L. intracellularis infection and pathogenesis have been studied in different animal models but understanding this disease has been hampered by the bacteria’s obligate intracellular nature, the competition with numerous and diverse commensal bacterial flora, and the complicated and often distinct biochemical environment in different parts of pig’s intestine. Its primary route of transmission is oral ingestion of contaminated feces from infected herd mates or rodents. Feces from mice experimentally infected with L. intracellularis were able to infect pigs thus showing the potential of rodents to be important factors in transmission of infection to pigs and possibly horses (10). The bacteria have adapted to survive the biochemical and immune defense challenges of a mammalian organism such as the low pH and pepsin in the stomach. The bacteria move with the chyme to the small intestine where the microenvironment is adequate for infection of enterocytes in the middle and distal jejunum and ileum. The possible reasons for preferring jejunum and ileum mucosa for invasion may be the favourable nutrient and pH environment, the presence of specific commensal bacterial flora and/or some yet uncharacterized receptors
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expressed on enterocytes in these segments but not in the duodenum or large intestine. The importance of commensal bacteria for L. intracellularis infection of pigs have been shown in an experiment where germ-free pigs were resistant to infection with pure L. intracellularis culture but were susceptible to infection when commensal flora was restored (11). Commensal bacteria could provide necessary nutrients, pH environment, immune regulation or other stimulatory signals that benefit L. intracellularis survival, attachment, and penetration in mucosal enterocytes.
Due to the complexity of commensal flora and region-specific distribution patterns, the determinants of infection remain unsolved.
L. intracellularis are obligate intracellular bacteria that must attach to and penetrate cells to be able to colonize and multiply (Fig. 1.3). These bacteria have an affinity towards immature and undifferentiated enterocytes inside jejunal and ileal crypts (Fig. 1.3). Upon infection, L.
intracellularis arrests epithelial cell differentiation and maturation thus keeping them in a state of continuous proliferation, which benefits bacterial division and spreading. These proliferating cells change the normal architecture of intestinal mucosa, leading to the absence of goblet cells, the destruction of brush border area, and the presence of many proliferating, undifferentiated enterocytes (Described in more detail in 1.3.4 Immune response to infection with L.
intracellularis). Together, these microscopic changes progress to macroscopic evidence of thickened, corrugated mucosal folds often covered with fibrinonecrotic membranes or blood in cases of PHE.
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Figure 1.3. Small intestine crypt epithelial cells and initial attachment of L. intracellularis to immature enterocytes. L. intracellularis are able to survive bactericidal molecules released by crypt intestinal cells and it can persist despite commensal competition. Their motility is controlled by flagella and, using an unknown mechanism, they can traverse through thick mucus and attach to immature epithelial cells inside the crypt. L. intracellularis have affinity towards immature epithelial cells, most likely transit amplifying cells (light blue), where they invade and multiply. They impact the eukaryotic cell cycle to inhibit differentiation while inducing proliferation of these immature cells.
There are several studies describing the progression of infection and PE lesions in hamsters but most clinically relevant studies were performed in pig models (12, 13, 7). Boutup and al. 2010 described the early pathogenesis of L. intracellularis infection and explored small intestinal loops as a model to study early infection. This group reported that L. intracellularis contacts the microvilli of mature enterocytes between 3 and 6 h after surgical inoculation into intestinal loops of pigs (12). In their small intestine loop model, they reported bacterial contact with jejunum enterocytes 6 h after bacterial inoculum into the loop (12). Although interesting, care must be taken to avoid over-interpretation of these results due to the intestinal loop model being an imperfect
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model. First, the route of infection is not natural as the bacteria are injected directly into the intestinal loop and this method of infection could impair normal intestinal peristalsis as it concentrates the bacteria in a small area. Second, the creation of ligated intestinal loops could impact local microvasculature and nerves thus potentially changing pH and oxygen concentration inside the loop, which may in turn impact L. intracellularis penetration and growth. Despite these limitations, this study shows the usefulness of this intestinal loop model and shows that it has the potential to be used to explore the properties of bacteria during early pathogenesis.
Guedes and al. 2017 (7) has presented the most comprehensive study to date of progression of gross and histologic lesions during L. intracellularis infection of 5 weeks old pigs from 1 to 35 days post-infection (dpi) with oral inoculation of a dose of 4.37 x 109 L. intracellularis. Three animals, two from the challenged group and one from the control group, were euthanized on days 1, 3, 5, 8, 11, 15, 19, 24, 29 and 35 dpi (7). Macroscopic and histologic lesions of PE were first identified 11 days after oral inoculation but the first detection of L. intracellularis antigen using immunohistochemistry (IHC) was at 5 dpi (7). These data indicate that bacteria establish themselves in the small intestine during the first 5 dpi and that they need at least 11 dpi to induce characteristic lesions in the small intestine mucosa. The lesions in small intestine on day 15 were characterized as a thickening and corrugation of the ileum and middle jejunum serosa (7). The mucosa of ileum and distal jejunum (1 m length) and colon (5 cm in length) were also affected with characteristic corrugations and thick folds covered with fibrin (7). The most severe lesions were observed in the middle jejunum with areas of mucosal necrosis and findings of fibronectin membranes. Between 9 and 28 dpi all infected animals had watery diarrhea with traces of blood, abdominal lymph nodes were 3 times the normal size and 200 ml of a yellowish transudate was present (7). The peak of gross mucosal lesions and the highest number of bacteria in enterocytes
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were recorded between 15 and 24 dpi (7). At necropsy on day 19 after infection, one pig showed progression of macroscopic lesions and spreading further into jejunum and colon. In correlation with the microscopic lesions and number of bacteria present in enterocytes, an apoptotic index was measured using the terminal deoxyuridine nick-end labeling (TUNEL) method and caspase-3 was measured using IHC (7). From 11 to 24 dpi, the apoptotic index increased and then returned to control values at 29 dpi (7). These data correspond well with previous findings by MacIntyre at al.
where the peak of infection and the spread of characteristic lesions were detected at the same period in 7 weeks old pigs infected orally with a dose of 2 x 108 of L. intracellularis (13). This group also explored the immune response 41 dpi and found evidence of immune down-regulation.
Guedes et al 2017 reported the detection of the highest titres of antigen-specific IgA in intestinal lavages between 15 and 29 dpi but they did not explore the correlation of protection and the importance of the local humoral response (7). L. intracellularis antigens in the intestine could be detected up to 29 dpi but macroscopic and microscopic lesions were resolved (7). Also, after 35 days, no bacterial DNA on intestinal samples could be detected by PCR analysis which indicates complete clearance of the pathogen 35 dpi (7). These studies provide insight into stages of L.
intracellularis infection and pathogenesis, which added to knowledge of the L. intracellularis life cycle.