| Antibody is acquired by immunization of Japanese rabbits using purified CSISV(tentative name,short for Chicken Stunt and Immunosupression Syndrome Virus).It is the antibody that with which an indirect immunohistochemistry is established to detect CSISV specially in tissues .The optimal work condition of the method are as following: firstly,4% neutral paraformaldehyde is used to fix tissue and polylysine is used as sticking reagent ,then the 5μm thick slices are commonly cut followed by baked at 58℃for about 5h, incubated for 30min using the solution of 3% H2O2-CH3OH in room temperature, and restored with microwave energy for 20 min in 0.01 mol/L PH 6.0 citrate buffer solution, then cultivated for 30min in wet box in room temperature with 5% BSA. Secondly,the dilution of the primary antibody is 1:200 and brooded for 60min at 37℃;the working concentration of the HRP-IgG is 1:35 and brooded for 40min at 37℃. Finally, 5 min are for coloration in DAB and 3 min for dye in Hematoxylin. The results of crossover trial,absorption test,substitution test,positive and negative control experiments indicated that this method has advantages of high sensitivity and specificity.The distribution regularity of CSISV in SPF chickens and Broilers has been studied after artificial infection using established method of immunohistochemistry (IH for short). The results of SPF chickens show that all organs appear negative reaction in 3h later after infection;the positive cells are detected in thymus and glandular stomach 6h later;CSISV are detected in glandular stomach, thymus, bursa, Kidney, duodenum, heart, liver, lung, pancreas, spleen, and jejunum 36h later;all organs presented strong positive 168h after infection. The positive are still strong in glandular stomach, thymus, bursa, Kidney, duodenum 216h after infection, however, other organs are not strong positive as before. The CSISV still can be detected in glandular stomach, thymus, bursa, kidney, duodenum 720h after infection through it can not be detected in all organs 960h later. In the whole process of the infection, no positive cells are detected in trachea though they are detected more or less in all other organs. The distribution rates of caecum and rectum are the lowest. With respect to SPF chickens'stool, the CSISV can be detected in stool 3h after infection as well as 720h later, whereas it can not be detected 960h after infection.The positive detection rates of CSISV antigen are high in the infectious chickens'glandular Stomach, thymus, bursa, duodenum and kidney. The glandular stomach's average detection rate is the highest, and 84% in thymus, 75% in bursa, 70.45% in kidney, 68.18% in duodenum. The average detection rates are 52.27%, 52.27%, 54.55%, 45.45%, 45.45%, 43.18%, respectively for liver, spleen, jejunum, lung, pancreatic, gland and ileum. The rate in heart is relatively low (29.55%), yet the lowest result appeared just in caecum (11.36%) and rectum (18.18%).The target cells of CSISV mainly include epithelial lining cells and lamina superficialis simple tubular gland in glandular stomach, thymic lymphocyte and macrophages in thymus, lymphocytes bursa in spleen, penal tubular epithelial cell ,foot cell and capillary endothelium cell in kidney, intestinal goblet cell, lymphocyte and capillary endothelium cell in duodenum ,hepatic cells and Kupffer cells in liver, alveolar epithelial cell, alveolar macrophage and capillary endothelium in lung, cardiac muscle cell and capillary Endothelium in heart. Epithelial cell and lymphocyte are the chief target cells of CSISV in all cells.The CSISV has the most strong tissue tropism to glandular stomach, thymus, bursa, kidney and duodenum. It indicates that these five tissues are CSISV's main target organs.In general, the CSISV's distribution regularity in Broilers is mostly the same with SPF chickens. The differences between them only lie in varieties and individuals.
|
PDF Full Text Request