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Download (9MB) A STUDY OF MASTITIS IN DAIRY HERDS WITH PARTICULAR REFERENCE TO STREPTOCOCCUS UBERIS by TIMOTHY RICHARD BARKER Thesis submitted for the degree of Master of Veterinary Medicine Faculty of Veterinary Medicine University of Glasgow Department of Veterinary Medicine University of Glasgow Copyright T. R. Barker November 1995 ProQuest Number: 11007782 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11007782 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 GLASGOW^ UNIVERSffl T.TBRARY _ 2 SUMMARY Streptococcus uberis (S. uberis) is the infectious agent in a significant proportion of cases of subclinical mastitis in dairy cows in the British Isles, and in some cases of clinical mastitis. It is a pathogen which can exist outwith the bovine mammary gland parenchyma, having been isolated from bovine skin, ruminal fluid, faeces and bedding. Using DNA typing of individual strains of S. uberis , it was hoped that an epidemiological survey of a herd infected with S. uberis could resolve whether all or only some strains of S. uberis (and Streptococcus parauberis) found on cows' skin and in the environment could gain access to the glandular tissue and cause mastitis. In order to find a farm where S. uberis was endemic in the dairy herd, milk and other samples were examined from seven herds in south-west Scotland that were likely to have a subclinical S. uberis problem. However, bacterial analysis of the milk samples collected showed that Staphylococcus aureus (Staph, aureus) was the predominant pathogen causing mastitis in the herds. The infrequent isolation of S. uberis from only one of the herds suggested the possibility that the cows' immunological control of S. uberis infection was incidental to having to deal with the constant Staph, aureus infection and, were the latter resolved, infection by S. uberis may become a more noticeable and serious problem. A protocol was developed to screen a large number of milk samples for the presence of S. uberis employing as few biochemical tests as possible, consistent with accurate identification; the tests were fermentation of inulin followed by haemolysis of blood agar and hydrolysis of aesculin and hippurate. In addition, a new "enrichment" broth consisting of equal volumes of Todd Hewitt and tryptose broths was found to produce a greater harvest of S. uberis colony forming units than conventional enrichment broths: The use of litmus milk as an enrichment broth was also discussed. 3 LIST OF CONTENTS Page Summary 2 List of Contents 3 List of Tables 12 List of Figures 13 List of Appendices 14 List of Abbreviations 16 Acknowledgements 17 Declaration 18 Chapter One A Review of the Literature 1.1 The Early History of Mastitis Investigation 19 1.1.1 The Genesis of Milk Microbiology 19 1.1.2 Route of Entry of Mastitis Pathogens 19 1.1.3 Types of Mastitis 20 1.1.4 Early Investigations into the Bacteriological Aetiologyof Mastitis 21 1.1.5 The Bacteria of "Normal" Milk and Udder Tissue 22 1.1.6 Genus Streptococcus 23 1.1.7 'Streptococcus brevis’ and 'Streptococcus longus' 24 1.1.8 Streptococcus lactis (Lactococcus lactis) 25 1.1.9 Classification of Streptococcal Species by Biochemical Tests 25 1.1.10 The Streptococci Isolated from Bovine Milk 27 1.1.11 Zoonotic Implications of Infected Milk in Human Disease and Public Health 29 4 1.1.12 Mastitis and Post-Calving Uterine Infection 31 1.1.13 The Pathogenicity of Staphylococcal Species 31 1.1.14 Haemolysis of Blood Agar by Streptococci 32 1.1.15 The Effect of Temperature on Bacterial Growth 34 1.1.16 The Limiting Hydrogen Ion Concentration of Streptococcal Isolates 35 1.1.17 Differentiation of Streptococcal Species by the Sodium Hippurate Test 36 1.1.18 Differentiation of Streptococcal Species by the Aesculin Hydrolysis Test 37 1.1.19 Differentiation of Streptococcal Species by the Inulin Fermentation Test 39 1.1.20 Differentiation of Streptococcal Species by Seriological Tests 40 1.1.21 Culture and Enrichment Media for Streptococci 41 1.1.21a Todd Hewitt Broth 41 1.1.21b Tryptose Media 42 1.1.21c Milk Enrichment Media 42 1.1.2 Id Litmus Milk 43 1.2 Characterisation of and Variation in Streptococcus uberis 44 1.2.1 Biochemical Properties of Streptococcus uberis 44 1.2.2 Typing Isolates of Streptococcus uberis 65 1.2.3 Differentiation of the Species into Streptococcus uberis and Streptococcus parauberis 67 1.3 Surveys of Streptococcus uberisInfection 68 1.4 Epidemiology of Streptococcus uberis 75 1.5 Defence Mechanisms of the Bovine Udder 83 1.5.1 The Teat Orifice 