Isolation Of Mycobacterium Avium Subsp. Paratuberculosis And The Establishment Of Mouse Infection Model

Paratuberculosis is a chronic intestinal inflammation caused by Mycobacterium avium subspecies paratuberculosis(MAP).It mainly infects ruminants and is widespread throughout the world.No country has announced the elimination of paratuberculosis at present.The course of paratuberculosis can be over years,and generally divided into subclinical and clinical stages.The former is mainly on cellular immunity,and the latter is mainly characterized by humoral immunity.The duration of the subclinical period is longer,infected animals carry bacteria but do not excrete,showing no clinical symptoms such as heavy diarrhea.At present,few reports has been presented on the prevalence of paratuberculosis in cattle of China.In order to understand the prevalence of paratuberculosis in cow in local areas of China,1023 cows from 3 dairy farms in Shandong Province were investigated using methods of delayed skin hypersensitivity(SDTH),enzyme-linked immunosorbent assay(ELISA)and PCR.The number of positive cows detected by SDTH was 113,and 17 were suspicious,positive rate of the group was 11.05%.The positive rates of different herds vary greatly,with the highest being 20.16% and the lowest being 6.45%.The number of positive cows detected by ELISA was 75,and the group positive rate was 7.33%.The highest antibody positive rate of 3 herds was 16.05%,and the lowest was 3.75%.Fecal samples were collected from cows with ELISA positive results,and the fecal genome was extracted for PCR detection of IS900-specific gene fragment.A total of 74 samples were tested,the positive rate was 4.05%.Epidemiological survey results show that paratuberculosis is common in cow farms in Shandong Province,and the positive rate of some cow farms is relatively high.Paratuberculosis has become one of the most important infectious diseases in China's cow farms.To strengthen the study on the pathogens of paratuberculosis,we conducted MAP isolation with 9 cows,including 2 cows which had been confirmed positive by SDTH,ELISA,and PCR,another 7 cows had been observed with heavy diarrhea.According to the MAP's characteristic of anti-acid,we improved method of sample processing,using chemical treatments combined with antibiotic treatment,successfully explored a method that can inhibit the growth of competitor bacteria,while maintaining the activity of MAP in the sample.By this method,4 strains of MAP were successfully isolated from two different farms.The isolated strains were verified by restriction analysis of the IS1311 specific genome fragment of the MAP,and it was confirmed that all the 4 isolates were S-type.These strains were named pTB-SD-01,pTB-SD-02,pTB-SD-03,pTB-SD-04.In order to understand the characteristics of animal infected by isolated strains,we used pTB-SD-01,pTB-SD-03,and MAP standard attenuated reference strain 316 F,MAP standard virulent reference strains III-V,infected C57B/L mice respectively.The transcriptional changes of IL-6,IL-10,IFN-?,and ?-actin in peripheral blood were detected at different time points.ELISA was used to detect the changes of MAP antibodies.From the 2 aspects of cellular immunity and humoral immunity,the biological characteristics of mice infected with different virulence MAP were comprehensively analyzed,established an animal model of MAP infection initially.