Animal Infection Assay And Setting-up Of Fluorescence Antibody Detection To Canine Coronavirus From Giant Pandas

Giant panda is a kind of precious animal throughout the world, which is under the threat of the existence due to its weak ability to survive and the worsening of environment by man-made factors. Disease, especially viral disease, is greatly menacing the survivability of pandas at all time. Canine coronavirus(CCV) is demonstrated in dogs with signs of acute gastroenteritis syndrome, which endangers the dog-raising industry. Three giant pandas were ill in the zoo of Chongqing in 1997, and two died after some treatments. A strain of virus was isolated from pandas' liver and spleen and was identified to be canine coronavirus, which was named as CCV DXMV. In order to probe what role that CCV DXMV played in the death of pandas several assays were carried out as followed.Pups were divided into three groups at random: test group(group A)was injected with CCV DXMV; positive group(group B) with standard strain CCV NL-18; negative group (group C) with CRFK cell culture. After taking virus orally, the tendency of temperature changing to group A and B were the same. Temperature rose to some extent, and then went down on a large scale. Rising extent of temperature was larger than that of falling extent. The main clinic symptoms: somnolent, anorexy and vomittd milk; always huddled together to get warmer with fluffy feather; excreted fetid loss stool with undigested milk-particles and blood mucus in it; excretion sticked perianal feather causing difficult defecation.Pups in group A and B all died in 5 days(group C normal). Anatomic changes were the same or similar in these pups: acute catarrhal enteritis, and some even hemorrhagic enteritis. Intestine dilated and intestinal wall became thinner and translucent that vessel could be seen clearly, in which was full of gray or flavo-green fetid liquid. There was much blood-like mucus to hemorrhagic enteritis; cardinal red blood flowed out from the cross-section of liver, heart, kidney and spleen; Gray or ravous focuses located on lung, which was almost lobular and fused into lobar sometime; Vascular net was clearer than normal on the surface or cross-section of the brain.Pathological changes: acute catarrhal enteritis in intestine, accompanied with epithelial cell proliferation in the glands of crypt; severe congestion in liver, central vein and capillary of perisinusoidal space hyperemia; splenic sinusoid hyperemia with some inflammatory cell infiltrate; interstitial nephritis in kidney; capillary of cardiac muscle fibers hyperemia andhemorrhage; nerve cell vacuolar degeneration with microglial nodules in brain; interstitial pneumonia in lung.In order to prove changes above were caused by CCV DXMV, CCV DXMV-FA and CCV NL-18-FA were applied to detect viruses in the tissue. Directed method of fluorescence antibody(FA) was preliminarily set up with positive frozen section(f.s.) from organs infected with CCV NL-18 and positive labeled antibody from CCV NL-18-FA. Testing titer of CCV DXMV-FA was 64 time diluted, and working titer was 32 time diluted; in specific test, there were not specific fluorescence in idofluorescence contrast and specific contrast 1-3, and bright flavo-green fluorescence could be seen in positive contrast and test samples.The results of f.s. dyed with CCV DXMV-FA showed that fluorescence only existed in cytoplasm successfully or focally, but there was no fluorescence in nucleus. In intestine, fluorescence mainly located in flbroblast and reticular cell of lamina propria of villi, and also in epicytes of proliferated crypt glands; in spleen, fluorescence mainly located in vascular endothelial cell and flbroblast of trabeculum and a little lymphocyte around them; in liver, fluorescence mainly located in endothelial cell of interlobular vein of portal area and fibroblasts around it. The research above could prove that CCV DXMV invaded the body and duplicated in the corresponding cells which led pups to death.CCV DXMV was proved to be a velogenic strain and had strong pathogenicity by animal infection test and fluorescence detection of CCV DXMV. It could be revealed that the virus was one of the reasons to the pandas' death. The study not only provided a new detection to CCV DXMV with rapid, specific, sensitive and effective characters, but also enriched the pathological contents of CCV.