SCREENING FOR RESIDUES OF ANTIBIOTICS AND CHEMOTHERAPEUTICS IN MILK AND ...

317 Pages·2010·3.52 MB·English

by  Sigrid Ooghe

To Eva and Amber

Cover photos: Wim Reybroeck
Printer: DCL Print & Sign, Zelzate. www.dclsigns.be

Promotors: Prof. Dr. Hubert F. De Brabander
Faculty of Veterinary Medicine, Department of Veterinary Public Health
and Food Safety, Laboratory of Chemical Analysis

Dr. Apr. Els Daeseleire
Institute for Agricultural and Fisheries Research, Technology and Food
Science Unit

Dr. Lieve Herman
Institute for Agricultural and Fisheries Research, Technology and Food
Science Unit

Dr. ir. Lynn Vanhaecke
Faculty of Veterinary Medicine, Department of Veterinary Public Health
and Food Safety, Laboratory of Chemical Analysis

Dean: Prof. Dr. Hubert F. De Brabander

Rector: Prof. Dr. Paul Van Cauwenberge

Copyright:
The author gives the authorization to consult and to copy parts of this work for
personal use only. Any other use is subject to Laws of Copyright.
Permission to reproduce any material contained in this work should be obtained from
the author.

ISBN 978-90-5864-244-8
SCREENING FOR RESIDUES OF ANTIBIOTICS AND
CHEMOTHERAPEUTICS IN MILK AND HONEY

Wim Reybroeck

Thesis submitted in fulfilment of the requirements
for the degree of Doctor (Ph. D.) in Veterinary Sciences,
Faculty of Veterinary Medicine, Ghent University, 2010

Promotor: Prof. Dr. H.F. De Brabander

Ghent University, Faculty of Veterinary Medicine
Department of Veterinary Public Health and Food Safety
Laboratory of Chemical Analysis

TABLE OF CONTENTS

Table of Contents
Abbreviations and Acronyms

Chapter 1 Legislative and Analytical Aspects,Residues of Antimicrobials in

Honey and Milk _______________________________________________________ 1
1.1 LEGISLATIVE AND ANALYTICAL ASPECTS _____________________________________________ 2
1.1.1 Legislation ___________________________________________________________________________ 2
1.1.2 Analytical aspects ______________________________________________________________________ 9
1.1.2.1 Analytical aspects of screening tests ___________________________________________________ 9
1.1.2.2 Microbiological screening tests _______________________________________________________ 9
1.1.2.3 Rapid screening tests ______________________________________________________________ 11
1.1.2.4 Chromatographic and confirmatory methods ___________________________________________ 12
1.1.2.5 Screening for antimicrobials in honey _________________________________________________ 13
1.1.2.6 Screening for antimicrobials in milk ___________________________________________________ 14
1.1.2.6.1 Integrated system for residue control in milk _______________________________________ 14
1.1.2.6.2 Testing of individual cows’ milk at the dairy farm ____________________________________ 16
1.1.2.6.3 Testing of ex-farm milk at the milk control stations __________________________________ 16
1.1.2.6.4 Testing of production or tanker milk at the processing establishment ____________________ 18
1.1.2.6.5 Monitoring of dairy products ____________________________________________________ 22
1.1.3 Anti-infectious agents _________________________________________________________________ 23
1.1.3.1 Sulfonamides ____________________________________________________________________ 23
1.1.3.2 Diaminopyrimidine derivatives ______________________________________________________ 23
1.1.3.3 Penicillins _______________________________________________________________________ 24
1.1.3.4 Cephalosporins ___________________________________________________________________ 24
1.1.3.5 Quinolones ______________________________________________________________________ 25
1.1.3.6 Macrolides_______________________________________________________________________ 25
1.1.3.7 Tetracyclines _____________________________________________________________________ 25
1.1.3.8 Lincosamides _____________________________________________________________________ 26
1.1.3.9 Aminoglycosides __________________________________________________________________ 26
1.1.3.10 Chloramphenicol and related drugs __________________________________________________ 26
1.1.3.11 Nitrofurans _____________________________________________________________________ 27
1.1.3.12 Nitroimidazoles __________________________________________________________________ 27
1.2 ANTIMICROBIALS IN HONEY ____________________________________________________ 28
1.2.1 Introduction _________________________________________________________________________ 28
1.2.2 Main bee diseases and pests ____________________________________________________________ 30
1.2.2.1 American foulbrood _______________________________________________________________ 30
1.2.2.2 European foulbrood _______________________________________________________________ 31
1.2.2.3 Nosemosis _______________________________________________________________________ 32
1.2.3 Use of antibiotics and chemotherapeutics in beekeeping _____________________________________ 32
1.2.4 Antibiotics and chemotherapeutics of interest in apiculture ___________________________________ 35
1.2.4.1 Tetracyclines _____________________________________________________________________ 35
1.2.4.2 Streptomycin _____________________________________________________________________ 36
1.2.4.3 Sulfonamides ____________________________________________________________________ 36
1.2.4.4 Tylosin __________________________________________________________________________ 37
1.2.4.5 Erythromycin _____________________________________________________________________ 37
1.2.4.6 Lincomycin ______________________________________________________________________ 38
1.2.4.7 Chloramphenicol __________________________________________________________________ 38
1.2.4.8 Nitrofurans ______________________________________________________________________ 39
TABLE OF CONTENTS

