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The Pennsylvania State University the Graduate School College Of The Pennsylvania State University The Graduate School College of Agricultural Sciences GENOMIC AND IMMUNOLOGICAL APPROACHES TO CONTROL GRAM- NEGATIVE INTRAMAMMARY INFECTIONS IN DAIRY CATTLE A Thesis in Animal Science by Annapoorani Chockalingam © 2006 Annapoorani Chockalingam Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2006 The thesis of Annapoorani Chockalingam was reviewed and approved* by the following: Cindy E. McKinney Assistant Professor of Transgenic Biology Thesis Advisor Co-Chair of Committee Douglas D. Bannerman Research Scientist, BFGL-ANRI, BARC, USDA-ARS Co-Chair of Committee Guy F. Barbato Associate Professor of Poultry Science Kenneth M. Weiss Evan Pugh Professor of Anthropology and Genetics Eric T. Harvill Assistant Professor of Veterinary Science Robert F. Paulson Associate Professor of Veterinary Science Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Dairy and Animal Science *Signatures are on file in the Graduate School iii ABSTRACT The incidence of mastitis due to Gram-negative bacteria continues to impact the dairy industry due to the ubiquitous nature of the pathogens. Gram-negative bacteria account for nearly 40% of clinical mastitis cases resulting in an estimated loss to the U.S. dairy industry of $800 million per year. The research presented here focuses on host- mediated genetic and immunological factors involved in Gram-negative bacteria induced mastitis during the early phases of disease onset. Array analysis of mammary gland gene expression in a global context in response to E. coli is currently unavailable. A mouse mastitis model was used to study the transcriptional response in the mammary gland. This study identified potential candidate genes for susceptibility to bovine mastitis. We identified bactericidal/permeability increasing protein (BPI) as a potential candidate gene for susceptibility to Gram-negative infections. Ten single nucleotide polymorphisms (SNPs) were identified within the BPI gene sequence encoding for the N-terminal region of the mature protein that exhibits bactericidal and LPS-neutralizing activity. A polymorphism located at +61 (G/A) resulted in a non-synonymous amino acid substitution from glycine to serine. No significant association was found between the identified SNP-marker allele and their haplotypes with SCS for a sample size of eighty- five Holstein dairy bulls. Current therapeutic and prophylactic measures have marginal efficacy against mastitis. The potency of human and bovine BPI-derived synthetic peptides was evaluated for its bactericidal and LPS-neutralizing activity in biological fluids from the bovine. In this study, a 24 amino acid (aa) sequence synthetic peptide of human (hBPIpep) and iv bovine BPI (bBPIpep) corresponding to domains 90-99 and 148-161 of both human and bovine mature BPI protein was found to exhibit 100% LPS-neutralizing activity at 100 µg/mL concentration. Complete bactericidal activity against E. coli was demonstrated in broth, serum and whey. However, only bacterial inhibitory activity was recorded in milk at the highest concentration tested (2 mg/mL). In the presence of EDTA (16mM concentration), a chelator of divalent cations, the hBPIpep exhibited complete bactericidal activity at the lowest concentration tested (10µg/mL). The other two bovine BPI peptides corresponding to amino acid 65-99 (bBPIpep-II) and 142-169 (bBPIpep-III) did not have any bactericidal activity. However, bBPIpep-III exhibited LPS-neutralizing activity equivalent to polymixin-B (concentration ≥ 30 µg/mL). The human and bovine BPI peptides were tested for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against clinical isolates of major Gram-negative mastitis pathogens. The innate immune response to Pseudomonas aeruginosa was studied to determine whether the induction pattern to different Gram-negative bacteria is conserved in bovine mammary gland immunity. We show that the cytokine pattern induced by P. aeruginosa and E. coli are conserved with respect to systemic and local innate immune responses. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................vii LIST OF TABLES....................................................................................................... ix ACKNOWLEDGMENTS ...........................................................................................xii Chapter 1 INTRODUCTION....................................................................................... 