Lawsonia intracellularis is an enteric pathogen that causes thickening of the intestinal mucosa layer resulting in malnutrition, diarrhea, and poor weight gain in swine. L. intracellularis increases intestinal cell proliferation in vivo. Swine enteroids are 3-dimensional in vitro models that can be used to study bacterial infections. The objective of this study was to evaluate cellular proliferation in swine enteroids infected by L. intracellularis. Ki-67 is a protein expressed by cells that are actively proliferating and is often used to document changes in proliferation. Swine enteroids were cultured in monolayers using transwells, infected with L. intracellularis (PHE/MN1-00), and harvested on 5, 7, and 14 d post infection (dpi). Enteroid monolayers were fixed in 4% formaldehyde overnight, and immunofluorescence for Ki-67 and L. intracellularis were performed. Experiments were replicated 3 times. Immunofluorescent staining using L. intracellularis antibodies demonstrated infection of enteroids. Cell proliferation was assessed by counting the number of Ki-67 positive cells under 20x magnification and comparing to the total number of cells as stained by 4′,6-diamidino-2-phenylindole (DAPI) in infected and non-infected enteroids. Cell counts were analyzed using paired t-test in GraphPad Prism comparing infected enteroids with uninfected enteroids for each dpi time. Cell proliferation between non-infected and infected enteroids were not different at 5 dpi (0.014±0.007 vs. 0.044±0.021, P = 0.21) or 14 dpi (0.101±0.10 vs. 0.061±0.034, P = 0.46). However, at 7 dpi, infected enteroids showed greater cell proliferation (0.67±0.056 vs. 0.078±0.58, P = .047). In infected pigs, it was reported that increased cell proliferation is observed at 7 dpi. In vitro swine enteroids reproduce the proliferative changes observed in vivo, validating their use as a model for L. intracellularis infection.

中文翻译：

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PSV-13 Lawsonia Intracellularis 增加体外猪肠道内的细胞增殖

胞内劳森氏菌是一种肠道病原体，会导致猪的肠粘膜层增厚，导致营养不良、腹泻和体重增加不良。L. intracellularis 增加体内肠细胞增殖。猪肠道是可用于研究细菌感染的 3 维体外模型。本研究的目的是评估被胞内乳杆菌感染的猪肠道内的细胞增殖。Ki-67 是一种由活跃增殖的细胞表达的蛋白质，通常用于记录增殖的变化。使用 transwells 在单层中培养猪肠道，用胞内乳杆菌 (PHE/MN1-00) 感染，并在感染后 5、7 和 14 天 (dpi) 收获。肠样单层在 4% 甲醛中固定过夜，免疫荧光检测 Ki-67 和 L。进行了胞内。实验重复 3 次。使用胞内乳杆菌抗体的免疫荧光染色证明了肠样的感染。通过在 20 倍放大倍数下计数 Ki-67 阳性细胞的数量并与感染和未感染的肠样中被 4',6-二脒基-2-苯基吲哚 (DAPI) 染色的细胞总数进行比较来评估细胞增殖。使用 GraphPad Prism 中的配对 t 检验分析细胞计数，比较每个 dpi 时间受感染的小肠与未感染的小肠。在 5 dpi（0.014±0.007 对 0.044±0.021，P = 0.21）或 14 dpi（0.101±0.10 对 0.061±0.034，P = 0.46）时，未感染和受感染肠样之间的细胞增殖没有差异。然而，在 7 dpi 时，受感染的小肠显示出更大的细胞增殖（0.67±0.056 对 0.078±0.58，P = .047）。在受感染的猪中，据报道，在 7 dpi 时观察到细胞增殖增加。体外猪肠道菌群重现了在体内观察到的增殖变化，验证了它们作为胞内乳杆菌感染模型的用途。