1.2.4.9 Nitroimidazoles ___________________________________________________________________ 39
1.2.4.10 Fluoroquinolones ________________________________________________________________ 39
1.2.4.11 Fumagillin ______________________________________________________________________ 39
1.2.4.12 Other antibiotics and chemotherapeutics _____________________________________________ 40
1.2.5 Stability and disposition of antimicrobials in honeybee hives __________________________________ 41
1.2.5.1 Introduction _____________________________________________________________________ 41
1.2.5.2 Tetracyclines _____________________________________________________________________ 41
1.2.5.3 Streptomycin _____________________________________________________________________ 42
1.2.5.4 Sulfonamides ____________________________________________________________________ 43
1.2.5.5 Tylosin __________________________________________________________________________ 43
1.2.5.6 Erythromycin _____________________________________________________________________ 44
1.2.5.7 Lincomycin ______________________________________________________________________ 44
1.2.5.8 Chloramphenicol __________________________________________________________________ 44
1.2.5.9 Nitrofurans ______________________________________________________________________ 45
1.2.5.10 Fluoroquinolones ________________________________________________________________ 45
1.2.5.11 Other antibiotics _________________________________________________________________ 45
1.2.5.12 Conclusions and reflections about the disposition of antimicrobials in hives _________________ 46
1.2.6 Occurrence of antimicrobial residues in honey on the Belgian and European market _______________ 47
1.3 ANTIMICROBIALS IN MILK _______________________________________________________ 53
1.3.1 Introduction _________________________________________________________________________ 53
1.3.2 The use of veterinary drugs in dairy cattle _________________________________________________ 55
1.3.3 Pharmacokinetics of veterinary drugs_____________________________________________________ 56
1.3.4 Veterinary drugs registered in Belgium for use in milk producing cows __________________________ 58
1.3.5 Sales and usage of antimicrobials ________________________________________________________ 60
1.3.6 Main causes of inhibitors in milk _________________________________________________________ 63
1.3.7 The occurrence of antimicrobial residues in milk ____________________________________________ 65
1.3.8 Drawbacks of residues of veterinary drugs in milk ___________________________________________ 66
1.3.9 Stability of antibiotics and chemotherapeutics in milk _______________________________________ 68
1.3.9.1 -Lactams _______________________________________________________________________ 68
1.3.9.2 Tetracyclines _____________________________________________________________________ 69
1.3.9.3 Aminoglycosides __________________________________________________________________ 70
1.3.9.4 Other antibiotics and sulfonamides ___________________________________________________ 70
1.4 REFERENCES __________________________________________________________________ 72
1.5 ANNEXES _____________________________________________________________________ 90

Chapter 2 Thesis Objectives _____________________________________________________ 99

Chapter 3 Validation of the Tetrasensor Honey Test Kit _________________________ 101
3.1 INTRODUCTION _______________________________________________________________ 103
3.2 MATERIALS AND METHODS _____________________________________________________ 105
3.2.1 Reagents and standards ______________________________________________________________ 105
3.2.2 Material ___________________________________________________________________________ 105
3.2.3 Test protocol and interpretation of the results ____________________________________________ 105
3.3 RESULTS AND DISCUSSION ______________________________________________________ 107
3.3.1 Stability of tetracyclines in honey _______________________________________________________ 107
3.3.2 Test and reader repeatability __________________________________________________________ 107
3.3.3 Specificity __________________________________________________________________________ 109
TABLE OF CONTENTS

