Mucosal epithelial cells are infected by a wide variety of pathogens and determining their response to infection is critical for understanding disease pathogenesis. A protocol was developed for culturing primary epithelial cells from fetal bovine intestine and the cultured cells were evaluated for susceptibility to an enteric viral infection. Immunohistochemical staining for cytokeratin confirmed that 60-75% of cultured cells were epithelial cells. Furthermore, following infection with bovine rotavirus (BRV) over 80% of cells in the ileal and jejunal cultures contained viral protein at 16 h post-infection. The intestinal epithelial cell cultures also contained fibroblasts so a jejunal fibroblast culture was established and infected with BRV. Viral protein was detected in jejunal fibroblasts but viral-induced cytopathology was delayed in fibroblast cultures when compared to epithelial cell cultures. This study describes an effective protocol for culturing bovine epithelial cells from fetal intestine and confirmed that the epithelial cells were susceptible to BRV infection. Ileal and jejunal cultures displayed limited growth following continuous passage but early passage epithelial cells provide competent target cells for studying host cell responses to an enteric viral pathogen.

. 2005. Microarray analysis of gene expression following preparation of sterile intestinal "loops" in calves. Can. J. Anim. Sci. 85: 13-22. The surgical preparation of multiple, sterile intestinal "loops" has provided an effective model for the analysis of vaccine-induced mucosal immune responses in ruminants. The objective of the present investigation was to evaluate intestinal "loops" as a model for genomic analyses of mucosal immune responses. Immunohistochemistry revealed no significant changes in mucosal epithelial cell architecture but microarray analyses were completed to determine if surgery and elimination of microflora significantly altered gene expression in the small intestine of one month-old calves. RNA was isolated from intestinal "loops" (Sample) and adjacent, intact intestine (Control) of each animal at 48 h (n = 3) and 12 d (n = 3) postsurgery. Total RNA from control and sample tissues was hybridized on Pyxis Genomics bovine cDNA microarrays (7884 ESTs) to identify differentially expressed genes. We observed only 2.3% (180/7884) of ESTs were significantly and differentially changed in expression at 48 h post-surgery and approximately 80% (143/180) of these genes were up-regulated. A subset of these differentially expressed genes was validated by quantitative Real Time PCR and these analyses demonstrated that many genes were returning to baseline expression levels at 12 d post-surgery. Notable among differentially expressed genes were gene clusters involved in apoptosis, cell cycle, and cell differentiation. Surgery and elimination of microflora in 1-mo-old calves significantly altered the expression of a relatively minor number of genes but these genes were implicated in important aspects of normal mucosal function. Therefore, intestinal "loops" that control for the effects of surgical manipulation and commensal microflora must be included when conducting mucosal gene expression analyses within this animal model. Can. J. Anim. Sci. 85: 13-22. La préparation de nombreux microéchantillons stériles de « boucles » intestinales par intervention chirurgicale s'est avérée fort utile pour analyser la réaction immune de la muqueuse induite par un vaccin chez les ruminants. La présente étude devait évaluer l'utilité des « boucles » intestinales comme modèle pour l'analyse génomique de la réaction immune de la muqueuse. L'immunohistochimie ne révèle pas de changement significatif à la structure de l'épithélium de la muqueuse, mais on a procédé à une analyse des jeux de microéchantillons pour savoir si l'intervention et la suppression de la microflore modifient de manière significative l'expression des gènes dans l'intestin grêle des veaux d'un mois. Les auteurs ont isolé l'ARN des « boucles » intestinales (sujet) et de l'intestin intact adjacent (témoin) de chaque animal 48 heures (n = 3) et 12 jours (n = 3) après l'intervention. Tout l'ARN des tissus du sujet et du témoin a été hybridé sur des jeux de microéchantillons d'ADNc bovin de Pyxis Genomics (7 884 séquences génomiques exprimées ou SGE) de manièr...