The Studies On Immuno-histochemical Techniques To Detect Riemerella Anatipestifer (RA) And Distribution Of RA Antigen And Pathological In Artifical Infected Duckling

A immuno-histochemical assay was developed for detection of R.A. in ducks .Using this method ,we can localized and find out the main target-cell of R.A. in ducks. It proved that the Indirect Immunoperoxidase Staining Methods (IISM) has high sensitivity and specificity by intercross assay ,absorption assay ,replacement assay and positive and negative matched control assay .Through optimization,IISM could get excellent result under such conditions :stablizing organs by citromint;treating clean slices by APES;keeping slices in 60@ for one hour;the diluted ratio of Rabbit anti-RA antibody is 1: 25; the one of the enzyme labelling goat anti-rabbit antibody(HRP) is 1:25;repairing antigens by 1% hydrochloric acid for 30mins;treating slices in 3% HaOa-carbinol for 20mins to eliminate the effect of endogenetic peroxidase;incubating the slices for 60mins by the first antibody,for 30mins by the enzyme labelling goat anti-rabbit antibody(HRP) for 30mins , for 30mins by blocking serum;at last ,staining slices by DAB for 1 min.Droping-nose and hyodermic injected to challenge two group unvaccined 24days-old ducks with Riemerella anatipestifer(RA) serotype 1 , result show two group ducks all growth with fibrinous pericarditis ,perihepatitis and airsacsis.Some ducks died at an early stage of the disease showed hyperemia and hemorrhage, inflammation of myocardium.After that ,dissolving and disappearing will happen in myocardium fibers; Hyperemia and hemorrhage was observed in liver and kidney,lung,spleen etc. granular degeneration and vacuolation ,necrosis widely existed in liver and kidney or immune organs(spleen ,thymus and bursa of fabricius).Some organs(such as heart ,lung and thymus etc) appeared fibrinous secretion, atrophy of intestinal gland and disruption of intestinal villus were observed.a few slides of brain showed hyperemia and infiltration of microglia.The detected positive percentage of two group in liver and intestine is the highest(duodenum 60.5%,liver 56.6%,rectum 53.7%,cecum 51.9%), the one in kidney -, lung and immune organs(spleen,bursa of Fabricius ,thymus) is high too(lung 45.7%,kidney 40%,spleen46.8%,bursa of fabricius 45%,thymus 41.7%),but the one in brain and glandular stomach is low(below 20%) .The experimental data show that the detected positive percentage go up with the incidence of disease.The staining antigen localized widely in liver cell , epithelial cells of intestine and renal glomerulus, airsac cell, reticular cell of spleen , epithelial cells of bursa of Fabricius. Analysing all above this , it can draw the conclusion that the main target-cell of R.A. is epithelial cell. RA is one kind of intracellular bacteria.