To Eva and Amber - Universiteit Gent

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



5.3.6 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



6.2.9 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



6.3.6 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

ABBREVIATIONS AND ACRONYMS

ADI acceptable daily intake AFB American foulbrood AFSSA Agence Française de Sécurité Sanitaire des Aliments AG aminoglycosides AG Aktiengesellschaft AHD 1-amino-hydantoin AMOZ 3-amino-5-morpholinomethyl-2-oxazolidinone AMGP antimicrobial growth promotors AN ansamycins Anon. anonymous AOAC Association of Official Analytical Chemists AOZ 3-amino-2-oxazolidinone B. Bacillus BA blood agar BCZ Belgische Confederatie van de zuivelindustrie vzw BDI Bureau of Dairy Industries BELAC Belgische Accreditatie-instelling BHI brain heart infusion broth BHIA brain heart infusion agar B.V. besloten vennootschap C control CAP chloramphenicol Cat. catalog CBL La Confédération Belge de l‟industrie Laitière CCα decision limit CC detection capability CE cephalosporins CFR code of federal regulations cfu colony forming unit CLP cyclic lipopeptide CIF Colour Impact Factor CNI Community Nutrition Institute Co. Company CRA-W Centre Wallon de Recherches Agronomiques CRL Community Reference Laboratory CTC chlortetracycline CV coefficient of variation CVMP Committee for Medicinal Products for Veterinary Use DA Delvotest Accelerator DAD diode array detector DC doxycycline DI diaminopyrimidine derivatives DNA deoxyribonucleic acid DMZ dimetridazole DSM Dutch State Mines E. Escherichia EC European Community EEC European Economic Community EFB European foulbrood EG Europese Gemeenschap


e.g. exempli gratia ELISA enzyme-linked immunosorbent assay EMA European Medicines Agency ESBL extended spectrum beta-lactamase ESI electrospray ionization et al. et alii etc. et cetera EU European Union exp. expiration FAO Food and Agriculture Organization of the United Nations FAMHP Federal Agency for Medicines and Health Products FASFC Federal Agency for the Safety of the Food Chain FDA Food and Drug Administration FEDESA Fédération Européenne de la Santé Animale FEEDM Fédération Européenne des Emballeurs et Distributeurs de Miel FEFANA EU Association of Specialty Feed Ingredients and their Mixtures FERA Food and Environment Research Agency FFD &CA Federal Food Drug and Cosmetic Act FIA fluorescent immunoassay FNZ Koninklijke Nederlandse Zuivelbond FTD furaltadone FZD furazolidone Glu glucuronide GmbH Gesellschaft mit beschränkter Haftung HCl hydrochloric acid HPLC high performance liquid chromatography H2O2 hydrogen peroxide IA immunoassay i.d. internal diameter IDF International Dairy Federation i.e. id est ILVO Institute for Agricultural and Fisheries Research IN -lactamase inhibitors Inc. Incorporated ISO International Organization for Standardization IPTG isopropyl-β-D-thio-galactoside-test JECFA Joint FAO/WHO Expert Committee on Food Additives KaHo Katholieke Hogeschool KGaA Kommanditgesellschaft auf Aktien K.O.I.V. Koninklijke Oost-Vlaamse Imkersvereniging L. Linnaeus LC liquid chromatography LFD lateral flow device LI lincosamides LMG Laboratory of Microbiology, Ghent University LOD limit of detection log P partition coefficient Ltd. Limited MA macrolides MA&L macrolides & lincosamides


MALDI matrix-assisted laser desorption ionization max maximum MBC minimal bactericidal concentration MCS milk control station MIC mimimum inhibitory concentration min minimum MIPS molecularly imprinted polymers MNZ metronidazole MPCA milk plate count agar MRL maximum residue limit MRPL minimum required performance limit MS mass spectrometry MSU meat, serum, and urine MUMS Minor Uses, Minor Species MWCO molecular weight cut-off [M + H]+ pseudo-molecular ion (in positive ion mode) m/z mass-to-charge ratio n or No number na no data available NA nutrient agar NaCl sodium chloride NaOH sodium hydroxide neg negative NFT nitrofurantoin NFZ nitrofurazone NO(A)EL no observable (adverse) effect level nt not tested OIE Office Internationale des Epizooties OSCN- hypothiocyanate anion OTC oxytetracycline O2 oxygen P. Pseudomonas PABA para-aminobenzoic acid PCA plate count agar PCB polychlorinated biphenyl PCLT Praktijkcentrum voor Land- en Tuinbouw PCR polymerase chain reaction PE penicillins P. l. Paenibacillus larvae PMO Pasteurized Milk Ordinance PO polymyxins pos positive QU fluoroquinolones RASFF Rapid Alert System for Food and Feed RCF g (gravitational)-force RIA radio immunoassay ROSA rapid one step assay RPA reference point for action RNA ribonucleic acid RNZ ronidazole


s.a. société anonyme SCC somatic cell count [SCN]- thiocyanate ion SD standard deviation SEM semicarbazide S.L. Sociedad de Responsabilidad Limitada S.p.A. Societa per Azioni SP-NT Sulfonamides Penicillins – New Test spp. species pluralis SPR surface plasmon resonance sr standard deviation of repeatability srl società a responsabilità limitata ssp. subspecies ST simple technology (SNAP) Str. Streptococcus SU sulfonamides subsp. subspecies T test T-AOZ sum of the concentrations of AOZ and parent furazolidone TBA tributyrin agar TC tetracycline TE tetracyclines TNZ tinidazole ToF time-of-flight TSA tryptone soya agar T&V Technology and Food Science unit UGent Ghent University UHT ultra-high temperature processing UPLC ultra-performance liquid chromatography UV ultraviolet v visual reading v. versus var. varians vis visual reading VRBA violet red bile agar vzw vereniging zonder winstoogmerk w/v weight/volume w/w weight/weight WHO World Health Organization X cloxacillin Zn zinc


°C degree Celsius meq milli-equivalents cm centimeter mg milligram d day min minute eV electronVolt ml milliliter € Euro mm millimeter g gram mS millisiemens h hour ng nanogram IU international units s second kDa kilodalton t ton kg kilogram V volt kJ kilojoule µg microgram l liter µS microsiemens M molair CA California MI Michigan CT Connecticut NJ New Jersey IN Indiana OH Ohio KS Kansas UK United Kingdom MA Massachusetts USA United States of America MD Maryland VT Vermont ME Maine

Chapter 1 Legislative and Analytical Aspects, Residues of Antimicrobials in Honey and Milk

1.1 Legislative and Analytical Aspects 2

1.1 LEGISLATIVE AND ANALYTICAL ASPECTS 1.1.1 Legislation The control and monitoring of residues of veterinary medicinal products in food of

animal origin is regulated by the European Union.

Council Directive 96/23/EC lays down the requirements that must be met in relation

to the planning and execution of national residue control plans for live animals and

products of animal origin. The principal objective of the legislation is to detect illegal

use of substances in animal production and the misuse of authorized veterinary

medicinal products and to ensure the implementation of appropriate actions to

minimize recurrence of all such residues in food of animal origin.

The Directive lays down measures requiring European Member States to monitor

the substances and groups of residues, listed in Annex I to the Directive. The

substances are classified in two main categories: Group A and Group B. Group A

lists substances having anabolic effect and unauthorized substances (i.e. prohibited

substances listed in Table 2 of the Annex to Commission Regulation (EU) No

37/2010). Group B contains a list of veterinary drugs and contaminants, i.e. residues

of many pharmacologically active substances which may be authorized for use in

food producing animals in the EU (i.e. allowed substances listed in Table 1 of the

Annex to Commission Regulation (EU) No 37/2010). It also comprises

organochlorine and organophosphate pesticides, and chemical elements such as

lead, cadmium, and mercury.

Annex II to Council Directive 96/23/EC lists for each commodity (e.g. honey, milk,

eggs, etc.) the Group A and Group B subgroups to be monitored. A summary of the

groups of residues or substances to be checked for in milk and honey is given in

Table 1.

Council Directive 96/23/EC requires Member States to draft a national residue

monitoring plan for the groups of substances detailed in Annex I. These plans must

comply with the sampling rules in Annex IV of the Directive. This Directive also

establishes the frequencies and level of sampling for each food commodity. Council

Directive 96/23/EC is amended by Council Regulation (EC) No 806/2003, Regulation

(EC) No 882/2004 and Corrigendum, and Council Directive 2006/104/EC.

http://www.fsai.ie/uploadedFiles/Legislation/Food_Legisation_Links/Veterinary_Medicines,_Animal_Remedies,_Control_of_Illegal_Substances_and_Po/Council_Directive_96_23_EC.pdf




Table 1. Annex II of Council Directive 96/23/EC: groups of residues or substances to be checked for in milk and honey. Group Group name milk honey

Group A. Substances having anabolic effect and unauthorized substances

A6 prohibited substances x

Group B. Veterinary drugs and contaminants

B1 antibacterial substances, including sulfonamides & quinolones x x

B2a other veterinary drugs - anthelmintics x

B2c other veterinary drugs - carbamates and pyrethroids x

B2e other veterinary drugs – non-steroidal anti-inflammatory drugs x

B3a other substances and environmental contaminants –

organochlorine compounds including PCBs x x

B3b other substances and environmental contaminants –

organophosphorus compounds x x

B3c other substances and environmental contaminants –

chemical elements x x

B3d other substances and environmental contaminants –

mycotoxins x

Notes: x, determination is mandatory.

Commission Decision 97/747/EC provides levels and frequencies of sampling in

order to monitor some substances and residues thereof for the animal products milk,

eggs, honey, rabbits, and game meat. For bovine milk, the annual number of

samples is 1 per 15,000 tonnes of the annual production of milk, with a minimum of

300 samples. The number of honey samples to be taken each year must at least be

equal to 10 per 300 t of the annual production for the first 3,000 t of production, and

one sample for each additional 300 t. Commission Decision 98/179/EC lays down

detailed rules for official sampling procedures and official treatment of samples until

they reach the laboratory responsible for analysis. Where checks demonstrate the

presence of unauthorized substances or products, or when maximum limits have

been exceeded, the provisions of Articles 19 to 22 of Regulation (EC) No

882/2004 will apply. The food will be placed under official detention till destruction,

special treatment, or re-dispatchment. In certain cases a recall can be requested if

the food is already placed on the market and the competent authority shall notify the

Commission and other Member States in accordance with the procedure provided


for in Article 50(3) of Regulation (EC) No 178/2002. The necessary measures are

taken to ensure that the rejected consignments will not be reintroduced into the

Community. All costs involved are for the food business operator responsible for the

consignment or its representative.

The establishment of maximum residue limits (MRLs) for pharmacologically active

substances of authorized veterinary medicinal products in foodstuffs of animal origin

is governed by Commission Regulation (EU) No 37/2010 and amendments,

repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC

and Regulation (EC) No 726/2004, both of the European Parliament and of the

Council. The classification of the pharmacologically active substances in

Commission Regulation (EU) No 37/2010 follows the classification foreseen in

Regulation (EC) No 470/2009. The substances are listed in alphabetical order in two

separate tables: one for allowed substances, integrating all substances listed in

Annexes I, II, and III of Council Regulation (EEC) No 2377/90, and one for prohibited

substances, listed in Annex IV to that Regulation. Since the list of allowed

substances is a positive list, the administration to food producing animals of

veterinary medicinal products containing pharmacologically active substances, which

are not listed in the table of allowed substances, is prohibited.

The term MRL may be defined as the maximum concentration of marker residue

(e.g. parent compound, metabolite, ...) resulting from the use of a veterinary drug,

expressed in µg kg-1, that is legally permitted or recognized as acceptable in or on a

food. The MRL is based on the type and amount of residue, considered to be without

any toxicological hazard for human health as expressed by the acceptable daily

intake (ADI). It also takes into account other relevant public health risks, as well as

food technological aspects. Furthermore, the MRL may be reduced to be consistent

with good practices in the use of veterinary drugs and to the extent that practical

analytical methods are available.

The ADI or the quantity that may be consumed daily without any harmful effect for

the whole lifespan, is the result of scientific risk assessment following

pharmacological and toxicological studies. The ADI is determined by the no-

observable-(adverse)-effect level (NO(A)EL), taking into account an uncertainty


NO OBSERVABLE ADVERSE EFFECT LEVEL

Uncertaintyfactor

AMOUNT OFANTIBIOTIC

OBSERVABLE

ADIMRL

EFFECT

(safety) factor (Figure 1). The uncertainty factor provides an adequate safety margin

for the consumer in the extrapolation of animal data to humans. Usually an

uncertainty factor of 10 for interspecies variation between animals and humans, and

a further factor of 10 that covers the variation in sensitivity within the human

population, is applied. This results in an overall uncertainty factor of 100.

MRL = NOEL x body weight / uncertainty factor x daily intake

In the calculation, an overall value of 60 kg is used for the body weight. Considering

a hypothetical daily consumption pattern (100 g liver, 300 g muscle (muscle and skin

for fish), 50 g kidney, 50 g fat (fat and skin for pork and poultry), 20 g honey, 1.5 l of

milk, and 100 g of egg), for each edible tissue a separate MRL has to be established

so that the total human consumption (daily intake) of the residues does not exceed

the ADI.

Figure 1. Schematic presentation how MRLs are established. Note that ADI is expressed in mg kg-1 body weight per day and MRL in µg kg-1 (Heeschen, 1997).

The list of the MRL values for anti-infectious agents fixed in bovine milk in

Commission Regulation (EU) No 37/2010 and amendments as of October, 12, 2010

is presented in Annex B of this Chapter. At this moment, there are no MRLs

established for anti-infectious agents in honey. Remark that the MRLs are mandatory

in the EU; other countries have taken over the European MRLs or have their own


regulatory limits. The Tolerance and/or Safe Levels for animal drug residues (partim

antimicrobials) in milk, as actually applied in the USA, are given in Annex C of this

Chapter.

The MRLs are taken into account for the setting of the withdrawal time. This is the

minimal period of time between the last treatment and the time the residues in the

foodstuff are decreased to levels below the MRL. In other words, the minimal time

after the last treatment needed to be respected, before milk can be delivered for

human collection or the honey can be harvested.

The regulatory limit for certain prohibited or unauthorized analytes is the Minimum

Required Performance Limit (MRPL) or the Reference Point for Action (RPA).

MRPLs are foreseen in Article 4 of Commission Decision 2002/657/EC. The

Commission Decisions 2003/181/EC and 2004/25/EC were setting harmonized

MRPLs for analytical methods employed to detect residues of substances whose use

is not authorized or is specifically prohibited in the Community in food of animal

origin. MRPLs are not based on toxicological studies but are in accordance with the

opinion of the Standing Committee on the Food Chain and Animal Health in

consultation with the Community Reference Laboratories, the National Reference

Laboratories, and the Member States in order to provide harmonized levels for the

control of those substances to ensure the same level of consumer protection in the

Community. So far MRPLs were set for chloramphenicol, medroxyprogesterone

acetate, nitrofuran metabolites (furazolidone, furaltadone, nitrofurantoin, and

nitrofurazone), and (leuco)malachite green. MRPL means the minimum content of an

analyte in a sample, which at least has to be detected and confirmed by the

laboratories. In the Statement to the minutes of the Standing Committee for the Food

Chain and Animal Health of 21 September 2004 (Anon., 2004b), it was moreover

agreed that MRPLs set according to Commission Decision 2002/657/EC, shall be

used as RPAs. This approach moreover means that any detection of substances

whose use is prohibited or not authorized in the Community, shall be followed by an

investigation into the source of the substance in question and appropriate

enforcement measures in particular aimed at the prevention of recurrence in the

case of documented illegal use.


Regulation (EC) No 470/2009 (Articles 25 & 29) lays down rules and procedures in

order to establish RPAs. A RPA is the level of a residue of a pharmacologically

active substance established for control reasons in the case of certain substances,

for which a maximum residue limit has not been laid down.

For substances without MRL, the Community Reference Laboratories (CRLs)

distributed a guidance paper providing recommended concentrations in order to

improve and harmonize the performance of analytical methods for national residue

control plans (Anon., 2007c). In some European countries, national action limits are

applied (e.g. Belgian action limits for residues of certain antibiotics and sulfonamides

in honey).

If third countries want to export products of animal origin towards the European

Union, they are required to submit a residue monitoring plan to the European

Commission on annual basis, setting out the guarantees that the residue monitoring

in their country is equivalent to the EU requirements on residues of veterinary

medicines, pesticides, and contaminants and in compliance with Council Directive

96/23/EC. Commission Decision 2004/432/EC is listing the approvals; the latest list

of approvals is annexed to Commission Decision 2010/327/EU. MRLs are applicable

to imported consignments (Commission Decision 2005/34/EC). The Commission

Decision 2005/34/EC harmonizes the levels for the control of residues of

substances, for which no permitted limit has been established in products of animal

origin imported from third countries by using MRPLs as reference points for action.

Quality criteria for residue analysis are described in Commission Decision

93/257/EEC, laying down the reference methods and the list of national reference

laboratories for detecting residues, as last amended by Commission Decision

2006/130/EC.

Commission Decision 2002/657/EC is implementing Council Directive 96/23/EC

concerning the performance of analytical methods and the interpretation of results.

The Decision provides performance criteria and other requirements for the analytical

methods to be used in the testing of official samples, to ensure the quality and

comparability of the analytical results generated by laboratories approved for official

residue control. The Decision is also laying down validation procedures for analytical

methods. The performance characteristics that have to be determined are different


for screening and confirmatory, and for qualitative and quantitative tests (Table 2).

Commission Decision 2002/657/EC was corrected by a Corrigendum and amended

by Commission Decisions 2003/181/EC and 2004/25/EC.

Table 2. Classification of analytical methods by the performance characteristics that have to be determined (Commission Decision 2002/657/EC). Detection

limit CC

Decision limit CCα

Trueness/ recovery

Precision Selectivity/ specificity

Applicability/ ruggedness/stability

qualitative

methods

Sa + - - - + +

Cb + + - - + +

quantitative

methods

Sa + - - + + +

Cb + + + + + +

Notes: Sa, screening methods; Cb, confirmatory methods; +, determination is mandatory.

Screening methods mean methods that are used to detect the presence of a

substance or class of substances at the level of interest. These methods have the

capability for a high sample throughput and are used to shift large numbers of

samples for potential non-compliant results. They are specifically designed to avoid

false compliant results (false compliant rate <5% at the level of interest (Commission

Decision 2002/657/EC)). Confirmatory methods mean methods that provide full or

complementary information enabling the substance to be unequivocally identified

and if necessary quantified at the level of interest.

The level of interest or the screening target concentration is the concentration at

which a screening test categorizes the sample as „positive‟ (potentially non-

compliant) and triggers a confirmatory test. For authorized analytes, the screening

target concentration is at or below the regulatory limit (MRL); for prohibited and

unauthorized analytes, the screening target concentration must be at or less than the

MRPL (or RPA); and for analytes for which MRLs have not been established, the

screening target concentration should wherever possible be at or less than the

recommended concentrations as described in the CRL Guidance Paper of

December 7, 2007 (Anon., 2007c).

The Community Reference Laboratories for residues (CRLs) provided guidelines for

the validation of screening methods for residues of veterinary medicines. The

guidelines cover two distinct phases in the validation process: the initial validation of


screening methods in the originating laboratory and the shortened or „abridged‟

validation of these methods in the receiving laboratory (Anon., 2010a).

1.1.2 Analytical aspects 1.1.2.1 Analytical aspects of screening tests

Analytical approaches can be broadly divided into screening tests, group specific

tests, and confirmatory methods. Screening tests are procedures intended for

primary examination of large numbers of samples for the possible presence of

residues. They should ideally be capable of detecting many compounds in a single

test procedure to minimize the number of tests required. A negative test should

reliably indicate that the product is free of residues. False-negative results at

violative level may not occur, however, in Commission Decision 2002/657/EC a false

compliant rate of 5% at the level of interested is accepted. False-positive tests, by

preference limited to a small number, are acceptable, provided that the false-

positives can be identified by further testing. In some cases, especially in milk

testing, product may be accepted or rejected solely on the basis of one or more

screening tests since the storage of raw milk is limited. Some organizations make

the difference between false-positive (positive test result when no detectable residue

is present) and false-violative results (positive test result when detectable residues

are present below violative levels). Screening tests ordinary require minimal sample

preparation and simple equipment (Moats, 1998).

The ideal screening test provides a positive response with a cut-off near the MRL, or

previously defined level of interest. A test which only yields positive results at values

considerably in excess of the MRL is of dubious value, unless there are no

alternative means of monitoring. Equally, a test kit that detects residues at

concentrations that are well below the MRL will result in an excessive number of

samples which require confirmatory testing in a regulatory programme (MacNeil and

Kay, 2000).

1.1.2.2 Microbiological screening tests

For primary screening for antimicrobial residues in food of animal origin, microbial

growth inhibition tests are generally used, since these tests are cheap and allow to

analyse a large number of samples in a short time, providing that no extraction is


included in the protocol. Hence, it is not probable that these tests will be replaced by

other techniques in the near future. Microbiological screening relies on the common

property of all antibacterials to inhibit the growth of the test organism. Inhibition tests

have been considered broad spectrum and unspecific but the microorganisms used

as test bacteria are of course not equally sensitive to all antibiotics. As a

consequence, they detect some substances better than others. Combinations of

different test bacteria, each in an optimal medium, are now considered as the best

tool to detect a large range of antibiotics up to the MRL levels. Optimal or very good

detection capabilities of β-lactam antibiotics are obtained with Geobacillus

stearothermophilus, Kocuria rhizophila, and Bacillus subtilis, while macrolides are

best detected with K. rhizophila. Low limits of detection (LODs) have been described

for tetracyclines with Bacillus cereus, and for quinolones with Escherichia coli and

Yersinia ruckeri. Sulfonamides are detected with G. stearothermophilus and Bacillus

pumilus. B. subtilis spores are added to media intended for aminoglycosides (De

Brabander et al., 2009).

In the traditional approach, sample (milk soaked in a filter disc) is placed on an agar

plate previously seeded with test bacteria and incubated. The presence of a clear

zone around the sample, where the bacterial growth is inhibited, indicates the

presence of inhibitory substances in the sample. If the identity of the antibiotic is

known, the residue can be quantified by measuring the zone of inhibition since the

diameter of the inhibition zone is proportional to the logarithm of the concentration of

the antibiotic. By comparing the diameter measured for the sample with those

obtained for different concentrations of the antibiotic, the concentration in the sample

can be calculated. Quantification is not possible with unknowns or if more than one

residue is present. In order to limit the interference of certain natural inhibitors, agar

diffusion tests were developed with the presence of a pH-indicator in the medium.

The acid production of the test organism during incubation is judged by the reading

of the colour of the test medium. These tests are usually in microplate or single

ampoule format. Other criteria for the interpretation of inhibition of growth could be

based on bioluminescence, conductivity, or turbidity measurements.

Some microbiological tests are more or less group specific by the selective

sensitivity of the test strain (B. cereus for tetracyclines; E. coli for quinolones,...);

another possibility to unveil the identity of the inhibitor is to add products with an


impact on the inhibition. In this way presence of -lactams or sulfonamides can be

confirmed by treating a replicate before testing with penicillinase or para-amino

benzoic acid (PABA), respectively. Within the -lactam family, benzylpenicillin,

ampicillin, and amoxicillin could be distinguished from the other penicillins and

cephalosporins by the addition of penase.

1.1.2.3 Rapid screening tests

To avoid the long incubation period inherent to microbiological inhibitor tests,

enzymatic, receptor, and immunological tests were developed for a rapid screening

of foodstuffs of animal origin on the presence of antimicrobials.

The first immunoassays (IA) were radio IAs (RIA) using radioactive isotope labeled

antigens. They were followed by ELISAs and fluorescent IAs (FIA). Immunoassays

can be rapid, selective, and sensitive and have shown considerable utility in some

areas of residue analysis.

The handling was simplified by the development of dipsticks and lateral flow

systems. On dipstick systems, the extract (milk or diluted honey) migrates on a

support coated with two zones of receptor ligands and/or antigens (indirect assay) or

two zones of receptors and/or immunoglobulins (direct assay). Just before

application, the sample is mixed with the soluble labeled reagent. After migration, the

presence or absence of the targeted analyte is indicated by the absence or the

development of a line at the test zone on the dipstick, respectively (Bergwerff, 2005).

In one-step strip tests, also known as lateral flow devices (LFD), the analyte-

containing solution is not mixed with reagents, but while migrating, matrix

components, including the analyte, are mixed with analyte-binding and reference-

binding molecules in a reagent pad just after application of the liquid sample

(Verheijen et al., 2000). For field tests, lateral flow devices remain the first choice.

Of the new technologies like biosensors, cellular bioassays, transcriptomics, and

proteomics, optical biosensors, based on surface plasmon resonance (SPR), have

attracted most attention in the field of antibiotic residue analysis (Gustavsson and

Sternesjo, 2004). First applications of molecularly imprinted polymers (MIP)

techniques in residue analysis are described (Andersson, 2000; Xu et al., 2004); in

most cases MIPs are applied as a selective sorbent in solid-phase extraction

methods for sample clean-up (Warriner et al., 2008). Novel electrochemical and


optical immunosensors (Marco et al., 2008), flow cytometric immunoassays

(Bienenmann-Ploum et al., 2008), and biochip array technology applications (Tohill

et al., 2008) for residue analysis are presently under evaluation. Instead of using

antibodies or receptors, new developments based on aptamers, artificial

oligonucleotides (RNA or DNA), or proteins with a high affinity for particular

molecular targets, can be expected in the future (Stead, 2008). Aptamers can be

produced in vitro for a wide range of molecular targets, including small molecules,

and have the ability to form defined tertiary structures to engage a specific target for

binding (Stead, 2008).

1.1.2.4 Chromatographic and confirmatory methods

The identity and quantity of the residue in a suspected sample cannot be determined

with a screening test. Hence the decision about the compliance of a sample cannot

be based on a screening result. Therefore there is a need for specific

chromatographic or other confirmatory methods.

To be useful, confirmatory methods must meet the stringent sensitivity and selectivity

requirements of regulatory agencies. Confirmatory methods shall provide information

on the chemical structure of the analyte. Consequently, methods based only on

chromatographic analysis, without the use of mass spectrometric detection, are not

suitable on their own for use as confirmatory methods for prohibited substances.

However, if a single technique lacks sufficient specificity, the desired specificity shall

be achieved by analytical procedures consisting of suitable combinations of clean-

up, chromatographic separation(s) and spectrometric detection (Commission

Decision 2002/657/EC). In general, physico-chemical methods for the confirmation of

anti-infectious agents are based on chromatographic separation of residues,

followed by spectroscopic quantification, such as UV, fluorescence, or MS detection.

The last years, despite the associated costs, mass spectrometric methods are more

and more used for very selective and specific multi-compound detection (Stolker et

al., 2008). In order to manage matrix-induced ion-suppression and/or deposition of

matrix debris in the ionization source affecting the stability of instrument operation,

advanced sample preparation is essential (Bergwerff, 2005).

Of the reported methods used for the determination of antibiotics in food (Web of

Knowledge database), grouped on the type of analytical technique applied, LC-MS is


the most employed analytical method (38%), followed by LC-UV (18%) and ELISA

(18%). The distribution is depicted in Figure 2 (Cháfer-Pericás et al., 2010).

other screening methods 12%

biosensors 8%

electrophoresis 6%ELISA 18%

LC-UV 18%

LC-MS 38%

Figure 2. Distribution of the analytical methods used for the determination of antibiotics in food (Cháfer-Pericás et al., 2010).

1.1.2.5 Screening for antimicrobials in honey

Due to the issue of zero tolerance for antimicrobial residues in honey by the absence

of MRLs and due to the high sugar content of honey, microbiological screening is not

often used for screening of honey for antibiotic residues. DSM Nutritional Products

(Geleen, the Netherlands) claims the PremiTest could be used for a broad-spectrum

detection of antimicrobials in honey. However, the detection capabilities do not meet

the action or reporting limits used in some European countries. Two methods for

microbiological detection of tetracyclines and one method for the microbiological

detection of tylosin in honey were published, based on the use of Bacillus cereus

(Gordon, 1989), Bacillus subtilis ATCC6633 (Khismatoullin et al., 2003), and

Micrococcus luteus ATCC 9341 (Khismatoullin et al., 2004), respectively.

For the detection of chloramphenicol, (dihydro)streptomycin, tetracyclines,

fluoroquinolones, and tylosin, several ELISA kits are commercially available. For

these compounds, except for fluoroquinolones, and also for the detection of

sulfonamides and macrolides (& lincosamides) in honey, Charm II receptor assays

(Charm Sciences Inc., Lawrence, MA) could be used. The sample preparation is

depending on the kit, while the assay itself takes about 30 minutes. Some biochip-

based methods like Biacore (GE Healthcare Europe GmbH, Freiburg, Germany) and


Anti Microbial Arrays (Randox Laboratories Limited, Crumlin, United Kingdom) allow

the detection of multiple drug residues in honey (McAleer et al., 2010). The Anti

Microbial Arrays system also includes the detection of nitrofuran metabolites

(O‟Mahony et al., 2010). The Biacore biosensor system is based on surface plasmon

resonance (SPR). With this system, a high throughput and a rapid (around five

minutes) multi-analyte screening in honey is possible (Weigel et al., 2005). However,

the instrument costs remain high.

There are also other detection possibilities without the need of expensive

instrumentation. The TetraSensor Honey (Unisensor s.a., Wandre, Belgium)

sensitively detects the four most important tetracyclines in honey in 30 minutes,

without any special equipment, making analysis at the production site possible. In

Chapter 3, the results of a validation study performed at ILVO-T&V are reported.

Only limited laboratory equipment is required to run the Sulfasensor (Unisensor s.a.),

a new generic monoclonal antibody test, for the detection of sulfonamides in honey

in 20 minutes. A 5-minute sample pretreatment is needed to release the

sulfonamides that are chemically bound to the sugars (Chabottaux et al., 2010a;

Reybroeck and Ooghe, 2010b).

Other on-site honey tests are the Chloramphenicol, Tetracyclines, and Quinolones

Residue Rapid Inspection Test Devices (Hangzhou Nankai Biotech Co., Ltd.,

Binjiang, China) and the Chloramphenicol, Tetracycline, Quinolones, Sulfadiazine,

and Penicillins Drug Residue Rapid Test Devices (SmarK!T, Zhejiang Huazheng

Import & export Co., Ltd., Hangzhou, China).

Other compounds such as nitroimidazoles (Polzer et al., 2010) and fumagillin (Tarbin

et al., 2010) are mostly directly screened using liquid chromatography-tandem mass

spectrometry (LC-MS/MS) detection, since no immunochemical methods for the

detection in honey have been developed yet.

1.1.2.6 Screening for antimicrobials in milk

1.1.2.6.1 Integrated system for residue control in milk

The dairy industry has always been interested to screen milk on the presence of

inhibitory substances in order to prevent technological problems when producing

cheese or yoghurt. Therefore the dairy sector has a very long tradition in

antimicrobial residue testing. Till now, in no other food sector the primary product is


group specification

confirmation(identification and quantification)

screening

MICROBIOLOGICAL

ELISA

HPLC

LC-MS/MS

MICROBIOL. + REAGENT

ELISA

RECEPTOR TEST

so extensively monitored on inhibitory substances as raw milk. In this sense it is not

surprising that several commercial tests for the screening of milk for antimicrobials

are available (Diserens et al., 2010).

To ensure a high technological quality of the milk and the safety of the consumers,

an integrated system (Heeschen and Suhren, 1996) is applied in Belgium, with

shared responsibilities of the dairy farmer, the processing establishment, and the

food inspection. A mixture of microbiological, immunological, and receptor screening

tests is used in practice since no single test is able to detect all anti-infectious agents

on MRL in milk. Positive results can give rise to a financial penalty for the farmer or

rejection of positive milk.