83 1.5.2 The Role of Keratin 85 5 1.5.3 The Role of the Cationic (Basic) Proteins in Keratin 86 1.5.4 The Role of Lipids in Keratin 88 1.5.5 The Natural Bacteriostatic Factors in Fresh Milk- Lactenins 89 1.5.6 Lactoferr in 90 1.5.7 The Lactoperoxidase, Thiocyanate, Hydrogen Peroxide System 90 1.5.8 The Defence Ability of Dry and Lactating Quarters Against Bacterial Infection 92 1.5.9 Humoral Immunity 95 1.5.10 Phagocytosis by Polymorphonuclear Leucocytes (Neutrophils) 96 1.6 Mastitis Control 100 1.6.1 The Early Influence of Milking Machines on Udder Infection 100 1.6.2 Milking Procedures and Segregation of Infected Cows 101 1.6.3 Age and Infection 102 1.6.4 Infection in Down-Calving Heifers 103 1.6.5 Subclinical Infection 105 1.6.6 Dry Period Infection 106 1.6.7 Teat Disinfection at the End of Lactation 108 1.6.8 Routine Milking Hygiene Measures 109 1.6.9 Antibacterial Therapy 110 1.6.10 Sulphonamide Therapy 113 1.6.11 Penicillin 114 1.6.12 Aqueous Versus Oil Based Preparations and the Development of Dry Cow Preparations 118 1.6.13 Comparison of Routine and Selective Dry Cow Therapy 124 1.6.14 Other Antibacterial Agents Used in Mastitis Treatment 126 1.6.14a Neomycin 126 1.6.14b Oxytetracycline 127 6 1.6.14c Erythromycin 128 1.6.14d Cephalosporins 128 1.6.15 Effects of Antibacterial Therapy on the Prevalence of Pathogenic Species 129 1.6.16 Intramammary Devices 130 1.6.17 An Early Trial in Passive Immunity 131 1.6.18 Vaccination 132 1.7 Milk Somatic Cell Counts 133 Chapter Two Materials and Methods 2.1 Introduction 143 2.2 On Farm Protocol 144 2.2.1 Access to Farms 144 2.2.2 Milk Somatic Cell Counts 145 2.2.3 On Farm Techniques 146 2.2.4 Collection of Milk Samples 146 2.2.5 Collection of Rectal and Vaginal Swabs 147 2.2.6 Collection of Udder Skin Swabs 147 2.2.7 Storages of Samples During Transportation and at the Laboratory 148 2.3. Laboratory Techniques for the Isolation and Identification of Streptococcus uberis 148 2.3.1 Blood Agar Media 148 2.3.2 Inulin Broth 149 7 2.3.3 Inulin Serum Agar 150 2.3.4 Modified Inulin Agar 151 2.3.5 Modified Inulin Agar Without Thallium Acetate 151 2.3.6 Agar Containerisation and Storage 152 2.3.7 Sodium Hippurate Media 152 2.3.8 Nutrient Broths 154 2.3.9 Broth Containerisation and Storage 155 2.3.10 Inoculation of Agar Plates 155 2.3.11 Inoculation of Broth Media 156 2.3.12 Inoculation of Both Agar and Broth Media 156 2.3.13 Confirmation of Identity of Isolates 156 2.3.14 Storage of Streptococcus uberis Isolates 157 2.4 Initial Procedures on Milk and Other Samples Collected at Farm Visits 157 2.5 Investigations into the Cultural Characteristics of Known Isolates of Streptococcus uberis 157 2.6 Staphylococcus aureus 158 2.6.1 Identification of Staphylococcus aureus 158 2.6.2 Antimicrobial Susceptibility Tests for Staphylococci 158 2.7 DNA Typing of Streptococcus uberis 159 2.7.1 The Protocol of Hill and Leigh (1989) and Subsequent Modifications 159 2.7.2 Isolation of Chromosomal DNA 160 2.7.3 Enzyme Digestion of DNA 161 2.7.4 Agarose Gel Electrophoresis 162 8 2.8 Other Investigations 163 2.8.1 Assessment of Three Nutrient Broths for the Growth of Streptococcus uberis 163 2.8.2 Assessment of the Number of Colony Forming Units in a Colony of Streptococcus uberis 164 Chapter Three Results 3.1 The Farms Visited 165 3.1.1 Access to Farms 165 3.1.2 General Description of the Farms Visited 166 3.1.3 Criteria for the Selection of Cows for Sampling 166 3.2 Laboratory Techniques for the Isolation and Identification of Streptococcus uberis 167 3.2.1 Differential Media Used to Isolate and Identify Streptococcus uberis 167 3.2.2 Blood Agar 168 3.2.3 Edwards Agar 168 3.2.4 Inulin Media 171 3.2.5 Sodium Hippurate Broth 171 3.2.6 Changes in the Isolation Protocol 174 3.3 Farm 1 176 3.3.1 Farm and Herd Management 176 3.3.2 Farm 1 Visit 1 18.03.1991 177 3.3.3 Selection of Cows for Sampling 177 3.3.4 Bacterial Isolation and Identification 178 3.3.5 Farm 1 Visit 2 22.04.1991 180 3.3.6 Selection of Cows for Sampling 180 3.3.7 Bacterial Isolation and Identification 180 3.3.8 Farm 1 Visit 3 13.05.1991 182 3.3.9 Selection of Cows for Sampling 182 3.3.10 Bacterial Isolation and Identification 182 3.3.11 Farm 1 Visit 4 29.08.1991 184 3.3.12 Selection of Cows for Sampling 184 3.3.13 Bacterial Isolation and Identification 184 3.3.14 Farm 1 Visit 5 11.09.1991 187 3.3.15 Selection of Cows for Sampling 187 3.3.16 Bacterial Isolation and Identification 187 3.4 Farm 2 188 3.4.1 Farm and Herd Management 188 3.4.2 Farm 2 Visit 1 15.04.1991 190 3.4.3
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