|Johnson, T - PURDUE UNIVERSITY|
|Johnson, T - PURDUE UNIVERSITY|
Submitted to: ARS Immunology Workshop
Publication Type: Abstract Only
Publication Acceptance Date: October 24, 2003
Publication Date: December 1, 2003
Citation: EICHER, S.D., JOHNSON, T.A., MCMUNN, K.A., JOHNSON, T.R. TOLL-LIKE RECEPTOR AND ACUTE PHASE CYTOKINE EXPRESSION IN NEONATAL DAIRY CALVES. ARS IMMUNOLOGY WORKSHOP. 2003. ABSTRACT P. 4. Technical Abstract: The well-being of farm animals is dependent on many factors, so that a multi-disciplinary approach is essential. The Livestock Behavior Research Unit uses behavior, physiology, and immunology measures to answer well-being questions for poultry, swine, and dairy cattle. The innate immune system is frequently the first immune responder during many stressors and acute phase cytokines affect behavior and learning. Toll-like receptors are the pathogen recognition molecules that initiate the cascade that increases the acute phase cytokines. The effect of stressors on the toll-like receptors of farm animals is not known. Studies have been completed that examine the effect of stimulants on RNA expression of toll-like receptors in young dairy calves. The objective of Study One was to assess the optimum concentration and times of incubation for expression of TLR2 and TLR4 after in-vitro stimulation of bovine monocyte derived macrophages with Saccharomyces cerevisiae (yeast cell-wall) beta-glucan (BG), Escherichia coli lipopolysaccharide (LPS), and Staphylococcus aureus peptidoglycan (PGN). Monocyte derived macrophages from 10 donor calves (5 to 6 weeks-of-age) were first stimulated with LPS or PGN at 1, 5, or 25mg/ml or BG at 20, 40, or 80mg/ml for one hour at 37°C. Then the optimum concentrations were used to incubate cells at 30, 60, or 90 min. Real-time RT-PCR was used to quantify RNA expression of TLR2 and TLR4. RNA from monocyte derived macrophage expressed more bovine TLR4 than TLR2. Sixty minutes appears to be an adequate time of stimulation to change TLR4 expression, but at least 90 minutes are needed to attain maximum changes in TLR2 expression. The objective of Study Two was to determine if in vitro stimulation of whole blood leukocytes with beta-glucan plus ascorbic acid (a potential immunomodulator) was dependent on the age of the calf. Blood samples were taken from 12 non-transported Holstein calves at 1, 3, 7, 10, 14, 18, 21, 24, and 28 days-of-age. Leukocytes were stimulated with ascorbic acid (0.3 ug/ml) plus beta-glucan (0.4 ug/ml) or media only for 1 hour and then RNA was extracted. The RNA was subjected to real-time RT-PCR for quantification of the expression of interleukin-1 (IL-1) and its receptor antagonist (IL-1Ra), and toll-like receptors 2 and 4 (TLR2 and TLR4). TLR2 and TLR4 had treatment effects (P<.05), but not day or treatment by day interactions. TLR2 was greater (P<.05) for treated cells on day 7, 14, and 24 and tended (P<.10) to be greater on day 10. In contrast, TLR4 was only greater for treated cells on day 7 (P<.05) and tended to be (P<.10) on day 24. IL-1 had a treatment main effect and a treatment by day interaction (P<.05), but IL-1Ra had main effects for treatment and day (P<.05), but only a trend (P<.10) for a treatment by day interaction. IL-1 was greater for treated cells (P<.05) on all but day 4. IL-1Ra was greater (P<.05) for treated cells only on days 1, 7, 10, and 24. Only IL-1 and its receptor antagonist expression were stimulated on day 1. On days 7 and 24 all tested receptors and cytokines had increased RNA expression. So, it appears that there are periods during which the blood leukocytes may be refractory for increased RNA expression of cytokines and toll-like receptors in response to beta-glucan and ascorbic acid stimulis. These data enhance our understanding of the biological kinetics of the developing neonatal calf immune system, so that management procedures can occur at the best possible times.