRSV was detected in the cytoplasm of the following cells: Macrophages which were scattered in alveolar interval, endothelial cells which were in the capillary vessel and blood vessel; Macrophages that were scattered in the epithelial tissue and around the small bronchia. PRRSV-antigen were mainly found at the germinal center and around splenic sinusoid in spleen. PRRSV-antigen was mainly distributed mostly in recess and its fringe in tonsil. Positive signals were also detected in cortex and thymus corpuscle. PRRSV-antigen in the lymph node was mainly distributed in medullary cord of lymph and around subcapsular sinus and medullary sinus; Sometimes could be found in subcapsular sinus and loose connective tissue around the lymphoid node. PRRSV-antigen was also detected in epithelium of intestine villus and germinal center of lymphoid nodule. PRRSV-antigen was also detected in the Kupffer cell in liver, and a few macrophage which is infused in the hepatic portal area.IHC is a specific, sensitive and visual staining method. It can be applied to diagnosethe PRRS, to detect the distribution of PRRSV antigen in tissues of the infected pigs, andin the retrospective diagnosis in formalin-fixed tissues.