When applying detection methods, the most cost-effective approach is to apply a

pyramidal structure (Figure 3). Screening of milk is started with microbiological

inhibitor tests due to their easy test performance, low price, and broad spectrum

detection capability. Compounds (e.g. chloramphenicol) not sensitively detectable

with bacteria, are usually screened by means of immunological (ELISA) assays.

Figure 3. Pyramidal structure for a cost-effective monitoring for antimicrobials in milk. Suspected samples can be analysed further in order to specify the family of residues

(group specification) by means of microbiological tests after addition of certain

substances (e.g. -lactamase for the identification of -lactam antibiotics), by means

of group-specific microbiological tests (e.g. Bacillus cereus-test for the detection of

tetracyclines), by means of group specific receptor tests (e.g. Charm II Milk assays),


or by immunological tests (e.g. Streptomycin EIA). Finally, once the family of the

residues is known, the identity and the concentration of the residue present can be

confirmed by a physico-chemical analysis (HPLC or LC-MS/MS).

1.1.2.6.2 Testing of individual cows‟ milk at the dairy farm

The farmer has the responsibility to use veterinary medicinal products correctly, to

keep and retain records on the veterinary medicinal products or other treatments

administered to the animals, dates of administration, and withdrawal periods

(Corrigendum to Regulation (EC) No 852/2004). The farmer must also ensure the

identification of animals undergoing medical treatment likely to transfer residues to

the milk, and respect the prescribed withholding time, in order to ensure that milk

obtained from treated animals before the end of the prescribed withdrawal period is

not used for human consumption (Corrigendum to Regulation (EC) No 853/2004).

More and more farmers are taking no risk and test the milk of each treated cow

individually, with a microbial inhibitor test at the end of the withholding period. For

that aim, several commercial tests like Delvotest SP-NT mini (DSM Food Specialties,

Delft, the Netherlands) and Copan Milk Test Single Test (DSM Food Specialties) are

on the market. These broad-spectrum tests, with individual test vials, are based on a

colour change of the pH-indicator (bromcresol purple) during incubation at 64°C from

purple to yellow when the milk is free from antimicrobials, due to the production of

acid by the test organism in the test agar. When the growth of the test organism is

inhibited, no acid is produced and the colour of the test agar remains purple. The

interpretation of the colour is performed visually. These tests are most sensitive for

penicillins, cephalosporins, and sulfonamides. More recently, some farmers are

using a rapid group-specific receptor test like eta-s.t.a.r..

1.1.2.6.3 Testing of ex-farm milk at the milk control stations

In accordance with Corrigendum to Regulation (EC) No 853/2004, a representative

number of raw milk samples, collected from milk production holdings taken by

random sampling, must be checked for that the raw milk placed on the market is not

containing antibiotic residues in a quantity that exceeds the levels authorized (MRLs)

under Council Regulation (EEC) No 2377/90 (now repealed by Commission


no penaltyCopan Milk TestScreeningneg

Post-screening

pos

no penaltylow quantity of -lactam

neg

eta-s.t.a.r. 25 cut-off

pospenalty-lactam

> cut-off

preheated milkCopan Milk Test

neg

penaltynon--lactam

negno penaltynatural inhibitors

+ penicillinaseCopan Milk Testpos pos

Cut-off values:

Copan Milk Test:CIF 4.5

eta-s.t.a.r. 25:ratio 0.040(Accuscan III)

Regulation (EU) No 37/2010), or that the combined total of residues of antibiotic

substances does not exceed any maximum permitted value.

In Belgium, each delivery of farm milk is automatically sampled and tested for

inhibitors. Screening is performed with the microbiological inhibitor test Copan Milk

Test (DSM Food Specialties), based on Geobacillus stearothermophilus var.

calidolactis, in microplate format. Colour interpretation, after incubation of the sealed

plates with milk on top of the agar, is performed by means of a flatbed scanner

(black cover) and special C-scan software. The reflectometric reading results in CIF

(Colour Impact Factor) values in the range from 0.1 (yellow) to 10.0 (purple).

Samples giving a CIF 4.5 are considered as positive and need to be further tested,

as shown in Figure 4.

Figure 4. Test procedure used by the Belgian milk control stations for testing of ex-farm milk on inhibitors, as part of the official quality control of raw milk.

As a first post-screening test, eta-s.t.a.r. 25 (Neogen Corporation, Lansing, MI) is

used in a semi-quantitative way with a ratio of 0.040 (Accuscan III reader) as cut-off

value. Samples giving a ratio 0.040 are considered to contain enough -lactam

residues to justify a penalty (price reduction). Samples giving a ratio >0.040 are

further checked on the presence of natural inhibitors and non--lactam antibiotics.

Therefore part of the milk is preheated for 10 min at 80°C to neutralize natural

inhibitors. If the preheated milk is testing negative, no penalty will be attributed since


the inhibition is caused by natural inhibitors. Another part of the milk is treated with

an active and broad-spectrum penicillinase for 20 min at 37°C to inactivate all -

lactam residues. A positive Copan Milk Test result for milk pretreated with

penicillinase will lead to a penalty for the responsible milk producer (presence of

non--lactam residues).

In Belgium, a penalty actually results in a price reduction of € 29.75 per 100 liter of

delivered milk and a temporary cancelling of the milk collection, until the proof is

given that the next farm milk is free from antimicrobials. Once a dairy producer is

penalized 4 times in a time span of 1 year, the collection of milk at his farm is

cancelled for 2 weeks.

It‟s worth noting that the result of the testing of ex-farm milk on antimicrobials by the

milk control stations is only known after the milk is also processed.

In most European countries, ex-farm milk is screened for antimicrobials as part of a

regulatory quality programme by means of a microbiological test. Besides the Copan

Milk Test, several tests are on the market to be used in routine by the milk control

stations. The most known are BR-Test AS Brilliant (DSM Food Specialties), BRT

Inhibitor Test (Analytik in Milch Produktions- und Vertriebs- GmbH, Munich,

Germany), Charm Blue-Yellow II (Charm Sciences Inc.), Delvotest SP-NT and SP 5-

pack NT DA (DSM Food Specialties), and Eclipse 3G (ZEU-INMUNOTEC S.L.,

Zaragoza, Spain). Delvotest Accelerator microplates are not incubated in a

waterbath or dry oven but on a special grid mounted on top of a flatbed scanner. The

colour change of the pH indicator in the agar of the wells of the microplate is followed

by reflectometric reading during incubation at 63°C. The end of incubation is

determined by the instrument once the colour in a sufficient number of wells turned

to yellow. With this system and reagents, the incubation time can be shortened to

105 to 140 minutes.

In some countries, milk control stations use a home-made test (improved tube

diffusion test (Vermunt et al., 1993), disc assay, ...).

1.1.2.6.4 Testing of production or tanker milk at the processing establishment

In accordance with the hygiene provisions, formulated in Regulation (EC) No

852/2004, food business operators have to comply with appropriate Community and


national legislative provisions, related to the control of hazards in primary production

and associated operations, including measures to control contamination arising from

veterinary medicinal products. Therefore, dairies have to perform autocontrol

programmes (Article 14 Council Directive 92/46/EEC; Corrigendum to Regulation

(EC) No 853/2004). The competent authorities have to monitor the checks carried

out on raw milk upon collection (Corrigendum to Regulation (EC) No 854/2004) and

to verify that the relevant requirements of food law are fulfilled by food business

operators at all stages of production, processing, and distribution (Regulation (EC)

No 178/2002). In accordance with Regulation (EC) No 853/2004, food business

operators in the dairy sector are not allowed to place on the market raw milk

containing levels of antibiotic residues exceeding the MRL. In order to comply with

those requirements, food business operators in the dairy sector carry out rapid

screening tests on milk before placing it on the market. Those tests are aimed at

determining the presence of antibiotic residues and have been designed to provide

positive results when such residues are close to the MRL but do not quantify the

actual level of residues present. Under those circumstances, only a test identifying

and quantifying the antibiotic residues can demonstrate that the MRL is not

exceeded. If such a confirmatory test is not carried out, milk showing a positive result

of a screening test is deemed to be unsafe (Commission Decision 2006/694/EC).

Raw milk containing antibiotic substances in excess of the regulatory level as laid

down in the Community MRL-legislation is unfit for human consumption and unsafe.

Such milk must be disposed of as an animal by-product of Category 2 as laid down

in Regulation (EC) No 1069/2009. Following the same legislation, milk from cows

which have been submitted to illegal treatment is classified as Category 1 material.

Before production, Belgian dairy processors must screen all milk on the presence of

antimicrobials (-lactams). Depending on the size of the production plant and the

system of milk collection and production, the check can be performed on production

milk, just before processing with a broad-spectrum microbiological inhibitor test

(Delvotest SP-NT or Copan Milk Test), or on tanker milk by means of a rapid -

lactam test. In this way, silo or tanker milk can be rejected after a positive result.

A protocol (Figure 5) was developed by the Belgian dairy sector in order to improve

uniform testing among all dairies. Since some cephalosporins are detected at levels


RAPID TEST ON TANKER LOAD

SAME RAPID TEST REPEATED(internal lab)

MICROBIOLOGICAL TEST

REJECTION (DESTRUCTION) HUMAN CONSUMPTION

+

+

+

-

-

-

far below MRL, the use of a microbiological test, like the Delvotest SP-NT, is

accepted for the final decision upon the rejection of a tanker load (Reybroeck and

Ooghe, 2004).

Figure 5. Belgian dairy sector protocol for testing of incoming tanker milk (Reybroeck and Ooghe, 2004).

Since testing with a rapid test can result in the destruction of the whole tanker load,

the assay should be accurate and reliable. Therefore, validation studies of

commercially available screening tests are very important (Suhren and Knappstein,

2007). For the daily check of the good performance of the residue tests (1st line

control), standards of non-fortified blank and fortified milk can be used. The Belgian

dairies also have the opportunity to participate in ring trials (3rd line control),

organized by ILVO-T&V. It‟s worth noting that some dairy companies are claiming

compensations for the costs of the destruction of a contaminated tanker load. In this

way a positive test for antibiotics in a tanker load of milk can be quite costly for the

producer responsible, who is asked to pay for the loss of the entire tanker load.

Since most occurring residues in milk are penicillins and cephalosporins, the tanker

milk is mostly only checked on the presence of -lactam antibiotics at the entrance of

the dairy. The testing of all incoming shipments of milk for -lactam antibiotics

became mandatory in the U.S., effective January 1, 1992 (Moats, 1998). This was

giving a boost to the development of rapid test procedures. The European Union is


more vague about how and when the testing of milk for antimicrobials must be

performed. In Spain, in 2008, a new legislation was introduced including the

obligation to screen tanker milk on the presence of tetracycline residues with a rapid

test on a routine basis (one test per five tanker loads) (Real Decreto 1728/2007).

The first fast test developed for screening of incoming loads of tanker milk for -

lactam antibiotics was the Penzym test (UCB-Bioproducts s.a., Braine-l‟Alleud,

Belgium), an enzymatic (carboxypeptidase) colorimetric test, giving a result in 20 min

(Knight et al., 1987). End of the 80‟s, early 90‟s, several screening tests with a total

test time below 10 minutes (receptor tests SNAP (Idexx Laboratories, Inc.,

Westbrook, ME), eta-s.t.a.r. (Neogen Corporation), Charm MRL Beta-lactam Test

(ROSA) (Charm Sciences Inc.), and the immunoassays Lactek (Idexx laboratories,

Inc.) and Parallux (Idexx Laboratories, Inc.)), became commercially available for

monitoring raw milk on the presence of -lactams (Reybroeck and Ooghe, 2004;

Diserens et al., 2010). More recently, some rapid tests (eta-s.t.a.r. 1+1 (Neogen

Corporation) and Charm MRL-3 (Charm Sciences Inc.) were adapted to give a test

result within 3 minutes, allowing screening of milk at the farm before collection.

Validation studies of these two tests are described in Chapters 5 and 6 of this thesis.

The last quarter of 2010, Idexx Laboratories, Inc. launched the SNAP Beta-Lactam

ST, a 7 minute receptor test with incubation at room temperature.

Also rapid tests in LFD or dipstick format for the detection of antimicrobials not

belonging to the -lactam family were developed. At present, for milk screening

purposes, kits are available for the detection of tetracyclines (SNAP Tetracycline

Test Kit (Idexx Laboratories, Inc.), TetraSensor Milk (Unisensor s.a.), Charm

Tetracyclines (ROSA) (Charm Sciences Inc.)), sulfamethazine (SNAP

Sulfamethazine Test Kit (Idexx Laboratories, Inc.), Charm Sulfamethazine Test

(Charm Sciences Inc.)), sulfonamides (Charm ROSA Sulfa Test (Charm Sciences

Inc.)), gentamicin (SNAP Gentamicin Test Kit (Idexx Laboratories, Inc.)),

enrofloxacin (Charm ROSA Enrofloxacin (Charm Sciences Inc.)), streptomycin

(Charm ROSA Streptomycin (Charm Sciences Inc.)), and CAP (Charm ROSA

Chloramphenicol (Charm Sciences Inc.)). Nowadays there are also kits on the

market for a simultaneous detection of -lactams and tetracyclines in milk

(TwinSensor Milk (Unisensor s.a.), TwinExpress Milk (Unisensor s.a.), Charm MRL


Beta-Lactam/Tetracycline (3 Minute) Combo Test (Charm Sciences Inc.), and SNAP

Duo (Idexx Laboratories, Inc.)). The generic Trisensor (Unisensor s.a.) dipstick

screening test allows the simultaneous detection of -lactams, tetracyclines, and

sulfonamides in milk. Recently, the detection of (fluoro)quinolones was implemented

on this multiplex lateral flow device (Chabottaux et al., 2010b), forming a quadric-

antibiotic families dipstick screening test. Finally, Parallux Milk Residue Testing

System (Medexx Co., Ltd., Bundang-Gu, Korea (South)) detects all six major beta-

lactams, tetracyclines, spectinomycin, neomycin, streptomycin, spiramycin, sulfa

drugs, and quinolones in one test in four minutes.

1.1.2.6.5 Monitoring of dairy products

In order to fulfil the requirements for the national monitoring plan, special sampling of

different dairy products is organized. Part of the monitoring programme is organized

and paid by the dairy sector itself, another part is performed by the Federal Agency

for the Safety of the Food Chain (FASFC). Monitoring is not only focusing on cows‟

milk (farm, tanker, and consumption milk), but also on milk powder, goats‟, ewes‟,

and mares‟ milk. The programme for residues of veterinary drugs includes, besides

the presence of antimicrobials, also antiparasitic agents (benzimidazoles and

macrocyclic lactones (avermectins)), nitro-imidazoles, coccidiostats, and non-

steroidal anti-inflammatory agents.

Since the routine testing by the milk control stations is not detecting all antimicrobials

at their respective MRL in milk, the monitoring on antimicrobials is based on different

screening tests: Delvotest SP-NT (broad-spectrum), Bacillus cereus-test (Suhren

and Heeschen, 1993) (tetracyclines), Escherichia coli-test (Suhren, 1997)

(fluoroquino-lones), and a β-lactam receptor test (detection of cefquinome, missed at

MRL by the Delvotest SP-NT). Part of the samples are checked by immunological

assays on the presence of chloramphenicol at the level of 0.1 µg kg-1 (CAP EIA

Chloramphenicol (EuroProxima B.V., Arnhem, the Netherlands)), on the presence of

3-amino-2-oxazolidone (AOZ), metabolite of the nitrofuran furazolidone (Ridascreen

Nitrofurans AOZ (R-Biopharm AG, Darmstadt, Germany)), and on the presence of

streptomycins (Streptomycins EIA (EuroProxima B.V.)).

Samples giving a positive result for a broad-spectrum test are further analysed with

group-specific Charm II Milk assays, in order to specify the group or family of the


unknown residue. Finally, the residue will be identified and quantified by LC-MS/MS

analysis, allowing to determine if the sample is compliant or not.

1.1.3 Anti-infectious agents Antimicrobial drugs are principally used in the treatment and prophylaxis of bacterial

infections. In addition, disinfectants and preservatives are used to kill or inhibit the

growth of micro-organisms. Although antibacterials are a very diverse class of

compounds, they are often classified in groups. They may be classified according to

their mode of action or spectrum of antimicrobial activity, but generally those with

similar chemical structure are grouped together (Anon., 2005a). The most simple

classification of anti-infectious agents is the division in antibiotics and

chemotherapeutics.

The most important groups of veterinary anti-infectious agents (Anon., 1998b; Anon.,

2005a) are discussed below in the sequence as tabulated in Council Regulation

(EEC) No 2377/90.

1.1.3.1 Sulfonamides

The sulfonamides are analogues of para-aminobenzoic acid (PABA). The first

sulfonamide of clinical importance was sulfanilamide. It was synthesized in Germany

in 1932. Many sulfonamides have since been synthesized. The sulfonamides have

been classified according to their rate of excretion. The short-acting sulfonamides

include sulfapyridine, sulfadiazine, and sulfadimidine. Sulfamethoxazole is a

medium-acting sulfonamide; sulfadimethoxine and sulfamethoxypyridazine are long-

acting sulfona-mides; while sulfadoxine and sulfamethopyrazine are ultra-long-acting

sulfonamides. Sulfonamides are usually bacteriostatic, and interfere with folic acid

synthesis of susceptible organisms. These drugs are competitive antagonists for

PABA, a precursor to folic acid. Folic acid is used in the biosynthesis of purines

needed for DNA production and is essential for bacterial growth.

1.1.3.2 Diaminopyrimidine derivatives

Diaminopyrimidine derivatives, such as trimethoprim and baquiloprim, can be used

as potentiators with sulfonamides to give a synergistic antimicrobial effect. Diamino-

pyrimidines also inhibit the biosynthesis of bioactive folic acid by interfering with the


metabolism of dihydrofolate to tetrahydrofolic acid. When diaminopyrimidines are

used along with sulfonamides, the combination is bactericidal.

1.1.3.3 Penicillins

All penicillins have the same ring structure and are monobasic acids that readily form

salts and esters. The penicillin nucleus, 6-aminopenicillanic acid, consists of a fused

thiazolidine ring and a β-lactam ring with an amino group at the 6-position. The

discovery of penicillin in 1928 was a milestone in medicine. Penicillin was originally

obtained from the mould Penicillium notatum. Better yields were achieved using P.

chrysogenum, and benzylpenicillin (penicillin G) was selectively produced by adding

the precursor phenylacetic acid to the fermentation medium. Phenoxymethylpenicillin

(penicillin V), with a phenoxyacetamido side-chain, is also a natural penicillin.

Ampicillin and amoxicillin belong to the aminopenicillins which show a broader

spectrum of activity by being also active against Gram-negative bacteria. The

isoxazolyl penicillins, cloxacillin, dicloxacillin, and oxacillin, are resistant to

penicillinase. Penicillins are bactericidal and act by inhibiting synthesis of the

bacterial cell wall.

1.1.3.4 Cephalosporins

Cephalosporins are semi-synthetic antibacterials derived from cephalosporin C, a

natural antibacterial. The active nucleus is very closely related to the penicillin

nucleus and consists of a β-lactam ring fused with a six-membered dihydrothiazine

ring, having an acetoxymethyl group at position 7. Cephalosporins are bactericidal

and, similarly to the penicillins, they act by inhibiting synthesis of the bacterial cell

wall. Together with the penicillins, they represent the -lactam group.

The most widely used system of classification of cephalosporins is by generations.

Cefalotin (developed in 1964), cefalonium, cefacetrile, cephapirin, cefalexin, and

cefazolin are examples of first-generation cephalosporins. To the second generation

(1978) belong cefamandole and cefuroxime. The third-generation cephalosporins

(1981), sometimes referred to as extended-spectrum cephalosporins, are more

stable to hydrolysis by -lactamases produced by Gram-negative bacteria and have

a wider spectrum and a greater potency of activity against Gram-negative bacteria.


Cefoperazone and ceftiofur are the best known drugs of that group. The newer

cephalosporins cefepime, cefpirome, and cefquinome are considered to be fourth-

generation because of their broad spectrum of activity. The carbapenem group, the

monobactams, and the carbacephems are structurally related to cephalosporins.

1.1.3.5 Quinolones

The 4-quinolones are a group of synthetic antibacterials structurally related to

nalidixic acid. The quinolones include a major subgroup, the fluoroquinolones. They

were produced by addition of the 7-piperazinyl group and a fluorine atom at position

6. The fluoroquinolones are bactericidal by inhibiting the bacterial DNA-gyrase,

having a role in the replication and transcription of DNA.

1.1.3.6 Macrolides

The macrolides are a large group of antibacterials mainly derived from Streptomyces

spp. and having a common macrocyclic lactone ring to which one or more sugars are

attached. They are all weak bases and only slightly soluble in water. The macrolides

are primarily bacteriostatic, but some are bactericidal at higher dosages for some

organisms. Macrolides bind to the 50S ribosomal subunit of susceptible species and

inhibit peptide formation. Macrolides have a post-antibiotic effect: that is,

antibacterial activity persists after concentrations have dropped below the minimum

inhibitory concentration. Erythromycin was discovered in 1952 and is the most widely

used macrolide.

1.1.3.7 Tetracyclines

The tetracyclines are a group of antibacterials, originally derived from certain

Streptomyces spp., having the same tetracycline nucleus. Tetracyclines are primarily

bacteriostatic antibiotics and have their main mechanism of action on inhibition of

protein synthesis in susceptible organisms. The first tetracycline to be introduced

was chlortetracycline in 1948.

Tetracycline, oxytetracycline, and chlortetracycline are natural products while the

more recent doxycycline is a semisynthetic derivative.


1.1.3.8 Lincosamides

Lincomycin is an antimicrobial produced by a strain of Streptomyces lincolnensis and

was first described in 1962. The lincosamides are bacteriostatic or bactericidal,

depending on the concentration. Although structurally not related to macrolides, the

lincosamides have similar antimicrobial activity and act at the same site on the

bacterial ribosome to suppress protein synthesis.

1.1.3.9 Aminoglycosides

Aminoglycosides are carbohydrates that contain one or more amino groups in their

glycosidic structure. Chemically, the aminoglycoside antibiotics can be divided into

two main groups: those containing streptidine (streptomycin and

dihydrostreptomycin), and those containing 2-deoxystreptamine. The 2-

deoxystreptamine antibiotics are further differentiated and can be broken down into

several subclasses. The aminoglycosides have a similar antimicrobial spectrum and

appear to act by interfering with bacterial protein synthesis, possibly by binding

irreversibly to the 30S and to some extent the 50S portions of the bacterial ribosome.

They are most active against Gram-negative rods. Streptomycin was the first

aminoglycoside to become commercially available and was isolated from a strain of

Streptomyces griseus in 1944.

1.1.3.10 Chloramphenicol and related drugs

Chloramphenicol is an antibacterial which was first isolated from cultures of

Streptomyces venezuelae in 1947 but is now produced synthetically. It has a

relatively simple structure and was the first broad-spectrum antibacterial to be

discovered. It acts by interfering with bacterial protein synthesis and is mainly

bacteriostatic. It is a relatively stable compound with high lipophilicity and a high

solubility in organic solvents. Shortly after its introduction, chloramphenicol was

found to have a serious and sometimes fatal depressant effect on the bone marrow.

Chloramphenicol could also cause the grey syndrome. As a result of this toxicity the

use of chloramphenicol has been restricted in many countries. Thiamphenicol and

florfenicol are still registered for veterinary use.


1.1.3.11 Nitrofurans

The nitrofurans are a group of antimicrobials (e.g. furazolidone (FZD), furaltadone

(FTD), nitrofurantoin (NFT), and nitrofurazone (NFZ)) with the defining structural

component consisting of a furan ring with a nitro group. Their use is banned in

Europe due to observed mutagenesis and carcinogenesis in laboratory animals. Due

to the rapid transformation of these substances, the monitoring of nitrofuran residues

has been focused on the detection of the protein-bound nitrofuran metabolites 3-

amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone

(AMOZ), 1-amino-hydantoin (AHD), and semicarbazide (SEM).

1.1.3.12 Nitroimidazoles

The nitroimidazoles include metronidazole, dimetridazole, ipronidazole, and

ronidazole. They have a N-1 methyl and a 5-nitro substituted imidazole ring in

common. The nitroimidazoles are antibacterial and antiprotozoal. The mechanism for

antiprotozoal activity is not understood. However, metrodinazole‟s antibacterial

activity is believed to result from the disruption of bacterial DNA and nucleic acid

synthesis in susceptible anaerobes via a polar metabolite. The 5-nitro group is

essential for the therapeutic action of nitroimidazoles (and also their mutagenic

potential). In Europe, the use of metronidazole, dimetridazole, and ronidazole in

food-producing animals is prohibited.

1.2 Antimicrobials in Honey 28

1.2 ANTIMICROBIALS IN HONEY 1.2.1 Introduction Honey is defined as the natural sweet substance produced by Apis mellifera bees

from the nectar of plants or from secretions of living parts of plants or excretions of

plant-sucking insects on the living parts of plants, which bees collect, transform by

combining with specific substances of their own, deposit, dehydrate, store, and leave

in honeycombs to ripen and mature (Council Directive 2001/110/EC).

The only honey allowed to be traded into the European Union has to be from Apis

mellifera, the European honeybee species, which has now spread all around the

world. However, regionally there are honeys made by other bee species which are

sometimes collected in considerable quantities, especially in parts of Asia from Apis

cerana, and to a lesser extent from Apis dorsata (Bogdanov, 2009). The main

sources for honey production are nectar and honeydew. Nectar is secreted in flower

nectaries, while honeydew is the secretion product of plant-sucking insects

(Hemiptera, mostly aphids). Both products are gathered by honey bees for their

sugar content, and stored in the wax honeycombs as energy source for periods

without foraging activity. Ripe honey is harvested by beekeepers and replaced by

sugar syrup solution as winter feed. Blossom honey can be differentiated from

honeydew honey by electrical conductivity. Accordingly blossom honeys should have

less, honeydew honeys more than 0.8 mS cm-1. Specific rotation measurements

could correct the classification for unifloral lime (Tilia spp.) and chestnut (Castanea

sativa) honey (Doberšek et al., 2006).

Honey is mainly containing sugars. The relative amount of the two monosaccharides,

fructose and glucose, useful for the classification of unifloral honeys (Bogdanov et

al., 2004), is depending on the floral source of the honey. An overview of the

composition of blossom and honeydew honey is given in Table 3.

Due to the high sugar content, crystallization of honey is a natural process. The

higher the glucose content (e.g. rape honey (Brassica spp.)), the faster the

crystallization. Some unifloral honeys with a high fructose content (e.g. black locust

honey (Robinia pseudoacacia L.)) remain liquid (Persano Oddo and Piro, 2004).

Ripe honey with a low moisture content, as removed from the hive by the beekeeper,

has a long shelf life and will not ferment.


Table 3. Composition of blossom and honeydew honey, values in g 100 g-1 (White, 1975; Anon., 1995).

Parameter Blossom honey Honeydew honey average min - max average min - max

water content 17.2 15-20 16.3 15-20 fructose 38.2 30-45 31.8 28-40 glucose 31.3 24-40 26.1 19-32 sucrose 0.7 0.1-4.8 0.5 0.1-4.7

other disaccharides 5.0 28 4.0 16 melizitose <0.1 4.0 0.3-22.0

erlose 0.8 0.56 1.0 0.16 other oligosaccharides 3.6 0.5-1.0 13.1 0.1-6.0

minerals 0.2 0.1-0.5 0.9 0.6-2.0 amino acids, proteins 0.3 0.2-0.4 0.6 0.4-0.7

pH 3.9 3.5-4.5 5.2 4.5-6.5 Notes: min, minimum; max, maximum. In 2008, the production of honey in the whole world, the European Union, and

Belgium amounted 1,496,416; 196,464; and 2,150 tonnes, respectively (Anon.,

2010i). The production of beeswax was 61,223; 4,169; and 50 tonnes, respectively

(Anon., 2010i). The EU has a honey deficit and usually has to import about half of

the honey consumed, self-sufficiency in 2004/05 being 54.2% (Anon., 2007a). The

EU imports approximately 150,000 t of honey each year.

Figure 6. Origin of the honey imported to Europe in 2007 (Anon., 2008a).


In 2007, Argentina remained the European Union‟s main supplier, followed by

Mexico, Uruguay, China, and Chili (Figure 6). Annual per capita honey consumption

in Europe was 0.7 kg in 2005 (Anon., 2007a).

1.2.2 Main bee diseases and pests Longtime ago, honeybees were domesticated in artificial hives for the production of

honey that was used as an important carbohydrate source and food sweetener.

Honeybees are social insects living in a colony of a single female queen bee, a

seasonally variable number of male drones to fertilize new queens, and about

20,000 to 40,000 female worker bees, responsible for brood rearing and nectar

collecting.

Like all living organisms, honeybees can be infested with diseases and pests. Bees

have two distinct life forms (brood and adult) and most diseases are specific to either

one stage or the other. Acarapiosis, American foulbrood, European foulbrood, small

hive beetle infestation (Aethina tumida), Tropilaelaps infestation, and Varroasis are

notifiable to Office Internationale des Epizooties (OIE) (Anon., 2008b). American

foulbrood, small hive beetle infestation, and Tropilaelaps infestation are notifiable

diseases within the European Community under Commission Regulation (EC) No

1398/2003, amending Annex A to Council Directive 92/65/EEC.

Only the bee diseases that could be treated with antibiotics or chemotherapeutics

will be shortly discussed beneath.

1.2.2.1 American foulbrood

American foulbrood or AFB is by far the most virulent brood disease known in honey

bees. The disease is caused by the spore-forming bacterium, Paenibacillus larvae

subsp. larvae. It attacks older larvae and young pupae, which are literally digested

by enzymes secreted by the bacterium. The comb has a pepper box appearance

where diseased larvae have been removed. Cappings may appear moist, sunken,

and perforated. Initially the dead larvae are slimy, and dry to form brown scales that

are highly infective. Because the spores can remain viable for years, many countries

imply bee colonies with AFB to be burned upon discovery. Other countries (USA,

Canada, ...) allow the use of antibiotics to keep the disease in check. Antibiotics can

only mitigate – not eliminate – the disease and therefore infected hives must be

treated constantly to prevent a foulbrood outbreak. Left unchecked, foulbrood


destroys the hive‟s bee population and can annihilate an apiary. If the infection is

moderate without clinical symptoms, a shook swarm method of treatment is

recommended (von der Ohe, 2003).

Figure 7. Honey bee larvae killed by American foulbrood, as seen in the cells. A, healthy larvae at the age when most of the brood dies of American Foulbrood. B-F, dead larvae in progressive stages of decomposition. The remains shown in F is called scale. G, longitudinal view of a scale (Root and Root, 1975).

1.2.2.2 European foulbrood

European foulbrood or EFB is closely related to AFB in symptomology. However, the

causative organism, the bacterium Melissococcus plutonius, does not form spores,

and so the disease is considered less problematic than AFB. The bacterium

generally only attacks younger larvae. The treatment of EFB is generally less drastic


than for AFB. Only in severe cases, eradication by burning is needed. Generally,

requeening with more resistant stock will clear up the disease. Also some

antimicrobials, e.g. oxytetracycline, showed to be effective to treat EFB.

1.2.2.3 Nosemosis

Nosemosis, caused by Nosema apis or N. ceranae, is by far the most damaging

adult bee disease. Infections are acquired by the uptake of spores during feeding or

grooming. Nosema apis infects the epithelial cells of the hind gut (ventriculus) of the

digestive tract of the adult bee, giving rise to large numbers of spores in a short

period of time and impairing the digestion of pollen thereby shortening the life of

bees. High incidences of Nosema are directly related to stress, such as periods of

long confinement or nutritional imbalance (Webster, 1993). Nosema ceranae was

originally a parasite of the Asian honey bee (Apis cerana) but now shows to be

widespread in some European regions, afflicting the adult bees and resulting in

depopulation and bee colony losses (Higes et al., 2006). Until recently, Nosema apis

had been considered to be a spore-forming microsporidian, a single-celled protozoan

but is now classified as fungus or fungi-related (Fischer and Jeffrey, 2005).

