In Vitro Study of an Orange Oil Derived Alternative to Antibiotics in the Treatment of Bovine Mastit

96 Pages·2015·0.96 MB·English

by  Matthew Hill

ABSTRACT

Title of Document: In Vitro Study of an Orange Oil Derived
Alternative to Antibiotics in the Treatment of
Bovine Mastitis

Cassandra Skye Federman, Masters of Animal
Science, 2015

Directed By: Dr. Debabrata Biswas, Animal Science

Bovine mastitis is a costly disease in the U.S. dairy industry. Its major causative
agent Staphylococcus aureus is often unresponsive to antibiotic therapy. Our first
study examined terpeneless, cold-pressed Valencia (CPV) orange oil as a possible
alternative to antibiotic therapy in the treatment of S. aureus associated bovine
mastitis. Orange oil showed significant inhibition of S. aureus growth and invasion
of bovine epithelial mammary cells, but only modest reductions in pre-formed
biofilms, which contribute to persistence of S. aureus infections. Our second study
examined major components of terpeneless, CPV orange oil. Of four major
compounds tested, only citral and linalool had significant inhibitory effects on S.
aureus growth. In addition, they were capable of reducing pre-formed biofilms as
well as association and invasion to bovine epithelial mammary cells. Part of this
inhibition was due to downregulation of virulence and biofilm genes.
In Vitro Study of an Orange Oil Derived Alternative to Antibiotics in the Treatment
of Bovine Mastitis

By

Cassandra Skye Federman

Thesis submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfillment
of the requirements for the degree of
Masters of Science
2015

Advisory Committee:
Dr. Debabrata Biswas, Chair
Dr. Zhengguo Xiao
Dr. Robert Peters
© Copyright by
Cassandra Skye Federman
2015
Dedication
For my fiancé, Robert, who always provided me with strength and encouragement,
and for my friends and family for all of their support.
ii
Acknowledgements
I would first like to acknowledge my advisory committee, Dr. Debabrata
Biswas, Dr. Zhengguo Xiao, and Dr. Robert Peters for their guidance in this project.
Their expertise and feedback have been incredibly valuable throughout this whole
process.
All of my lab members have offered a great deal of help and support. I cannot
imagine being able to get as far as I have without them. Particularly, I would like to
thank Jose Alejandro Almario, Nityananda Chowdhury, Christopher Ma, and Serajus
Salaheen for their assistance in these projects.
Firmenich Citrus Center has my appreciation for helping to contribute to the
study and providing supplies.
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Table of Contents

