Haptoglobin-Matrix Metalloproteinase 9 Complex as a Biomarker for Acute Inflammation in Cattle THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Charles Austin Hinds D.V.M Graduate Program in Veterinary Clinical Sciences The Ohio State University 2011 Master's Examination Committee: Jeffrey Lakritz, Advisor Andrew Niehaus Christopher Premanandan Paivi Rajala-Schultz D. Michael Rings Copyrighted by Charles Austin Hinds 2011 Abstract Bovine respiratory disease (BRD) is a major cause of economic loss in feedlots in the United Sates. These losses are associated not only with morbidity and mortality, but also the expense of using antimicrobial drugs unnecessarily. One of the recurring problems is the inability to diagnose and therefore treat respiratory disease appropriately. An Hp-MMP 9 protein complex has been identified in neutrophil granules and in the serum of cattle with acute bacterial sepsis. The purpose of this project was to evaluate the utility of an Hp-MMP 9 complex ELISA in the diagnosis of acute septic inflammation in cattle. Three experiments were performed. The first experiment was designed to determine whether Hp-MMP 9 could be used in the prediction of BRD in calves recently admitted to feedlots. Using health, treatment and weight gain data, our aim was to determine whether Hp-MMP 9 could predict which calves would be identified with clinical respiratory disease and would require therapy in the days following sample collection. We compared serum concentrations of Hp to Hp-MMP 9 to assess how well the complex performed in these animals. Our results clearly show that serum Hp and Hp-MMP 9 complex are present in cattle admitted to a feedlot. It appears that the presence of these two analytes are independent; however, due to the inability to obtain health and treatment records, we were not able to draw any conclusions about Hp-MMP 9 as a predictor for respiratory or other diseases or reduced ADG in feedlot cattle. ii The second experiment was designed to evaluate the utility of Hp-MMP9 complex ELISA in comparison to ELISA for total Hp or MMP 9 alone as an indicator of acute septic inflammatory disease in cattle. Animals were classified as being healthy, having acute inflammation, or having chronic inflammation. Serum Hp, MMP 9, and Hp-MMP 9 concentrations were measured from each animal and compared to disease status. The results of this experiment demonstrated significant differences in serum Hp- MMP 9 concentrations observed in cattle with acute septic disease compared to those animals with chronic inflammatory/metabolic disease or healthy animals. Total Hp concentrations were higher in diseased animals but not different between acute and chronic disease. Serum MMP 9 concentrations were not different between any groups. The final experiment evaluated the serum concentrations of Hp-MMP-9 during an LPS challenge as a surrogate of an inflammatory event. For this experiment, we administered E. coli lipopolysaccharide IV to calves and measured the Hp-MMP 9 complexes in serum over time (-24 hrs to 96 hrs) by serum ELISA. The concentration versus-time curves for Hp-MMP 9 concentration in the calves in this study, albeit, much lower than serum haptoglobin, were remarkably very similar to the concentration versus- time curves of serum Hp concentration. It is reasonable to assume that a majority of the Hp detected by the Hp ELISA is neutrophil in origin. These experiments indicate that Hp-MMP 9 is an indicator of acute inflammation in cattle. Further study is needed to determine the usefulness of this test in the field. iii Acknowledgments I would like to thank Dr. Lakritz for his guidance in not only this project, but also in my clinical and academic endeavors throughout my residency. I would like to thank the rest of my Master’s committee Drs. Niehaus, Premanandan, Rajala-Schultz, and Rings. I would also like to thank the entire Food and Fiber section for their support throughout my residency. iv Vita May 2000 .......................................................Saltillo High School, Saltillo, MS 2007................................................................D.V.M., Mississippi State University 2007-2008 .....................................................Intern, Food and Fiber Medicine and Surgery, The Ohio State University 2007-2008 .....................................................Resident, Food and Fiber Medicine, The Ohio State University Publications Gregory A. Bannikov, C. Austin Hinds, Paivi J. Rajala-Schultz, Chris Premanandan, D. Michael Rings. Serum Haptoglobin-Matrix Metalloproteinase 9 (Hp-MMP 9) Complex as a Biomarker of Systemic Inflammation in Cattle. Vet Immunol Immunopathol 2011;139:41-49. Fields of Study Major Field: Veterinary Clinical Sciences v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Chapter 1: Literature Review ............................................................................................. 1 Chapter 2: The Utility of Hp-MMP 9 Complex ELISA vs. Hp ELISA in Predicting Bovine Respiratory Disease in Feedlot Cattle .................................................................. 25 Chapter 3: Serum Hp-MMP 9 Complex in Cattle with Clinically Defined Diseases. ...... 33 Chapter 4: Serum Hp-MMP 9 Complex Response Following a Single Intravenous Dose of Lipopolysaccharide ....................................................................................................... 48 References ......................................................................................................................... 60 Appendix A: Tables .......................................................................................................... 70 Appendix B: Figures ......................................................................................................... 79 vi List of Tables Table 1. Calves grouped by number of positive Hp time-points ...................................... 71 Table 2. Calves grouped by number of positive Hp-MMP 9 time-points ........................ 72 Table 3. Samples grouped by Hp concentration and corresponding ADG ....................... 73 Table 4. Samples grouped by Hp-MMP 9 concentration and corresponding ADG ......... 74 Table 5. Disease Classification 1 Acute with Hp, MMP 9, and Hp-MMP 9 .................... 75 Table 6. Disease Classification 2, Chronic with Hp, MMP 9, Hp-MMP 9 ...................... 76 Table 7. Disease Classification 3, Healthy with Hp, MMP 9, Hp-MMP 9 ...................... 77 Table 8. Mean Hp, MMP 9, Hp-MMP 9 for disease classifications ................................. 78 vii List of Figures Figure 1. Correlation of Hp, Hp-MMP 9 in all samples ................................................... 80 Figure 2. Correlation of Hp, Hp-MMP 9 excluding Hp=0 ............................................... 81 Figure 3. Hp-MMP 9 concentration when Hp=0 vs. Hp>0 .............................................. 82 Figure 4. ADG when Hp was positive at 0, 1, >1 time-points .......................................... 83 Figure 5. ADG when Hp-MMP 9 was positive at 0, 1, 2, >2 time-points ........................ 84 Figure 6. ADG compared to concentration of Hp............................................................. 85 Figure 7. ADG compared to concentration of Hp-MMP 9 ............................................... 86 Figure 8. Hp-MMP 9 and MMP 9 ELISA specificity ...................................................... 87 Figure 9. Hp, MMP 9, and Hp-MMP 9 concentrations compared to diseases status ....... 88 Figure 10. Mean respiratory rates of calves during endotoxin trial .................................. 89 Figure 11. Mean rectal temperature of calves during endotoxin trial ............................... 90 Figure 12. Mean heart rate of calves during endotoxin trial ............................................. 91 Figure 13. MeanWBC of calves during endotoxin trial .................................................... 92 Figure 14. Mean band neutrophils of calves during endotoxin trial ................................. 93 Figure 15. Mean Hp of calves during endotoxin trial ....................................................... 94 Figure 16. Mean Hp-MMP 9 of calves during endotoxin trial ......................................... 95 Figure 17. Hp, Hp-MMP 9 over time ............................................................................... 96 Figure 18. Hp-MMP 9, respiratory rate over time ............................................................ 97 viii Chapter 1: Literature Review Introduction Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle in the United States. Roughly 45% of death losses in cattle in 2005 were due to inflammatory disease, 60% of which was due to BRD.1 A review of the relevant literature indicates losses in average