1.2.3 Use of antibiotics and chemotherapeutics in beekeeping

Honeybees are classified as food producing animals in the EU, thus the

establishment of a Maximum Residue Limit (MRL) for honey is necessary before a

marketing authorization for a veterinary medicinal product can be granted. In view of

the lack of metabolism in the beehive, an elimination of residues over a certain

period of time as defined for other food producing species, would not occur. So, in

principle only medicinal products which do not give residues in the honey after use

(zero day withdrawal period) could be authorized for bees as indicated by the

Committee for Medicinal Products for Veterinary Use (CVMP). So far, no MRLs have

been established for antibiotics and sulfonamides in honey, what theoretically means

that the use of antibiotics in beekeeping is not permitted in the EU. The resulting

„zero tolerance‟ policy of residues causes significant trade problems as clear,

harmonized rules do not exist with regard to acceptable control methods, limits of

detection, or sampling methods, resulting in a different interpretation by EU Member

States. In the absence of either EU MRLs or reference points for action (RPAs), the

presence of any detectable (and confirmed) residue in honey imported into the EU


would mean that those consignments cannot legally be placed on the market in the

EU (Regulation (EC) No 470/2009). In that view, the European Federation of Honey

Packers and Distributors (FEEDM) requested the establishment of RPAs in order to

allow for control of honey imported from third countries. In the meantime, some EU

Member States and Switzerland established action limits, non-conformity or

tolerance levels. The EC Reference Laboratory (Anon., 2007c) proposed

recommended concentrations for screening for antibiotics and sulfonamides in honey

for national residue control plans established in accordance with Council Directive

96/23/EC. An overview of the limits applied is given in Table 4.

Table 4. Limits (in µg kg-1) applied for antibiotics and chemotherapeutics in honey in various European countries.

Antibiotic or chemotherapeutic

Limits applied in various countries (in µg kg-1) Belgium (action limit)a

France (non-

conformity limit)b

Switzerland (tolerance)c

(till January 1, 2009)

UK (reporting

limit)d

EU (recommended concentration for screening)e

streptomycin 20 10 20 20 40 tetracyclines 20 10 20 20 20 sulfonamides 20 - 50 50 50 macrolides

(erythromycin) - - - 2 20

macrolides (tylosin) - 15 - 2 20 chloramphenicol 0.1 - - - 0.3 (MRPLf)

nitrofurans 1 - - - 1g (MRPLf) Notes:a Anon., 2001; b Martel, 2010; c Diserens, 2010; d Anon., 2003; e Anon., 2007c; f MRPL, Minimum Required Performance Limit (Commission Decision No 2003/181/EC); g MRPL set for poultry meat and aquaculture products (Commission Decision No 2003/181/EC), applicable on honey (Anon., 2007c).

At present, the Swiss tolerance limits are no longer valid (Diserens, 2010) and in the

UK, concentrations above the decision limit (CCα) of the confirmatory method are

now reported as non-compliant instead of the concentrations mentioned in Table 4

(Sharman, 2010).

Despite the lack of MRLs for anti-infectious agents in honey, antibiotics and

chemotherapeutics could be used in the EU in apiculture based on the „cascade‟

system as described in Article 11 of Directive 2001/82/EC, as amended by Directive

2004/28/EC. The cascade system was introduced to solve the general problem of


availability of veterinary medicinal products for minor species. The cascade system

is open to all animal species, including honeybees (Anon., 2007b), provided that the

active substance concerned has been included in Annex I, II or III of Council

Regulation (EEC) No 2377/90 (recently repealed by Table 1 in the Annex of

Commission Regulation (EU) No 37/2010)) and the prescribing veterinarian specifies

a withdrawal period. Hence, the use of oxytetracycline is allowed in the UK under the

cascade for the treatment of EFB with a withdrawal period of at least 6 months

(Anon., 2010b). In France, the treatment of AFB with antibiotics is accepted,

provided that the disease is not yet largely developed and the honey and wax are

destroyed afterwards (Anon., 2005c).

The problem of availability of veterinary medicines to treat honeybees has been

discussed extensively at a workshop held in December 2009 at the European

Medicines Agency (EMA) in London, United Kingdom (Anon., 2010b). At the 19th

session of Codex Committee on Residues of Veterinary Drugs in Foods that took

place in August-September 2010 in Burlington, VT, recommendations were made to

consider MRLs in honey and to work out a Codex guideline on Good Veterinary

Practice in honey production. Such a guideline could provide harmonized guidance

that would ensure the safety of bee products and enable fair trade practices (Anon.,

2010l). When considering honey intake for MRL setting, the JECFA standard diet

assumes a consumption of 20 g per person per day. At the 70th JECFA meeting a

revised consumption value of 50 g per person per day was proposed (Anon., 2010l).

In the USA, antibiotic drugs authorized for treatment of bees include oxytetracycline,

tylosin, and bicyclohexylammonium fumagillin. However, the use of these antibiotics

is to be discontinued sufficiently in advance of the honey flow in order to prevent

residues in the honey since there are no authorized residues of these antibiotics in

honey. Even so, there are no authorizations or tolerances for other drugs like

sulfonamides, erythromycin, or streptomycin in treating bees. Fluoroquinolones are

prohibited for use in treating honeybees (Anon., 2010m & Stutsman, 2010).


1.2.4 Antibiotics and chemotherapeutics of interest in apiculture 1.2.4.1 Tetracyclines

Tetracyclines are broad-spectrum bacteriostatic antibiotics with a long history in

veterinary medicine and are used for the treatment and control of a wide variety of

bacterial infections. Oxytetracycline (OTC), usually in its hydrochloride form, is used

in apiculture since the early fifties for the treatment of bacterial brood diseases like

AFB (Hopingarner and Nelson, 1987; Spivak, 2000) and EFB (Oldroyd et al., 1989;

Waite et al., 2003; Thompson et al., 2005). Three modes of OTC application have

been commonly used: a dusting with OTC in powdered sugar, repeated several

times at weekly intervals; a solution of OTC in syrup fed to the bees; and now most

common, an „extender patty‟ consisting of OTC, sugar, and vegetable shortening

(Kochansky, 2000). Terramycin® (OTC hydrochloride) (Phibro Animal Health,

Ridgefield Park, NJ) has been the only approved drug treatment for the foulbrood

diseases for a long period in the United States (Anon., 2010m). Terramycin® is

existing in different application forms. Application of Terramycin® in powder form is

likely to result in lower initial residue levels, compared to an application in liquid form

(Anon., 2002c). Nowadays in the USA, many other animal drug products based on

OTC as ingredient are approved by FDA for use in apiculture. The application should

be finished and all product removed at least 6 weeks prior to main honey flow

(Anon., 2010m).

Despite the fact that no MRL for tetracyclines has been fixed in honey,

oxytetracycline is used in the UK in the statutory treatment of EFB, since this is

considered by the authorities as within the cascade system for veterinary medicines

under Minor Uses, Minor Species (MUMS) (Thompson et al., 2005). The use is only

permitted under certain circumstances, i.e. under veterinary supervision and

applying long withdrawal periods. In September 2009, approximately 4,660 hives

were treated with OTC in response to an outbreak of EFB in eastern Scotland

(Anon., 2010q).

The product Oxypharm plv. sol.® (Pharmagal s.r.o., Nitra, Slovakia), containing

oxytetracycline hydrochloride, is authorized in Slovakia for treatment or protection

application to the winter feed of EFB (Anon., 2009b).

The intensive use of tetracyclines in professional beekeeping resulted in tetracycline-

resistant Paenibacillus strains in the US (Miyagi et al., 2000), Canada (Colter, 2000),


and Argentina (Alippi, 2000). There is now general concern about widespread

resistance.

1.2.4.2 Streptomycin

Streptomycin is an aminoglycoside antibiotic used in apiculture to protect bees

against a variety of brood diseases. Despite the drug is not authorized in most

countries (EU, USA), the use is often suggested in bee forums and in beekeeping

handbooks (Mutinelli, 2003). In China, streptomycin and chloramphenicol were

preferred antibiotics to control a large AFB outbreak in 1997, instead of eradication

(Ortelli et al., 2004). At the Apimondia meeting in 1997 (Antwerp, Belgium) it was

mentioned that Mexican beekeepers provided the bees reinforcing products

containing streptomycin directly in the beehives (Bogdanov and Fluri, 2000).

Streptomycin shows a partial effect against the fire blight bacterium Erwinia

amylovora. Plantomycin® or Fructocin®, streptomycin-containing agents, are

sometimes used in fruit plantations during flowering season. Honey can be

contaminated with streptomycin in limited concentrations up to 20 µg kg-1 after

contact of the foraging bees with Plantomycin® treated blossom (Brasse, 2001;

Kölbener et al., 2003). In the USA, streptomycin is authorized as a pesticide for

treatment in apples and pears during the blossom period. Nevertheless, there are no

pesticide tolerances for streptomycin in honey (Stutsman, 2010).There are reports

about the natural occurrence of low levels of streptomycin in honey from Zambia.

The streptomycin could possibly be produced by bacteria belonging to the genus

Streptomyces (Bradbear et al., 2005).


Sulfonamides play an important role as effective chemotherapeutics for bacterial and

protozoal diseases in veterinary medicine. They are frequently administered in

combination with dihydrofolate reductase inhibitors of the group of

diaminopyrimidines. The use of sulfonamides to protect honey bees against bacterial

diseases became a common practice in commercial beekeeping after Haseman and

Childers (1944) learned that sulfa drugs, particularly sulfathiazole, could prevent the

spread of AFB. The compound sulfathiazole provided a short-term control by

suppressing the symptoms of the bee disease caused by Paenibacillus larvae. It also


prevented the reproductive spores from germinating. The use of sulfa drugs in the

bees‟ food in spring and fall was also encouraged by other authors (Eckert, 1947;

Reinhardt, 1947; Johnson, 1948; Katznelson and Gooderham, 1949; Katznelson,

1950). Despite the effectiveness of sulfonamides against AFB, their stability and

consequent residues in honey caused problems, and the registration was allowed to

lapse in the seventies (Shimanuki and Knox, 1994).

Some beekeepers also apply sulfonamides against nosemosis in a prophylactic way

by addition to winter feed sugar solution (Lourdes, 2002). This practice, as

suggested in beekeeping manuals (e.g. Anon., 2010h) and in publications regarding

the treatment of infections due to microsporidia (Liu and Weller, 1996; Didier, 1998;

Conteas et al., 2000), increased after fumagillin became less available in the EU .

1.2.4.4 Tylosin

Tylosin, a macrolide antibiotic, has been used globally in beekeeping. Its efficacy

was proven by different authors (Hitchcock et al., 1970; Moffett et al., 1970; Peng et

al., 1996; Pettis and Feldlaufer, 2005). Tylosin was found to be more stable in sugar

syrup than OTC (Kochansky et al., 1999). In October 2005, Tylan (tylosin tartrate)

Soluble® (Elanco Animal Health, Indianapolis, IN) received approval in the US from

the Food and Drug Adminstration (FDA) for the treatment of active AFB, but not for

preventative use in healthy colonies. The use of tylosin should be discontinued at

least 4 weeks before the honey flow (Anon, 2010m). The use of the dust method of

tylosin is greatly favoured over the patty method in countries with infestations of

small hive beetles, since their populations significantly increased in all colonies

treated with patties (Elzen et al., 2002). As efficacy, safety, and residue studies were

available, the UK beekeepers association requested the originator to apply for a

„global marketing authorization‟ as created by Article 5 of Directive 2004/28.

However, such a marketing authorization would prevent data protection for the

dossier that would immediately become available for the generic competition and so

preventing return on investment. The use of tylosin against AFB was promoted after

Paenibacillus had shown resistance against tetracyclines.

1.2.4.5 Erythromycin

Erythromycin, another macrolide, was first tested in 1955 (Katznelson et al., 1955;

Katznelson, 1956). Depending on the literature, erythromycin has been reported to


be effective against AFB (Machova, 1970; Okayama et al.,1996) and EFB (Wilson

and Moffet, 1957; Wilson, 1962), while other authors found it to be ineffective against

AFB (Katznelson et al., 1955; Moffett et al., 1958). Despite the doubt about its

effectiveness, erythromycin was used by professional beekeepers in the Southern

Marmara region of Turkey (Gunes et al., 2008).

1.2.4.6 Lincomycin

Lincomycin belongs to the group of lincosamides. Its activity against Paenibacillus

larvae strains has been reported by some authors (Okayama et al., 1996; Kochansky

et al., 2001). Lincomycin was, along with tylosin, tested as potential drug for FDA

approval to control tetracycline-resistant AFB disease. Lincomycin was effective in

controlling AFB, when applied to honeybee colonies as a dust in confectioners‟ sugar

(Feldlaufer et al., 2001). The FDA approval itself is still an in-progress project.

1.2.4.7 Chloramphenicol

Chloramphenicol (CAP) is a potent, broad-spectrum antibiotic and a potential

carcinogen that is banned from use in food producing animals, including honey bees,

in the European Union since 1994 (Commission Regulation (EC) 1430/94). The

consumption of CAP contaminated food may pose human health risks associated

with the development of a potentially life-threatening blood disorder, called aplastic

anaemia. In China in 1997-1998, hundreds of thousands of beehives were infected

by AFB and treated by the beekeepers with CAP or streptomycin to save their hives

and their industry (Ortelli et al., 2004). In these years, also in other sectors (poultry

and shrimp production) CAP was still applied in south-east Asian countries. In

January 2002, concern regarding „serious deficiencies of the Chinese residue control

system and problems related to the use of banned substances in the veterinary field‟,

lead to the European Union to issue a suspension of imports of all products of animal

origin from China (Anon., 2002b). The ban on import of honey from China was

released in July 2004 (Anon., 2004a). Meanwhile, a growing trend in irregularities in

trade practices with Chinese honey were noticed. Besides dumping on the

international market for prices below production costs, Chinese honey was also

shipped through other countries to disguise its origin and to avoid border inspections

(Anon., 2009a).


1.2.4.8 Nitrofurans

There are only few publications mentioning that nitrofurans are used in the

maintenance of bees for honey production. In Europe, nitrofurans are prohibited

substances for all food producing animals (Commission (EU) Regulation No

37/2010). Nitrofurans are rapidly metabolized and covalently bound with proteins or

peptides. Results of a simultaneous analysis of the metabolites of four nitrofuran

veterinary drugs, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone, in

honey have shown that furazolidone is the main nitrofuran antibiotic administered to

treat bacterial diseases of bees (Khong et al., 2004).

1.2.4.9 Nitroimidazoles

Dimetridazole (DMZ), metronidazole (MNZ), and ronidazole (RNZ) are classified in

Europe as prohibited substances for all food producing species (Commission

Regulation (EU) No 37/2010). Zhou et al. (2007) claim that in China in recent years,

5-nitroimidazoles have been commonly used to prevent and control Nosema apis in

hives. Chinese beekeepers consider it as a cheap alternative for fumagillin. The use

of MNZ, DMZ, and RNZ is now prohibited in food animals in China. Tinidazole (TNZ)

has never been authorized as a veterinary drug, and is also considered as a banned

substance in China. The most found nitroimidazole residue in honey is MNZ.

1.2.4.10 Fluoroquinolones

The majority of the clinical use of quinolones is in the form of fluoroquinolones. The

base chemical in quinolones is nalidixic acid. Despite the missing of scientific data

showing their efficacy, the application of fluoroquinolones in apiculture, especially in

Asia, as a prophylaxis of bee diseases is highly conceivable and has increased

during the last few years. This use was confirmed by the detection of residues in

honey from that area (Savoy Perroud, 2009). Residue testing in honey is showing

enrofloxacin (ciprofloxacin) and norfloxacin as the main fluoroquinolone antibiotics

administered.

1.2.4.11 Fumagillin

To prevent and control nosemosis, fumagillin was commonly used in beekeeping in

several parts of the world. Fumagillin, an antibiotic prepared from Aspergillus flavus,

was found to be effective by Katznelson and Jamieson in 1952. Treatment with the


antibiotic fumagillin inhibits the spores reproducing in the ventriculus but does not kill

the spores (Bailey, 1953; Webster, 1994). Fumidil B® (bicyclohexylammonium

fumagillin) (Mid-Continent Agrimarketing Inc., Overland Park, KS) is approved in the

US by FDA for use in beekeeping to prevent Nosema disease. In order to prevent

residues in honey, fumagillin should not be fed immediately before or during the

honey flow (Anon., 2010m).

Fumagillin was on the EU market since 1970. However, as MRLs could not be

recommended by the CVMP due to the inadequacy of data available to ensure

consumer safety, no marketing authorization could be maintained or issued for bees.

The CVMP acknowledged that fumagillin would be an essential substance for

veterinary medicine for bees (Anon., 2000). Since then and in a new regulatory

context considering MUMS data requirements to establish and having obtained free

scientific advice for MUMS, new work on toxicity studies is ongoing by the animal

health company that produces fumagillin (Anon., 2010b). In the meantime, the use of

fumagillin in the EU is not permitted. Nevertheless, Fumidil B® (CEVA Animal Health

Ltd., Chesham, United Kingdom) with fumagillin bicyclohexylamine salt as active

ingredient, is still in the UK an authorized product in syrup (20 mg g-1) for the control

of Nosema in honeybees (Anon., 2009b). Studies by Stanimirovic et al. (2007)

suggested that fumagillin has genotoxic (clastogenic) potential in mammals in vivo.

Other studies (Stevanovic et al., 2008) indicated that fumagillin is clastogenic and

cytotoxic to cultured human lymphocytes. Since no other antibiotics or

chemotherapeutics are registered for the treatment of nosemosis, prevention

procedures need to be applied. Some beekeepers experiment with more natural

substances as essential oils (Maistrello, 2008).

1.2.4.12 Other antibiotics and chemotherapeutics

Machova (1970) obtained good sensitivity of Paenibacillus larvae to bacitracin, a

polypeptide antibiotic. The isolates of Paenibacillus larvae tested by Okayama et al.

(1996) were most susceptible to penicillins, macrolides, and lincomycin.

Microsamicin among the macrolides, and ampicillin among the penicillins, appeared

to be the most effective agents. Ampicillin was also tested in beehives, where it

resulted in high residues in honey but only in very low levels in larvae, casting doubt

on its utility in disease control (Nakajima et al., 1997 and 1998). Kochansky and

Pettis (2005) reported later that all -lactams (penicillins and cephalosporins), while


active in vitro, are apparently not effective in the field. Kochansky et al. (2001)

screened alternative antibiotics against OTC-resistant Paenibacillus larvae.

Rifampicin, a bactericidal antibiotic drug of the rifamycin group, was by far the most

active antibiotic tested; monensin (an ionophore antibiotic), and the earlier described

erythromycin, tylosin, and lincomycin showed also to be active in vitro to resistant

strains of P. l. larvae. In a later study (Kochansky and Pettis, 2005), more

antimicrobials were tested. The ionophore antibiotics narasin, lasalocid, salinomycin,

laidlomycin, and maduramycin had a high in vitro activity but they were inactive in

the field. The lincosamide pirlimycin and the pleuromutiline antibiotic tiamulin showed

high activity in vitro but share the mode of action of tylosin, and therefore offer no

benefit. The effectiveness of the macrolide antibiotic tilmicosin against AFB in vitro

and in vivo was reported by Reynaldi et al. (2008).

The list of effective alternatives for oxytetracycline and tylosin to treat AFB is very

limited. It is essential that these veterinary drugs, in the countries where their use is

authorized, are utilized in a manner that will delay the onset of resistance, and that

other methods of dealing with AFB are explored (Kochansky and Pettis, 2005).

Despite some drugs seem to represent potential alternative treatments for AFB,

there are so far no signs of use of bacitracin, microsamicin, rifamycin, monensin,

pirlimycin, tiamulin, and tilmicosin in beekeeping.

1.2.5 Stability and disposition of antimicrobials in honeybee hives 1.2.5.1 Introduction

The stability of different antibiotics in honey at a temperature of 34°C (hive

temperature) was followed for 9 months by Landerkin and Katznelson (1957) to have

an idea how long antibiotic residues may remain in the hive and on the comb.

Streptomycin (the most stable compound tested), tetracycline, and chlortetracycline

showed some activity at the end of the test period, while erythromycin and

oxytetracycline were scarcely detectable after 2 months.


In the study of Martel et al. (2006), tetracycline was very stable in honey: the half-life

of tetracycline hydrochloride in honey stored at 20 and 35°C in dark was 242 and

121 days, respectively. In other studies tetracyclines were degrading fast in honey.

Half-life of OTC in incurred honey at 34°C was reported as 12 days stored at 34°C in


the laboratory (Argauer and Moats, 1991), and 2 to 4 days when remained

undisturbed in the cells of the comb within the active bee colony (Gilliam and

Argauer, 1981a). Münstedt (2009) stored in dark at room temperature honey fortified

with 500 µg kg-1 of oxytetracycline, tetracycline, and chlortetracycline, respectively.

After 7 months, the residues of oxytetracycline were below 10 µg kg-1, while only

epimers of tetracycline and chlortetracycline were found.

There are several studies published regarding the depletion of oxytetracycline in

beehives (Gilliam and Argauer, 1981a & b; Matsuka and Nakamura, 1990; Lodesani

et al., 1994). Also more recent publications (Anon., 2002c; Thompson et al., 2005;

Martel et al., 2006) show that, when used in beekeeping, important concentrations of

tetracyclines up to mg kg-1 could be found in the honey of the treated hives with a

slow depletion and degradation (a half-life for oxytetracycline residues of 9 to 44

days (Thompson et al., 2005) or 11 to 14 days (Anon., 2002c), and 65 days for

tetracycline hydrochloride in honey from supers (Martel et al., 2006)). In the study

performed at FERA (Anon., 2002c), application of Terramycin® (1 g of

oxytetracycline, single dose) in liquid form resulted in very high residue levels in

honey with residues of 3.7 mg kg-1 8 weeks after application. Thompson et al. (2005)

suggested a withdrawal period of up to 16 weeks required for colonies treated with

oxytetracycline in liquid sucrose, and up to 18 weeks is required for those colonies

treated in icing sugar, considering a reporting limit of 50 µg kg-1. Münstedt (2009)

treated hives with 200 mg of chlor- and oxytetracycline three times, with an interval

of 7 days. Nine weeks after the last dosing, the sum of chlortetracycline and epi-

chlortetracycline in the honey ranged from 311 to 512 µg kg-1. Only in one sample of

the honey from the five hives treated with oxytetracycline, residues were found (13

µg kg-1).

1.2.5.3 Streptomycin

Pang et al. (2004) found streptomycin to be stable stored for a period of more than 4

months at room temperature, without any occurrence of disintegration or metabolic

reaction. In a study of the Food and Environment Research Agency (FERA, Sand

Hutton, United Kingdom), no significant decline in concentration of streptomycin in

honey over 161 days at room temperature was observed (Anon., 2006b). In the

same study, the stability of lincomycin in honey over 28 days at room temperature

was proven.


The distribution of streptomycin was followed after dosage of 1 g per hive (single

dose in sucrose solution) to bee colonies. The highest mean concentration of

streptomycin found in honey was 124 mg kg-1, 7 days after dosing. The

concentration declined to 8.0 mg kg-1 at day 28, with a final concentration of 6.5 mg

kg-1 at day 332 (Anon., 2006b).


Sulfamethazine residues were found in Flemish honey samples at ppm level the year

after the use of sulfa drugs in the winter feed by the beekeepers to prevent

nosemosis (Reybroeck et al., 2004). Even after removal of all frames with stock of

winter feed or honey, it took two years before the concentration of sulfa residues

dropped below the Belgian action limit of 20 µg kg-1 (Reybroeck, unpublished data).

1.2.5.5 Tylosin

It has been recognized that the parent compound, tylosin A, degrades in acidic

media such as honey, to yield the antimicrobially active degradation product,

desmycosin (tylosin B) with a half-life of approximately 4 months at 34°C

(Kochansky, 2004). During storage of 16 weeks at ambient temperature,

approximately 20% of the tylosin A had degraded to desmycosin (Thompson et al.,

2007). In honey sampled from hives approximately 9 months after the last treatment

with tylosin, a relatively constant ratio of tylosin A to desmycosin (overall average of

1.2) was observed. In other incurred honey samples this ratio was in the range of 0.9

to 1.6 (Thompson et al., 2007). Desmycosin seemed to be quite stable in honey, the

sum of both tylosin and desmycosin decreased only slightly over 9 months

(Kochansky, 2004). It has been demonstrated that honey destined for human

consumption should be analysed for both tylosin A and desmycosin, rather than for

the parent antibiotic alone (Kochansky 2004; Thompson et al., 2007).

Tylosin was applied by Feldlaufer et al. (2004) to honey bee colonies in a

confectioner‟s sugar dust (200 mg of active compound) three times, with an interval

of 7 days. Tylosin concentrations in surplus honey from treated colonies declined

from an average of 1.31 mg kg-1 during the treatment period, to 160 µg kg-1 three

weeks after the last treatment. Elzen et al. (2002) demonstrated that the application

of a grease patty versus dust application of tylosin, resulted in increased residues of

tylosin in the hive products wax and honey. Different results were obtained for pollen


patties by Thompson et al. (2007). In their experiments, pollen patty treatments

contributed to a substantially lower tylosin residue production in honey, in

comparison to sugar dusting treatments.

In another study, tylosin was dosed to hives (1 g per hive, single dose) and the

distribution of both tylosin A and desmycosin in the honey was followed. The highest

mean concentration of tylosin A found in honey was 17 mg kg-1, 3 days after dosing.

The concentration declined to 6.1 mg kg-1 on day 28, with a final concentration of

930 µg kg-1 at day 238. The concentration of desmycosin remained relatively

consistent at 2.3 mg kg-1 at day 3, 2.6 mg kg-1 on day 28, and 1.0 mg kg-1 on day

238 (Adams et al., 2007; Anon., 2006b).

1.2.5.6 Erythromycin

An erythromycin-fortified cake was fed to bees by Gunes et al. (2008). In this test

hive, the erythromycin residue level in honey was approximately 28 µg kg-1, 3

months after dosing.

1.2.5.7 Lincomycin

Bee colonies treated with 1.2 g lincomycin hydrochloride per hive resulted in a

highest mean concentration of lincomycin in honey of 24 mg kg-1 3 days after

treatment, a mean of 3.5 mg kg-1 after 129 days. Lincomycin was persistent in the

hive and detected in all over wintered samples of honey, 290 days after dosing

(Adams et al., 2009).

1.2.5.8 Chloramphenicol

CAP and CAP-Glu are stable in solvent and in fortified matrix (milk powder, shrimp,

kidney) at room temperature tested for at least 20 weeks (Ashwin et al., 2005). CAP

did not form significant concentrations of glucosides in honey. Consequently, free

CAP is a suitable marker compound for determination and quantification of CAP

residues in honey (Adams et al., 2008).

Adams et al. (2008) followed the distribution of CAP after dosage of 1 g per hive

(single dose in sucrose solution) to bee colonies. The highest mean concentration of

CAP found in honey was 26 mg kg-1 at 7 days after dosing. This concentration

declined to 2.0 mg kg-1 at day 28, with a final concentration of 1.0 mg kg-1 on day

332 („over wintered‟ sample). Even when the shook swarm procedure was used, in


an attempt to „clean‟ the bee colonies, CAP was still detected in honey 332 days

after dosage (100 µg kg-1). The dosage also resulted in CAP residues in beeswax

and royal jelly (highest mean concentration of 6.8 mg kg-1 and 3.0 mg kg-1 at 7 days,

respectively) (Adams et al., 2008).

1.2.5.9 Nitrofurans

Furazolidone in honey, stored at room temperature, showed a rapid decline (> 90%)

over 14 days, while its metabolite AOZ remained stable. Therefore AOZ is

considered to be the most suitable marker compound to detect the use of

furazolidone in apiculture (Anon., 2006b).

At the Food and Environment Research Agency, the distribution of furazolidone after

dosage (1 g of active compound per hive) in beehives was followed (Anon., 2006b).

On one hand, this was resulting in a dilution of the drug in the honey flow; on the

other hand, furazolidone was degrading to AOZ. In super honey, the highest mean

residue concentrations were measured seven days after dosing (2.5 mg kg-1 of

furazolidone and 5.8 mg kg-1 T-AOZ (sum of the concentrations of AOZ and parent

furazolidone)). The results also confirmed that furazolidone and T-AOZ can still be

detected in honey 332 days after dosing with furazolidone (mean concentration: 440

µg kg-1 of furazolidone, and 530 µg kg-1 of T-AOZ).

1.2.5.10 Fluoroquinolones

Ciprofloxacin was administered to bee colonies. The highest concentration of

ciprofloxacin was >10 mg kg-1 3 days after dosing. The average concentrations of

ciprofloxacin in honey at 18 weeks were between 622-1,370 µg kg-1 (Fussell et al.,

2010).

1.2.5.11 Other antibiotics

Mirosamicin, mixed in a pollen-substitute paste, was administered to honeybee

colonies continuously during one week, at a dosage of 200 mg/hive/week. A

relatively low distribution of mirosamicin in honey was observed (Nakajima et al.,

1998). One day dosing of mirosamicin in sucrose syrup resulted in a very high and

long lasting residue in honey (Nakajima et al., 1998).

Comparable results were obtained for the disposition of ampicillin: a single dose of

30 mg ampicillin per hive administered in syrup resulted in high drug residue levels in


honey and residual residues beyond the detection limit for more than 14 days. In the

hives given ampicillin (30 mg per hive) in pollen substitute paste, relatively low honey

residues were found (Nakajima et al., 1997).

1.2.5.12 Conclusions and reflections about the disposition of antimicrobials in hives

In general, the highest residue concentrations in honey are found within one week

after dosage. Afterwards, residue levels in honey from supers diminish by a dilution

effect of the honey flow and for some compounds (oxytetracycline, tylosin,

furazolidone,...) by a degradation of the parent drug. Residues of the marker

compounds for most drugs are still detected in honey, harvested the year after the

drug application, even when the shook swarm method after the dosing was applied.

This is mainly caused by the fact that antimicrobials are not actively metabolized by

the honeybees. Consequently, all food needs to be consumed by the bees, in order

to get an elimination of the residues in the hive. In this view, applications in syrup are

least desirable, since the syrup is stored directly by the bees. Any sort of application

during nectar flow, when supply honey is being stored, also poses a residue risk. In

view of the zero-tolerance for residues of antimicrobials in honey in many countries,

very long withdrawal periods together with other biotechnical measures need to be

considered.

Assessment of residues in honey is more complex than in mammalian or avian

tissues. In honey matrix, there is no time dependent depletion/elimination of residues

as a result of pharmacokinetics. Residues, once present in honey, largely remain

there. Apart from the possible chemical decay of a substance in honey matrix over

time, the main variation responsible for the level of residues at harvest time is the

honey yield (dilution effect), which in large parts depends on the production site

(geographical area) and weather conditions at flowering time. Therefore, the only

feasible withdrawal period in honey is a „zero‟ withdrawal period, i.e. that there is no

need to specify an interval between last treatment and harvest of honey

(Anonymous, 2010b).

According the new MRL regulation, if the metabolism and depletion of the substance

cannot be assessed, the scientific risk assessment may take into account monitoring

data or exposure data (Anonymous, 2010b).


1.2.6 Occurrence of antimicrobial residues in honey on the Belgian and European market Honey is generally considered as a natural healthy product. However, in the early

years 2000, the frequency of residues of anti-infectious agents in foreign table and

industrial honey present on the Belgian market was remarkably high (Table 5). Many

honey samples contained even different residues at the same time and residues

were also found in organic honey despite an official bio-control label.

Chloramphenicol was also found in Chinese royal jelly (Reybroeck, 2003).

Streptomycin and chloramphenicol were often detected in honey, due to the blending

with honey from Chinese origin.

Table 5. Results of the detection of streptomycins, tetracyclines, sulfonamides, -lactams, and chloramphenicol in imported table and industrial honey on the Belgian market in the period 2000-2002, except for CAP that was only determined in 2002 (Reybroeck, 2003).

