The Study Of The Distribution And Location Of Rabies Virus In Suckling Mice And Canines Brain Tissues

The 6 days old mice were infected artificially with SRV-9 rabies virus strain via intracerebral inoculation.When clinical signs were presented,all mouse were killed humanely in the biological safety and sterile situation.Some of the brain tissues from the mice and natural infected canines brains were fixed in the 4%paraform for immunohistochemistry,and the others were detected by nested RT-PCR and direct fluorescence antibody test.A nested RT-PCR was developed to detect rabies virus,and there was not any cross reaction with two other canine viruses including CDV and CPV.The positive mouse brain infected with the SRV-9 virus strain and the natural infected canines brains were amplified a specific 344 bp fragment.In addition,the direct fluorescence antibody test was used to detect the RV antigen in the brain swabbing slices,and green fluorescent light was observed under the fluorescence microscope.An indirect immunohistochemistry test was established to investigate the distribution and location of the SRV-9 virus strain in the mouse-infected brain and natural infected canines brains. Several conditions were optimized,for example,the antibody was prepared and purified with the methods of salt precipitation,DEAE52 chromatography and protein A sepharose chromatography.The concentration of the primary antibody was 2.216mg/ml,and the rabbit anti-horse IgG antibody was 1.452mg/ml.After labelling with the alkali phosphatase,the concentration was 0.97mg/ml,the titer was 1:3200.The standard,procedures of indirect immunohistochemistry test was established according to the following steps.The retrieval buffer was citrate buffer solution(0.01M PH6.0);the retrieval antigen was used by the high temperature and pressure;the first antibody of horse anti-RV serum was diluted 50 time in antibody diluent solution and the second antibody of rabbit anti-horse IgG-AP were diluted 100 time.An indirect immunohistochemistry,which has the highly sensitivity,strongly stability and highly repeatability,was established.The method was used to investigate the distribution and location of rabies virus in the infected mice brain,and had the better results,compared with the histopathologic HE stain.The histopathologic HE stain did not find the eosinophilic inclusion in the mice brain,and four of eight canines brain have the inclusion in the cytoplasmic of Purkinje cell bodies,but the result of indirect immunoistochemistry test all were positive and weakly cytoplasmic signals within neurons of the hippocampus CA1,CA3,(excluding the dentate gyms) in the mice brain but it was strong signals in the canines brain.A strong cytoplasmic signal was apparent within Purkinje cell bodies and within primarily pyramidal neurons of the cerebral cortex and in the cerebellum in the mice and caines brains.This study can establish the basis for the etiopathogenesis and the distribution in the infected animals organism of the rabies virus.