3.3.4 Detection capability __________________________________________________________________ 109
3.3.5 Test ruggedness _____________________________________________________________________ 111
3.3.5.1 Impact of the nature of the honey on the detection capability ____________________________ 111
3.3.5.2 Batch-to-batch differences and reagents’ stability regarding the detection capability__________ 115
3.3.5.3 Impact of drying of the strips _______________________________________________________ 117
3.3.5.4 Test for false-negative/false-positive results ___________________________________________ 117
3.4 REFERENCES _________________________________________________________________ 120

Chapter 4 Transfer of Sulfamethazine from Contaminated Beeswax to Honey __ 123
4.1 INTRODUCTION _______________________________________________________________ 125
4.2 MATERIALS AND METHODS _____________________________________________________ 128
4.2.1 Reagents and standards ______________________________________________________________ 128
4.2.2 Apparatus __________________________________________________________________________ 129
4.2.3 Sample preparation __________________________________________________________________ 129
4.2.4 LC-MS/MS analysis ___________________________________________________________________ 131
4.2.5 Calculation of partition coefficients _____________________________________________________ 132
4.2.6 Experiments and sampling ____________________________________________________________ 132
4.3 RESULTS AND DISCUSSION ______________________________________________________ 134
4.4 CONCLUSIONS ________________________________________________________________ 141
4.5 REFERENCES _________________________________________________________________ 142

Chapter 5 Validation of the eta-s.t.a.r. 1+1 ____________________________________ 147
5.1 INTRODUCTION _______________________________________________________________ 149
5.2 MATERIALS AND METHODS _____________________________________________________ 151
5.2.1 Reagents and standards ______________________________________________________________ 151
5.2.2 Material ___________________________________________________________________________ 153
5.2.3 Test procedure and interpretation of the results ___________________________________________ 153
5.2.4 Test and reader repeatability __________________________________________________________ 154
5.2.5 Test selectivity ______________________________________________________________________ 154
5.2.6 Detection capability __________________________________________________________________ 154
5.2.7 Test robustness _____________________________________________________________________ 155
5.2.7.1 Length of incubation ______________________________________________________________ 155
5.2.7.2 Influence of waiting time on reader results____________________________________________ 155
5.2.8 Milk influences ______________________________________________________________________ 156
5.2.8.1 Milk quality and composition _______________________________________________________ 156
5.2.8.2 Type of milk and animal species_____________________________________________________ 156
5.2.9 Test for false-positive/false-negative results ______________________________________________ 157
5.2.10 Reagent influence (batch differences) __________________________________________________ 157
5.2.11 Inter-laboratory testing ______________________________________________________________ 157
5.2.12 Daily control samples ________________________________________________________________ 158
5.3 RESULTS AND DISCUSSION ______________________________________________________ 158
5.3.1 Test and reader repeatability __________________________________________________________ 158
5.3.2 Test selectivity ______________________________________________________________________ 159
5.3.3 Detection capability __________________________________________________________________ 159
5.3.4 Test robustness _____________________________________________________________________ 161
TABLE OF CONTENTS

5.3.4.1 Length of incubation ______________________________________________________________ 161
5.3.4.2 Influence of waiting time on reader results____________________________________________ 162
5.3.5 Milk influences ______________________________________________________________________ 162
5.3.5.1 Milk quality and composition _______________________________________________________ 162
5.3.5.2 Type of milk and animal species_____________________________________________________ 164
5.3.6 Test for false-positive/false-negative results ______________________________________________ 167
5.3.7 Reagent influence (batch differences) ___________________________________________________ 167
5.3.8 Interlaboratory testing _______________________________________________________________ 168
5.3.9 Daily control samples _________________________________________________________________ 169
5.4 CONCLUSIONS ________________________________________________________________ 170
5.5 REFERENCES _________________________________________________________________ 171

