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Document Type

Thesis - University Access Only

Award Date

2015

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Radhey Shyam Kaushik

Abstract

Intestinal epithelial cells play an important role in innate immune responses against enteric pathogens. No intestinal epithelial cell lines derived from young calves are currently available. Many enteric pathogens that cause diarrhea chiefly infect young calves. The main goal of this study is to establish a stable bovine intestinal epithelial cell line that can be maintained in continuous culture. This cell line will serve as a model system for studying innate immune responses against enteric pathogens. A primary bovine intestinal epithelial cell (BIEC) line, designated as BIEC-c4 was established from ileum of a young calf using limiting dilution method. Immunohistochemistry confirmed its epithelial phenotype, as cytokeratin was strongly expressed in these cells. The established BIEC-c4 cell line was immortalized using simian virus 40 large T antigen (SV40), human telomerase reverse transcriptase (hTERT) and human papilloma virus (HPV) E6/E7 genes. These immortalized BIECs also expressed cytokeratin protein, confirming the epithelial phenotype. Confirmation of expression of SV40, hTERT and E6/E7 genes was done by polymerase chain reaction. Immunohistochemistry and immunofluorescence assays confirmed the expression of these proteins in the immortalized BIECs. Growth kinetics analysis indicated that there were no significant differences in doubling time of the three immortalized BIEC cell lines as compared to BIEC-c4 cells. xvii Intestinal epithelial cells express various cell surface sugars. All the four BIEC cell lines were characterized for the expression of various sugar moieties by assessing their lectin binding profile. MALII and SNA lectins recognize and specifically bind to sialic acids with (α-2, 3) linkage and (α-2, 6) linkage respectively. Enteric pathogens, including rotavirus and coronaviruses bind to sialoglycoconjugate receptors on epithelial cells. Using flow cytometry, 23 lectins were tested for their ability to bind to different sugars present on all four BIEC types. Both MALII and SNA showed strong staining on BIEC-c4 cells, as well as on SV40, hTERT and HPV immortalized BIECs. In this study, BIECs were characterized for the expression of Toll-like receptors (TLRs) that play an important role in sensing pathogens. The expression levels of various TLRs (1-10) mRNA were compared among all the four BIEC cell types using real-time reverse transcriptase polymerase chain reaction (qRT-PCR). As a positive control, various TLRs mRNA expression was studied in bovine jejunum tissue. This study showed that while bovine jejunal tissues expressed many TLRs, the four BIEC cell lines did not express mRNA for any of the TLRs. The products from real time qRT-PCR were also resolved on agarose gel and both the size as well as intensity of bands were in accordance with the Ct values obtained from PCR for the bovine jejunal sample. However, no products were detected in BIEC samples. This study indicates that the various BIEC cell lines may not be very suitable for studying TLR-mediated innate immune response as they did not express any TLR mRNA at the detectable levels. Studies on primary BIEC infectivity and pathogenesis were performed using three different enteric viruses that cause diarrhea in young calves with high fatality: bovine rotavirus (BRV), bovine coronavirus (BCV) and bovine viral diarrhea virus (BVDV). xviii Using immunological and molecular techniques, it was shown that while BRV and BCV infected BIEC cells, neither of them were able to replicate productively. Based on morphological study, no cytopathic effect was observed after 2 passages for any of the three viruses. Immunofluorescence and viral titer assays using FITC conjugated antibodies specific to virus proteins confirmed lack of virus replication in the infected BIEC cells. This was also confirmed by real-time qRT-PCR analysis for bovine rotaviruses in culture supernatants. Concluding remarks: Primary culture of bovine intestinal epithelial cells from ileum of a young calf was successfully established. The BIEC cells were immortalized using SV40, hTERT and E6/E7 genes. Flow cytometry was used to study lectin binding profile in these cells. All the four BIEC cell types expressed sialoglycoconjugate receptors based on the binding of MALII and SNA lectins. Real-time qRT-PCR showed that the established BIEC cell lines do not express any TLR. Immunofluorescence assay, virus titer assay and real-time qRT-PCR analysis confirmed that while BRV and BCV infect BIEC cells, neither of them were able to replicate in these cells. Therefore, intestinal epithelial cell lines from young calf developed in this study may not be a suitable model for studying TLR mediated innate immunity or pathogenesis with BRV, BCV and BVDV.

Library of Congress Subject Headings

Calves--Immunology
Epithelial cells
Ileum
Cell culture

Description

Includes bibliographical references (pages 138-150)

Format

application/pdf

Number of Pages

168

Publisher

South Dakota State University

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Rights Statement

In Copyright