Identification Of Cryptosporidium Isolated From Dogs And The Effects Of Immune Indices On Infected Mice

Cryptosporidium was one of the important prozotoan parasite,which infected people and animals together. It was found in experimental mice gland in 1907. Cryptosporidisis is a worldwild distribution anthropozoonosis with the main clinical signs of diarrhea.The disease has severely harm to human health and animal production and been recognized as one of the cause of mortality in association with the acquired immunodeficiency syndrome(AIDS).The control and prevention centre of USA has classified the disease as B etiology. This disease was also classified as one of the two kinds of important parasitic disease that our country took precautions against especially in 2003. In recent years,cryptosporidium was studied and made a lot of important achievements in many respects such as aetiology,morphology,epidemiology, pathogenic molecular biology,diagnosis and integrated control technology ;But as the morphologic features among differential genus often do not markedly,And in present lack of consistency in the clasiification of protozoan parasite in general in domestic and abroad.With the development of the technology of molecular biology,these reports constantly rush about the new genetic of Cryptosporidium,Which make the precise number of Cryptosporidium unknown.We confronted great difficult in preventive and control this disease.the host immune function appears to be less present report, therefore characteristics of the host immunity has been our concern.The subject has taking an epidemic investigate of cryptosporidium to in part farm of hefei in Anhui province , survey the postive dogs of infected cryptosporidium , observing the morphologic oocyst of Cryptosporidium. Morphology and Technology of molecular biology were applied to identify the havested oocysts: Attempt to determine genotypes of all Cryptosporidium in dogs to expound mechanism of Cryptosporidium molecule epidemiology; Take the oocyst to construct an animal model in order to offer the theoretical base for elucidating the pathogenic mechanism and researching the prevention and regulation.Firstly,the 232 fecal specimen in dogs was detected by satturated white sugar, we preliminaryly classified the Cryptosporidium harvested in hefei as Cryptosporidium parvum by the oocyst characteristics such as shape constitution, size and the index number of size. The infection rates of the other three dairy farms is greatly different. Counted and analyied infection of dog in different age,each age dog could infect cryptosporidium, but different age dog with different situation. Puppies's infection rate was higher than the other age of dogs notably.Meanwhile, different age dogs are different in infected intensity.sencondly,we applied the discontinous sucrose gradients to purified oocysts, which was shaked by shaker for 15 minutes, added plitting liquid(2%DTT,1×TE),freezed and melted for three times in -70℃, water-bath for 20 minutes in 90℃, extracted DNA from oocysts finally. We designed and synthetized the specific primer of cryptosporidium according to the crytposporidium 18SrRNA then amplifing the specific 540bp fragment. In the same time, we designed and synthetized the specific primer of cryptosporidium according to the crytposporidium HSP70 then amplifing the specific 448bp fragment The reagent box of PCR was used to purified PCR products and the products was sequenced by shanghai sangon company. According to the homology sequence in NCBI, we take the biological sofe such as DNAStar analyze the sequence and construct phylogentic trees, confiring that the Cryptosporidium is Cryptosporidium parvum in hefei for the molecular level.Lastly ,we constructed animal models, 15 BALB / C mice were randomly divided into five groups , A, B, C, D and E meanwhile every group was feed in different cages. The number of mice in every groups is 3. The belly of mice in group A were inoculated 60μL Cryptosporidium with syringe by the channel of mouth ,those mice drinked water with dexamethasone and the does is 1.5mg/100ml , renoveatured water every day until the end of this study . The group B are same as A,excepting to add the drug of immunosuppression and the does is suppression, 1.0mg/100ml; The group C as an non-immunosuppressive group, the way of inoculation Cryptosporidium as A and B,meawile ,the way of drinking without the drug of immunosuppression. The group D as an immunosuppressive group add the drug of immunosuppression, and the does is 1.0mg/100ml; The group E is an non-immunosuppressive group.We count the number of oocyste get from the mice feacal and record the law of discharge oocysts and others like the intensity of oocysts. When the mice died,we dissect dead body to observe the changes in endosomatic immune organ. During of feeding ,we took a little blood from eyes, separating serum form blood and knowing the total amount protein, albumin and globulin in serum, knowing changes of immunosuppressive agents in infected animals immune function.Berifly ,the result we get froming the study provided with some significant reference for prevention and cure Cryptosporidium in hefei; and monitored Cryptosporidiosis. And provied some improtant reference for studying the changes in endosomatic immune organ when was infected and studying the pathogenic mechanism of Cryptosporidium and choosing drug for curing the disease.