Dedication ..................................................................................................................... ii
Acknowledgements ...................................................................................................... iii
Table of Contents ......................................................................................................... iv
List of Tables .............................................................................................................. vii
List of Figures ............................................................................................................ viii
Chapter 1: Introduction ................................................................................................. 1
Bovine Mastitis ......................................................................................................... 1
Infection in Dairy Cattle ....................................................................................... 1
Causative Agents .................................................................................................. 2
Various Types of Mastitis ..................................................................................... 2
Treatments/Therapeutics ....................................................................................... 3
Cost to Dairy Industry ........................................................................................... 4
S. aureus .................................................................................................................... 5
General Information .............................................................................................. 5
S. aureus Associated Mastitis ............................................................................... 6
Treatment of S. aureus Associated Mastitis ......................................................... 7
Alternative Therapies .......................................................................................... 10
Orange Oil ............................................................................................................... 11
Composition ........................................................................................................ 11
Antimicrobial Activity ........................................................................................ 12
As an Alternative to Antibiotics ......................................................................... 13
Goals and Objectives .............................................................................................. 14
Chapter 2: Terpeneless, Cold-pressed Valencia Orange Oil and its Effects on
Staphylococcus aureus, Bovine Epithelial Mammary Cells, and Infection................ 16
Abstract ................................................................................................................... 16
Introduction ............................................................................................................. 17
Methods and Materials ............................................................................................ 19
Bacterial Strains and Growth Conditions ........................................................... 19
Preparation of Orange Oil ................................................................................... 19
Determination of Minimum Inhibitory Concentration (MIC) ............................ 20
Growth Inhibition Assay ..................................................................................... 20
Inhibition of Pre-Formed Biofilms ..................................................................... 21
Mammalian Cell Culture and Monolayer Formation.......................................... 22
Inhibition Assay for S. aureus Association to and Invasion of MAC-T cells .... 22
Detection of Cytotoxicity of Orange Oil on MAC-T cells ................................. 23
Statistical Analysis .............................................................................................. 24
Results ..................................................................................................................... 24
Minimum Inhibitory Concentration of CPV ....................................................... 24
Growth Inhibition of S. aureus by CPV.............................................................. 24
Effect of CPV on Pre-Formed Biofilms.............................................................. 25
Cytotoxicity of CPV on MAC-T cells ................................................................ 26
Inhibition of S. aureus Association to and Invasion of MAC-T cells ................ 26
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Discussion ............................................................................................................... 27
Chapter 3: Major Components of Terpeneless, Cold-pressed Valencia Orange Oil and
its Effects on Staphylococcus aureus, Bovine Epithelial Mammary Cells, and
Infection ...................................................................................................................... 32
Abstract ................................................................................................................... 32
Introduction ............................................................................................................. 33
Methods and Materials ............................................................................................ 35
Bacterial Strains and Growth Conditions ........................................................... 35
Preparation of Orange Oil Components .............................................................. 35
Determination of Minimum Inhibitory Concentration ....................................... 36
Growth Inhibition Assay ..................................................................................... 36
Inhibition of Pre-Formed Biofilms ..................................................................... 36
Mammalian Cell Culture and Monolayer Formation.......................................... 37
Detection of Cytotoxicity of Citral and Linalool on MAC-T cells ..................... 38
S. aureus Association and Invasion Assay for MAC-T cells.............................. 38
Effect on Gene Expression of S. aureus Virulence and Biofilm Genes ............. 39
Statistical Analysis .............................................................................................. 40
Results ..................................................................................................................... 41
MICs of Citral, Linalool, Decanal, and Valencene Required to Inhibit S. aureus
............................................................................................................................. 41
Time Dependent Inhibitory Effects of Citral and Linalool on S. aureus Growth41
Inhibition of Pre-formed S. aureus Biofilms with Citral and Linalool ............... 42
Cytotoxic Effects of Minimum Concentration of Citral and Linalool on MAC-T
cells ..................................................................................................................... 42
Role of Citral and Linalool in Adhesion to and Invasion into MAC-T cells by S.
aureus .................................................................................................................. 43
Effects of Citral and Linalool on Expression of MAC-T Cytokine Genes and S.
aureus Genes Involved in Virulence and Biofilms ............................................. 43
Discussion ............................................................................................................... 44
Chapter 4: Conclusion................................................................................................. 50
Chapter 5: List of Tables............................................................................................ 52
Table 1: Primers used for cytokine gene expression in this study. ......................... 52
Table 2: Primers used for S. aureus virulence gene expression in this study. ........ 53
Table 3: Primers used for S. aureus biofilm gene expression in this study. ........... 54
Table 4: Minimum Inhibitory Concentrations (MIC) of Citral, Linalool, Decanal,
and Valencene on S. aureus .................................................................................... 55
Chapter 6: List of Figures .......................................................................................... 56
Figure 1: Growth Inhibition Assay of Cold-Pressed, Terpeneless Valencia Orange
Oil on S. aureus....................................................................................................... 56
Figure 2: Inhibition of Pre-Formed S. aureus Biofilms by Cold-Pressed,
Terpeneless Valencia Orange Oil ........................................................................... 57
Figure 3: Relative Cytotoxicity of Orange Oil on MAC-T Cells ........................... 58
Figure 4: Inhibition of S. aureus Association to MAC-T cells by Cold-Pressed,
Terpeneless Valencia Orange Oil ........................................................................... 59
Figure 5: Inhibition of S. aureus Invasion of MAC-T cells by Cold-Pressed,
Terpeneless Valencia Orange Oil ........................................................................... 60
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Figure 6: Growth Inhibition Assay of Citral and Linalool on S. aureus over 72 h 61
Figure 7: Growth Inhibition Assay of 0.04% Citral and 0.24% Linalool on S.
aureus over 3 h........................................................................................................ 62
Figure 8: Inhibition of Pre-Formed S. aureus Biofilms by Citral and Linalool ..... 63
Figure 9: Relative Cytotoxicity of Citral on MAC-T cells ..................................... 64
Figure 10: Relative Cytotoxicity of Linalool on MAC-T Cells .............................. 65
Figure 11: Inhibition of S. aureus Association to MAC-T cells by Citral and
Linalool ................................................................................................................... 66
Figure 12: Inhibition of S. aureus Invasion of MAC-T cells by Citral and Linalool
................................................................................................................................. 67
Figure 13: Relative Expression of MAC-T Cytokine Genes after Infection and
Treatment ................................................................................................................ 68
Figure 14: Relative Expression of S. aureus Virulence Genes During Infection of
MAC-T Cells After Being Treated with Citral and Linalool .................................. 69
Figure 15: Relative Expression of S. aureus Biofilm Genes After Being Treated
with Citral and Linalool for 24 h ............................................................................ 70
Bibliography ............................................................................................................... 71