Chapter 6 Validation of the Charm MRL-3 ______________________________________ 175
6.1 INTRODUCTION _______________________________________________________________ 177
6.2 MATERIALS AND METHODS _____________________________________________________ 178
6.2.1 Reagents and standards ______________________________________________________________ 178
6.2.2 Material ___________________________________________________________________________ 180
6.2.3 Test procedure and interpretation of the results ___________________________________________ 180
6.2.4 Test and reader repeatability __________________________________________________________ 181
6.2.5 Test selectivity ______________________________________________________________________ 182
6.2.6 Detection capability __________________________________________________________________ 182
6.2.7 Test robustness _____________________________________________________________________ 183
6.2.7.1 Length of incubation ______________________________________________________________ 183
6.2.7.2 Influence of waiting time on reader results____________________________________________ 183
6.2.8 Milk influences ______________________________________________________________________ 183
6.2.8.1 Milk quality and composition _______________________________________________________ 183
6.2.8.2 Type of milk and animal species_____________________________________________________ 184
6.2.9 Test for false-positive/false-negative results ______________________________________________ 184
6.2.10 Reagent influence (batch differences) __________________________________________________ 185
6.2.11 Inter-laboratory testing ______________________________________________________________ 185
6.2.12 Daily control samples ________________________________________________________________ 185
6.3 RESULTS AND DISCUSSION ______________________________________________________ 186
6.3.1 Test and reader repeatability __________________________________________________________ 186
6.3.2 Test selectivity ______________________________________________________________________ 186
6.3.3 Detection capability __________________________________________________________________ 187
6.3.4 Test robustness _____________________________________________________________________ 189
6.3.4.1 Length of incubation ______________________________________________________________ 189
6.3.4.2 Influence of waiting time on reader results____________________________________________ 190
6.3.5 Milk influences ______________________________________________________________________ 190
6.3.5.1 Milk quality and composition _______________________________________________________ 190
6.3.5.2 Type of milk and animal species_____________________________________________________ 192
6.3.6 Test for false-positive/false-negative results ______________________________________________ 194
6.3.7 Reagent influence (batch differences) ___________________________________________________ 195
6.3.8 Interlaboratory testing _______________________________________________________________ 196
6.3.9 Daily control samples _________________________________________________________________ 197
6.4 CONCLUSIONS ________________________________________________________________ 199
6.5 REFERENCES _________________________________________________________________ 200
TABLE OF CONTENTS

Chapter 7 Inhibitory Effects by Pseudomonas spp. in Raw Milk on
Microbiological Inhibitor Assays _____________________________________________ 203
7.1 INTRODUCTION _______________________________________________________________ 205
7.2 MATERIALS AND METHODS _____________________________________________________ 210
7.2.1 Milk sampling _______________________________________________________________________ 210
7.2.2 Antimicrobial testing by microbiological inhibitor tests and receptor assays _____________________ 210
7.2.3 Assessment of milk quality ____________________________________________________________ 212
7.2.3.1 Composition parameters and pH ____________________________________________________ 212
7.2.3.2 Total bacterial count, enumeration of psychrotrophic and lipophylic bacteria _______________ 212
7.2.3.3 Determination of lipolysis, fat oxidation and proteolysis _________________________________ 212
7.2.4 Further bacteriological testing _________________________________________________________ 213
7.2.4.1 Isolation, characterization and identifications of strains _________________________________ 213
7.2.4.2 Phylogenetic analysis _____________________________________________________________ 213
7.2.4.3 Growth and bacterial inhibitor production ____________________________________________ 214
7.2.4.4 Bacterial inhibitor characterization assays ____________________________________________ 214
7.2.4.4.1 Estimation of the molecular weight ______________________________________________ 214
7.2.4.4.2 Heat tolerance of bacterial inhibitor and fatty acids _________________________________ 215
7.2.4.4.3 Inhibition spectrum of the bacterial inhibitor ______________________________________ 215
7.2.4.4.4 Impact of test medium on the inhibition __________________________________________ 216
7.3 RESULTS _____________________________________________________________________ 216
7.3.1 Screening of milk for antibiotic residues __________________________________________________ 216
7.3.2 Assessment of milk quality ____________________________________________________________ 217
7.3.3 Isolation, characterization, and identification of strains _____________________________________ 219
7.3.4 Growth and bacterial inhibitor production ________________________________________________ 219
7.3.5 Bacterial inhibitor characterization assays ________________________________________________ 222
7.3.5.1 Dialysis experiments ____________________________________________________________ 222
7.3.5.2 Heat tolerance ________________________________________________________________ 224
7.3.5.3 Inhibitory spectrum ____________________________________________________________ 224
7.3.5.4 Impact of test medium on the inhibition ____________________________________________ 225
7.3.5.5 Technological significance _______________________________________________________ 225
7.3.6 Experiments with fatty acids ___________________________________________________________ 225
7.3.6.1 Effect of fatty acids on microbiological inhibitor assays __________________________________ 225
7.3.6.2 Dialysis experiments ______________________________________________________________ 227
7.3.6.3 Effect of heat-treatment __________________________________________________________ 227
7.4 DISCUSSION __________________________________________________________________ 228
7.5 FINAL CONCLUSIONS ___________________________________________________________ 232
7.6 REFERENCES _________________________________________________________________ 234

General Discussion _____________________________________________________________ 241
Summary ______________________________________________________________________ 253
Samenvatting __________________________________________________________________ 261
Curriculum Vitae _______________________________________________________________ 269
Acknowledgements