Group n n Positive % Positive streptomycins 108 51 47 tetracyclines 98 29 30 sulfonamides 98 31 32 -Lactams 18 0 0

chloramphenicol 85 40 47

Table 6. Trends in antimicrobial residues in commercial table honey on the European market from 2002 to 2009 (Reybroeck et al., 2006; Reybroeck and Ooghe, 2010a).

Country 2002 2005 2009

n n pos % pos

n n pos % pos

%a pos

n n pos % pos

%a pos

Belgium 20 13 65 21 4 19 14 21 2 10 5 Italy 20 3 15 19 6 32 11 15 2 13 7 Portugal 20 10 50 20 7 35 35 20 4 20 20 Spain 20 7 35 20 0 0 0 15 3 20 7 Switzerland ---b ---b ---b ---b ---b ---b ---b 15 1 7 7 UK ---b ---b ---b 20 3 15 0 ---b ---b ---b ---b Notes: %a pos, based on the results for streptomycin, sulfonamides, tetracyclines, and chloramphenicol only (groups looked for in 2002); ---b: no analysis.


Monitoring of residues of antimicrobials in honey was set up and some countries

introduced action limits to allow honey with limited contamination on the market. The

high occurrence of antibiotic residues took the interest of consumers‟ organizations.

In a first study based on a sampling in 2001 of commercial table honeys on the

Belgian market, 25 upon 32 samples (78%) contained residues of streptomycin,

sulfonamides, and/or tetracyclines. The results of the samplings in 2002, 2005, and

2009 of commercial table honey are presented in Table 6.

More detailed data of antimicrobial residues in commercial table honey on the

European market from 2001 to 2009, with sorting of the residues per family, are

given in Table 7 (Reybroeck et al., 2006; Reybroeck and Ooghe, 2010a).

Table 7. Trends in antimicrobial residues in commercial table honey on the European market from 2001 to 2009, classified per group of compounds (Reybroeck et al., 2006; Reybroeck and Ooghe, 2010a). Country Year n n Pos STR SU TE CAP MA&L QU Belgium 2001 32 25 21 9 9 nt nt nt

2002 20 13 6 1 2 9 nt nt 2005 21 4 3 2 0 0 1 nt 2009 21 2 0 1 0 0 0 1

Italy 2002 20 3 2 0 1 0 nt nt 2005 19 6 0 2 0 0 4 nt 2009 15 2 0 1 0 0 1 0

Portugal 2002 20 10 3 5 3 4 nt nt 2005 20 7 0 7 2 0 0 nt 2009 20 4 0 4 0 0 0 0

Spain 2002 20 7 3 5 1 4 nt nt 2005 20 0 0 0 0 0 0 nt 2009 15 3 1 0 0 0 1 1

Switzerland 2009 15 1 1 0 0 0 0 0 UK 2005 20 3 0 0 0 0 3 nt Notes: nt, not tested; STR, streptomycin; SU, sulfonamides; TE, tetracyclines; CAP, chloramphenicol; MA&L, macrolides and lincosamides; QU, fluoroquinolones.

In the period 2002-2009, the percentage of positive honey samples dropped in all

countries participating in the study. The concentrations found in the most recent

study were in general lower than in the previous studies, and there were no longer

samples containing different residues at the same time. In 2009, the samples were

also checked on the presence of nitrofuran metabolites (SEM, AMOZ, AOZ, and

AHD), and all tested negative (Reybroeck and Ooghe, 2010a).


Since the results for the honeys, bought in Belgium in 2001, date from the period

before Belgian action limits for antimicrobial residues in honey were fixed (Anon.,

2001), all 25 positive samples need to be considered as non-compliant. Eight out of

the 20 honeys sampled in Belgium in 2002 were also non-compliant. They contained

streptomycin (23 and 33 µg kg-1), sulfathiazole (390 µg kg-1), tetracycline (40 µg

kg-1), chloramphenicol (0.17, 0.27, and 1.90 µg kg-1), or the combination of

tetracycline (40 µg kg-1) and chloramphenicol (0.19 µg kg-1), respectively. Of the

honeys bought in Belgium in 2005 and 2009, there was, in both cases, one sample

non-compliant, containing tylosin B (4 µg kg-1), and the combination of ciprofloxacin

(5 µg kg-1) and norfloxacin (3 µg kg-1), respectively.

In locally (Flanders) produced honey, the frequency of residues of anti-infectious

agents in honey was low in the period 2000-2003 (Table 8). For the few

contaminations of Flemish honey with streptomycin and tetracyclines, in some cases

the responsible beekeeper admitted the addition of foreign honey to his own

production (Reybroeck, 2003). High concentrations of sulfa drugs in some local

honey samples were caused by the addition of sulfa drugs to the winter feed. The

source of contamination of some honey samples with low concentrations of

sulfonamides (Table 9) could not directly be indicated. This will be further discussed

in Chapter 4. From 2004 on, antimicrobial residues were no longer found in Flemish

honey.

Table 8. Trends in antimicrobial residues in locally produced Flemish honey from 2000 to 2010 (Reybroeck et al., 2008 and unpublished data).

Substance Positive honey samples / number of samples

2000- 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

streptomycin 4/248 0/90 --- 0/10 0/20 0/15 0/15 0/25 0/20 0/20 tetracyclines 2/72 0/90 --- 0/10 0/20 0/15 0/15 0/25 0/20 0/20

sulfonamides 3/72 3/91 12/203 0/20 0/20 0/15 0/15 0/35 0/30 0/30

chloramphenicol 0/93 0/226 --- 0/10 0/20 0/15 0/15 0/25 0/20 0/20 -lactam 0/50 --- --- --- --- --- --- --- --- ---

macrolides & lincosamides --- --- --- --- --- 0/15 0/15 0/25 0/20 0/20

fluoroquinolones --- --- --- --- --- 0/15 0/15 0/25 0/20 0/20 Note: ---: no analysis.


Table 9. Concentrations of sulfonamides in Flemish honey in 2002 and 2003 (Reybroeck et al., 2004).

Year Sulfamethazine (µg kg-1) or sulfathiazolea (µg kg-1) 2002 1,564 6,184 b

2003 9 11 15 15 + 9a 32 116 151 458 + 1,229a

588 713 3,075 13,194

Notes: a sulfathiazole; b sulfamerazine and sulfadiazine (not quantified).

At European level, a lot of information about antimicrobial residues in honey could be

gathered from the Rapid Alert System for Food and Feed (RASFF) notifications

(Anon., 2010k). The notifications for the period 2002-2010 are gathered in Table 10.

Alert notifications are sent when a food presenting a serious health risk is on the

market and when rapid action is required. Border rejections concern food

consignments that have been tested and rejected at the external borders of the EU

when health risk has been found. Information notifications are used when a risk has

been identified about food placed on the market but the other members do not have

to take action.

In the early 2000, most notifications concerned chloramphenicol, streptomycin, and

sulfonamides. Along the years, antimicrobial residues belonging to other groups of

antimicrobials showed up. From 2007 on, there were no longer RASFF notifications

communicated regarding chloramphenicol in honey. This was the result of a policy of

intensive monitoring, border inspections, autocontrol by the sector, in combination

with rejection of contaminated products. In most cases the concentration of

antimicrobials in the honey was rather limited and far below the MRLs set for

authorized compounds in other food matrices like meat or milk.

In the category of residues of veterinary medicinal products, it is obvious that

nitrofuran metabolites are still the most-notified hazard in the RASFF in 2008, the

large majority of findings made in crustaceans. In the second place come the

problems reported for residues in honey (Anon., 2010k).


Table 10. RASFF notifications for residues of antimicrobials in honey in the period 2002 - November 20, 2010 (Anon., 2010k). Year Type n Residues 2002 alert 11 chloramphenicol (9x, 0.9-38.7 µg kg-1), streptomycin (5x, 2.6-

500 µg kg-1) information 27 chloramphenicol (17x, 0.2-4,430 µg kg-1), streptomycin (7x, 20-

1,300 µg kg-1), sulfadiazine (3x, 5-11,000 µg kg-1), sulfathiazole (2x, 28.8-67 µg kg-1), tetracycline (2x, 50-50 µg kg-1), oxytetracycline (18,000 µg kg-1), dihydrostreptomycin (30 µg kg-1), sulfamethazine (28.3 µg kg-1), sulfamethoxazole (4.4 µg kg-1), trimethoprim (5 µg kg-1)

2003 alert 5 chloramphenicol (5x, 0.5-1.6 µg kg-1), streptomycin (1x, 30 µg kg-1)

information 32 sulfathiazole (13x, 3.5-130.3 µg kg-1), streptomycin (10x, 0.3-220 µg kg-1), chloramphenicol (6x, 0.3-3.4 µg kg-1), sulfamerazine (5x, 9.9-63.7 µg kg-1), sulfamethazine (4x, 25.4-109 µg kg-1), AOZ (2x, 0.1-0.9 µg kg-1), tetracycline (2x, 24-195 µg kg-1), doxycycline (26.5 µg kg-1), sulfamethoxine (14.6 µg kg-1), sulfadimethoxine (2.5 µg kg-1),

2004 alert 9 chloramphenicol (2x, 0.5-20 µg kg-1), sulfadimidine (2x, 9.3-140 µg kg-1), sulfathiazole (11.4 µg kg-1), sulfadiazine (5.7 µg kg-1), sulfamerazine (100 µg kg-1), sulfonamide (not specified, a), streptomycin (105b µg kg-1), AOZ (1.4 µg kg-1), AMOZ (1.4 µg kg-1)

information 24 tylosin (8x, 3.7-30 µg kg-1), streptomycin (4x, 72-190 µg kg-1), chloramphenicol (3x,>0.01-0.6 µg kg-1), AOZ (3x, 0.3-2.1 µg kg-1), sulfathiazole (3x, 16.6-222 µg kg-1), sulfadimidine (3x, 23.7-28.3 µg kg-1), sulfamethoxazole (2x, 9-60 µg kg-1)

2005 alert 7 chloramphenicol (3x, 0.3-2.1 µg kg-1), tylosin (3x, 0.1-2.5 µg kg-1), oxytetracycline (1x, 21.5b µg kg-1), streptomycin (14 µg kg-1), sulfonamide (not specified, 10 µg kg-1)

information 32 sulfonamide (not specified, 9x, 32-1,602 µg kg-1), streptomycin (7x,11-46 µg kg-1), chloramphenicol (6x, 0.32-1.5 µg kg-1), sulfa-methazine (5x, 20-170 µg kg-1), oxytetracycline (3x, 16-71), SEM (2x, 1.1-2.3 µg kg-1), AOZ (2x, <2-3.4 µg kg-1), sulfathiazole (2x, 46-182 µg kg-1), antibiotic (not specified, 2x), tylosin (36 µg kg-1), tetracycline (1x, 13b µg kg-1), sulfa-methoxazole (5 µg kg-1)

2006 alert 3 sulfadimidine (2x, 17-50 µg kg-1), streptomycin (47.7 µg kg-1) information 10 sulfathiazole (3x, 7-157 µg kg-1), chloramphenicol (2x, 0.82-1.8

µg kg-1), AMOZ (2.5 µg kg-1), sulfadimidine (41 µg kg-1), tetracycline (24.6 µg kg-1), streptomycin (13 µg kg-1), trimethoprim (7 µg kg-1), tylosin (2.0 µg kg-1),

information 24 tylosin (8x, 3.7-30 µg kg-1), streptomycin (4x, 72-190 µg kg-1), chloramphenicol (3x,>0.01-0.6 µg kg-1), AOZ (3x, 0.3-2.1 µg kg-1), sulfathiazole (3x, 16.6-222 µg kg-1), sulfadimidine (3x, 23.7-28.3 µg kg-1), sulfamethoxazole (2x, 9-60 µg kg-1)


Year Type n Residues 2007 alert 5 sulfathiazole (3x, 1.1-39 µg kg-1), sulfadimidine (2x, 2.1-2.4 µg

kg-1), tylosin (30.1 µg kg-1), sulfadimethoxine (1.3 µg kg-1) information 20 sulfamethoxazole (7x, a-1,316 µg kg-1), trimethoprim (7x, a-239

µg kg-1), tetracycline (5x, a-33 µg kg-1), ciprofloxacin (5x, a-6.4 µg kg-1), tylosin (4x, 0.4-2.1 µg kg-1), sulfadiazine (4x, a), sulfadimidine (2x, 27-1,500b µg kg-1), sulfathiazole (119 µg kg-1), oxytetracycline (31 µg kg-1), lincomycin (10 µg kg-1), streptomycin (179 µg kg-1), SEM (1.9 µg kg-1), AOZ (1.2 µg kg-1)

2008 alert 2 sulfathiazole (1x, 252b µg kg-1), SEM (1.6 µg kg-1) border rejection

15 erythromycin (9x, 0.1-1.7 µg kg-1), oxytetracycline (4x, 12-67,600 µg kg-1), streptomycin (2x, 3-16.9 µg kg-1), ciprofloxacin (2x, 5.4-6.1 µg kg-1)

information 11 sulfathiazole (3x, 5.5-44 µg kg-1), sulfadimidine (2x, 19-301 µg kg-1), sulfamethazine (2x, 22->40 µg kg-1), tetracycline (2x, 16-17.8 µg kg-1), oxytetracycline (14.8 µg kg-1), sulfadiazine ( 9.2 µg kg-1)

2009 alert 3 SEM (3x, 4.2-11 µg kg-1) border rejection

1 sulfadimidine (42 µg kg-1)

information 8 streptomycin (2x,10-22 µg kg-1), tetracycline (2x, 12-12.3 µg kg-1), oxytetracycline (2x, 13-15 µg kg-1), tylosin (4.3 µg kg-1), lincomycin (4.1 µg kg-1)

2010 (up to Nov. 20)

alert 3 sulfadimethoxine (5b µg kg-1), enrofloxacin (49 µg kg-1), metronida-zole (a)

border rejection

2 lincomycin (2x, 2.4-2.4 µg kg-1), erythromycin (2 µg kg-1)

information 4 streptomycin (2x, 43-49 µg kg-1), lincomycin (1x, 2.3b µg kg-1), sulfonamide (not specified, 18 µg kg-1)

Notes: a concentration not specified; b mean. Additional information about residues of antimicrobials in honey is available in year

reports (e.g. Anon. 2010n) and publications (Heering et al., 1998; Kaufmann et al.,

2002; van Bruijnsvoort et al., 2004; Bogdanov, 2006; Baggio et al., 2009). Regarding

the situation outside Europe, websites with information about recalls, market

withdrawals, and safety alerts can be consulted (e.g. FDA (Anon., 2010o) and the

Canadian Food Inspection Agency (Anon., 2010p)).

1.3 Antimicrobials in Milk 53

1.3 ANTIMICROBIALS IN MILK 1.3.1 Introduction Milk is a translucent white liquid produced by the mammary glands of mammals. Its

role is to nourish and provide immunological protection for the mammalian young.

The nutritional value of milk is high. Milk contains significant amounts of saturated

fat, protein, lactose, and calcium, as well as vitamin C. The composition of milk is

affected by animal species and breed variations (Table 11).

Table 11. Average composition of milks of various mammals, per 100 grams (Johnson, 1974; McCance and Widdowson, 2002). Constituents Unit Cow Goat Ewe Mare Buffalo Water g 86.61 87.00 80.71 89.04 81.1 Fat g 4.14 4.25 7.90 1.59 8.0 Protein g 3.58 3.52 5.23 2.69 4.5 Lactose g 4.96 4.27 4.81 6.14 4.9 Ash g 0.71 0.86 0.90 0.51 nd Nonfat solids g 9.25 8.75 11.39 9.37 nd Energy kJ 275 253 396 nd 463 Cholesterol mg 14 10 11 nd 8 Calcium IU 120 100 170 nd 195 Saturated fatty acids g 2.4 2.3 3.8 nd 4.2 Monounsaturated fatty acids g 1.1 0.8 1.5 nd 1.7 Polyunsaturated fatty acids g 0.1 0.1 0.3 nd 0.2 Note: nd, no data. Milk is a colloid suspension of casein micelles, globular proteins, and lipoprotein

particles within a water-based fluid. The fat globules are the largest particles in milk.

Each fat globule is surrounded by a membrane, consisting of phospholipids and

proteins. Milk fat is a mixture of various fatty acid esters, mostly triglycerides. Fatty

acids containing double bonds in the acyl chain are referred to as unsaturated fatty

acids.

The most important fatty acids of milk include the saturated acids: butyric (4:0),

caproic (6:0), caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), palmitic

(16:0), and stearic acid (18:0); and the unsaturated palmitoleic (16:1n-7), oleic

(18:1n-9), linoleic (18:2n-6), and α-linolenic (18:3n-3) acid (Table 12). Myristic,

palmitic, stearic, and oleic acids predominate (Mantere-Alhonen, 1995).

http://en.wikipedia.org/wiki/Cow

http://en.wikipedia.org/wiki/Goat

http://en.wikipedia.org/wiki/Sheep

http://en.wikipedia.org/wiki/Water_Buffalo

http://en.wikipedia.org/wiki/Phospholipid


(B)

(E)

(D)

Table 12. Typical fatty acid composition of milk originating from different species (Calder and Burdge, 2004).

Source Fatty acid (g per 100 g total fatty acids) ≤12:0 14:0 16:0 18:0 16:1n-7 18:1n-9 18:2n-6 18:3n-3

cows‟ milk 13 12 26 11 3 28 2 1 goats‟ milk 21 11 27 10 2 26 2 1 ewes‟ milk 24 12 25 9 3 20 2 1

The rumen bacteria play a central role in the synthesis of triglycerides, being able to

alter the composition of the fatty acids in the fat derived from the feed. The

proportions of different fatty acids can be considerably affected by the nature of the

feed (Calder and Burdge, 2004). The milk lipids always include a small amount of

free fatty acids, secreted from the mammary gland. The content of free fatty acids

increases when milk is stored, due to lipolysis, caused either by lipases in milk,

originating from the udder, or by microbial enzymes hydrolysing glycerides (Mantere-

Alhonen, 1995).

Figure 9. The casein micelle structure. A casein micelle consists of a complex of submicelles (Heyndrickx et al., 2010).

The largest structures in the fluid portion of milk are casein protein micelles:

aggregates of protein molecules, bonded by calcium phosphate (Figure 9). Other

proteins present in milk, like lactoglobulin, are more water-soluble than the caseins

and do not form larger structures. These proteins are called whey proteins since they

http://en.wikipedia.org/wiki/Casein

http://en.wikipedia.org/wiki/Micelle

http://en.wikipedia.org/wiki/Calcium_phosphate


remain suspended in the whey, while the caseins coagulate into curds.

Approximately 80% of the milk protein is casein, the remaining 20% is whey protein.

Lactoferrin and transferrin are the two proteins in milk which bind iron. Due to their

ability to bind iron they are antibacterial proteins. Milk also contains enzymes

originating either from the mammary gland contaminating flora in the udder or from

production after milking. The most important ones are peroxidase, phosphatase,

catalase, lipase, protease, and lysozyme (Mantere-Alhonen, 1995). Lipase releases

fatty acids and causes a rancid taste to develop (Figure 8).

Figure 8. Enzymatic reaction of a lipase catalysing hydrolysis of a triacylglycerol substrate. The carbon atoms in the glycerol core are annotated with numbers (Heyndrickx et al., 2010).

Milk production is an important sector within the agricultural business. In Belgium, in

2009, there were 12,056 milk producers with a total herd of 982,933 cows (503,796

dairy cows and 479,137 suckler cows). In 2009, a total of 100 million liter milk was

delivered by 9,963 milk producers to dairies. The average fat and protein content of

the delivered milk was 42.15 and 34.49 g l-1, respectively (Anon., 2010c). In Europe

in 2009, there were 23,665,000 dairy cows, good for the production of 134,799,000

tonnes of milk.

1.3.2 The use of veterinary drugs in dairy cattle The main reasons for the use of anti-infectious agents in dairy cattle are the control

of bovine mastitis and dry cow therapy. In economic terms, mastitis, a microbial

infection of the mammary gland, can generally be considered as the most serious

disease of dairy cattle (Taponen and Myllys, 1995). The economic losses are a result

of the lowered milk production, the decreased fat content, the poor-quality milk

http://en.wikipedia.org/wiki/Whey


(increased somatic cell count, increased quantity of free fatty acids (short-chained

fatty acids (C4-C12)), increased amount of whey proteins), the necessary culling of

infected cows, and the added expense of drugs and veterinary bills. Cows with

clinical mastitis are, according to normal practice, treated with intra-mammary

preparations. Dry cow therapy, the intra-mammary infusion of antibiotics immediately

after the last milking of lactation, is considered as an integral part of mastitis control

and aims eliminating the existing intra-mammary infections and preventing new

infections (Neave et al., 1966). Dry cow therapy has following advantages over

lactation therapy: a higher cure rate, the drug is not milked out, and in addition, the

risk of contaminating milk with drug residue is reduced, and there are no economic

losses due to the discard of antibiotic containing milk (Sandholm and Pyörälä, 1995).

However, some authors claim that the suggested superiority of systematic

administration at drying off, compared with conventional intra-mammary treatment,

has not been proven in practice (Jánosi and Huszenicza, 2001). Dry cow antibiotic

preparations require good activity against Staphylococcus aureus, including -

lactamase producing strains. Intra-mammary injectors, containing narrow spectrum

penicillins (penicillin, cloxacillin, nafcillin), cephalosporins, and spiramycin are

therefore widely used (Jánosi and Huszenicza, 2001).

In a Swiss study on the antibiotics used in lactating cows, in 2003-2004, 74.6% of

the treatment consisted of the application of intra-mammary infusions (mastitis

treatment during lactation (30.4%) and dry cow therapy (44.2%)). The other

treatments with antibiotics were for calving (10%), the digestive tract (5.5%), the

teats (1.5%), the lungs (1.3%), the locomotive system (1.3%), and others (3.1%)

(Schaeren, 2006).

1.3.3 Pharmacokinetics of veterinary drugs The pharmacokinetics of antimicrobials in the lactating cow are mainly related to the

route of administration (MacDiarmid, 1983), the dose and the number of treatments,

certain physicochemical properties of the excipient, the milk production, and the

condition of the udder (Sandholm, 1995). The percentage of the dose excreted as

residues in the milk, after intramuscular administration, is only 0.001% for

benzylpenicillin (penicillin G) or cloxacillin, 0.02% for neomycin, 0.07% for

(oxy)tetracycline, 0.08% for ampicillin, 1.4% for lincomycin, 2.6% for tylosin, and


3.8% for erythromycin (Sandholm, 1995, Honkanen-Buzalski and Reybroeck, 1997).

Also oral, subcutaneous, intravenous, or intra-uterine administration of penicillin is

resulting in low penicillin concentrations in the milk (Cannon et al., 1962; Dubreuil et

al., 2001).

Figure 10. Compartmental modelling. Blood is the central compartment which delivers the drugs to tissues, metabolizing and excretory organisms (Sandholm, 1995).

Table 13. Theoretical calculation of the dilution of the residues during transport of the milk from the farm to the dairy, after intra-mammary (tube of 0.8 g of benzylpenicilline, assumption that 50% of the benzylpenicillin from the tube was excreted in milk) and intramuscular administration (syringe of 0.8 g of benzylpenicillin, assumption that 0.001% of the benzylpenicillin from the injection was excreted in milk), respectively. Calculation for a farm of 20 lactating cows. Detection capability of the residue test for benzylpenicillin is 2 µg kg-1 (e.g. Delvotest SP-NT, Copan Milk Test). Milk transport Volume

(dilution) Intra-mammary Intramuscular

content in milk (µg kg-1)

residue test

content in milk (µg kg-1)

residue test

treated cow 25 l per day 16,000 + 0.32 - farm silo 500 l per day 800 + 0.016 - tanker 15,000 l 26.7 + 5.3 x 10-4 - dairy silo 200,000 l 2.0 + 4.0 x 10-5 -

Intra-mammary application is resulting in higher percentages of residues in the milk,

especially the first milking after treatment (Hovmand et al., 1954). Hargrove et al.


(1950) found that 26 to 49% of penicillin infused into the udder, and 39 to 58% of

infused streptomycin was excreted via the milk after treatment. The penicillin

excretion of the first milking following treatment was 37.4% of the quantity that was

administered intra-mammary (Hovmand et al., 1954). So large volumes of milk, like

a tanker load, can only get contaminated above MRL by intra-mammary use of

veterinary drugs (Table 13).

1.3.4 Veterinary drugs registered in Belgium for use in milk producing cows In total, 82 different veterinary drugs (brand/trade names) with an anti-infectious

agent as active substance are registered in Belgium for use in milk producing cows.

The complete list is given in Annex A of this Chapter. A classification of these drugs

on the type of the pharmacologically active substance is shown in Table 14.

Table 14. Classification per pharmacotherapeutic group of the veterinary drugs with an anti-infectious agent as active substance for use in milk producing cows in Belgium. Status on August 5, 2010 (Anon., 2010j).

Drug family n brands

Drug family (-ies) n brands

penicillins 21 ansamycins 1 cephalosporins 17 polymyxins 2

tetracyclines 12 sulfonamides + diaminopyrimidine derivatives

4

aminoglycosides 2 penicillins + aminoglycosides 5 macrolides 3 cephalosporins + aminoglycosides 1

lincosamides 1 penicillins + -lactamase inhibitors 1 quinolones 10 aminoglycosides + lincosamides 2

Forty-five of the antimicrobial drugs or 54.9% of the brands contain a -lactam

antibiotic. Tetracyclines and quinolones are present in 12 (14.6%) and 10 (12.2%)

brands, respectively.

As shown in Table 15, most veterinary drugs are registered for intramuscular use,

followed by intra-mammary and intravenous route of administration.


Table 15. Classification on the route of administration of the veterinary drugs with an anti-infectious agent as active substance for use in milk producing cows in Belgium. Status on August 5th, 2010 (Anon., 2010j).

Administration n Antimicrobial family

PE CE TE AG MA LI QU AN PO SU +DI

PE +AG

CE +AG

PE +IN

AG +LI

intra-mammary 22 7 8 1 1 1 3 1 1 intramuscular 44 13 7 5 2 3 5 1 4 2 1 1 intravenous 16 1 1 1 2 7 4 subcutaneous 20 4 3 1 1 1 6 2 1 1 intra-uterine 7 1 1 4 1 cutaneous 3 3 per os 1 1 peritoneal 1 1 Total 114 27 20 13 5 6 1 18 1 2 10 6 1 1 3

Notes: n, number of brands; PE, penicillins; CE, cephalosporins; TE, tetracyclines; AG, aminoglycosides; MA, macrolides; LI, lincosamides; QU, quinolones; AN, ansamycins; PO, polymyxins; SU, sulfonamides; DI, diaminopyrimidine derivatives; IN, -lactamase inhibitors.

In Belgium, the number of pharmacologically active substances that can be applied

in milk producing cows by the use of a registered veterinary drug is limited to 34. A

summary of these substances is given in Table 16.

Table 16. Classification on the active compound of the in Belgium registered veterinary drugs with an anti-infectious agent for administration to milk producing cows. Status on August 5, 2010 (Anon., 2010j).

Drug family Active substance n Drug family Active substance n penicillins amoxicillin 7 tetracyclines chlortetracycline 6

ampicillin 4 oxytetracycline 6 benzylpenicillin 9 aminoglycosides dihydrostreptomycin 3

cloxacillin 7 gentamicin 1 nafcillin 2 kanamycin 1

penethamate 2 neomycin 3 cephalosporins cefalexin 4 spectinomycin 2

cefalonium 1 quinolones danofloxacin 2 cefazolin 2 enrofloxacin 6

cefoperazone 1 marbofloxacin 2 cefquinome 4 ansamycins rifaximin 1

ceftiofur 5 polymyxins colistin 2 cephapirin 1 sulfonamides sulfadiazine 1

macrolides spiramycin 1 sulfadimethoxine 1 tylosin 2 sulfadoxine 2

lincosamides xx

lincomycin 2 -lactamase inhibitors clavulanic acid 1 pirlimycin 1 diaminopyrimidine derivatives trimethoprim 4


It concerns 6 penicillins (amoxicillin, ampicillin, benzylpenicillin, cloxacillin, nafcillin,

and penethamate), 7 cephalosporins (cefalexin, cefalonium, cefazolin, cefoperazone,

cefquinome, ceftiofur, and cephapirin), 2 macrolides (spiramycin and tylosin), 2

lincosamides (lincomycin and pirlimycin), 2 tetracyclines (chlor- and oxytetracycline),

5 aminoglycosides (dihydrostreptomycin, gentamicin, kanamycin, neomycin, and

spectinomycin), 3 quinolones (dano-, enro-, and marbofloxacin), 1 ansamycin

(rifaximin), 1 polymyxin (colistin), 3 sulfonamides (sulfadiazine, sulfadimethoxine,

and sulfadoxine), 1 -lactamase inhibitor (clavulanic acid), and 1 diaminopyrimidine

derivative (trimethoprim) (Anon., 2010j).

Validation studies and a residue control plan for residues of anti-infectious agents in

milk should mainly focus on these compounds and on prohibited substances for all

food producing animals (Regulation (EC) No 470/2009; Commission Regulation (EU)

No 37/2010). However, illegal and off-label use of antimicrobials with no MRL fixed in

milk, could also result in residues in milk.

Benzylpenicillin, occurring in 9 brand names, is the most abundant active substance,

followed by amoxicillin and cloxacillin (both 7 brands), and chlor- and oxytetracycline

and enrofloxacin (6 brands) (Table 16). Also in literature, it is stated that penicillin is

most often the antibiotic of choice, since this drug is effective in the control of

streptococci and micrococci, the organisms responsible for most cases of mastitis

(Albright et al., 1961; Pyörälä, 1995; Sandholm, 1995). Other authors report a

frequent use of benzylpenicillin at off-label dosages, because it is inexpensive and

widely available (Moats, 1998).

1.3.5 Sales and usage of antimicrobials Only limited recent information on the usage or sales of veterinary drugs is available.

Data on the consumption of antimicrobial agents in the EU member states (including

Switzerland) have been published by FEDESA/FEFANA for the year 1997 (Anon.,

1998a). Reportedly, 3,994 tonnes of antimicrobial agents have been used in 1997,

for which forty percent amounts for the practice of growth enhancement in food

producing animals. The concern for the cross-resistance to glycopeptides,

macrolides, and streptogramins used in human medicine, the aim to retain the

efficacy of Zn-bacitracin as a therapeutic agent in human medicine, and toxicological

aspects in the case of quinoxalins were the main reasons for the ban or withdrawal


0

100

200

300

400

500

600

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

kg a

ctiv

e su

bsta

nce

x 1,

000

YearAMGP Antibiotics Total

of most substances used for growth promotion by January 1, 1999 (Schwarz et al.,

2001). As from 2006 on, antimicrobial growth promotors were entirely banned

(Regulation (EC) No 1831/2003). The ban on the use of antimicrobial growth

promotors did not result in a decrease of total consumption of antibiotics, as could be

seen for the Netherlands in Figure 11. It was shown that in the same period the total

size of the livestock did not change significantly.

Figure 11. Total sales of antibiotics in the Netherlands, 1998 to 2008. AMPG, antimicrobial growth promotors (Anon., 2010g). In the Netherlands, the antibiotic usage on prescription expressed in terms per

grams per kg live weight has even doubled in 2007 compared to 1999, but

decreased in 2008 (Anon. 2010g).

In 2009, the total European (including Swiss) sales of animal health products

amounted to € 4,210,467,000. This represented 31.1% of the global market in 2009.

The sales of antimicrobials in Europe amounted to € 802,446,000 or 19.1% of the

European animal health market, with 53.6% of the antimicrobials as injectables and

46.4% in oral forms (Anon., 2010f).