1 REFERENCES ............................................................................................................22 Chapter 2 A MICROARRAY SCREEN FOR IDENTIFICATION OF POTENTIAL CANDIDATE GENES FOR GRAM-NEGATIVE INTRAMAMMARY PATHOGENESIS IN A MOUSE MASTITIS MODEL .........32 REFERENCES ............................................................................................................50 Chapter 3 SINGLE NUCLEOTIDE POLYMORPHISMS AND HAPLOTYPES WITHIN THE BOVINE BACTERICIDAL/PERMEABILITY-INCREASING PROTEIN: A CANDIDATE GENE FOR MASTITIS ...............................................52 REFERENCES ............................................................................................................68 Chapter 4 EFFICACY OF HUMAN BACTERICIDAL/PERMEABILITY- INCREASING PROTEIN DERIVED PEPTIDE AGAINST MAJOR GRAM- NEGATIVE MASTITIS PATHOGENS .....................................................................72 REFERENCES ............................................................................................................93 Chapter 5 EFFICACY OF BOVINE BACTERICIDAL/PERMEABILITY- INCREASING PROTEIN DERIVED PEPTIDE AGAINST MAJOR GRAM- NEGATIVE MASTITIS PATHOGENS .....................................................................97 REFERENCES ............................................................................................................120 vi Chapter 6 THE BOVINE INNATE IMMUNE RESPONSE DURING EXPERIMENTALLY INDUCED Pseudomonas aeruginosa AND Escherichia coli MASTITIS ............................................................................................................125 REFERENCES ............................................................................................................157 Chapter 7 SUMMARY AND FUTURE DIRECTIONS .............................................163 APPENDIX .................................................................................................................167 vii LIST OF FIGURES Chapter 1 Figures: Figure 1: Schematic representation of bovine mammary gland. 3 Figure 2A: Cross section of cow udder from an uninfected quarter. 5 Figure 2B: Cross section of cow udder 72 hour post-infection with E. coli. 5 Chapter 2 Figures: Figure 1: Cross section of murine mammary gland (A) Saline Infused (B & C) E. coli treated. 40 Figure 2A: Density plot: Perfect match and mismatch probes for 22,690 genes. 42 Figure 2B: Box plot: Perfect match and mismatch probes for 22,690 genes. 42 Figure 3: Differentially expressed genes of unknown function co-expressed with genes of known function with gene node correlation r = 0.92. 46 Chapter 3 Figures: Figure 1: Chromatogram of bBPI gene Exon 1 sequenced with reverse primer. 59 Figure 2A: SignalP prediction of signal peptide sequence for glycine at position +21. 62 Figure 2B: SignalP prediction of signal peptide sequence for serine at position +21. 62 Chapter 4 Figures: Figure 1: Bactericidal activity of human BPI synthetic peptide hBPIpep in milk in the presence of EDTA. 84 Figure 2: Percent inhibition of purified LPS by human BPI derived synthetic peptide. 86 Chapter 5 Figures: Figure1: Percent inhibition of purified LPS by bovine BPI derived synthetic peptides. 112 viii Chapter 6 Figures: Figure 1: Bacterial growth of P. aeruginosa and E. coli following experimental challenge. 136 Figure 2: Effect of P. aeruginosa intramammary infection on systemic response. 138 Figure 3: Effect of bacterial infection on milk somatic cell counts (SCC). 140 Figure 4: Effect of intramammary bacterial infection on mammary vascular permeability. 141 Figure 5: Effect of intramammary infection on complement activation and pro- inflammatory cytokine levels in milk. 143 Figure 6: Effect of intramammary infection with E. coli on TNF-α and IL-8 levels in milk. 145 Figure 7: Effect of P. aeruginosa infection on anti-inflammatory cytokine levels in milk. 147 Figure 8: Effect of E. coli infection on anti-inflammatory cytokine levels in milk. 148 Figure 9: Intramammary challenge with P. aeruginosa increases milk levels of sCD14 and LBP. 149 ix LIST OF TABLES Chapter 1 Tables: Table 1. Quantitative trait loci for mastitis and somatic cell score (SCC). 9 Table 2. Major cytokines: their sources and functions. 12 Table 3. List of antibiotics approved for mastitis treatment in dairy cattle. 20 Chapter 2 Tables: Table 1.List of differentially expressed genes between uninfected and E. coli infected mammary tissue classified on their molecular function in the disease process. 43 Table 2. Validation of differentially expressed immune related candidate genes for mastitis by Q-PCR 45 Chapter 3 Tables: Table 1. Forward and reverse primer sequence and product size for the five exons of bovine BPI gene. 57 Table 2. Single nucleotide polymorphisms identified within the bovine BPI gene. 59 Table 3. Estimated bBPI haplotype frequencies for SNPs for BPI gene in Holstein dairy breed. 60 Chapter 4
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