关键词: 副结核分枝杆菌, 分离培养, 亚型鉴定, 羊, 内蒙古

Abstract: 【Objective】 Paratuberculosis is listed in the must-report at the list of OIE diseases, infections and Invasion by the World Organization for Animal Health (OIE). It is classified as the second 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 sub-lineages 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. 【Method】 28 MAP-positive sheep disease samples collected in Inner Mongolia were isolated and cultured by MAP, and the colonies were stained with Ziehl-Neelsen. The positive bacteria were propagated and the genomic DNA was extracted. IS900 gene, IS1311 gene and DMC gene were amplified, sequenced and analyzed. The PCR products of IS1311 gene were identified by Hinf I and Mse I double digestion. 【Result】 28 samples were cultured for 7 - 12 weeks, a total of 9 mediums grew colonies, the colonies were translucent milky white, smooth surface. Single colonies were selected for acid-fast staining, and irregular (single or branched), red-stained Brevibacterium was observed under the microscope, which was consistent with the morphological characteristics and acid-fast staining characteristics of Mycobacterium. The PCR products of IS900, IS1311 and DMC genes of 9 isolates were consistent with the expected size of the target gene fragment. 9 isolates were identified as MAP strains, named MAP-NM1 to MAP-NM9. DMC gene amplification product size of 310bp, consistent with type II MAP characteristics. IS1311 gene amplification products were digested by Hinf I and Mse I restriction endonucleases, and 4 target bands were obtained in 9 strains of MAP, which were consistent with type II MAP. The sequencing results of IS1311 gene and the analysis of MAP representative strains of type I, type II, type III, Indian Buffalo and American Buffalo showed that the nucleotide sites at positions 64, 65, 68, 223, 236, 422, 527 and 628 of the nine MAP IS1311 gene fragments conformed to the characteristics of type C and type B MAP. Sequence analysis of IS900 gene sequencing results showed that the 169th and 216th nucleotides of the nine MAP IS900 gene fragments were C (cytosine) and A (adenine), and accord with Type II and type III MAP. The phylogenetic tree of 17 MAP IS900 gene reference sequences from GenBank database with 9 isolates in this study showed that the 9 isolates in this study were all in the type II MAP branch. Blast online analysis was performed on the sequencing results of the three genes. The reference sequences with the highest homology with the isolates obtained in this study were all type II MAP, and the homology was higher than 98%. In conclusion, all the 9 MAP isolates were type II MAP. 【Conclusion】 To the best of our knowledge, this is the first isolate of the MAP type Ⅱ strains in sheep in Inner Mongolia. 

Key words: Mycobacterium avium , subsp. , paratuberculosis,  , isolated, typing,  , sheep,  , Inner Mongolia