In 2010, Europe was setting up the European Surveillance of Veterinary

Antimicrobial Consumption (ESVAC) project to collect, to analyse, and to report data

on the use of antimicrobial agents in animals. These data are essential to identify


0.96

0.10

1.70

0.140.340.680.01

2.530.10

and quantify risk factors for the potential development and spread of antimicrobial

resistance in animals. But so far, no data about the European consumption of

antibiotics per pharmacotherapeutic group became available. Recent data are

available from the Netherlands (Anon., 2010g). On the Dutch dairy farms,

tetracyclines are accounting for more than half of all veterinary therapeutic antibiotic

sales in 2008. Other frequently used classes of antimicrobials include the

combination of sulfonamides and trimethoprim, penicillins/cephalosporins, and

macrolides/lincosamides (Anon., 2010g). These substances exhibiting antimicrobial

activity are used in animals in three ways: namely therapeutic, metaphylactive, and

prophylactive use (Schwarz et al., 2001).

Concerning the exposure to antibiotics, expressed in terms of daily dosages per

animal per year for the period 2004-2008, for dairy farms, an annual variation with

increased usage from 2006 was noticed in the Netherlands (Anon., 2010g). The

average dairy cow received 6.6 daily dosages per year in 2008 (Figure 12). Thirty-

eight % of the total antibiotic use on dairy farms consisted of combination therapy

(mostly penicillins with aminoglycosides for intra-mammary therapy), and 26%

originated from the administration of penicillins. Further analysis of the data revealed

a tendency to an increased use of third- and fourth-generation cephalosporins, more

specifically of ceftiofur. Sixty-seven percent of the intra-mammary use was destined

for drying off, which means that on average more than 90% of the dairy cows have

received dry cow treatment in all four quarters (Anon., 2010g).

Figure 12. Antibiotic use on Dutch dairy farms in daily dosages per animal per year in 2008 (Anon., 2010g).


1.3.6 Main causes of inhibitors in milk In a study on drug residues in dairy cattle industry, conducted by Kaneene and Ahl

(1987), 30% of the farm milk tank failures for residues were related to insufficient

knowledge about withdrawal periods, 23% were due to mistakes from occasional

employees, 14% due to poor identification or record of animals treated, 12% to

metritis treatment, 7% to dry cow treatment for mastitis, 4% by incorrect label

reading, and for 10% no cause could be determined. These 10% of undetermined

causes could include cows that individually have prolonged penicillin clearance times

(McQueen, 1987), and/or accidents as contamination of the bedding by the urine of

treated cows causing residues in the milk of neighbouring untreated cows (Vanos,

1973).

In France, a study was carried out by Fabre and co-workers (1995) on farms where

the milk at the official control was positive. In 17% of the cases the cause was not

identified. The distribution of the incriminated categories is shown in Table 17. Within

each category the cause was tried to be identified.

Table 17. Incriminated categories of farm tank milk failure. The total exceeds 100% as in certain cases 2 categories were incriminated at the same farm (Fabre et al., 1995). Categories n %

treatment of clinical mastitis 330 64 dry cow therapy 122 24

teat hygiene and care 18 3 pathologies other than mammary 55 11

medicated feeds or anti-parasitic treatments 5 1 poor cleaning of milking equipment 4 1

Total cases with at least one mistake identified 516

For the category of clinical mastitis treatment (n=330) following mistakes were

identified: milking mistake (accidental milking of a treated cow) (56%), no respect of

dose and/or withdrawal times (38%), incorrect use (product intended for

intramuscular route, used intra-mammary; or drying off treatment used to treat

clinical mastitis) (12%), abnormal use of the treatments by parenteral route (no

respect of dose or withdrawal times) (9%), poor rinsing of the milking claw after the

milking of a treated cow (8%), and the use of too small containers for milking of

treated cows (contaminated milk went into the tank) (6%). When a drying off


treatment was incriminated (n=122), accidental milking of a non-identified dry cow

(66%) and not respecting the withholding period (41%) could be identified as major

causes.

Other common reasons for antibiotic contamination include the purchase of lactating

cows where the new owner was unaware of recent antibiotic treatments prior to sale,

cows drinking from a medicated footbath, off-label drug use, and antibiotic-treated

cows were milked last but the milk line was not diverted from the bulk tank. Finally,

cross- contamination by the use of a dirty syringe by the farmer or veterinarian and

criminal acts could also be added to the list.

In 2001 an investigation was performed in Flanders by a milk control station at 101

farms with bulk tank milk failure (Anon., 2002a). At 26 farms, no antimicrobials were

used during the last 2 weeks. At 60 farms, veterinary drugs were administrated for

the udder health (clinical mastitis (38 farms), dry off (17 farms), and other (5 farms)).

Surgery, muscle diseases, intestinal diseases, and metritis therapy were the other

reasons for the application of antimicrobials at 10, 3, 1, and 1 farm(s), respectively.

The presumptive major reasons for the presence of antimicrobial residues in the bulk

tank milk are summarized in Table 18.

Table 18. Presumptive major reasons for the presence of antimicrobial residues in the bulk tank milk of 101 Flemish farms with bulk tank failure (Anon., 2002a).

Reason positive result n % too early delivery, no further reason 24 23.8

too early delivery, no testing after treatment 25 24.8 mistake in milking technique 25 24.8

too short dry off period (<6 weeks) 9 8.9 medication not registered for lactating cows 6 6.0

other milker 5 5.0 no idea 4 4.0

just respecting the withdrawal period 3 3.0

Several shortages in farm management were remarked which could increase the

chance on accidents and human mistakes: no identification of treated cows (18%),

no recording of the dry cow therapy treatment (48%), no respect of the prescribed

withdrawal period (8%), no separate or last milking of treated cows (45%), and no

adequate rinsing of the milking equipment after milking of antibiotic-treated cows

(81%). Fifty-one percent of the farmers in this study were not monitoring the milk of


the antibiotic-treated cows with an antibiotic residue test, before adding the milk to

the bulk tank (Anon., 2002a).

The risk of residue occurrence decreased in association with the use of milk residue

test kits and when tie stall and pipeline milking were used rather than milking parlors

(McEwen et al., 1991).

1.3.7 The occurrence of antimicrobial residues in milk In 2009, of the 1,374,801 ex-farm milk samples in Belgium analysed in the

framework of official payment control, 872 samples or 0.06% were found to be

positive by the milk control stations (Anon., 2010d & e). In most cases, residues of β-

lactam substances were the main reason for bulk tank milk failure. In an identification

study, performed at the Technology and Food Science Unit of the Institute for

Agricultural and Fisheries Research (ILVO-T&V), on all 181 positive Flemish ex-farm

milk samples leading to penalization in the months May and June of 2003, 79% of

the samples contained natural (benzylpenicillin) or aminopenicillins (ampicillin or

amoxicillin), 8% isoxazolyl penicillins or cephalopsporins, and 4% a combination of

β-lactam and non-β-lactam residues. In total, therefore, 91% of the samples

contained β-lactams (Reybroeck and Daeseleire, 2003). In 11 samples (5.5%) the

concentration was exceeding ten times the respective MRL: 9 of these samples

contained benzylpenicillin with a concentration ranging from 42 to 12,000 µg kg-1,

while the other two samples contained cloxacillin (308 and 16,518 µg kg-1). The

concentration of penicillins in these samples was high enough to contaminate a

complete tanker load above MRL. Rather similar data about the dominance of -

lactam residues were given in the year report of 2005 of Comité du Lait (Walloon

region): in 2003 the percentage of penalizations due to β-lactams was 94.5%, 90.0%

in 2004, and 83.9% in 2005 (Anon., 2006a). It is worth noting that these data are

influenced by the detection capabilities of the applied screening test.

In Germany, β-lactams could, in 95% of the cases, be identified in inhibitor-positive

milk samples (Kress et al., 2007). Benzylpenicillin was still the predominant antibiotic

detected (74.6%) during regulatory control, followed by ceftiofur (11%),

ampicillin/amoxicillin (6.3%), and isoxazolyl penicillins (3.2%).

In 2006, a large screening was performed in Belgium (unpublished data) on the

presence of tetracyclines upon MRL on farm milk level. The milk of each milk


producer was checked with a microbiological test (Nouws et al., 1995). Only 5

samples upon 12,885 Belgian ex-farm milk samples (0.04%) contained tetracyclines.

In spring 2007 (12,377 samples) and 2009 (9,818 samples), the same study was

repeated and all Belgian ex-farm milk samples tested negative (Anon., 2010d and

unpublished data). In November 2008, a large number of Belgian ex-farm milk

samples (5,573 samples) were screened on the presence of fluoroquinolones by

means of E. coli-test in microplate format (Suhren, 1997). Despite the availability on

the Belgian market of several brands with fluoroquinolones as active substance, no

quinolone residues were detected (unpublished data).

In Belgium in 2006, the percentage of positive tanker loads upon arrival was 0.073%

(initial testing with a rapid test); in 0.044% of the tanker loads the presence of

antimicrobial residues was confirmed by a microbiological test.

On European level, in the period January 2002 - November 20, 2010, there were 12

alert RASFF notifications for residues of veterinary medicinal products in milk and

milk products. In all cases it concerned the prohibited substance chloramphenicol. In

the same period there were no border rejection notifications and 30 information

notifications; in 26 cases it concerned chloramphenicol, and in 4 cases bacterial

inhibitors (Anon., 2010k).

1.3.8 Drawbacks of residues of veterinary drugs in milk The presence of antimicrobial residues in milk can have several drawbacks:

inhibition of dairy starter cultures used in the production of cheese and yoghurt,

health aspects like possible hypersensitivity reaction by the consumer, and

contribution to the development of antibiotic resistance.

Many starter cultures used in the production of fermented products, e.g. yoghurt and

cheese, may be inhibited by antimicrobial substances. In general, concentrations

exceeding MRL are needed for total inhibition of mesophilic (Gouda, Cheddar,

Edam, or Camembert) and thermophilic (Emmenthal, Gruyère, or yoghurt) starter

cultures. However, product quality may be already impaired by low antibiotic levels

(Mäyrä-Mäkinen, 1995; Suhren, 1996; Reybroeck, 1997; Grunwald, 2002). To

prevent techno-logical problems, all milk coming into the dairy should have been

tested before unloading into the huge silos or before production.

The likelihood of an acute toxicity from veterinary drugs or their metabolites,

originating from the consumption of milk, is extremely low, since the low occurrence


of antimicrobial residues in consumption milk. Moreover, most of the drugs used in

dairy cattle production are also approved for human use. The amount of intake as a

primary drug will largely exceed the unintentially consumption of residue-containing

milk. The use of veterinary drugs with high potential to cause toxic effects like

chloramphenicol, nitrofurans, etc. has been prohibited in all food producing animals

(Commission Regulation (EU) No 37/2010). Chloramphenicol is known to be a

possible causative agent of fatal aplastic blood anaemia. Nitrofurans are potentially

carcinogenic or mutagenic. Besides direct toxic effects, also other negative effects

due to the ingestion of residues of antibiotics or chemotherapeutics have been

described. Tetracycline residues are deposited in bones and teeth and hence can

slow down the growth of the skeleton and irreversibly discolour the teeth of children.

Other residues can affect the immune system, the human gut flora or cause

hypersensitive reactions. Hypersensitivity to penicillin is the most common side effect

experienced by human patients with an incidence ranging from 0.7% to 10% of the

population. Hypersensitivity reactions to penicillin are idiosyncratic, not dose-related,

and have no inheritance pattern (Jones and Seymour, 1988). People can suffer

allergic reactions such as skin rashes, hives, asthma or anaphylactic shock, even

caused by very low concentrations of antibiotics and metabolites (Anon., 2006b).

Another side effect is the potential build-up of antibiotic resistant organisms in

humans, since the food chain is the predominant way of reaching humans of

antibiotic resistant zoonotic bacteria (Witte, 1998). Resistance of S. aureus strains

isolated from mastitis infections to penicillins have been stated in different countries.

This resistance is less in the Nordic European countries than in many other countries

(Sandholm, 1995). In dairy cows, resistance in E. coli has been traditionally low in

the Netherlands but is increasing alarmingly fast in a few years time. In 2005,

multidrug resistance was rarely observed, while in 2008, 11% of the isolates were

resistant to two or more antibiotic classes, and resistance up to eight classes has

been seen in individual isolates (Anon., 2010g). Highest resistance levels in E. coli

strains isolated from mastitis milk samples from dairy cows were observed against

tetracycline (16.2%), streptomycin (13.1%), and ampicillin (11.1%). In coliform

bacteria, resistance levels against the first two mentioned antibiotics were similar.

However, resistance levels in coliform bacteria against ampicillin were much higher

(85%), besides a 22% resistance against the combination of amoxicillin with

clavulanic acid. Both in E. coli and coliform strains, isolated from milk samples,


resistance against second- (cefuroxime) and third- (cefoperazone) generation of

cephalosporins was detected. Also resistance against fourth-generation

cephalosporins (cefquinome) in E. coli isolates from mastitis samples was found,

which is indicative for the presence of Extended Spectrum Beta-Lactamase (ESBL)

(Anon., 2010g).

1.3.9 Stability of antibiotics and chemotherapeutics in milk

1.3.9.1 -Lactams

Different stability studies of penicillins in milk have been published. Krienke and

Fouts (1950) did not find any detectable loss of penicillin potency upon 9 to 10 days

refrigerated storage of whole milk and condensed milk and upon 10 weeks storage

of skimmed milk powder at room temperature. Kersey et al. (1953) observed eight

antibiotics to be stable in milk held at 8 to 10°C for at least 1 week. A loss in potency

was found by Shahani et al. (1956) when following the antibiotic activity of potassium

penicillin spiked in raw milk and stored for a period of 7 days at 1 to 3°C. In a stability

study performed by Gaudin et al. (2001) by using the STAR protocol and measuring

the inhibition zones, benzylpenicillin (8 µg kg-1) and oxacillin (30 µg kg-1) doped in

milk and stored at -20°C were stable during a period of 27 days, while some loss in

activity was observed for cefazolin (50 µg kg-1). In the framework of a proficiency

testing study organized by the Community Reference Laboratory (CRL) in Fougères

(France), the stability at -20°C of benzylpenicillin (5 µg kg-1) and cloxacillin (60 µg

kg-1) doped in milk was checked by using a LC-MS/MS procedure during a period of

48 to 55 days. No problem of depletion was observed by Fuselier et al. (2004).

The stability of five major β-lactam antibiotics (amoxicillin, ampicillin, cloxacillin,

oxacillin, and benzylpenicillin) in fortified milk and in milk extracts prepared for LC-

ESI-MS/MS analysis was studied by Riediker et al. (2004) at varying cold

temperatures (4, -20, and -76°C). Storage of milk samples at 4°C resulted in

measurable losses of all β-lactams after 6 days (>50% in most cases). Slow

degradation of benzylpenicillin, cloxacillin, and oxacillin was observed in milk stored

at -20°C, but no losses were recorded at -76°C over 4 weeks. All antibiotics in milk

extracts for LC-ESI-MS/MS analysis showed good stability at all temperatures

tested.


In a proficiency study by CRL in Fougères (Fuselier et al., 2008) no problem of

degradation was significantly observed during storage at -20°C during 6 weeks for

benzylpenicillin at 6 µg kg-1, cloxacillin at 40 µg kg-1, cefquinome at 50 µg kg-1, and

cefalonium at 20 µg kg-1 in raw milk.

Okerman et al. (2007) studied the stability of frozen stock solutions of penicillins and

cephalosporins using an agar diffusion test with Bacillus subtilis as a test strain. After

6 months of preservation at -20°C, benzylpenicillin and amoxicillin lost nearly half of

their potency, the cephalosporins ceftiofur and cephapirin one quarter, but ampicillin

was more stable.

Internal standard samples, consisting of respectively, benzylpenicillin and cloxacillin

spiked in raw milk and stored for 2 months below -20°C did not demonstrate any

problems with stability. Storing raw milk in the refrigerator (2-8°C), however, can

result in stability problems with β-lactams due to the possible formation of

penicillinase by certain milk bacteria (Guay et al., 1987).


As described in the literature, tetracyclines are susceptible to conformational

degradation to their 4-epimeres as a function of pH and temperature. The

epimerization occurs in the pH range of approximately two to six (McCormick et al.,

1957; Andersen et al., 2005). Calcium, as well as calcium-rich foods such as milk

products, may impair absorption of tetracycline antibiotics because of chelate

formation and therefore diminish their effectiveness (Jung et al., 1997).

In some publications a loss in activity of tetracyclines doped in milk was described:

Gaudin et al. (2001) reported some loss for chlortetracycline (400 µg kg-1) in milk

stored at -20 for 27 days; Okerman et al. (2007) found for frozen stock solutions

preserved for 6 months at -20°C a loss of less than 10% for doxycycline and

chlortetracycline, while oxytetracycline and tetracycline lost about 25% of their

potency.

On the other side, Fuselier et al. (2004) did not observe degradation after a period of

48 to 55 days at -20°C for tetracycline (180 µg kg-1) and oxytetracycline (240 µg kg-1)


doped in milk. Oxytetracycline doped at 100 and 200 µg kg-1 in raw milk was 5

weeks stable at -20°C (Gaudin et al., 2010).

Also at ILVO-T&V, problems with the stability of the standard of oxytetracycline

doped in raw milk and stored for 2 months below -20°C were never encountered, nor

with stock solutions of tetracyclines stored at 1-4°C for maximum one week.

1.3.9.3 Aminoglycosides

Aminoglycosides are characterized by thermal stability and non-volatility.

Aminoglycosides are extremely hydrophilic compounds that do not bind strongly to

protein in the matrix. Gentamicin consists of five isomers, i.e. C1, C1a, C2, C2a, and

C2b.

Considering publications on aminoglycosides, most data on streptomycin and

neomycin show a good stability during storage (Haagsma, 1993). In a stability study

performed by Gaudin et al. (2001), neomycin at 2,000 µg kg-1 and framycetin at

1,000 µg kg-1 doped in milk were stable during a period of 27 days when stored at

-20°C, while some loss in activity was noticed for dihydrostreptomycin (5,000 µg

kg-1). In the proficiency study regarding the analysis of aminoglycoside residues in

milk by ELISA, gentamicin at 50 and 150 µg kg-1, streptomycin at 100 and 300 µg

kg-1, dihydrostreptomycin at 100 and 300 µg kg-1, and neomycin at 75 and 225 µg

kg-1 were stable during a period of 1 month stored at -20°C (Gaudin and Sanders,

2003). Neomycin at 2,400 µg kg-1 showed to be stable for a period of 49 to 55 days

at -20°C in the proficiency testing organized by CRL in Fougères (Fuselier et al.,

2004).

In a study conducted by Zhu et al. (2008), stock solutions of 13 aminoglycosides

were stable for at least 6 months in standard dilution solution in plastic tubes at

2-4°C while the extracts for analysis were stable only for 1 week when stored in the

same conditions.

1.3.9.4 Other antibiotics and sulfonamides

Macrolides are often inactivated in basic (pH>10) as well as in acidic environments

(pH <4 for erythromycin).

Gaudin et al. (2001) found erythromycin (80 µg kg-1), pirlimycin (200 µg kg-1), and

novobiocine (5,000 µg kg-1) doped in milk and stored at -20°C, stable during a period


of 27 days. In the same study some loss in activity was noticed for spiramycin (400

µg kg-1), ciprofloxacin (100 µg kg-1), and sulfadimethoxine (200 µg kg-1).

Chloramphenicol materials at 0.5 and 2.5 µg l-1 in milk were stable for a period of 29

days when stored at -20°C (Gaudin and Maris, 2002).

Noa et al. (2002) tested the stability of 6 sulfonamides (sulfamethoxazole,

sulfachloropyridazine, sulfathiazole, sulfamonomethoxine, sulfamerazine, and

sulfamethazine), 3 nitrofurans (nitrofurazone, furazolidone, and furaltadone), and

chloramphenicol in preserved raw milk samples. During 5 days of cold storage all

antimicrobials tested, except sulfathiazole, remained stable in raw milk samples

preserved with 0.1% K2Cr2O7 or 0.05% HgCl2.

In a study of Wu et al. (2007), the stability of the stock standard solutions of

sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxypyridazine, sulfamono-

methoxine, sulfamethoxazole, sulfadimethoxine, and sulfaquinoxaline in methanol

was at least 6 months at 4°C. The recoveries of sulfadiazine and sulfathiazole in milk

spiked at 10 µg l-1 and stored at -20°C for three weeks, obviously decreased while no

effect was observed for the other sulfonamides tested (Wu et al., 2007).

In the study of Okerman et al. (2007) on frozen stock solutions, the quinolones

flumequine, enrofloxacin, and marbofloxacin were relatively stable; the loss of

activity was less than 10% after 6 months of preservation at -20°C.

Roca et al. (2010) studied the thermostability of quinolones in milk. Their results

showed that quinolones are very resistant to different heat treatments with maximum

losses of concentration of 12.7% for ciprofloxacin and 12.0% for norfloxacin at 120°C

and 20 min.

1.4 References 72

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1.5 Annexes 90

1.5 ANNEXES Annex A Veterinary drugs with a registration in Belgium for use on lactating cows (Anon. 2010j, status on August 5, 2010). 1 For intra-mammary administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder CODILAC penicillins cloxacillin Codifar DRYCLOXA-KEL penicillins cloxacillin Kela Laboratoria KLOXERATE DC penicillins cloxacillin Pfizer Animal

Health ORBENIN EXTRA DRY COW 600 mg

penicillins cloxacillin Pfizer Animal Health

ORBENIN LONG ACTING

penicillins cloxacillin Pfizer Animal Health

AMPICLOX QUICK RELEASE

penicillins ampicillin, cloxacillin Pfizer Animal Health

KLOXERATE DRY COW PLUS

penicillins ampicillin, cloxacillin Pfizer Animal Health

RILEXINE 200 LACTATING COW

cephalosporins cefalexin Virbac

RILEXINE 500 DRY COW

cephalosporins cefalexin Virbac

CEPRAVIN DRY COW cephalosporins cefalonium Intervet CEFOVET TARISSEMENT

cephalosporins cefazolin Merial

CEFOVET LACTATION cephalosporins cefazolin Merial PATHOZONE cephalosporins cefoperazone Pfizer Animal

Health COBACTAM LC INTRAMAMMAIRE

cephalosporins cefquinome Intervet

VIRBACTAN (ex COBACTAN DC)

cephalosporins cefquinome Virbac

PIRSUE 5 mg/ml macrolides pirlimycin Pfizer Animal Health

FATROX ansamycins rifaximin Vétoquinol MASTI-KEL penicillins +

aminoglycosides benzylpenicillin, neomycin Kela Laboratoria

NAFPENZAL DC (ex Nafpenzal N)

penicillins + aminoglycosides

benzylpenicillin, nafcillin, dihydrostreptomycin

Intervet

NAFPENZAL LC (ex NAFPENZAL 72)

penicillins + aminoglycosides

benzylpenicillin, nafcillin, dihydrostreptomycin

Intervet

UBROLEXIN cephalosporins + aminoglycosides

cefalexin, kanamycin Boehringer Ingelheim

LINCOCIN INTRAMAMAIRE

aminoglycosides, lincosamides

neomycin, lincomycin Pfizer Animal Health

1.5 Annexes 91

2 For intramuscular administration (dairy cattle)

Brand/Trade name Drug family Antimicrobial chemical Authorization holder

CLAMOXYL Ready To Use

penicillins amoxicillin Pfizer Animal Health

CODIMOX LA penicillins amoxicillin Codifar DOKAMOX 150 penicillins amoxicillin Emdoka

DUPHAMOX LA penicillins amoxicillin Pfizer Animal

Health LONGAMOX penicillins amoxicillin Vétoquinol VETRIMOXIN Long Acting

penicillins amoxicillin CEVA Santé animale

AMPILUX 200 mg/ml penicillins ampicillin Alfasan Int

MAMMYZINE 10 g penicillins penethamate Boehringer

Ingelheim

MAMMYZINE 5 g penicillins penethamate Boehringer

Ingelheim DEPOSIL penicillins benzylpenicillin Eurovet

DUPHAPEN penicillins benzylpenicillin Pfizer Animal

Health PEN-30 penicillins benzylpenicillin VMD PENI-kel 300.000 IE/ml penicillins benzylpenicillin Kela Laboratoria CEPOREX INJECT cephalosporins cefalexin Intervet COBACTAN 2,5% cephalosporins cefquinome Intervet COBACTAN 4,5% cephalosporins cefquinome Intervet CEFENIL cephalosporins ceftiofur Norbrook Lab

CEVAXEL 50 mg/ml cephalosporins ceftiofur CEVA Santé

Animale

EXCENEL cephalosporins ceftiofur Pfizer Animal

Health

EXCENEL RTU cephalosporins ceftiofur Pfizer Animal

Health CODICYCLINE 200 LA tetracyclines oxytetracycline Codifar

DUPHACYCLINE 100 tetracyclines oxytetracycline Pfizer Animal

Health DUPHACYCLINE 30% LA

tetracyclines oxytetracycline Pfizer Animal Health

DUPHACYCLINE LA tetracyclines oxytetracycline Pfizer Animal

Health

TERRAMYCINE LA tetracyclines oxytetracycline Pfizer Animal

Health EMDOGENT 50 aminoglycosides gentamicin Emdoka

SPECTAM INJECT aminoglycosides spectinomycin CEVA Santé

Animale SUANOVIL 20 macrolides spiramycin Merial TYLAN 200 macrolides tylosin Eli Lilly TYLO-kel 20% macrolides tylosin Kela Laboratoria

ADVOCIN 2.5% quinolones danofloxacin Pfizer Animal

Health FLOXADIL 100 mg/ml quinolones enrofloxacin Emdoka FLOXADIL 50 mg/ml quinolones enrofloxacin Emdoka MARBOCYL 10% quinolones marbofloxacin Vétoquinol MARBOCYL S10% quinolones marbofloxacin Vétoquinol COLISTINE SULFAAT 1 MIO IE/ml

polymyxins colistin VMD

NOROCLAV INJECT penicillins, -lactamase inhibitors

amoxicillin, clavulanic acid Norbrook Lab

1.5 Annexes 92


DUPHAPEN STREP penicillins, aminoglycosides

benzylpenicillin, dihydrostreptomycin

Pfizer Animal Health

NEOPEN penicillins, aminoglycosides

benzylpenicillin, neomycin Intervet

LINCO-SPECTIN lincosamides, aminoglycosides

lincomycin, spectinomycin Pfizer Animal Health

DUPHATROXIM sulfa drugs, diaminopyrimidine derivatives

sulfadiazine, trimethoprim Pfizer Animal Health

VETITRIM sulfa drugs, diaminopyrimidine derivatives

sulfadimethoxine, trimethoprim

Eurovet

BORGAL 24% sulfa drugs, diaminopyrimidine derivatives

sulfadoxine, trimethoprim Virbac

DOFATRIM-JECT sulfa drugs, diaminopyrimidine derivatives

sulfadoxine, trimethoprim Dopharma Research

3 For intravenous administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder AMPI-DRY 3 g, 5 g penicillins ampicillin Prodivet COBACTAN 4,5% cephalosporins cefquinome Intervet EMDOGENT 50 aminoglycosides gentamicin Emdoka TYLAN 200 macrolides tylosin Eli Lilly TYLO-kel 20% macrolides tylosin Kela Laboratoria

ADVOCIN 180 quinolones danofloxacin Pfizer Animal

Health

ADVOCIN 2.5% quinolones danofloxacin Pfizer Animal

Health BAYTRIL 10% INJECT OPL

quinolones enrofloxacin Bayer

BAYTRIL 5% INJECT OPL

quinolones enrofloxacin Bayer

FLOXADIL 100 mg/ml quinolones enrofloxacin Emdoka FLOXADIL 50 mg/ml quinolones enrofloxacin Emdoka MARBOCYL 10% quinolones marbofloxacin Vétoquinol

DUPHATROXIM sulfa drugs, diaminopyrimidine derivatives sulfadiazine, trimethoprim

Pfizer Animal Health

VETITRIM sulfa drugs, diaminopyrimidine derivatives

sulfadimethoxine, trimethoprim

Eurovet

BORGAL 24% sulfa drugs, diaminopyrimidine derivatives sulfadoxine, trimethoprim

Virbac

DOFATRIM-JECT sulfa drugs, diaminopyrimidine derivatives sulfadoxine, trimethoprim

Dopharma Research

4 For subcutaneous administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder CLAMOXYL Ready To Use

penicillins amoxicillin Pfizer Animal Health

CODIMOX LA penicillins amoxicillin Codifar AMPILUX 200 mg/ml penicillins ampicillin Alfasan Int DEPOSIL penicillins benzylpenicillin Eurovet CEPOREX INJECT cephalosporins cefalexin Intervet EXCENEL RTU cephalosporins ceftiofur Pfizer Animal

Health NAXCEL RUNDVEE cephalosporins ceftiofur Pfizer Animal

Health

1.5 Annexes 93


CODICYCLINE 200 LA tetracyclines oxytetracycline Codifar EMDOGENT 50 aminoglycosides gentamicin Emdoka TYLO-kel 20% macrolides tylosin Kela Laboratoria ADVOCIN 180 quinolones danofloxacin Pfizer Animal

Health ADVOCIN 2,5% quinolones danofloxacin Pfizer Animal

Health BAYTRIL 100 quinolones enrofloxacin Bayer ENROXIL MAX 100 mg/ml INJECT OPL

quinolones enrofloxacin KRKA

FLOXADIL 100 mg/ml quinolones enrofloxacin Emdoka MARBOCYL 10% quinolones marbofloxacin Vétoquinol NEOPEN penicillins,

aminoglycosides benzylpenicillin, neomycin Intervet

LINCO-SPECTIN lincosamides, aminoglycosides

lincomycin, spectinomycin Pfizer Animal Health

BORGAL 24% sulfa drugs, diaminopyrimidine derivatives

sulfadoxine, trimethoprim Virbac

DOFATRIM-JECT sulfa drugs, diaminopyrimidine derivatives

sulfadoxine, trimethoprim Dopharma Research

5 For intra-uterine administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder DOKAMOX 150 penicillins amoxicillin Emdoka METRICURE cephalosporins cefapirin Intervet CHLOORTETRACY-CLINE 2000 U-STAAF ERNST

tetracyclines chlortetracycline Friedrich Ernst

EMDOMETRIN 2000 tetracyclines chlortetracycline Emdoka GYNAECOLOGISCHE OBLETTEN

tetracyclines chlortetracycline VMD

METRICYCLIN tetracyclines chlortetracycline Kela Laboratoria EMDOGENT 50 aminoglycosides gentamicin Emdoka 6 For peritoneal administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder DEPOSIL penicillins benzylpenicillin Eurovet 7 For per os administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder ENDOCOLIN 10% polymyxins colistin Emdoka 8 For cutaneous administration (dairy cattle) Brand/Trade name Drug family Antimicrobial chemical Authorization

holder CHLORTETRA SPRAY tetracyclines chlortetracycline OR&B Consulting CYCLOSPRAY tetracyclines chlortetracycline Eurovet OXYTEM SPRAY tetracyclines oxytetracycline Emdoka

1.5 Annexes 94

Annex B Maximum residue limits (MRLs, Commission Regulation (EU) No 37/2010 and amendments), minimum required performance limits (MRPLs, Commission Decision 2003/181/EC) and recommended concentrations (Anon., 2007c) of anti-infectious agents in bovine milk. Status on October 12, 2010. Allowed substances (Annex, Table 1) Group Pharmacologically

active substance Marker residue MRL

(µg kg-1) Other provisions

sulfonamides group parent drug 100 the combined total residues should not exceed 100 µg/kg

diaminopyrimidine derivatives

baquiloprim baquiloprim 30 trimethoprim trimethoprim 50

penicillins benzylpenicillin benzylpenicillin 4 ampicillin ampicillin 4 amoxicillin amoxicillin 4 oxacillin oxacillin 30 cloxacillin cloxacillin 30 dicloxacillin dicloxacillin 30 nafcillin nafcillin 30 for intra-mammary use

only penethamate benzylpenicillin 4

cephalosporins

ceftiofur

sum of all residues retaining the -lactam structure expressed as desfuroylceftiofur

100

cefquinome cefquinome 20 cefazolin cefazolin 50

cephapirin sum of cephapirin & desacetylcephapirin 60

cefacetrile cefacetrile 125 for intra-mammary use only

cefoperazone cefoperazone 50 for intra-mammary use in lactating cows only

cefalexin cefalexin 100 cefalonium cefalonium 20 for intra-mammary use

and eye treatment only quinolones marbofloxacin marbofloxacin 75

danofloxacin danofloxacin 30

difloxacin difloxacin ---a not for use in animals from which milk is produced for human production

enrofloxacin sum of enrofloxacin and ciprofloxacin 100

flumequine flumequine 50

oxolinic acid oxolinic acid ---a not for use in animals from which milk is produced for human production

1.5 Annexes 95

Group Pharmacologically active substance

Marker residue MRL (µg kg-1)

Other provisions

macrolides spiramycin sum of spiramycin and neospiramycin 200

tylosin tylosin A 50 erythromycin erythromycin A 40 tilmicosin tilmicosin 50

tulathromycin tulathromycin equivalents b ---a

not for use in animals from which milk is produced for human production

gamithromycin gamithromycin ---a


tildipirosine tildipirosine ---a not for use in animals from which milk is produced for human production

florfenicol and related compounds florfenicol

sum of florfenicol and its metabolites measured as florfenicol-amine

---a


thiamphenicol thiamfenicol 50 tetracyclines tetracycline sum of parent drug and

its 4-epimer 100

oxytetracycline sum of parent drug and its 4-epimer 100

chlortetracycline sum of parent drug and its 4-epimer 100

doxycycline doxycycline ---a not for use in animals from which milk is produced for human production

naftalene-ringed ansamycin rifaximin rifaximin 60

lincosamides lincomycin lincomycin 150 pirlimycin pirlimycin 100

aminoglycosides spectinomycin spectinomycin 200 streptomycin streptomycin 200 dihydrostreptomycin dihydrostreptomycin 200 gentamicin sum of gentamicin C1,

C1a, C2, and C2a 100

neomycin (+framycetin) neomycin B 1500 kanamycin kanamycin A 150

apramycin apramycin ---a not for use in animals from which milk is produced for human production

paromomycin paromomycin ---a


1.5 Annexes 96


Marker residue MRL (µg kg-1)

Other provisions

other antibiotics novobiocin novobiocin 50 for intra-mammary use only

polypeptides bacitracin sum of bacitracin A, B, and C 100

-lactamase inhibitors clavulanic acid clavulanic acid 200 polymyxins colistin colistin 50 ionofors monensin monensin A 2 Notes:

a use not allowed, zero tolerance.

b(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10,13-tetra-hydroxy-3,5,8,10,12,14-

hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)--D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopent-decan-15-one expressed as tulathromycin equivalents.

