The entry of Mycobacterium avium subsp. paratuberculosis (MAP) to the intestinal mucosa is critical to the pathogenesis and control of paratuberculosis in animals, which is poorly understood. An in-vivo multiple-intestinal loop model involving Peyer's patch (PP) and non-Peyer's patch (non-PP) areas was developed in lambs to examine (i) the attachment and uptake of MAP across the intestinal mucosa, (ii) the variation in the uptake of MAP in different segments of the small intestine and (iii) the ability of various strains of MAP to invade the mucosa. By using a number of methods including polymerase chain reaction, immuohistochemistry (IHC), in-situ hybridization (ISH), histology and transmission electron microscopy, it was observed that MAP entered the intestinal mucosa through follicular M cells (fM cells) in PP as well as possibly through villous M cells (vM cells) in non-PP areas. The fM cells are the specifically and functionally modified cells lining the follicular associated epithelium (FAE) over Peyer's patch areas, whereas the vM cells are originally the transformed enterocytes present throughout the intestinal mucosa. The observation of more number of bacteria, bacterial antigen or bacterial genome in the ileal mucosa lined with continuous PP in comparison to the jejunal mucosa with and without discrete PP suggested that translocation of MAP across the fM cells were more efficient than the vM cells. The field strains of MAP isolated from cattle and goat showed greater ability (P<0.05) for invasion into the small intestinal mucosa of the lambs than that of the vaccine strain. The demonstration of MAP genome by ISH and its antigen by ICH in the intestinal mucosa, and the inability to isolate the bacteria from the mucosal homogenate of infected loop tissues suggested that the bacteria could transform into the cell-wall deficient forms after the invasion. This could be significant from early pathogenesis point of view.