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List of Tables
Table 1: Primers used for cytokine gene expression in this study. ......................... 52
Table 2: Primers used for S. aureus virulence gene expression in this study. ........ 53
Table 3: Primers used for S. aureus biofilm gene expression in this study. ........... 54
Table 4: Minimum Inhibitory Concentrations (MIC) of Citral, Linalool, Decanal,
and Valencene on S. aureus .................................................................................... 55

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List of Figures
Figure 1: Growth Inhibition Assay of Cold-Pressed, Terpeneless Valencia Orange
Oil on S. aureus....................................................................................................... 56
Figure 2: Inhibition of Pre-formed S.aureus Biofilms by Cold-Pressed, Terpeneless
Valencia Orange Oil ............................................................................................... 57
Figure 3: Relative Cytotoxicity of Orange Oil on MAC-T Cells ........................... 58
Figure 4: Inhibition of S. aureus Association to MAC-T cells by Cold-Pressed,
Terpeneless Valencia Orange Oil ........................................................................... 59
Figure 5: Inhibition of S. aureus Invasion of MAC-T cells by Cold-Pressed,
Terpeneless Valencia Orange Oil ........................................................................... 60
Figure 6: Growth Inhibition Assay of Citral and Linalool on S. aureus over 72 h 61
Figure 7: Growth Inhibition Assay of 0.04% Citral and 0.24% Linalool on S.
aureus over 3 h........................................................................................................ 62
Figure 8: Inhibition of Pre-formed S. aureus Biofilms by Citral and Linalool ...... 63
Figure 9: Relative Cytotoxicity of Citral on MAC-T cells ..................................... 64
Figure 10: Relative Cytotoxicity of Linalool on MAC-T Cells .............................. 65
Figure 11: Inhibition of S. aureus Association to MAC-T cells by Citral and
Linalool ................................................................................................................... 66
Figure 12: Inhibition of S. aureus Invasion of MAC-T cells by Citral and Linalool
................................................................................................................................. 67
Figure 13: Relative Expression of MAC-T Cytokine Genes after Infection and
Treatment ................................................................................................................ 68
Figure 14: Relative Expression of S. aureus Virulence Genes During Infection of
MAC-T cells After Being Treated with Citral and Linalool ................................... 69
Figure 15: Relative Expression of S. aureus Biofilm Genes After Being Treated
with Citral and Linalool for 24 h ............................................................................ 70

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