Establish And Primarily Application Of Bovine Paratubrculosis Detection Methods

Bovine paratuberculosis is a chronic digestive system diseases caused by Mycobacterium avium.subsp paratuberculosis(MAP),as a result of the serious symptoms of dairy cattle infected with MAP, such as the decline of milk production, lose flesh continuously,intractable diarrhea and even death, Bovine paratuberculosis brings a great economic loss to animal husbandry. There's also evidence shows that it has relationships with Chron's disease,researchers brings more attentions on this disease. MAP is an intracellular bacterium and has a strong environmental resistance. Bovine paratuberculosis in young calves is usually through the oral route and have a long subclinical stage.The clinical symptoms accurs at the late period. Currently there is no effective treatment for Bovine paratuberculosis, find and knock out the infection bovines is the effective way to purification the dairy farm. Because the body after infected with the bacteria have the features of the separation of cellular immunity and humoral immunity, it brings a lot of trouble to detect the disease in time. At the early stage of Bovine paratuberculosis, cellular immunity is the main immune response with bacterium excretion intermittently, while the humoral immunity is enhanced with bacterium excretion gradually increased after a 2-5 years subclinical stage.The sensitivity and specificity of differernt detection methods varies a lot aiming at different stages after infection, together with the highly homologous between MAP and Mycobacterium bovies, there's a big trouble in desease dignosis. Establish Bovine paratuberculosis detection method is beneficial to find cattle in early infection and eliminate in time, to greatly help purify the dairy farm. Research of diagnosis methods of Bovine paratuberculosis and different diagnosis suitable for different stages after infection is of great importance in terms of immune law of Bovine paratuberculosis.This article is mainly focused on pathogen detection and immune detection of Bovine paratuberculosis, and compare these detection methods established with traditional STDH.In pathogen detection, we recoveried Mycobacterium phlei using Glycerol AGAR medium, and then transfer to Glycerin broth medium, after a series of distillation and extraction, mycobactin was successfully achieved. Add the mycobactin at 1% into the medium, we product paratuberculosis culture medium successfully. The detection results of recovery of P18 showed that the medium is of good culture ability. A specific PCR detection methods was successfully established according to MAP specific insert sequence IS900 by apair of primers, and the sensitivity and specificity of this method was verified by Mycobacterium bovies, Mycobacterium phlei, Mycobacterium avium and Mycobacterium intracellulare, the detection result of 300 fecal samples shows that there were 38 positive samples.In serological detection, we expressed five MAP specific protein including MAP0855,MAP0862, MAP1345, MAP2154, MAP2963. These specific protein obtained by prokaryotic expression were used as coating antigen after purifed and quantification, five indiret ELISA methods were primary established after a series of optimization. The detection results of these ELISA methods showed that MAP0862 and MAP1345 are of good diagnostic value, and the coincide rate with IDEXX kit were 94.3% and 93.7%. In this study, we also researched the serological detection results of MAP0862 and MAP1345 in cooperation, the result showed that the coincide rate with IDEXX kit is 92.7%. These ELISA methods primarily established were used to detect bovine serums in different times after infection and research change ruler of antibody to different specific antigen.Through analyzing the structure of the specific protein expression, the strong epitopes were selected to chemical synthesis, MAP2# and MAP3# based on MAP0862 and MAP145 are of good diagnostic value.The detection results of 300 young cattle from a dairy in Shandong using PCR and ELISA methods established in this research, traditional STDH and commercial ELISA kit showed that using deferent detection methods there exist difference.This study preliminarily established bacterial culture, PCR detection and antibody diagnostic method suitable for Bovine paratuberculosis.