2 Prohibited substances (Annex, Table 2) (MRL cannot be established) Group Pharmacologically

active substance Marker residue MRPL

(µg kg-1) Recommended concentration

(µg kg-1) florfenicol & related compounds chloramphenicol chloramphenicol 0.3

(0.1)a 0.3 (MRPL)

nitrofurans nitrofurans (+furazolidone)

metabolites AMOZ, AHD, SEM, AOZ 1b 1

nitroimidazoles ronidazole hydroxymetabolites 3 dimetridazole hydroxymetabolites 3 metronidazole hydroxymetabolites 3

sulfones dapsone dapsone 5 Notes: a Belgian MRPL. b MRPL set for poultry meat and aquaculture products (Commission Decision 2003/181/EC).

1.5 Annexes 97

Annex C FDA Tolerance and/or Safe Levels of animal drug residues in milk; M-I-05-5, September 27, 2005, partim antimicrobials (Anon., 2005b).


Tolerance (µg kg-1)

Safe level (µg kg-1)

sulfonamides sulfadimethoxine 10 penicillins penicillin 0 5 ampicillin 10 amoxicillin 10 cloxacillin 10 cephaloporins ceftiofur 100a cephapirin 20 macrolides tylosin 50 erythromycin 0 50 tetracyclines group and individually 300b lincosamides pirlimycin 400 aminoglycosides dihydrostreptomycin 125 neomycin 150 other antibiotics novobiocin 100 polypeptides bacitracin 500

Notes: a The tolerance was established for the marker residue, not the parent compound. b This tolerance includes both the sum and the individual residues of chlortetra-cycline, oxytetracycline and tetracycline.

Appendix N of the PMO states: "Safe levels" are used by FDA as guides for prosecutorial discretion. They do not legalize residues found in milk that are below the safe level. In short, FDA uses the "safe levels" as prosecutional guidelines and in full consistency with CNI v. Young stating, in direct and unequivocal language, that the "safe levels" are not binding. They do not dictate any result; they do not limit the Agency's discretion in any way; and that they do not protect milk producers, or milk from court enforcement action. "Safe levels" are not and cannot be transformed into tolerances that are established for animal drugs under Section 512 (b) of the FFD&CA as amended. "Safe levels" do not:

1. Bind the courts, the public, including milk producers, or the Agency, including individual FDA employees; and

2. Do not have the "force of law" of tolerances, or of binding rules. Under CNI v. Young, FDA is not precluded from taking action against a product with a residue level below the safe level, nor is FDA unable to exercise enforcement discretion when the safe levels are exceeded. FDA will make this determination depending on circumstances and available evidence on a case-by-case basis. Extra-label use of sulfonamide drugs in lactating dairy cattle (except approved use of sulfadimethoxine, sulfabromomethazine and sulfaethoxypyridazine) is prohibited by 21 CFR 530.41.

.

Chapter 2 Thesis objectives

2 Thesis Objectives 100

Thesis objectives Antimicrobials are used in agriculture and can result in residues in food of animal

origin. Reliable methods are needed to screen honey and milk for residues of

veterinary drugs. Performance characteristics of qualitative screening methods like

selectivity, detection capability, applicability, and ruggedness have to be validated

before the methods can be used in surveillance programmes like official residue

monitoring control plans. The first objective of this thesis was to validate three

screening tests following Commission Decision 2002/657/EC. It concerns the

Tetrasensor Honey test kit for screening for tetracyclines in honey (Chapter 3) and

two rapid dipstick screening tests for detection of -lactam residues in milk, namely,

the eta-s.t.a.r. 1+1 (Chapter 5) and the Charm MRL-3 (Chapter 6).

Residues in honey and milk can result in financial penalties, market withdrawals,

food recalls, and safety alerts. However, in some cases the food producer

(beekeeper or dairy farmer) claims not to have used antimicrobials. This was the

case for some Flemish producers of honey samples containing small amounts of

sulfa residues (<50 µg kg-1 sulfamethazine). Residue monitoring on beeswax

intended for the fabrication of wax foundations revealed some contaminations with

sulfonamides. Therefore, a migration test was set up to study if sulfa-containing

beeswax foundations could result in contaminated honey. In a small lab experiment,

medicated syrup was brought in contact with beeswax to investigate the transfer of

sulfamethazine (Chapter 4).

At two farms with frequent problems of false-positive Delvotest results as part of the

regulatory quality programme, milk was sampled for further study to indicate the

interfering inhibitors in the milk (Chapter 7).

3 Validation

Chapter 3 Validation of the Tetrasensor Honey Test Kit

Adapted from:

Reybroeck W., Ooghe S., De Brabander H., Daeseleire E. 2007. Validation of the

Tetrasensor Honey test kit for the screening of tetracyclines in honey. Journal of

Agricultural and Food Chemistry 55: 8359-8366.

3 Validation of the Tetrasensor Honey Test Kit 102

Validation of the Tetrasensor Honey test kit for the screening for tetracyclines in honey

Abstract Regarding anti-infectious agents, no Maximum Residue Limits are fixed for honey in

the European legislation. Some Member States established action limits and

discussions are being conducted in order to set reference points for action at

European level.

The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid

screening (30 min) of honey on the presence of tetracyclines. The test was validated

according to Commission Decision 2002/657/EC. The test detects tetracycline,

oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and

sensitive way. Depending on the type of tetracycline, detection capabilities (CC)

between 6 and 12 µg kg-1 were obtained (4-7 µg kg-1 for dried dipsticks). The test is

rugged and participation with the test in an international ring trial gave correct

results.

It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test

that even can be used at the production site.


3.1 INTRODUCTION Honey is generally considered as a natural and healthy product. The addition of

additives or conserving agents to honey is not allowed. However, in recent years, the

problem of residues of antibiotics in honey has been mentioned in some publications

(Reybroeck, 2003; Bogdanov, 2006). Antibiotics, for example tetracyclines, are used

in apiculture for the treatment of bacterial brood diseases like American foulbrood

(Paenibacillus larvae subsp. larvae) (Hopingarner and Nelson, 1987; Spivak, 2000)

and European foulbrood (Melissococcus plutonius) (Waite et al., 2003; Thompson et

al., 2005). This practice is illegal in Europe. However, oxytetracycline is used in the

UK in the statutory treatment of European foulbrood since this is considered by the

authorities as within the cascade system for veterinary medicines under minor uses,

minor species (Thompson et al., 2005). The intensive use of tetracyclines in

professional beekeeping in the United States and South America resulted in

tetracycline-resistant Paenibacillus strains (Alippi, 2000; Miyagi et al., 2000).

Tetracyclines are broad-spectrum bacteriostatic antibiotics with a long history in

veterinary medicine and are used for the treatment and control of a wide variety of

bacterial infections. When used in beekeeping, important concentrations up to

milligram per kilogram level could be found in the honey of the treated hives

(Thompson et al., 2005; Martel et al., 2006) with a slow depletion and degradation (a

half-life time for oxytetracycline of 9 to 44 days (Thompson et al., 2005) and a half-

life time of 65 days for tetracycline in honey from supers (Martel et al., 2006)). The

research conducted by Adams et al. (2006) on the fate of chloramphenicol,

furazolidone, streptomycin, and tylosin in honey after administration to bee colonies

resulted in similar conclusions.

Reliable screening methods are needed in order to check honey for the presence of

antibiotics. In general, Charm II receptor assays are used, but the rate of false-

positive results could be relatively high (Reybroeck, 2003; Beaune and Garcet, 2004;

Beaune et al., 2005). A new rapid receptor-based screening test for the detection of

tetracyclines in honey was developed by Unisensor s.a. (Wandre, Belgium), namely,

the Tetrasensor Honey.

The use of antibiotics in apiculture is not authorized in the European Union. No

Maximum Residue Limits (MRLs) are fixed for tetracyclines in honey in the European


legislation (Commission Regulation (EU) No 37/2010 and amendments). Some

Member States established action limits or tolerance levels in order to make the

situation more clear for honey producers, traders, and food inspectors. In Belgium,

action limits for residues of antibiotics and sulfonamides in honey were introduced in

2002, taking into account analytical possibilities and available toxicological data.

During the first period of 6 months, the action limit for the group of tetracyclines was

preliminarily set at 50 µg kg-1. Since July 1, 2002, this value has been fixed at 20 µg

kg-1 (Anon., 2001). France applies since 2009 a non-conformity limit for tetracyclines

in honey of 10 µg kg-1 (Martel, 2010) and at present, the screening target in UK is 20

µg kg-1 but samples with a residue concentration above the decision limit (CCα) of

the confirmatory method are reported as non-compliant (Sharman, 2010). In

Switzerland, for tetracyclines in honey a tolerance level of 20 µg kg-1 was applied till

January 1, 2009 (Diserens, 2010). Discussions are being held at the European level

to set working limits for residues of antibiotics in honey. The Community Reference

Laboratories proposed 20 µg kg-1 as the recommended concentration for screening

for tetracyclines in honey (Anon., 2007).

At present, it is generally accepted that the screening level for tetracyclines in honey

should lie within the range of 10-20 µg kg-1. To adapt their screening test to this

level, Unisensor improved the sensitivity of the Tetrasensor Honey test kit in 2004.

All data in this study are based on test kits with an improved detection capability

(second generation). The kits of this generation carry „Detection limit at 10 µg kg-1‟

on the label.

The aim of this work was to perform a validation study of the Tetrasensor Honey

(second generation) screening method based on the validation criteria set in

Commission Decision 2002/657/EC. Since the Tetrasensor Honey is a qualitative

screening kit for tetracyclines, only the following parameters had to be investigated

for validation purposes: the specificity of the test kit, the detection capability, and the

test ruggedness.

Parts of the results of this paper were presented at the 39th Apimondia International

Apicultural Congress in Dublin, Ireland (Reybroeck, 2005).


3.2 MATERIALS AND METHODS 3.2.1 Reagents and standards The tetracycline (T3383), oxytetracycline (O5875), chlortetracycline (C4881),

doxycycline (D9891), penicillin G (benzylpenicillin, PENNA), cephapirin (C8270),

sulfadiazine (S8626), neomycin (N1876), and erythromycin (E6376) were all from

Sigma-Aldrich (Bornem, Belgium). The enrofloxacin (17849) was from BioChemika

(Bornem, Belgium).

Standard stock solutions of 100 mg l-1 were made in water and kept below 4°C.

Dilutions of 1 mg l-1 and 0.1 mg l-1 were freshly prepared on a daily basis.

The Tetrasensor Honey kits were from Unisensor s.a.. In general, lot TH00616-

042405/4 (expiration date November 23, 2005) was used for the evaluation study; for

some parts, such as the study of batch-to-batch differences and the stability of the

reagents, lot TH00624-041907/2 (expiration date January 19, 2006) was also used.

Charm II Tetracyclines Honey kits were from Charm Sciences Inc. (Lawrence, MA).

A mixture of different honey samples of known (organic) origin and of different

composition (liquid and solid, flower and honeydew, Belgian, and imported) was

used as blank honey. Each honey from the blank mixture was tested individually as

negative with the Charm II Tetracyclines Honey (detection capability for tetracycline,

oxytetracycline , chlortetracycline, and doxycycline in honey ≤10 µg kg-1).

3.2.2 Material For the instrumental reading a QuantiSensor (Matest Systemtechnik GmbH,

Mössingen, Germany), a small reader device with specially designed QuantiSensor

software (release 345, version 2003), was used. A QuantiSensor Control dipstick

(batch 051307/01, expiration date July 13, 2008) was daily used to check whether

the reader system functioned properly.

3.2.3 Test protocol and interpretation of the results For liquid and semisolid honey, there is no sample preparation requested. Solid

honey can be liquidified by heating in a closed glass test tube in a water bath at

37°C. The lid of the plastic vial is filled with honey so that a correct amount of honey

(around 600 mg) is diluted with the buffer content of the vial (1.8 ml). A total of 200 µl

of diluted honey sample is added to the lyophilized receptor present in a glass vial

and incubated at room temperature (20 5°C) for 15 min. During this first incubation


period, tetracyclines possibly present in the honey bind with the specific receptor.

After 15 min, the dipstick is dipped into the vial, and a second incubation at room

temperature takes place for 15 min. When the liquid passes through the green

capture lines, a red colour appears. The first line captures the remaining active

receptor, and the second line takes a certain amount of the excess reagent that

passed through the first line. The second line serves as a control line and always has

to become visible; otherwise, the test is invalid. This is shown in Figure 1.

Figure 1. Visual interpretation of Tetrasensor Honey dipsticks.

Results were read both visually and using the Quantisensor, comparing the colour

intensity of both capture lines. The visual interpretation is as follows: when the colour

of the test line is more intensive than the colour of the control line, the honey sample

is negative („vis neg‟). In all other cases the honey is contaminated with tetracyclines

(„vis pos‟). The visual interpretation is always done before the instrumental reading in

order not to influence the judging by the technician.

For the instrumental reading the intensity of the colour formation is measured, and

the result is expressed as the ratio of the colour intensity of the test line to the colour

intensity of the control line. Honey samples with a ratio of 1.40 are free of

tetracyclines („neg‟); honey samples with a ratio 0.90 and <1.40 are slightly

contaminated („low pos‟), and honey samples with a ratio <0.90 are more heavily

contaminated („pos‟). When testing honey samples in routine, samples giving a ratio

invalid neg pos

CTRL TEST


≥1.40 are considered free from residues of tetracyclines; samples giving a ratio

<1.40 are considered suspect for the presence of tetracyclines.

3.3 RESULTS AND DISCUSSION The EU legislation concerning residue analysis (Council Regulation (EEC) No

2377/90 and amendments) valid in 2006, was used as a guideline for the validation

of the method.

3.3.1 Stability of tetracyclines in honey Tetracycline is rather stable in honey so long as the honey is stored in dark, since

tetracyclines are light-sensitive. As part of a collaborative trial, Martel et al. (2005)

implemented a stability study by storing honey with an analyte (tetracycline) for 2

months at 4°C. No loss of analyte content could be observed. Münstedt et al. (2002)

spiked honey with 500 µg kg-1 of oxytetracycline, chlortetracycline, and tetracycline.

Its high-performance liquid chromatographic (HPLC) analysis after 10 months of

storage at ambient temperatures still showed more than half of the original

concentration of chlortetracycline and tetracycline, but no detectable oxytetracycline,

proving an instability of oxytetracycline in honey.

In the study about the false-positive rate for a new Charm II Tetracyclines Honey kit

with adapted sensitivity, the incurred samples of the ring trial (Beaune and Garcet,

2004) were retested after 1 year of storage in the dark in a cool room by LC-MS/MS,

and nearly identical concentrations of tetracycline were measured (data not shown).

3.3.2 Test and reader repeatability A blank and four incurred positive honey samples (import table honey) were

analysed 20 times. The colour of the test line was evaluated at the end of each

assay (wet dipstick) and a second time after 30 min (dry dipstick). The results were

used to calculate the test repeatability on the basis of wet or dry dipstick readings.

To calculate the repeatability of the reader, only dry dipsticks were measured 20

times since the ratio still shifts slowly during the drying of the strips. This was done at

three different contamination levels, namely, for a blank, a low, and a high positive

strip. The results are shown in Table 1.


Table 1. Test and reader repeatability (wet and dry dipstick reading).

Sample Conta-minationa

Test repeatability (n=20) (wet dipstick reading) Minimum ratio Maximum ratio Mean ratio bsr

honey 1 blank 2.72 3.99 3.28 0.38 honey 2 low pos 0.84 1.26 1.09 0.11 honey 3 low pos 0.50 1.40 1.06 0.23 honey 4 high pos 0.16 0.93 0.58 0.21 honey 5 high pos 0.02 0.07 0.05 0.01


Test repeatability (n=20) (dry dipstick reading) Minimum ratio Maximum ratio Mean ratio bsr

honey 1 blank 1.90 3.74 2.96 0.44 honey 2 low pos 0.55 0.82 0.69 0.07 honey 3 low pos 0.24 0.65 0.49 0.12 honey 4 high pos 0.19 0.68 0.40 0.14 honey 5 high pos 0.01 0.05 0.03 0.01


Reader repeatability (n=20) (dry dipstick reading) Minimum ratio Maximum ratio Mean ratio bsr

honey 6 blank 1.64 2.15 1.80 0.14 honey 7 low pos 0.85 1.08 0.98 0.07 honey 8 high pos 0.06 0.16 0.11 0.03 Notes: a contamination level based on mean ratio (wet dipstick reading). b sr, standard deviation of repeatability. The test repeatability was good and even improved as the concentration of

tetracyclines in the honey increased. In general, the standard deviations of

repeatability decreased when the dry dipstick readings were considered in

comparison to the wet dipstick reading, except for the blank honey sample. The

reader repeatability also improved as the concentration of tetracyclines in the honey

increased and lower ratio values were obtained.

The consistency in visual judging by the technicians was also checked. It needs to

be emphasized that the technicians all received training in the reading of dipsticks as

part of the accreditation procedure and that they all had very much experience in

colour interpretation of analogue dipsticks (βeta-s.t.a.r. and Tetrasensor Tissue).

Real negative and positive honey samples were never wrongly classified by any

technician; only very occasionally and only for samples giving a borderline result

(both test lines equal in intensity) a different result between the readings by two

persons was obtained (data not shown).


3.3.3 Specificity The specificity or the ability of the method to distinguish between the analyte being

measured (tetracycline residues) and other substances was first investigated by

spiking blank honey in duplo with some other relatively high concentration anti-

infectious agents (antibiotics and chemotherapeutics). The Tetrasensor Honey test

kit was used for the analysis. One substance was chosen from each of the most

important groups: benzylpenicillin (penicillins), cephapirin (cephalosporins),

sulfadiazine (sulfonamides), enrofloxacin (quinolones), neomycin (aminoglycosides),

and erythromycin (macrolides); spiking was performed at 100 times the Belgian

action limit for tetracyclines (=2 mg kg-1). The colour of the test line was evaluated

directly at the end of the assay and after 30 min (dry dipstick).

All honey samples doped with sulfonamides or antibiotics other than tetracyclines

provided negative ratios and also visually the results were also all interpreted as

negative. In general, following the drying of the dipsticks, the ratio values dropped,

but the results all remained negative.

From the results, it can be concluded that the analysis is not disturbed by anti-

infectious agents, which are different from tetracyclines. The Tetrasensor Honey kit

is very specific for the analysis of tetracyclines.

3.3.4 Detection capability Another important validation parameter is the detection capability for the most

important tetracyclines (tetracycline (TC), oxytetracycline (OTC), chlortetracycline

(CTC), and doxycycline (DC)).

Therefore, starting from the detection capability concentrations obtained from the

manufacturer, blank honey was spiked with the investigated tetracycline at different

concentrations: in the range of 1-10 µg kg-1 in steps of 1 µg kg-1 and in the range of

10-20 µg kg-1 in steps of 2 µg kg-1. The spiked samples were blind coded before

analysis. For each investigated tetracycline, the lowest concentration giving 19 (low)

positive test results on 20 test results was determined. When a certain concentration

tested negative two times, a higher concentration was directly tested since 19

positive test results on 20 test results was no longer achievable, in order to save time

and reagents.

Since the strips were read both visually and by using a reader system, the detection

capability was determined for both means of strip reading. Moreover, the strips were


not only read immediately (wet dipstick reading), but also after 30 min of drying (dry

dipstick reading). The results are shown in Tables 2 and 3.

Table 2. Visual and instrumental reading of the testing of honey spiked with the most important tetracyclines, wet dipstick reading.

Substance spiked in blank

honey

Concen-tration

(µg kg-1)

Visual reading

(n positive/ n analysed)

Instrumental reading n (low)

positive/ n analysed

Average ratio

Lowest ratio

Highest ratio

tetracycline 8 1/5 2/5 1.44 1.12 1.61 9 20/20 20/20 1.09 0.84 1.26 oxytetracycline 10 2/5 3/5 1.37 1.02 1.70 12 20/20 20/20 1.06 0.50 1.39 chlortetracycline 4 13/20 14/20 1.23 0.59 1.74 5 20/20 20/20 0.88 0.50 1.06 doxycycline 5 3/5 3/5 1.34 1.13 1.64 6 20/20 20/20 0.93 0.58 1.27

First of all, no differences in detection capability were noticed between the visual and

the instrumental reading. Second, the detection capabilities obtained from dry

dipstick reading were lower than those obtained from wet dipstick reading. So, by

postponing the reading, the sensitivity of the test increased. A summary of the

detection capabilities is given in Table 4. The detection capabilities in this study are

better than these obtained by Alfredsson et al. (2005), as could be expected since

the sensitivity of the kit was improved by Unisensor in 2004.

Table 3. Visual and instrumental reading of the testing of honey spiked with the most important tetracyclines, dry dipstick reading (after 30’).

Substance spiked in blank

honey

Concen-tration

(µg kg-1)

Visual reading

(n positive/ n analysed)



Average ratio

Lowest ratio

Highest ratio

tetracycline 6 6/9 6/9 1.28 1.01 1.60 7 20/20 20/20 1.14 0.37 1.36 oxytetracycline 6 13/15 13/15 1.27 1.03 1.50 7 19/20 19/20 1.22 1.05 1.49 chlortetracycline 3 3/9 4/9 1.43 0.73 1.85 4 20/20 20/20 0.90 0.41 1.25 doxycycline 3 0/2 0/2 1.50 1.44 1.56 4 20/20 20/20 1.12 0.96 1.31


Table 4. Detection capability (CC) of the Tetrasensor Honey test kit for the most important tetracyclines: wet dipstick reading and dry dipstick reading (after 30’).

Substance spiked in blank honey

Detection capability (µg kg-1) (visual and instrumental reading)

Wet dipstick reading Dry dipstick reading tetracycline 9 7 oxytetracycline 12 7 chlortetracycline 5 4 doxycycline 6 4 3.3.5 Test ruggedness

Honey is a complex matrix with a large variety in composition due to different

proportions of the possible sources, nectar and/or honeydew, coming from a great

variety of plants. So it is important to check the robustness of the Tetrasensor Honey

test kit on different unifloral and multifloral honeys.

3.3.5.1 Impact of the nature (type, origin, physical parameters, etc.) of the honey on

the detection capability

As a starting point, we took the detection capability for tetracycline (TC) of 9 µg kg-1

(wet dipstick reading, Table 4), since this concentration is just at the top of the dose-

response curve. Within the group of tetracyclines, tetracycline was the most obvious

choice since it is the most frequently detected tetracycline in honey on the Belgian

market (Reybroeck, 2003).

When testing different types of honey, a comparison was run to see whether the

same test detection capability was obtained.

The following types of honey were compared in this study: Belgian versus imported

honey (Table 5), blossom versus honeydew honey (Table 6), rape (Brassica spp.)

(high glucose content) versus black locust (Robinia pseudoacacia L.) honey (high

fructose content) (Table 7), and solid versus liquid honey (Table 8).

Regarding flower honey, no significant differences were observed between the

Belgian and the imported honey, since all the honey samples (spiked with 9 µg kg-1

TC) gave low positive to positive results (Table 5). For the Belgian honey samples,

this ratio ranged from 0.58 to 1.15; for the imported honey samples from 0.67 to 1.27

(both wet dipstick readings).


Table 5. Visual and instrumental reading of the testing of Belgian honey versus imported honey.

Origin

Blank honey TC 9 µg kg-1 spiked in blank honey

Ratio on t=0

Vis on t=0

Ratio on t=30'

Vis on t=30'

Ratio on t=0

Vis on t=0 Ratio on t=30' Vis on

t=30' Flower honey

Belgium 1 2.83 (neg) v neg 2.13 (neg) v neg 0.84 (pos) v pos 0.52 (pos) v pos 1.13 (low pos) v pos 0.48 (pos) v pos Belgium 2 3.17 (neg) v neg 2.22 (neg) v neg 0.58 (pos) v pos 0.36 (pos) v pos 0.67 (pos) v pos 0.34 (pos) v pos Belgium 3 3.69 (neg) v neg 2.32 (neg) v neg 1.15 (low pos) v pos 0.57 (pos) v pos 0.88 (pos) v pos 0.44 (pos) v pos Cuba 4.68 (neg) v neg 3.38 (neg) v neg 1.07 (low pos) v pos 0.54 (pos) v pos 1.23 (low pos) v pos 0.57 (pos) v pos Chili 2.77 (neg) v neg 2.49 (neg) v neg 1.27 (low pos) v pos 0.66 (pos) v pos 1.15 (low pos) v pos 0.65 (pos) v pos India 2.26 (neg) v neg 1.43 (neg) v neg 0.67 (pos) v pos 0.28 (pos) v pos 0.75 (pos) v pos 0.30 (pos) v pos Honeydew honey

Belgium 1 2.43 (neg) v neg 1.61 (neg) v neg 0.54 (pos) v pos 0.27 (pos) v pos 0.41 (pos) v pos 0.27 (pos) v pos Belgium 2 2.48 (neg) v neg 2.03 (neg) v neg 0.71 (pos) v pos 0.48 (pos) v pos 1.05 (low pos) v pos 0.57 (pos) v pos Belgium 3 3.42 (neg) v neg 2.04 (neg) v neg 0.95 (low pos) v pos 0.57 (pos) v pos 1.07 (low pos) v pos 0.56 (pos) v pos Spain 2.99 (neg) v neg 2.80 (neg) v neg 1.43 (neg) v neg 1.17 (low pos) v pos 1.61 (neg) v neg 1.21 (low pos) v pos Notes: Vis, visual reading; v , visual reading; neg, negative; pos, positive.

Regarding honeydew honey, a difference was observed between the Belgian and

the imported honeydew honey (Table 5). All Belgian honeydew honey samples

spiked with 9 µg kg-1 TC gave low positive to positive results, while the Spanish

honeydew honey sample spiked with 9 µg kg-1 TC gave a negative result (wet

dipstick reading). So, the detection capability of 9 µg kg-1 tetracycline was not valid

for the Spanish honeydew honey. It is worth noting that the dry reading results

became „low pos‟.

Electrical conductivity could be used for differentiation between honeydew and

blossom honeys (except chestnut honey) since electrical conductivity correlates well

with the mineral content of honey (Bogdanov et al., 2004). Regarding the

composition criteria for honey (Council Directive 2001/110/EC) blossom honey

should have an electrical conductivity below 0.8 mS cm-1 while the electrical

conductivity of honeydew and chestnut honey should be higher than 0.8 mS cm-1. In


the validation study, we compared the detection of tetracycline in blossom and

honeydew honeys (Table 6). No differences were observed between the blossom

honey and the honeydew honey: all honey samples spiked with 9 µg kg-1 TC gave

low positive to positive results. For the blossom honey samples, this ratio ranged

from 0.58 to 1.15; for the honeydew honey samples, from 0.41 to 1.07 (both wet

dipstick reading).

Table 6. Influence of the botanical origin of the honey on the detection capability: blossom honey versus honeydew honey, both of Belgian origin.

Identi-fication

Con-duc-tivity

(µS cm-1)

Blank honey TC 9 µg kg-1 spiked in blank honey

Ratio on t=0

Vis on t=0

Ratio on t=30'

Vis on t=30'

Ratio on t=0

Vis on t=0

Ratio on t=30'

Vis on t=30'

Blossom honey

sample 1 167 2.83 (neg) v neg 2.13 (neg) v neg 0.84 (pos) v pos 0.52 (pos) v pos 1.13 (low pos) v pos 0.48 (pos) v pos sample 2 395 3.17 (neg) v neg 2.22 (neg) v neg 0.58 (pos) v pos 0.36 (pos) v pos 0.67 (pos) v pos 0.34 (pos) v pos sample 3 241 3.69 (neg) v neg 2.32 (neg) v neg 1.15 (low pos) v pos 0.57 (pos) v pos 0.88 (pos) v pos 0.44 (pos) v pos Honeydew honey

sample 1 988 2.43 (neg) v neg 1.61 (neg) v neg 0.54 (pos) v pos 0.27 (pos) v pos 0.41 (pos) v pos 0.27 (pos) v pos sample 2 1091 2.48 (neg) v neg 2.03 (neg) v neg 0.71 (pos) v pos 0.48 (pos) v pos 1.05 (low pos) v pos 0.57 (pos) v pos sample 3 1063 3.42 (neg) v neg 2.04 (neg) v neg 0.95 (low pos) v pos 0.57 (pos) v pos 1.07 (low pos) v pos 0.56 (pos) v pos Notes: Vis, visual reading; v , visual reading; neg, negative; pos, positive.

Among the European unifloral honeys, rape honey (Brassica spp.) and black locust

honey (Robinia pseudoacacia L.) differ in composition to an extreme extent. Rape

honey is light in colour and always comes in a crystallized form (solid). Rape honey

contains an average of 40.5% (w/w) glucose and 38.3% (w/w) fructose, and the

mean fructose/glucose ratio amounts to 0.95. Black locust honey is very light in

colour and flavour with 26.5% (w/w) glucose and 42.7% (w/w) fructose and a

fructose/glucose ratio of 1.61 (Persano Oddo and Piro, 2004).

No differences were observed in detection capability (CC) in the examination of

rape and black locust honey, despite the serious differences in composition and

texture of both honeys (Table 7).


Table 7. Influence of the type of the honey on the detection capability: rape honey versus black locust honey.

