Establishment Of Animal Model With Fox Source Canine Adenovirus Type I And Real-time PCR For Detection In Fox

Canine Adenovirus Type I can cause canine animal infectious hepatitis bear and for encephalitis.This disease has strong pathogenic.The disease which have been reported in most countries around the world.This disease is characterized by strong pathogenicity,infecting a wide range of animals and threatening fur animals seriously like foxes.Once the foxes are infected by the disease they are not easy to be cured and continue to expel toxin up to nine mouths.This disease has a strong ability to resist the outside.Therefore,research on CAV-1 is extremely important.In order to research for the pathogenicity of Canine Adenovirus Type I,the virus was identified by projection electron microscopy.We can see the virus particle structure completed around 80 nm by electron microscope.Then we cultivate canine kidney cells with cell culture flask by adsorption method.We can see the cells appear a typical type of cell lesion that are grape bunches gathered and rounded.Canine type Adenovirus can agglutinate chicken,human O-type blood and the Canine Adenovirus can agglutinate guinea pig`s red blood cells,so we verified different PH PBS on guinea pig RBC aggregation effect.The result is when PH value of 7.2 a better aggregation can occur.In order to detect the pathogenic of Canine Adenovirus Type 1.We use Canine Adenovirus Type 1 artificially infect silver fox and observe incidence of the animals' disease.We determined the ID50 of silver fox which inffected Canine Adenovirus Type 1 Virus is 10-4.63/mL by designed a method that use the same attack dose but different dilution times of the original virus to attack the fox.We used Canine Adenovirus Type 1 Virus to infect the silver foxes of different ages.The results showed that with the silver foxes months age increasing the immune function of the foxes also enhanced.1TCID50 dose of F1301 strains can`t make silver foxes whose age are more than 5 months completely dead.From the clinical symptoms the silver foxes` average body temperature has risen to 39.7 within 48 hours? of the onset of illness,during this time they are quiet and don't eat anything and accompanied by the symptoms of nervous twitch.It is distinctly see the liver congestion swelling,interstitial brittle and with red and yellow,alternating on the surfare by vivisection.Meanwhile we can find the spleen enlargement and some hemorrhagic spots on spleen and lungs.Most of liver cells are necrosis by observe the pathological.In order to detect the contend of CAV-1 in Silver Fox.We designed the primers according to the unique E3 region of CAV-1.The CAV-1 gene fragment was amplified by conventional PCR method and Ligated With pEASY?Blunt Simple Cloning Kit Vector,they were transformed into competent cells.The Standard were prepared by picking bacteria,shaking the bacteria and soon,finally we established the corresponding standard curve.The result showed that each curve appears a good linear relationship with each other through the climatic amplification curve.The correlation coefficient R2=1,it indict that the standard curve is linearly good.A single peak can be seen from the dynamic amplification curve and the melting cure.It indicts that a better specificity of the establishment of the curve.The coefficient of variation(CV%)of the three repeat tests was no more than 2%.This indicates that the curve was reproducible.The clinical samples were detected by the established Fluorescence Quantitative PCR Assay and the routine PCR detection method.The results showed that the sensitive of the routine PCR detection method was lower that the Fluorescence Quantitative PCR detection method.The established fluorescence detection method can quantitative analyze CAV-1 virus effectively and quickly.It provides a convenient method of detection CAV-1.