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Veterinary/zoology session

Interleukin-6 production by lipopolysaccharide stimulated cultured rumen epithelial cells
Mitze Stefanie - year 5
SzIE, Faculty of Veterinary Science, Department of Physiology and Biochemistry
Supervisors: Zsuzsanna Neogrády DVM, Gálfi Péter DVM


The subacute ruminal acidosis (SARA) which occurs mostly in high yielding dairy cows after parturition at the beginning of the lactation is usually triggered by a high intake of easily fermentable carbohydrates in conjunction with low fiber consumption. As a consequence, increased formation of volatile fatty acids (VFA) can be observed within the rumen. The ruminal pH drops for several hours per day under 5.5-5.6 resulting in the overgrowth of acidophil Gram-positive bacteria producing further VFA and the parallel destruction of the usually predominant Gram-negative cellulolytic bacteria. This destruction leads to an increase of lipopolysaccharides (LPS), the cell wall components of Gram-negative bacteria in the ruminal fluid. It has been reported that the increase in LPS results in the simultaneous production of inflammatory cytokines, like interleukins (IL) in the liver and supposed by us also in the rumen which can serve as mediators of the endotoxin type effects of LPS.

To examine the role of LPS in the SARA, in vitro rumen epithelial cultures have been prepared as described earlier. For this purpose, three Merino sheep have been used, fed only hay ad libitum. After slaughter and exsanguation pieces of the rumen mucosa were cut out and cells have been isolated with fractionated trypsinization. The fractions containing mostly cells of stratum spinosum and basale have been used in the first two experiments. In the first experiment isolated rumen epithelial cells have been resuspended in Medium-199 and mixed with LPS of Escherichia coli (O55:B5), using concentrations of 50-10-5-1-0 µg/ml LPS for 4 hrs and 10µg/ml for 24 hrs, and incubated at 37° C. The IL-6 secretion was measured by ELISA method. After 4 and 24 hrs stimulations slight, non significant elevation of IL-6 production was measured. Secondly, isolated rumen epithelial cells were grown in MEM Hanks’ medium with 15 or 5% fetal bovine serum (FBS) on collagen I coated plastic tissue culture dishes for 21 days, and were stimulated with 10 µg/ml LPS for 18 hrs at 37° C. FBS had been deprived 8 hours prior to the stimulation. As a result, calculated for mg cell protein 183.9±19.4 pg/mg protein (control: 0.01pg/mg) IL-6 production was measured. Thirdly, mucosal tissue culture has been established by seeding of papillae in tissue culture dishes after removal of epithelial cells by trypsinization. The mostly fibroblast-like cells have been cultured for 14 days without collagen coating in MEM-Hanks’ medium and without FBS prior to the experiment for 8 hrs. The stimulation with 10 µg/ml LPS for 18 hours at 37° C did not result in significant increase of IL-6.

We suppose that the cells of the rumen mucosa produce inflammatory IL, like IL-6, and mostly the epithelial cells are responsible for this secretion, probably leading to certain inflammatory response in SARA.

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