Identi-fication

Blank honey TC 9 µg kg-1 spiked in blank honey Ratio on

t=0 Vis on

t=0 Ratio on

t=30' Vis on t=30'

Ratio on t=0

Vis on t=0

Ratio on t=30'

Vis on t=30'

rape 3.79 (neg) vis neg 2.40 (neg) vis neg 0.89 (pos) vis pos 0.62 (pos) vis pos 0.75 (pos) vis pos 0.50 (pos) vis pos

black locust 4.27 (neg) vis neg 2.98 (neg) vis neg 1.17(low pos) vis pos 0.45 (pos) vis pos

0.81 (pos) vis pos 0.32 (pos) vis pos Notes: Vis, visual reading; neg, negative; pos, positive.

Honey normally becomes solid due to a natural crystallization process caused by the

high percentage of sugars, especially glucose. In the validation study, the detection

in honey of both liquid and solid forms was studied (Table 8). For the solid honey

samples, the ratios obtained ranged from 0.75 to 1.15; for the liquid honey samples,

the ratios ranged from 0.58 to 1.37 (both wet dipstick reading). No differences were

observed between the solid honey and the liquid honey: all honey samples spiked

with 9 µg kg-1 TC gave low positive to positive results. It is worth noting that, while

the liquid honey sample 2, which when not spiked, gave an extremely high ratio of

6.55, a (low) positive result was also obtained when it was spiked with 9 µg kg-1 of

tetracycline.

Table 8. Influence of a physical parameter of the honey (form) on the detection capability: solid honey versus liquid honey, both from Belgian origin.

Identi-fication

Blank honey TC 9 µg kg-1 spiked in blank honey Ratio on

t=0 Vis on

t=0 Ratio on

t=30' Vis on t=30'

Ratio on t=0

Vis on t=0

Ratio on t=30'

Vis on t=30'

solid 1 2.83 (neg) vis neg 2.13 (neg) vis neg 0.84 (pos) vis pos 0.52 (pos) vis pos 1.13 (low pos) vis pos 0.48 (pos) vis pos solid 2 3.69 (neg) vis neg 2.32 (neg) vis neg 1.15 (low pos) vis pos 0.57 (pos) vis pos 0.88 (pos) vis pos 0.44 (pos) vis pos solid 3 3.86 (neg) vis neg 1.98 (neg) vis neg 0.75 (pos) vis pos 0.28 (pos) vis pos 0.83 (pos) vis pos 0.28 (pos) vis pos liquid 1 3.17 (neg) vis neg 2.22 (neg) vis neg 0.58 (pos) vis pos 0.36 (pos) vis pos 0.67 (pos) vis pos 0.34 (pos) vis pos liquid 2 6.55 (neg) vis neg 3.59 (neg) vis neg 1.12 (low pos) vis pos 0.67 (pos) vis pos 1.33 (low pos) vis pos 0.79 (pos) vis pos liquid 3 4.90 (neg) vis neg 2.82 (neg) vis neg 0.80 (pos) vis pos 0.48 (pos) vis pos 1.37 (low pos) vis pos 0.68 (pos) vis pos

Notes: Vis, visual reading; neg, negative; pos, positive.


3.3.5.2 Batch-to-batch differences and reagents‟ stability regarding the detection

capability

It was examined whether the same detection capabilities for the four most important

tetracyclines were obtained when using two completely different batches (reagents

and strips). The results are shown in Tables 9 and 10. Lot B, used shortly after

production, gave a mean ratio value of 3.98 (wet dipstick reading) and 3.42 (dry

dipstick reading) for the blank honey.

Table 9. Batch to batch differences and kit stability regarding the detection capability, wet dipstick reading.

Substance spiked in blank honey, Lot of reagents

Con-centration (µg kg-1)



Average ratio

Lowest ratio

Highest ratio

blank, Lot A -- 0/10 4.18 2.42 5.00 blank, Lot B „fresh‟ -- 0/10 4.52 2.74 5.71 blank, Lot B „old‟ -- 0/10 3.98 2.38 5.13 tetracycline, Lot A 9 20/20 1.09 0.84 1.26 tetracycline, Lot B „fresh‟ 9 8/20 1.41 1.02 1.79 tetracycline, Lot B „old‟ 9 20/20 0.81 0.21 1.39 oxytetracycline, Lot A 12 20/20 1.06 0.50 1.39 oxytetracycline, Lot B „fresh‟ 12 18/20 1.23 0.96 1.52 oxytetracycline, Lot B „old‟ 12 20/20 0.75 0.19 1.35 chlortetracycline, Lot A 5 20/20 0.88 0.50 1.06 chlortetracycline, Lot B „fresh‟ 5 17/20 1.05 0.36 1.78 chlortetracycline, Lot B „old‟ 5 20/20 0.43 0.19 1.09 doxycycline, Lot A 6 20/20 0.93 0.58 1.27 doxycycline, Lot B „fresh‟ 6 20/20 0.58 0.16 0.93 doxycycline, Lot B „old‟ 6 20/20 0.63 0.22 1.02 Notes: Lot A, TH00616-042405/4; Lot B, TH000624-041907/2.

In the first experiment, spiked honey samples were tested on the same day with two

different batches, namely Lot TH00616-042405/4 (A) and Lot TH000624-041907/2

(B). It is worth noting that lot B was used shortly after production, and was marked as

„fresh‟, while lot A was already several months old. From Tables 9 and 10, it can be

concluded that in this experiment, differences in detection capability were obtained

for tetracycline, oxytetracycline, and chlortetracycline (wet dipstick reading). The

detection capabilities claimed in Table 2 were not reached with lot B „fresh‟.

However, the ratio values were close to the cut-off value of 1.40 (the highest ratio

value obtained for wet dipstick reading is 1.79). All ratio values for doped honey


samples were far below the ratio values for the blank honey (wet dipstick reading: lot

A, mean ratio = 4.18; lot B, mean ratio = 4.52 „fresh‟). So the differences in test

capability between the 2 different batches remained limited.

Table 10. Batch to batch differences and kit stability regarding the detection capability, dry dipstick reading.

Substance spiked in blank honey, Lot of reagents

Con-centration (µg kg-1)



Average ratio

Lowest ratio

Highest ratio

blank, Lot A -- 0/10 3.36 1.91 4.34 blank, Lot B „fresh‟ -- 0/10 3.65 2.12 4.96 blank, Lot B „old‟ -- 0/10 3.24 1.82 4.42 tetracycline, Lot A 9 20/20 0.69 0.55 0.82 tetracycline, Lot B „fresh‟ 9 20/20 0.87 0.56 1.22 tetracycline, Lot B „old‟ 9 20/20 0.47 0.35 1.12 oxytetracycline, Lot A 12 20/20 0.49 0.24 0.65 oxytetracycline, Lot B „fresh‟ 12 20/20 0.62 0.50 0.76 oxytetracycline, Lot B „old‟ 12 20/20 0.39 0.02 0.89 chlortetracycline, Lot A 5 20/20 0.64 0.38 0.78 chlortetracycline, Lot B „fresh‟ 5 20/20 0.72 0.38 1.08 chlortetracycline, Lot B „old‟ 5 20/20 0.39 0.02 0.89 doxycycline, Lot A 6 20/20 0.58 0.40 0.85 doxycycline, Lot B „fresh‟ 6 20/20 0.40 0.19 0.68 doxycycline, Lot B „old‟ 6 20/20 0.29 0.11 0.57 Notes: Lot A, TH00616-042405/4; Lot B, TH000624-041907/2.

In this experiment, both tested batches had a different production date, so it should

be further made clear whether the small differences are related to a different

production or related to a different age at the moment of use. So it was decided to

store batch B for more than a year at 4°C and to retest the same concentrations of

tetracyclines spiked in the same blank honey just before the expiration date of the

reagents. The results of this additional testing with the reagents marked as „old‟ are

also summarized in Tables 9 and 10.

From the stability testing data, we remarked a tendency of a small improvement of

the testing capacity of the reagents during the shelf life. The reagents of lot B, used

just before the expiration date, gave results comparable to the results obtained with

lot A.


3.3.5.3 Impact of drying of the strips

The impact on the test result of drying of the strips was investigated throughout the

validation by comparing direct (wet dipstick) readings of the test strips and reading

after at least 30 min of drying.

Detailed results are provided in the separate tables. The ratio values always

decreased when the reading was postponed (longer time for the colour formation

and the dipsticks become dry); so the detection capability of the test increased by

postponing the reading. At the same time, throughout the drying of the strips, the

colour formation at both capture lines became more pronounced, which facilitated

visual reading.

3.3.5.4 Test for false-negative/false-positive results

To investigate the possibility of false-negative results, naturally incurred honey

samples from our collection were retested using the Tetrasensor Honey test kit. All

samples with a known concentration above the detection capability also gave

positive screening results when using the Tetrasensor Honey test kit.

The test, BELAC accredited since the end of 2004, has also been used at ILVO-T&V

routinely over the past 3 years. Positively screened samples were sent to an external

laboratory for confirmation. Out of the concentrations obtained in the positively

screened honey samples for tetracyclines, there was no indication that honeys with

tetracyclines above the detection limits found in this validation study, were missed in

the screening. Moreover, the concentration determined by LC-MS is sometimes far

below the detection limit of the Tetrasensor Honey screening test.

In 2004, our laboratory used the Tetrasensor Honey test kit in an international

proficiency test regarding tetracyclines, organized by Famille Michaud Apiculteurs

(Gan, France). Our Tetrasensor Honey results in this proficiency test (Beaune and

Garcet, 2004) are shown in Table 11, with exceptions from the 10 blanks, which

were all found to be negative (no false-positive samples).

The blind-coded positive honey samples in the proficiency test, which were naturally

contaminated with 4 µg kg-1 tetracycline or higher, all gave (low) positive results (wet

dipstick reading) for the Tetrasensor Honey test kit. Only one honey sample,

naturally contaminated with 3 µg kg-1 tetracycline, gave a negative result (wet


dipstick reading); however, when the dipstick was interpreted after 30 min, the same

sample already gave a low positive result.

Table 11. Tetrasensor Honey results of an international proficiency test: control of false-negative results of honey contaminated naturally with tetracyclines. Concentration of

tetracycline (in µg kg-1,LC-MS)

Tetrasensor Honey Result

Ratio on t=0 Vis on t=0 Ratio on t=30' Vis on t=30'

3 1.90 (negative) negative 1.34 (low positive) negative 4 1.12 (low positive) positive 0.47 (positive) positive 6 1.37 (low positive) positive 0.54 (positive) positive 10 0.48 (positive) positive 0.13 (positive) positive 25 0.23 (positive) positive 0.04 (positive) positive 38 0.05 (positive) positive 0.02 (positive) positive 72 0.00 (positive) positive 0.02 (positive) positive 77 0.10 (positive) positive 0.03 (positive) positive

Notes: Vis, visual reading; LC-MS results by Jean-Marc Diserens, Lausanne, Switzerland. In 2005, our laboratory also participated in another international collaborative trial on

antibiotic residues in honey, organized by the Laboratoire d‟Etudes et de

Recherches sur la Pathologie des Petits Ruminants et des Abeilles de l‟Anses

(Sophia Antipolis, France) (Martel et al., 2005). Three samples contained no

residues above the limit of detection of the LC-MS reference analysis. These

samples tested all negative for Tetrasensor Honey. In this collaborative trial, sample

6, containing 8.7 µg kg-1 tetracycline, yielded a negative result directly at the end of

the test (wet reading). However the result became „low positive‟ after half an hour

(dry dipstick reading). The other positive samples with concentrations of tetracycline

ranging from 19.8 to 31.7 µg kg-1 (LC-MS) were all detected as positive even during

wet reading. Five out of 7 laboratories using HPLC/Diode Array Detection (DAD)

reported sample 6 as negative; of the 22 laboratories using LC-MS, two laboratories

reported sample 6 as not detected and one laboratory as <5 µg kg-1.

So in both trials, no false-negative or false-positive results were obtained with the

Tetrasensor Honey test kit. In the 2004 proficiency test, even concentrations of

tetracycline below the detection capability of 9 µg kg-1 yielded „low positive‟ results

with the Tetrasensor Honey test kit. This could be explained by the dose–response

results as shown in Tables 2 and 3: concentrations just below the detection

capability could sometimes result in a positive result. Another explanation could also


be the time delay between the Tetrasensor analysis and the physicochemical

confirmation, which possibly resulted in a degradation of incurred tetracycline in

honey to degradation products with sterical similarity to the parent compound

(Münstedt et al., 2002). Finally, it‟s worth noting that Tetrasensor Honey test kit

detects not only the parent compounds but also the epimers.

If the data of the screening of 100 table honeys from different countries are

considered (Reybroeck et al., 2006), no false-positive or false-negative results were

obtained when the Tetrasensor Honey test kit was used as screening method,

whereas a rate of 8% false-positive results were obtained for the same honey when

the Charm II Tetracyclines Honey was used. In this study, 2 samples with a

tetracycline concentration below the detection capability tested positive for

Tetrasensor Honey; the presence of tetracyclines in both samples (4 and 6 µg

tetracycline kg-1) was confirmed by LC-MS/MS.

From this work, it can be concluded that Tetrasensor honey is a suitable test kit for

the rapid screening for tetracyclines in honey. The test takes 30 min. In general, no

differences were noticed between a visual and an instrumental reading of the

dipsticks. So a reader system is not required for screening purposes. Since no

special equipment (incubator, reader, etc.) is required, the test can be performed at

the production site even by the beekeeper himself.

The test detects the tetracyclines in honey in a specific and sensitive way.

Depending on the type of tetracycline concerned, a detection capability between 6

and 12 µg kg-1 was obtained directly after the second incubation period. When

reading dry dipsticks, detection capabilities between 4 to 7 µg kg-1 were obtained.

So, when the dipstick becomes dry, the detection capabilities improve.

The test procedure is very simple and the test is rugged. No influence on the test

capability was noticed, with regard to the geographical or botanical origin or by

physical parameters (solid versus liquid). Only small problems were encountered

with a Spanish honeydew honey.

We noticed differences in test capability between 2 different batches. However, the

differences remained limited, and when the tests were repeated 16 months later

using the second test kit, significant differences were no longer observed. A stability

study showed a slight increase of the test sensitivity during storage.


No false-negative and no false-positive results were obtained in two international

proficiency tests and a study of 100 table honey samples.

Acknowledgement The authors thank the company Unisensor s.a. for kindly supplying the test kits. The

authors wish to thank Kurt Hullebusch and Veronique Ottoy (ILVO-T&V) for practical

assistance during the validation experiments.

3.4 REFERENCES Adams S., Heinrich K., Caldow M., Sharma A., Ashwin H., Homer V., Stead S., Fusell R., Kelly M., Wilkins S., Thompson H., Sharman M. 2006. Investigation of the fate of veterinary drugs used in apiculture. Proceedings of the 5th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, Belgium, May 16-19, 2006: 27. Alfredsson G., Branzell C., Granelli K., Lundström A. 2005. Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC–MS/MS. Anal. Chimica Acta 529(1-2): 47-51. Alippi A.M. 2000. Is Terramycin losing its effectiveness against AFB. Bee Biz 11: 27-29. Anonymous. 2001. Advies 2001/11: Betreft: Residuen van antibiotica en sulfonamiden in honing (dossier Sc Com 2001/11). Wetenschappelijk Comité van FAVV. http://www.favv.be/home/com-sci/avis01_nl.asp. Anonymous. 2007. CRLs view on state of the art analytical methods for national residue control plans. CRL Guidance Paper (December 7, 2007): 1-8. Beaune P., Diserens J.M., Reybroeck W. 2005. Proficiency Testing of Charm II Tests for Residue Control of honey. Proceedings of the 39th Apimondia International Agricultural Congress, Dublin, Ireland, August 21-26, 2005: 33-34. Beaune P., Garcet C. 2004. Report Charm II Tetracycline ring trial. Famille Michaud Apiculteurs, Gan, France: 1-11. Bogdanov S. 2006. Contaminants of bee products. Apidologie 37: 1-18. Bogdanov S., Ruoff K., Persano Oddo L. 2004. Physico-chemical methods for the charac-terization of unifloral honeys: a review. Apidologie 35: 4-17. Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Comm. 2002, L 221: 8-36. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010 L15: 1-72. Council Directive 2001/110/EC of 20 December 2001 relating to honey. Off. J. Eur. Comm. 2002, L10: 47-52.


Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Comm. 1990, L224: 1-8. Diserens J.-M. 2010. Personal communication. Hopingarner R., Nelson K. 1987. American foulbrood cleanup rate using three terramycin treatments. Am. Bee J. 128: 120-121. Martel A.C. 2010. Personal communication. Martel A.C., Zeggane S., Halimi C. 2005. Report Results of collaborative trial on antibiotic residues in Honey-2005. International Honey Commission: 1-40. Martel A.C., Zeggane S., Drajnudel P., Faucon J.P., Aubert M. 2006. Tetracycline residues in honey after hive treatment. Food Addit. Contam. 23: 265-273. Miyagi T., Peng C.Y.S., Chuang R.Y., Mussen E.C., Spivak M.S., Doi R.H. 2000. Verification of oxytetracycline-resistant American foulbrood pathogen Paenibacillus larvae in the United States. J. Invertebr. Pathol. 75: 95-96. Münstedt T., Rademacher E., Petz M. 2002. Chlortetracycline and oxytetracycline residues in honey after administration to honey-bees. Proceedings of the 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, Belgium, June 4-7, 2002: 147. Persano Oddo L., Piro R. 2004. Main European unifloral honeys: descriptive sheets. Apidologie 35: 38-81. Reybroeck W. 2003. Residues of antibiotics and sulphonamides in honey on the Belgian market. Apiacta 38: 23-30. Reybroeck W. 2005. Validation of the Tetrasensor Honey for the screening of tetracyclines in honey. Proceedings 39th Apimondia International Apicultural Congress in Dublin, Ireland, August 21-26, 2005: 34. Reybroeck W., Ooghe S., Daeseleire E. 2006. Presence of antibiotics and sulfonamides in honey and royal jelly on the European market. Proceedings of the Second European Conference of Apidology EurBee 2006, Prague, Czech Republic, September 10-14, 2006: 117. Sharman M. 2010. Personal communication. Spivak M. 2000. Preventative antibiotic treatments for honey bees. A. Bee J. 140: 867-868. Thompson H.M., Waite R.J., Wilkins S., Brown M.A., Bigwood T., Shaw M., Ridgway C., Sharman M. 2005. Effects of European foulbrood treatment regime on oxytetracycline levels in honey extracted from treated honeybee (Apis mellifera) colonies and toxicity to brood. Food Addit. Contam. 22: 573-578. Waite R.J., Brown M.A., Thompson H.M., Brew M.H. 2003. Controlling European foulbrood with the shook swarm method and oxytetracycline in the UK. Bee World 82: 130-138.

Tran

Chapter 4 Transfer of Sulfamethazine from Contaminated Beeswax to Honey

Adapted from:

Reybroeck W., Jacobs F.J., De Brabander H.F., Daeseleire E. 2010. Transfer of

sulfamethazine from contaminated beeswax to honey. Journal of Agricultural and

Food Chemistry 58: 7258-7265.

4 Transfer of Sulfamethazine from Contaminated Beeswax to Honey 124

Transfer of sulfamethazine from contaminated beeswax to honey

Abstract

A liquid chromatographic-tandem mass spectrometric method for the determination

of sulfa drugs in beeswax was developed. When monitoring beeswax intended for

the fabrication of wax foundations, residues of sulfonamides were found.

A migration test was set up to study if sulfonamide-containing beeswax foundations

could lead to contamination of honey. The higher the concentration of

sulfamethazine doped in the wax, the higher the concentration of sulfamethazine

found in the honey. The maximum transfer of the initial amount spiked in the wax

foundation was 15.6, 56.9, and 29.5%, respectively.

In a second experiment, the percentage of sulfamethazine migrating from medicated

winter feed to beeswax in relation to the concentration in the syrup and the contact

time was studied. The maximum transfer of sulfamethazine from medicated sucrose

syrup to beeswax was 3.1%.


4.1 INTRODUCTION The use of sulfonamides to protect honey bees against bacterial diseases became a

common practice in commercial beekeeping after Haseman and Childers (1944)

learned that sulfa drugs, particularly sulfathiazole, could prevent the spread of

American foulbrood (AFB). The compound sulfathiazole provided a short-term

control by suppressing the symptoms of the bee disease caused by Paenibacillus

larvae. It also prevented the reproductive spores from germinating. The use of sulfa

drugs in the bees‟ food in spring and fall was also encouraged by other authors

(Eckert, 1947; Reinhardt, 1947; Johnson,1948; Katznelson and Gooderham, 1949;

Katznelson, 1950). Despite the effectiveness of sulfonamides against AFB, their

stability and consequent residues in honey caused problems, and the registration

was allowed to lapse in the 1970s (Shimanuki and Knox, 1994).

Another important bee pathogen is Nosema apis, which causes nosemosis, the most

widespread of all adult honey bee diseases. Until recently, Nosema apis had been

considered to be a microsporidian, a single-celled protozoan, but is now classified as

fungus or fungi-related (Fischer and Jeffrey, 2005). Recently, Nosema ceranae

showed to be widespread in some European regions, afflicting the adult bees and

resulting in depopulation and bee colony losses (Higes et al., 2006). The

effectiveness of fumagillin, an antibiotic prepared from Aspergillus flavus, was found

to be effective in 1952 by Katznelson and Jamieson (1952). It is commonly used in

beekeeping to prevent and control nosemosis in several parts of the world. In the

EU, fumagillin is no longer available (Piro and Mutinelli, 2003). Some publications or

manuals mention that sulfa drugs can be used against nosemosis (Lourdes, 2002;

Anon., 2010a) or to treat infections due to microsporidia (Liu and Weller, 1996;

Didier, 1998; Conteas et al., 2000).

Sulfonamides play an important role as effective chemotherapeutics for bacterial and

protozoal diseases in veterinary medicine. They are frequently administered in

combination with dihydrofolate reductase inhibitors of the group of

diaminopyrimidines (Anon., 2005). In Europe, sulfonamides were classified in Annex

I of Council Regulation (EEC) No 2377/90 with a maximum residue limit (MRL) fixed

at 100 µg kg-1 for the combined residues of all compounds in the sulfonamide group

in meat, fat, liver, and kidney of all food-producing species (since 1992) and in

bovine, ovine, and caprine milk (since 1994). In the current legislation, they are

classified as allowed substances with the same MRLs (Regulation (EC) No


470/2009; Commission Regulation (EU) No 37/2010). However, no MRLs are set for

sulfonamides in honey. As a consequence, a zero tolerance policy regarding

residues of sulfonamides in honey is applied. Belgium established an action limit for

sulfonamides in honey at 20 µg kg-1 (Anon., 2001) and in Switzerland, for

sulfonamides in honey a tolerance level of 50 µg kg-1 was applied till January 1, 2009

(Diserens, 2010). An action limit takes analytical possibilities and available

toxicological data into account in order to facilitate international trade. The European

Community Reference Laboratories for residues proposed 50 µg kg-1 as the

recommended concentration for screening for sulfonamides in honey (Anon., 2007b).

Despite the lack of MRLs for sulfonamides in honey, sulfa drugs could be used in the

EU in apiculture based on the cascade system as described in article 11 of Directive

2001/82/EC, as amended by Directive 2004/28/EC. The cascade system was

introduced to solve the general problem of availability of veterinary medicinal

products for minor species. The cascade system is open to all animal species,

including honeybees, provided that the active substance concerned has been

included in Annex I, II, or III of Council Regulation (EEC) No 2377/90 and the

prescribing veterinarian specifies the withdrawal period (Anon., 2007a).

In the framework of a Flemish honey quality programme, locally produced honey has

been examined at ILVO-T&V for the presence of residues of antimicrobials since the

year 2000. In the period 2000-2001, 4 out of 248 samples contained streptomycins,

2 out of 72 samples were positive for tetracyclines, and 3 out of 72 samples had

sulfa drug residues. For the streptomycin and tetracycline contamination, in most

cases the beekeeper admitted having added foreign honey to his production

(Reybroeck, 2003). In 2002, sulfa drugs were found in 3 out of 91 samples and in

2003, 12 out of 203 samples contained residues of sulfonamides. In most cases it

concerned sulfamethazine (up to 13,000 µg kg-1) and in one case, a combination of

sulfamethazine (458 µg kg-1) and sulfathiazole (1,229 µg kg-1) was found (Reybroeck

et al., 2004). Since 2004, no antimicrobial residues were found in locally produced

honey examined for the presence of (dihydro)streptomycin, tetracyclines,

sulfonamides, chloramphenicol, macrolides (since 2006), lincosamides (since 2006),

and fluoroquinolones (since 2006)) (Reybroeck et al., 2008b and unpublished data).

Questioning of the beekeeper proved that the high concentrations found in 2002 and

2003 were caused by the use of sulfonamides to prevent or to treat nosemosis.

However, in five honey samples, the contamination of sulfonamides was limited to


concentrations below 50 µg kg-1. The five producers involved all claimed not to have

used preparations containing sulfonamides. Hence, external sources of

contamination needed to be considered. Other authors reported about the presence

of residues of sulfonamides in honey (Heering et al., 1998; Martel and Zeggane,

2003; Morlot and Beaune, 2003; Posyniak et al., 2003; Wallner, 2003; Kaufmann

and Känzig, 2004; Sheridan et al., 2008). In 2008, there were 9 European

notifications in the Rapid Alert System for Food and Feed (RASFF) from the

Directorate-General for Health & Consumers about residues of sulfonamides in

honey (Anon., 2010b). It concerned sulfathiazole in honey from Hungary, Lithuania

(2 notifications), and Portugal; sulfamethazine in honey from Turkey (3 notifications)

and Egypt; and sulfadiazine in honey from China. In 2009 there was only 1

notification regarding sulfamethazine in honey from Turkey.

At ILVO-T&V, screening of honey samples on the presence of residues of sulfa

drugs is performed by Charm II Sulfonamides Honey (Charm Sciences Inc.,

Lawrence, MA). Confirmation of suspect samples is performed by a LC-MS/MS

method validated according to Commission Decision 2002/657/EC for seven

sulfonamides (sulfapyridine, sulfadiazine, sulfamethoxazole, sulfathiazole,

sulfamerazine, sulfamethazine, and sulfaquinoxaline), using sulfachloropyridazine as

internal standard.

Besides honey, beeswax is also an important bee product. Beeswax is a natural wax

produced by the worker bees in their wax-producing mirror glands on the inner sides

of the sternites on abdominal segments 4 to 7. The new wax scales are masticated

by the worker bees and used to build honeycomb cells in which their young are

raised and honey and pollen are stored. Ripened honey is also capped with wax.

Beeswax is an extremely complex material that contains over 300 different

substances. Its main components are palmitate, palmitoleic acid, hydroxypalmitate,

and oleate esters of long-chain aliphatic alcohols. Beeswax has a high melting point,

ranging from 61 to 65°C (Bogdanov, 2004). Beeswax is often contaminated with

residues of pesticides and acaricides that then contaminate the honey (Bogdanov et

al., 2003; Bogdanov, 2006). A LC-MS/MS method was developed for the

determination of sulfonamides in beeswax. When performing residue control on 10

samples of beeswax intended for the fabrication of wax foundations, residues of

sulfonamides were found in imported beeswax as well as in local beeswax. Crude

beeswax from India contained 325 µg kg-1 sulfadiazine, while crude Belgian beeswax


from hives treated with sulfonamides was contaminated with 62 µg kg-1 sulfadiazine.

The wax of a honeycomb of a Flemish apiary where sulfa-containing honey was

found, contained 14 µg kg-1 sulfamethazine (Reybroeck et al., 2004). To our

knowledge, no studies were performed in the past to check if sulfa-containing

beeswax could lead to contamination of the honey. A migration test was set up to

investigate this possibility.

A second experiment was set up to determine the residue concentrations build up in

beeswax after different contact times with sulfa-medicated syrup.

Parts of the results of this paper were presented at the 40th Apimondia International

Apicultural Congress in Melbourne, Australia (Reybroeck et al., 2007) and at

EuroResidue VI, Conference on residues of Veterinary Drugs in Food in Egmond

aan Zee, the Netherlands (Reybroeck et al., 2008a).

4.2 MATERIALS AND METHODS 4.2.1 Reagents and standards Sulfapyridine (S-6252), sulfadiazine (S-8626), sulfamethoxazole (S-7507),

sulfathiazole (S-0127), sulfamerazine (S-9001), sulfamethazine (S-6256),

sulfaquinoxaline (S-7382), and sulfachloropyridazine (S-9882, internal standard) all

came from Sigma-Aldrich (Bornem, Belgium). Individual standard stock solutions

were prepared by weighing approximately 5 mg of standard in a glass tube and

adding an appropriate amount of acetonitrile/water (50:50, v/v) to reach a

concentration of 1 mg ml-1. Stock solutions were stored at -18°C. Working solution 1,

containing the seven sulfa drugs at 10 µg ml-1, was prepared daily by diluting the

stock solutions in a mixture of acetonitrile-water (50:50, v/v). The composite working

solution 2 of 1 µg ml-1 was prepared by diluting working solution 1 in water.

n-Hexane (082906), acetonitrile (012078), methanol (136878), and formic acid

(069178) came from Biosolve (Valkenswaard, the Netherlands). Sodium acetate

(62648), sodium sulfate (106647), and acetic acid (1.00063) were from Merck KGaA

(Darmstadt, Germany). Hydrochloric acid (2M) (403872) came from Carlo Erba

(Milan, Italy). Sodium hydroxide (B405348019) came from BDH Laboratory Supplies

(Poole, United Kingdom). The MSU extraction buffer came from Charm Sciences

Inc.


Water was HPLC grade (generated by an ELGA purification system). Filters (Millex

GV, 0.22 µm, SLGVX13) for filtration of the extract were from Millipore (Billerica,

MA). Solid Phase Extraction columns were Sep-Pak tC18 columns (500 mg, 6 ml,

WAT036790) from Waters (Millford, MA). Syrup was prepared from Ultra Grade

sucrose (S7903) from Sigma-Aldrich. A syrup concentration of 67% w/v (2:1

sucrose/water by weight) was used. The blank beeswax, obtained from a Flemish

hobby beekeeper, had no evidence of any sulfa drug residue as tested by LC-

MS/MS analysis. The plastic Petri dishes of 14 cm diameter were from Plastiques-

Gosselin (Hazebrouck, France).

4.2.2 Apparatus The LC-MS/MS system for the honey and beeswax analyses consisted of an

Alliance Separations Module 2695 system from Waters coupled to Quattro LC (triple

quadrupole) of Micromass (Manchester, United Kingdom) equipped with the Z-spray

system. The MS system was controlled by version 3.3 of the MassLynx software

(Waters). Chromatography for honey and beeswax analysis was performed on a

XTerra MS C18 column (186000546, Waters). The column (particle size 5 µm, 150

mm x 2.1 mm i.d.) was protected by a guard column containing the same material.

The following small lab equipements were used: a shaking device KS250 from IKA

Werke GmbH & Co.KG (Staufen, Germany), a centrifuge Centra-CL3 from Thermo

IEC (Needham Heights, MA), an incubator BD240 with natural convection from

Binder GmbH (Tittlingen, Germany), a water bath and heating circulator from Julabo

Labortechnik GmbH (Seelbach, Germany), and an ultrasonic bath Branson 2200

Ultrasonic Cleaner from Branson Ultrasonics Corporation (Danbury, CT).

4.2.3 Sample preparation Honey. For honey, the clean-up was based on the method described by Maudens et

al. (2004). Honey was homogenized by manual stirring. An aliquot of 1.5 g of the

homogenized honey sample was weighed in a centrifuge tube of 50 ml. At this stage,

50 µg kg-1 of sulfachloropyridazine (Internal Standard) was added, and the sample

was allowed to stand for 20 min. After the addition of 1.5 ml of a 2M HCl solution, the

tube was placed on a shaking device for 30 min. After adjustment of the pH to 5.0

with 550 µl of 5M NaOH and 750 µl of 1.2M sodium acetate, 8 ml of acetonitrile was

added and the tube was placed again on the shaking device for 30 min. After


centrifugation for 30 min at 1800 RCF, the upper layer was decanted into a second

centrifuge tube and 5 g of sodium acetate was added. After shaking for 5 min, the

tube was centrifuged for 10 min at 1800 RCF and the liquid layer was transferred to

a glass tube. The extract was evaporated under nitrogen in a water bath of 45°C.

