Isolation,Identification Of Mycobacterium Avium Subsp.Paratuberculosis From Sheep And The Establishment Of Mouse Infection Mode In Inner Mongolia

Paratuberculosis is listed in the must-report at the list of WOAH diseases,infections and Invasion by the World Organization for Animal Health(WOAH).It is classified as the third kind of animal disease in China.It causes chronic and proliferative enteritis in many ruminants.Infected animals become a continuous source of infection in farms through intestinal intermittent excretion,which has brought great economic losses to aquaculture.The pathogen of Mycobacterium avium subsp.paratuberculosis(MAP)belongs to intracellular parasitic Gram-positive bacteria,and is a third group of zoological pathogenic microorganisms,including type C(also designated as type II)and type S.Type C also includes type B.Type S can be further subdivided into sub-group types I and III.And sublineages of camelid isolates Studies have shown that each subtype of MAP has no host specificity,but is regional.Inner Mongolia is the first region of the disease in China.It is of great significance to obtain and accurately identify the subtype and genetic characteristics of MAP strains in Inner Mongolia for the prevention and control of paratuberculosis.In this study,MAP isolation culture,colony morphology identification and ZiehlNeelsen staining were performed on twenty-eigh MAP-positive sheep derived disease samples from Inner Mongolia.Amplification,sequencing and sequence analysis of IS900 gene,IS1311 gene and DMC gene were performed on the genome DNA of propagated bacteria.At the same time,the PCR products of IS1311 gene were digested by Hinf I and Mse I.Twenty-eight samples were cultured for 7-12 weeks,and colonies grew in nine medium.Colonies are translucent opalescent and smooth.A single colony was Ziehl-Neelsen staining,and the results showed that the bacteria were red-stained and slightly curved,with irregular arrangement,single or branched.The PCR amplification products of the IS900,IS1311 and DMC genes of the nine isolates were consistent with the expected size of the target gene fragment.The nine isolates were identified as MAP strains,which were named MAP-NM1 to MAP-NM9.The amplified product size of DMC gene was 310 bp,which conforms to the characteristics of type II MAP.The amplification products of nine strains of MAP IS1311 gene were digested by Hinf Ⅰ and Mse Ⅰ to obtain four bands,which were consistent with type Ⅱ MAP.The results of IS1311 gene sequencing showed that the base loci were conforms to the characteristics of type C and B MAP.The results of IS900 gene sequencing were consistent with MAP characteristics of type Ⅱ and type Ⅲ.The phylogenetic tree of IS900 genes showed that they were all divided into type Ⅱ MAP branches.The results of 3 gene sequencing were analyzed online by Blast,and the highest homology with this study was type Ⅱ MAP,and the homology was higher than 98%.In summary,all the nine MAP isolates in this study were type Ⅱ MAP.The BABL/c mouse infection model was established using isolated and cultured MAP strain.Over the course of eight weeks,the average body weight of mice in the low-dose group and the control group increased steadily,while the average body weight of mice in the high-dose group began to decrease at the 8th week.The stool was tested for the IS900 gene and all came back negative.Tissue organs are tested for the IS900 gene,the highest detection rate was found in the intestines of mice in the low-dose group and in the liver and intestines of mice in the high-dose group.The histopathological results showed that the intestinal villus epithelial cells of the experimental group were shed,epithelioid cells were observed in the spleen,and granular degeneration occurred in hepatocytes and renal tubule epithelial cells.The average transcription levels of cytokines such as IFN-γ,IL-6 and IL-10 in peripheral blood of mice were detected weekly.The results showed that the average transcription levels of the three cytokines were affected by MAP inoculation dose and infection time.In this study,for the first time,nine MAP strains from sheep in Inner Mongolia were isolated,cultured and identified.Based on this,the BALB/c mouse of MAP infection model was established.It can provide a basis for follow-up research on Paratuberculosis.