Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

Abstract

BackgroundPorcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI). However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions.MethodsA ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1) wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2) crude vaccine bacteria (Enterisol® Ileitis Vet), and 3) vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively.ResultsAlthough at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border.ConclusionsThe ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates the early intestinal invasion of L. intracellularis in vivo.

Recent Advances in Understanding the Pathogenesis ofLawsonia intracellularisInfections

“…The peak of the infection, where the majority of cells are heavily infected (greater than 30 bacteria in the cytoplasm of each cell), occurs between 5 and 7 days postinoculation, reaching 80% to 90% of infected cells. 24 Clinical and subclinical disease has been previously reproduced using pure cultures of L. intracellularis at low passages (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) in cell culture. 27,64,69,109,110 Lack of clinical and pathological changes was demonstrated in pigs experimentally infected with a porcine isolate passed 40 times in vitro, indicating that the attenuation process occurs between passages 20 and 40.…”

“…A ligated intestinal loop model was applied to study early stages of PE, but it is not feasible for investigating the molecular pathogenesis involved in the cell proliferation. 8 A microarray-based study of fibroblastic-like cells in vitro identified altered transcription of genes related to cell cycle and cell differentiation. 76 However, the main phenotypic changes characterized by cell proliferation were not reproduced, and the results need to be confirmed in vivo.…”

In vitro screening of non-antibiotic components to mitigate intestinal lesions caused by Brachyspira hyodysenteriae, Lawsonia intracellularis and Salmonella enterica serovar Typhimurium.

“…This observation may be due to the short period of in vitro incubation which may lead to a low level of bacteria infecting and propagating inside the epithelial cells. Previous authors reported that the pathogen may take up to 12 hours to invade cells after oral inoculation, or 6 hours when ligated intestinal loops were infected directly with vaccine inoculum (71,72). The ability of the vaccine strain to induce such changes is also questionable, but it has been shown to do so in vivo (73).…”

In Vitro Screening of Non-Antibiotic Components to Mitigate Intestinal Lesions Caused by Brachyspira hyodysenteriae, Lawsonia intracellularis and Salmonella enterica Serovar Typhimurium

“…This observation may be due to the short period of in vitro incubation which may lead to a low level of bacteria infecting and propagating inside the epithelial cells. Previous authors reported that the pathogen may take up to 12 h to invade cells after oral inoculation, or 6 h when ligated intestinal loops were infected directly with vaccine inoculum [ 70 , 71 ]. The ability of the attenuated vaccine strain to induce such changes is also questionable, but it has been shown to do so in vivo [ 37 ].…”