3.1 How does Lawsonia intracellularis infect the intestinal cells?

Lawsonia intracellularis has only recently been recognized as a pathogen and, therefore, its virulence factors are as yet not well characterized. Its major pathogenic mechanism is infection and hyperplasia of enterocytes (Lawson and Gebhart 2000). Generally, no significant inflammatory response is reported and the infection remains localized in the enterocytes.

Attachment and entry of this bacterium into immature epithelial cells occurs at the apical surface. Specific adhesins or receptors for L.intracellularis have not been characterized yet; however, attachment and entry appear to require specific bacterium-host cell interaction (McOrist et al. 1995). The process of invasion does not depend on L.intracellularis viability as eukaryotic cells could still internalize formalin-fixed organisms (Lawson et al. 1995). In the same study, invasion was significantly reduced by blockage of cellular metabolism and cytoskeleton rearrangement by cytochalasin D. However, other mechanisms of cell entry may be involved as many cells still became infected despite treatment with cytochalasin D. L.intracellularis subsequently escapes its membrane-bound vacuole and lies free in the cytoplasm. This mechanism of escape from membrane-bound vacuoles into the cytoplasm and avoidance of the damaging effects of phagolysosomal fusion is also observed in several other species of intracellular bacteria, such as: Shigella, Listeria, Rickettsia spp. and Clostridium piliforme. Lytic toxins (cytolysin or hemolysins) confer this activity for those bacteria species. Cytolytic (hemolytic) activity was observed in L.intracellularis infection in vitro (Hannigan 1997). Recently, a hemolysin protein that might be involved in attachment and invasion was expressed by L.intracellularis in vitro and in vivo (McCluskey et al. 2002).

Cell proliferation, an important feature of proliferative enteropathy, has not been reproduced in vitro. As a result, most studies on the pathogenesis of L.intracellularis have been conducted in vivo. The mechanism by which L.intracellularis induces cell proliferation is unknown. In vivo, proliferating enterocytes show poor major his­tocompatibility complex class II expression. This loss of antigen-presenting function may provide an immunological safe environment for L.intra-cellularis to grow (McOrist et al. 1992, 1995). Temporary reduction of apoptosis induced by L.intracellularis infection might be one of the mechanisms involved in enterocyte proliferation. In one study, the absence of the bacteria was associated with resumption of apoptotic events in the intestinal mucosa and an increase of normal epithelial cells in recovering lesions (McOrist et al. 1996). However, no quantitative or statistical evaluations of the apoptotic events were done in affected and nonaffected cases. Machuca et al. (1999) showed an increase of apoptosis in hyperplastic crypts and villi of pigs with naturally occurring proliferative enteropathy. Hyperplastic crypts in the ileum, presumably highly infected by L.intracellularis, had significantly more apoptotic cells, detected by caspase-3 immunohistochemistry stain (p<0.0002), than normal crypts (Guedes 2002). It appears that cell proliferation, a characteristic feature of proliferative enteropathy, is not caused by reduction of apoptosis. Future studies will bring insights into the relation between L.intra-cellularis infection and cell proliferation.

Morphological studies of early lesions in experimentally infected animals have indicated that enterocyte hyperplasia is directly preceded by the presence of the intracellular organism (Jacoby 1978, Johnson and Jacoby 1978, Guedes 2002). In vivo, the onset of hyperplasia associated with proliferative enteropathy follows an increase in intracellular L.intracellularis numbers in the enterocytes. Likewise, resolution of the lesions is closely related to the disappear­ance of the intracellular organisms, indicating a close association between the two events (Lawson and Gebhart 2000). The means by which L.intracellularis produces hyperplasia is unknown.

Multiplication of the bacteria by binary fission free in the cytoplasm was observed from two to six days after cell culture infection. Drugs that inhibit cell growth also inhibit multiplication of L.intracellularis, indicating that cell division is required for bacterial multiplication (Lawson et al. 1995). Six days after infection, highly infected eukaryotic cells have balloon-like, cytoplasmic protrusions, packed with bacteria that were then released from the cell (McOrist et al. 1995).
(Microfold) M cells may be involved in the pathogenesis of L. intracellularis infection as proliferative enteropathy lesions in early stages of infection appear in the Peyer’s patch area of the intestine (McOrist et al. 1993).