The residue was dissolved in 2 ml of a 1% acetic acid solution, and the tube was

placed for 5 min in an ultrasonic bath. Solid phase extraction columns (SPE C18)

were conditioned with 5 ml methanol and 2 x 5 ml of water. The honey extract was

brought onto the column and was allowed to flow through the column at a slow flow

rate. The column was washed with 5 ml of water and after drying, the sulfonamides

were eluted from the column with 1.3 ml of methanol. The extract was again

evaporated under nitrogen in a water bath of 45°C. The residues were dissolved in

500 µl of the mixture of acetonitrile/water (50:50, v/v) and 0.1% formic acid, and after

filtration, the extract was brought into a HPLC vial and 40 µl was injected into the

LC-MS/MS system. With each series of samples, a blank sample, samples spiked at

2, 5, 10, 20, 50, and 100 µg kg-1 sulfamethazine (calibration curve), and a second

line control sample (spiked by laboratory responsible or other technician) were

analysed.

Beeswax. Five grams of beeswax, cut into small pieces, was weighed in a centrifuge

tube of 50 ml. After the addition of 200 µg kg-1 sulfachloropyridazine as internal

standard, the beeswax was dissolved in 30 ml of hexane by shaking on a shaking

device at 500 rpm. Afterwards, 20 ml of MSU extraction buffer was added. The tube

was manually shaken for 2 min, sonicated for 5 min in an ultrasonic bath, and again

manually shaken for 2 min. After centrifugation for 10 min at 2850 RCF, the

supernatant was discarded and the water phase was transferred to another

centrifuge tube of 50 ml. The solid phase extraction column (SPE C18) was

conditioned with 5 ml of methanol and 2 x 5 ml of HPLC water. The extract was

allowed to flow through the column at a slow flow rate. The column was washed with

5 ml of water, and after drying, the sulfonamides were eluted from the column with

2.5 ml of methanol. The methanol was evaporated under nitrogen at 45°C, and the

residues were dissolved in 500 µl acetonitrile/water (50:50, v/v) containing 0.1%

formic acid, and after filtration, we brought the extract into a HPLC vial and injected

40 µl into the LC-MS/MS system. With each series of samples, a blank sample,

samples spiked at 100, 200, 500, 750, and 1000 µg kg-1 sulfamethazine (calibration

curve), and a second line control sample were analysed.


4.2.4 LC-MS/MS analysis The LC separation was performed on a reversed-phase column with an organic

mobile phase. The mobile phase consisted of water (A) and acetonitrile (B), both

containing 0.1 % formic acid. The gradient conditions were as follows: from 0-0.5

min, held 100 % A; ramped over 0.1 min to 55 % A and 45 % B; ramped over 7.9

min to 35 % A and 65 % B; ramped over 0.1 min to 100 % B; held for 10 min;

ramped over 1.4 min to 100 % A; held 100 % A for 10 min to reequilibrate the

system. The flow rate was 0.25 ml min-1. The abundant parent ions [M + H]+

produced by positive electrospray ionization were selected for collisional dissociation

with argon. When analysing honey or beeswax from unknown origin, for each of the

7 sulfa drugs the transition of the precursor ion into at least two product ions was

followed in multiple reaction monitoring in order to obtain enough identification

points, as required in Commission Decision 2002/657/EC. All compounds could be

detected in one run. A summary of the cone voltages, collision energies, precursor

and product ions, and retention times of the 7 compounds is presented in Table 1.

Table 1. Summary of tuning and chromatographic parameters for analysis of sulfonamides in honey. Compound Precursor

ion (m/z) Cone

Voltage (V) Product

ions (m/z) Collision energy

(eV)

Retention time (min)

sulfamethazine 279.06 35 155.94 91.99a 124.15

20 30 27

7.57

sulfapyridine 250.00 30 156.00a 92.10

108.00

15 28 25

7.35

sulfadiazine 250.94 27 156.06a 92.18

107.95

16 25 22

7.35

sulfamethoxazole 253.97 24 156.10 91.99a 108.07

15 25 25

8.21

sulfathiazole 256.07 26 156.19 92.11a 107.95

15 27 25

7.28

sulfamerazine 265.10 25 156.04a 92.01

108.00

17 28 25

7.50

sulfaquinoxaline 301.07 25 156.18a 92.00

108.04

17 30 28

8.49

sulfachloropyridazine 285.02 27 156.00 15 7.99 Note: a most abundant product ion.


The Quattro LCZ mass spectrometer was operated in the ESI-MS/MS positive ion

mode. High-purity nitrogen was used as drying gas and as ESI nebulising gas. Argon

was used as collision gas to obtain product ions. Dwell time and interchannel delay

were optimized with standard solutions and were set at 0.5 and 0.03, respectively.

The source block and desolvation temperature were set at 120 and 300°C,

respectively.

4.2.5 Calculation of partition coefficients The partition coefficient of some sulfonamides was calculated using Advanced

Chemistry Development (ACD/Laboratories) Software, version 8.14 (1994-2010

ACD/Laboratories, Toronto, Canada).

4.2.6 Experiments and sampling In a first experiment, we placed sulfamethazine-containing wax foundations in

beehives in order to see whether this practice could lead to contamination of the

produced honey. A wax foundation is a thin sheet of beeswax that is embossed with

the hexagonal shape that the bees naturally form for their honeycomb. The bees

draw out the comb by adding wax on top of the foundation to create hexagonal

cells, where the honey and pollen are stored, and the eggs are laid by the queen. In

general, beekeepers buy the wax foundations at a beekeepers‟ shop. The wax

foundations for the experiment were made by molding wax by means of negative

silicone templates of a beeswax sheet, pouring hot wax of 80°C into it and closing

the mold and allowing the wax to cool. To prepare sulfamethazine-containing wax

foundations, liquid blank beeswax was spiked with sulfamethazine at three different

levels, namely 1, 10, and 100 mg kg-1. Sulfamethazine was dissolved in 5 ml of

methanol, added to 120 g of liquid beeswax, and mixed by hand with a stainless

steel stirrer. After molding, a small amount of each wax foundation was sampled for

sulfamethazine residue analysis by LC-MS/MS. The spiked wax foundations were

placed in rectangular wooden frames with wires across the vertical centre to hold

the wax foundations in place. In mid-June, the start of the summer blossoming

season, each frame with a spiked wax foundation was placed close to the brood

nest in a different hive of the experimental apiary of UGent, to let the honeybees

(Apis mellifera L.) draw out the spiked wax foundation to honeycomb. The three


free-flying colonies were housed in double brood boxes with 11 Simplex standard

frames (34.0 cm by 19.6 cm of comb; 2.3 cm thickness of the wooden frame) and

one super box. After 1 week, the frames were transferred to the super box of the

hives to prevent the queen from laying eggs in the combs. The supers were

separated from the rest of the hives by a queen excluder. At four weeks from the

start, the combs contained capped honey, and the frames were removed from the

hives for a first sampling of honey. The honey combs were further incubated for 3

months in the laboratory in an incubator with natural convection at 35°C,

corresponding to the temperature in the hive, and sampled monthly. The sampling

of honey was performed by spooning from both sides of the comb. In the sampled

honey, the quantity of sulfamethazine was determined by LC-MS/MS. Honey was

also sampled from other frames present in the supers of the hives participating in

the trial. These control samples were analysed on the presence of residues of

sulfonamides. Before analysis, honey and wax were separated by sieving at a

temperature of 30-35°C to prevent interference by small wax particles present in the

honey.

A second experiment investigated the possible level of contamination of beeswax

caused by transfer from medicated syrup solution. Large Petri dishes were filled with

30 ml (=29 g) of blank beeswax. Winter feed solution of 66% w/v sucrose was

prepared and spiked with sulfamethazine at three different concentrations, namely

30, 150, and 750 mg l-1. In each Petri dish, 50 ml of medicated syrup was poured on

top of the 0.2 mm layer of beeswax, the plates were closed and sealed to prevent

evaporation, and placed undisturbed in an incubator at 35°C for 3 months. After 14

days, 1, 2, and 3 months, four Petri dishes per sulfamethazine concentration were

opened. The syrup was discarded, and the beeswax was thoroughly washed with

distilled water and dried with absorbent paper. The amount of sulfamethazine in the

wax samples was determined by LC-MS/MS.


4.3 RESULTS AND DISCUSSION The results for the determination of sulfa drugs in honey and wax are given in Table

2.

Table 2. Mean value (in µg kg-1), standard deviation (in µg kg-1), and coefficient of variation (in %) for the determination of sulfonamides in honey (n=6) at 2 and 4 µg kg-1, and sulfamethazine in beeswax (n=4) at 25 and 100 µg kg-1. Honey

Compound Honey spiked at 2 µg kg-1 Honey spiked at 4 µg kg-1

Mean (µg kg-1)

SDa (µg kg-1)

CVb (%) Mean

(µg kg-1) SDa

(µg kg-1) CVb (%)

sulfamethazine 2.00 0.14 7.00 4.27 0.17 3.92 sulfapyridine 2.09 0.08 3.85 4.11 0.19 4.64 sulfadiazine 2.02 0.16 8.04 4.07 0.13 3.31 sulfamethoxazole 2.08 0.07 3.43 4.10 0.24 5.88 sulfathiazole 2.01 0.25 12.23 3.97 0.22 5.49 sulfamerazine 2.07 0.09 4.47 4.24 0.11 2.48 sulfaquinoxaline 2.00 0.14 7.00 4.10 0.12 3.00 Beeswax

Compound Wax spiked at 25 µg kg-1 Wax spiked at 100 µg kg-1

Mean (µg kg-1)

SDa (µg kg-1)

CVb (%) Mean

(µg kg-1) SDa

(µg kg-1) CVb (%)

sulfamethazine 26.90 0.86 3.20 117.57 8.46 7.19 Notes: a SD, standard deviation. b CV, coefficient of variation. For the 7 sulfa drugs tested in honey, the decision limit (CCα) was ≤2 µg kg-1, and

depending on the type of sulfa drug, the detection capability (CC) was in the range

of 2.2-2.4 µg kg-1. The mean value, standard deviation, and coefficient of variation

for six blank honey samples spiked at 2 µg kg-1(CCα) and 4 µg kg-1 (2x CCα) were

determined. The decision limit (CCα) for sulfamethazine and sulfadiazine in beeswax

was 20 µg kg-1; the detection capability (CC) was 25 µg kg-1. The mean value,

standard deviation, and coefficient of variation for four blank beeswax samples

spiked at 25 and 100 µg kg-1 were calculated.

All residue analyses were performed at ILVO-T&V. Since the spiking with

sulfamethazine took place in heated liquid beeswax at 80°C, leading to a certain

degradation of the sulfa-drug, parts of the spiked wax foundations were analysed by

LC-MS/MS to determine the concentration of sulfamethazine in the wax foundations.


Quattro LC

Time 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

%

0

100 Comb B 1:100 2: MRM of 1 Channel ES+

285.02 > 156 1.22e4

Area

8.01 2175

Comb B 1:100 1: MRM of 3 Channels ES+ 279.06 > 124.15

1.21e5 Area

7.60 20133


1.05e5 Area

7.60 17050


1.53e5 Area

7.60 27349

I.S.: sulfachloropyridazine

sulfamethazine

sulfamethazine

sulfamethazine

Figure 1 shows the chromatogram of the detection of sulfamethazine in the wax

foundation of comb B, 100 times diluted.

Figure 1. Chromatogram of the wax foundation of comb B, 100 times diluted. x axis, retention time in min; y axis, abundance (%). Sulfachloropyridazine = internal standard.

The wax foundations used in the migration experiment contained 590 (comb A),

3,760 (comb B), and 73,000 (comb C) µg kg-1 sulfamethazine. This indicates that 41,

62, and 27% of the initial spiked concentration, respectively, was lost due to heat

degradation. The differences in degradation could possibly be explained by the

longer time spent to mold the wax foundation spiked at 10 mg kg-1 sulfamethazine.

The weight of a wax foundation was approximately 60 g; hence, the total amount of

sulfamethazine present in the wax foundations was 35, 226 and 4,380 µg,

respectively.

The results of the migration of sulfamethazine to the honey during storage of the

honey in the combs are shown in Table 3. The higher the concentration of

sulfamethazine in the wax, the more residues were found in the honey. Migration of

residues from the beeswax to the honey in combs B and C was still continuing in the

first month of storage of the frames in the incubator.


Table 3. Concentration (in µg kg-1) and amount (in µg) of sulfamethazine in honey and percentage of transfer (in %) of sulfamethazine from beeswax at different sampling dates (n=1)a.

Time after start of

experiment (months)

Sulfamethazine concentration (µg kg-1)

in honey

Sulfamethazine (µg) in honey

Transfer (%)b of sulfamethazine

Comb A Comb B Comb C Comb A Comb B Comb C Comb A Comb B Comb C 1 <2 10 176 <4 18 323 <10.4 8.1 7.4 2 3 52 628 6 95 1,152 15.6 42.3 26.3 3 2 58 704 4 106 1,292 10.4 47.2 29.5 4 3 70 605 6 128 1,110 15.6 56.9 25.3

Hive A Hive B Hive C control comb <2c <2c 4c Notes: a Comb A, B, and C with 590; 3,760; and 73,000 µg kg-1 sulfamethazine in the wax

foundation, respectively. b Percentages of transfer are based on 60 g of wax foundation and 1.835 kg of honey

per comb. c Concentration (in µg kg-1) of sulfamethazine in honey sampled from the control

combs with residue-free beeswax from the beehives involved in the transfer experiment.

From the second sampling on, rather steady residue values were found in the honey.

A level of 590 µg kg-1 sulfamethazine in the wax foundation resulted in measurable

contaminations in the honey, while 3,760 µg kg-1 sulfamethazine in the wax

foundation resulted in sulfamethazine contaminations in the honey, surpassing the

recommended concentration for the screening of 50 µg kg-1 as proposed by the EU

Community Reference Laboratories for residues. The control honey samples from

hives A and B were free of sulfa residues, while 4 µg kg-1 of sulfamethazine was

found in the control sample from the third hive. The control honey results excluded

external sources of contamination and indicated that the sulfamethazine in the honey

samples fully originated from the spiked beeswax. Regarding the low contamination

of 4 µg kg-1 of sulfamethazine in the comb of hive C, it is known that bees always

recuperate and move material. Therefore, it‟s possible that bees of that hive were

transferring small quantities of contaminated honey to other honey combs in the

same super.

The sampling of honey by spooning was always performed at places where the

comb was fully built out by the bees to the thickness of the wooden frame (2.3 cm)

and filled with capped honey. A frame completely filled with honey contained

approximately 1.84 kg of honey. At all sampling sites on the comb, the wax/honey


ratio was consequently always 60 g/1.84 kg. When these conditions (60 g of wax

foundation and 1.84 kg of honey) were taken into account, the maximum amount of

sulfamethazine that migrated from the three spiked wax foundations to the honey

was 15.6, 56.9, and 29.5%, respectively. These data show that sulfamethazine is not

that lipophilic and is quite easily released from the wax to migrate to the honey in the

combs. In our experiment, sulfamethazine was used since this sulfonamide was the

most commonly identified in Flemish honey. The probability that other sulfonamides

also transfer from contaminated beeswax to honey is very high. The percentage of

transfer is depending on the lipophilicity of the substance. The octanol/water partition

coefficient for some sulfa drugs was calculated with specialized software. The log P

values or the logarithm of the ratio of the concentrations of the un-ionized sulfa drugs

in the solvents water and octanol are shown in Table 4. Chemicals with low log P

values (e.g., less than 1) may be considered relatively hydrophilic; conversely,

chemicals with high log P values (e.g., greater than 4) are very hydrophobic. The log

P value for sulfamethazine (0.803, relatively hydrophilic) explains the high

percentages of transfer of sulfamethazine from beeswax to honey. In comparison to

sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfachloropyridazine are

somewhat more lipophilic, while sulfapyridine, sulfadiazine, sulfathiazole, and

sulfamerazine are more hydrophilic.

Table 4. log P Values for some sulfa drugs.

Compound CAS number log P sulfamethazine 57-68-1 0.8030.259 sulfapyridine 144-83-2 0.0340.318 sulfadiazine 68-35-9 -0.1170.255

sulfamethoxazole 723-46-6 0.8870.419 sulfathiazole 72-14-0 0.0470.396

sulfamerazine 127-79-7 0.3430.257 sulfaquinoxaline 59-40-5 1.3050.385

sulfachloropyridazine 80-32-0 1.0180.622

The transfer results of the in vivo experiment indicate that the purchase and the use

of contaminated wax foundations by the beekeeper can lead to low-level

sulfonamide contaminations in honey. Beekeepers can be advized to recycle their

own beeswax for the fabrication of the wax foundations or to ask for a certificate

when buying wax foundations from wax transforming factories that are mostly using

wax from unknown origins. Under practical conditions, wax is recycled from different


types of comb material, namely, brood combs, honey combs, and cappings. At wax

transforming factories, a single lot of highly contaminated wax could cause a high

residual concentration in all resulting foundations (Lodesani et al., 2003). The use of

a synthetic foundation wax like Syncera® (Jacobs and Remon, 2001) could also be

an effective alternative. Fully drawn plastic foundations, with the hexagonal worker

cells embedded in the plastic, are also available. This study also indicates that in

case authorities are considering a zero-tolerance for antimicrobial residues in honey,

beekeepers could lose their honey production just by using wax foundations bought

on the market. The use of action limits could be an answer to this problem.

However, not all cases of low-level sulfonamide contamination in Flemish honey can

be claimed to be caused by the use of sulfa-contaminated wax foundations. Other

sources of sulfonamide contamination could be the robbery by bees of contaminated

honey from hives of other apiaries (Seiler and Kaufmann, 2002), the feeding of

sulfonamide-contaminated honey to bee colonies by the beekeeper, and the mixing

of privately produced honey with external honey of dubious quality. There is also the

possibility that the source of contamination with sulfonamides is related to

agricultural practices, e.g., bees can collect sulfa-medicated drinking water from

poultry farms, rabbit cages, or race-pigeon lofts. The manure of animals (pigs, or

cows) treated with sulfonamides or sulfa-containing surface water could also be the

vector (Richter et al., 2005). In the case of the low contamination of Flemish honey,

the link to agriculture cannot be completely ruled out. However, it is considered to be

rather unlikely for some sulfa drugs without a registration in Belgium, e.g.

sulfamethazine. Contamination of honey with residues of sulfanilamide could also be

caused by the collection by bees of nectar from meadows treated with the herbicide

asulam. Such honey is not only contaminated by asulam but also by its degradation

product sulfanilamide (Kaufmann and Känzig, 2004).

Beeswax and propolis are most likely to be contaminated with synthetic acaricides

since varroacides have to be used for long-term Varroa destructor control. Synthetic

acaricides like bromopropylate, coumaphos, fluvalinate, and flumethrin are mostly

fat-soluble and persist in wax. After acaricide treatments, they accumulate in wax

and contaminate honey to a much lesser extent (Bogdanov et al., 1998; Wallner,

1999). The rate of transfer of acaricides to honey is directly related to their


lipophilicity. Recycling of wax by melting of combs has no degradation effect on

acaricide concentration (Bogdanov et al., 1998). The use of CheckMite+®

(coumaphos) for the chemical treatment of the small hive beetle (Aethina tumida)

even results in higher residue levels in wax and honey than those found after the use

of coumaphos against Varroa (Nasr and Wallner, 2003). para-Dichlorobenzene and

naphthalene are sometimes used for wax moth (Galleria mellonella and Achroia

grisella) control. These practices result in residues of these compounds in the wax

and honey (Bogdanov, 2006). But the experiment described in this paper is the first

to report that contaminated beeswax could be the vector of honey contamination with

antimicrobial residues. There is a high persistence of sulfonamide residues in honey

and in beeswax inside the hive. In 1950, a patent was given to the invention of bee

comb foundation with sulfathiazole incorporated in the beeswax to protect the hives

against AFB (Small, 1950). Sulfa drugs are known to remain effective at a hive

temperature of 34°C for at least 3 years (Katznelson and Jamieson, 1954). No

information was available on the persistence of sulfa drugs in recycled beeswax. The

persistence depends on the lipophilicity of the substances and their stability at

temperatures above the melting point of beeswax. In our experiment, beeswax was

melted on a hotplate at 80°C, and a maximum loss of 62% was stated. In wax

transforming factories where large amounts of beeswax are melted, the heating time

will be much longer than the time applied for the molding of the three spiked wax

foundations in our experiment. On the other hand, there are serious indications that

re-melting of wax is not eliminating all sulfa residues.

In order to check to what extent the beeswax can be contaminated by the application

of sulfa drugs, the transfer from medicated syrup solution to blank beeswax was

investigated in an in vitro experiment. The migration of sulfamethazine to beeswax

from 3 syrup solutions with a different sulfamethazine concentration was followed for

3 months. The results of the sulfamethazine concentrations measured by LC-MS/MS

in the beeswax after 14 days, 1, 2, and 3 months of contact with 50 ml of medicated

sucrose syrup are summarized in Table 5.


Table 5. Concentration (in µg kg-1) and amount (in µg) of sulfamethazine in 30 ml of beeswax and percentage of transfer (in %) of sulfamethazine from syrup to blank beeswax after 14 days, 1, 2, and 3 months of contact with 50 ml of medicated syrup solution (n=1)a.

Incu-bation

Sulfamethazine concentration

(µg kg-1) in beeswax

Sulfamethazine (µg) in beeswax

Transfer (%) of sulfamethazine

Syrup A Syrup B Syrup C Syrup A Syrup B Syrup C Syrup A Syrup B Syrup C 14 days 109 500 2,000 3 15 58 0.2 0.2 0.2 1 month 115 2,000 5,000 3 58 145 0.2 0.8 0.4 2 months 96 8,000 6,000 3 232 174 0.2 3.1 0.5 3 months 101 7,000 24,000 3 203 696 0.2 2.7 1.9 Note: a Syrup A, B, and C spiked with 30,000; 150,000; and 750,000 µg l-1 sulfamethazine, respectively.

A concentration of 30 mg kg-1 sulfamethazine in the syrup resulted in a maximum

sulfamethazine concentration of 115 µg kg-1 in the beeswax, and a plateau was

reached after 2 weeks of contact. Concentrations of 150 and 750 mg kg-1

sulfamethazine in the syrup resulted in maximum 8,000 and 24,000 µg kg-1

sulfamethazine in the beeswax, respectively. The maximum level was reached after

2 and 3 months of contact, respectively. The total amount of sulfamethazine in the

syrup on top of the wax was 1.5 mg (syrup A), 7.5 mg (syrup B), and 37.5 mg (syrup

C), respectively. The maximal amount of sulfamethazine transferred was 3, 232, and

696 µg or 0.2, 3.1, and 1.9%, respectively. There is no direct explanation for the

rather big differences in transfer percentages. As could be expected for the rather

hydrophilic sulfamethazine (log P=0.803), these data indicate that beeswax has a

certain ability to retain sulfamethazine. Based on the log P values mentioned in

Table 4, the same could be supposed for other sulfa drugs.

In Europe, the provision of winter feed is normally given by the beekeepers before

the end of September. This feed is stored by the bees in the combs and will be

consumed during wintertime and in spring. The contact time is in practice as long as

or even longer than the three months applied in the experiment. Mutinelli (2003)

mentions an application of sulfathiazole in USA against European foulbrood. During

three gorging treatments at 4 to 5 day intervals, they administered medicated syrup

containing 265 mg l-1 sulfathiazole (equivalent to 1 g of sulfathiazole per gallon). This

is in the range of the concentrations applied in the experiment. In reality, the ratio of

volume of stored syrup in capped cells in relation to the weight of beeswax is much


higher than the ratio of 50 ml/29 g in the experiment. Therefore, in reality higher

transfer percentages could be expected.

This experiment indicates that after application of sulfamethazine, high

concentrations of sulfamethazine residues can be expected in the wax. This is likely

to be the case for other sulfa drugs as well. However, the monitoring data reveal

rather low sulfa concentrations (62 and 325 µg kg-1 of sulfadiazine in bulk beeswax

and only 14 µg kg-1 of sulfamethazine in wax from a honey comb of a hive with

production of sulfa- contaminated honey). A possible explanation could be that the

contaminated beeswax is largely diluted by pure beeswax coming from different

apiaries. Concerning the wax of the individual honey comb, there is no certainty that

this comb was already present in the hive during the sulfa application in the winter

feed of the previous year. It‟s possible that the comb sampled in summer was

originating from a wax foundation given in spring.

4.4 CONCLUSIONS The presence of residues of sulfonamides in beeswax was confirmed by a newly-

developed LC-MS/MS method. A migration experiment followed the transfer of

sulfamethazine from beeswax to honey. The higher the concentration of

sulfamethazine doped in the wax, the higher the concentration of sulfamethazine

found in the honey. The maximum transfer of the initial amount spiked in the wax

foundation to honey was 15.6, 56.9, and 29.5%, respectively.

A second in vitro experiment determined the percentage of sulfamethazine migrating

from medicated winter feed to beeswax in relation to the concentration in the syrup

and the contact time. The maximum transfer of sulfamethazine from medicated

sucrose syrup to beeswax was 3.1%. The higher the concentration of sulfamethazine

doped in the syrup, the longer it takes to get the highest percentage of transfer.

The results of both experiments indicate that after the use of sulfonamides in the

hive, a pool of residues remains in the wax of the combs. Residues remaining in the

beeswax can contaminate the honey during the next honey season. Therefore, the

harvest and the destruction of the first honey production are not sufficient measures

to guarantee residue-free honey in later productions. Especially in the countries

without tolerance levels for sulfa drugs in honey, low concentrations of sulfonamides

in honey could have serious implications. Our findings also imply that veterinarians

prescribing sulfonamides in apiculture based on the cascade system may find it


difficult to indicate withdrawal periods. These consequences should also be kept in

mind should the European Commission choose to fix MRLs for anti-infectious agents

in honey. The results also indicate serious implications regarding the recycling of

beeswax.

Acknowledgement We appreciate the excellent practical work performed by Petra De Neve, Kurt

Hullebusch, Katleen Vander Straeten, and Patricia Van Herreweghe (ILVO-T&V) and

the language correction of the manuscript by Miriam Levenson (ILVO) .

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Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010 L15: 1-72. Conteas C.N., Berlin O.G.W., Ash L.R., Pruthi J.S. 2000. Therapy for human gastrointestinal microsporidiosis. Am. J. Trop. Med. Hyg. 63(3): 121-127. Council Regulation (EEC) No 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Comm. 1990 L224: 1-8. Didier E.S. 1998. Microsporidiosis. Clin. Infect. Dis. 27: 1-8. Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products. Off. J. Eur. Comm. 2001 L311: 1-66. Directive 2004/28/EC of the European Parliament and of the Council of 31 March 2004 amending Directive 2001/82/EC on the Community code relating to veterinary medicinal products. Off. J. Eur. Union 2004 L136: 58-84. Diserens J.-M. 2010. Personal communication. Eckert J.E. (1947) Use of sulfa drugs in the treatment of American foulbrood disease of honeybees. J. Econ. Entomol. 40: 41-44. Fischer M.W., Jeffrey D.P. 2005. Evidence from small-subunit ribosomal RNA sequences for a fungal origin of Microsporidia. Mol. Phylogenet. Evol. 36: 606-622. Haseman L., Childers L.F. 1944. Controlling American foulbrood with sulfa drugs. Univ. Missouri Agric. Exp. Sta. Bull. 482: 3-16. Heering W., Usleber E., Dietrich R., Märtlbauer E. 1998. Immunochemical screening for antimicrobial drugresidues in commercial honey. Analyst 123(12): 2759-2762. Higes M., Martin R., Meana A. 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92(2): 93-95. Jacobs F.J., Remon J.P. 2001. The discovery and practical use of a substitute for bee-wax. Poster presentation Apimondia International Apicultural Congress, Durban, Republic of South Africa. Johnson J.P. 1948. Sulfa drugs for American foulbrood of honeybees: Third Report. J. Econ. Ent. 41(2): 314-318. Katznelson H. 1950. The influence of antibiotics and sulpha drugs on Bacillus larvae, cause of American foulbrood of the honeybee, in vitro and in vivo. J. Bacteriol. 59: 471-479. Katznelson H., Gooderman C.B. 1949. Sulfathiazole in relation to American foulbrood. Sci. Agric. 32: 180-184. Katznelson H., Jamieson C.A. 1952. Control of Nosema disease of honeybee with fumagillin. Science 115: 70-71.


Katznelson H., Jamieson C.A. 1954. Note on current studies on the chemotherapy of American foulbrood of the honeybee and on the stability of sulpha drugs in honey. Sci. Agr. 34: 120. Kaufmann A., Känzig A. 2004. Contamination of honey by the herbicide asulam and its antibacterial active metabolite sulphanilamide. Food Addit. Contam. 21(6): 564–571. Liu L.X., Weller P.F. 1996. Antiparasitic drugs. N. Eng. J. Med. 334(18): 1178-1182. Lodesani M., Costa C., Bigliardi M., Colombo R. 2003. Acaricide residues in bee wax and organic beekeeping. Apiacta 38: 31-33. Lourdes J. 2002. Evaluaciόn de un méthodo de análisis de residuos de sulfamidas, en miel de abejas (Apis mellifera L.), a través de cromatografía líquida de alta precisiόn (HPLC), en fase reversa. Thesis Universidad Austral de Chile, Facultad de Ciencias Agrarias, Escuela de Ingeniería en Alimentos, Valdivia, Chili. Martel A.C., Zeggane S. 2003. HPLC determination of sulfathiazole in French honeys, J. Liq. Chromatogr. Relat. Technol. 26: 953-961. Maudens K., Zhang G.-F., Lambert W. 2004. Quantitative analysis of twelve sulphonamides in honey after acidic hydrolysis by high-performance liquid chromatography with post-column derivatization and fluorescence detection. J. Chromatogr. 1047(1): 85-92. Morlot M., Beaune P. 2003. An experience with Charm II system. Apiacta 38: 15-20. Mutinelli F. 2003. Practical application of antibacterial drugs for the control of honey bee diseases. Apiacta 38: 149-155. Nasr M.E., Wallner K. 2003. Miticides residues in honey and wax in North America. Am. Bee J. 143: 322. Piro R., Mutinelli F. 2003. The EU legislation for honey residue control. Apiacta 38: 226-234. Posyniak A., Zmudzki J., Niedzielska J., Sniegocki T., Grzebalska A. 2003. Sulfonamide residues in honey. Control and Development of analytical procedure. Apiacta 38: 249-256. Reinhardt J.F. 1947. The sulfathiazole cure of American foulbrood: an explanatory theory. J. Econ. Entomol. 40: 45-48. Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Union 2009 L152: 11-22. Reybroeck W. 2003. Residues of antibiotics and sulphonamides in honey on the Belgian market. Apiacta 38: 23-30. Reybroeck W., Daeseleire E., Ooghe S., Jacobs F. 2004. Sulpha drugs in honey and other bee products. Abstract Apimondia symposium 2004 “Prevention of Residues in Honey 2”, Celle, Germany, April 27-28, 2004.


Reybroeck W., Daeseleire E., Jacobs F. 2007. Residue formation of sulfonamides in honey by migration from contaminated beeswax. Oral presentation on September 19, 2007 at 40th Apimondia International Apicultural Congress, Melbourne, Australia, September 9-13, 2007. Programme & Abstracts: 206. Reybroeck W., Daeseleire E., Jacobs F. 2008a. Can sulfa-contaminated beeswax lead to residues in honey? Oral presentation on May 19, 2008 at EuroResidue VI, Conference on residues of Veterinary Drugs in Food, Egmont aan Zee, the Netherlands, May 19-21, 2008. Proceedings edited by L.A. Van Ginkel & A.A. Bergwerff, ISBN 989-90-804925-3-0: 43-45. Reybroeck W., Ooghe S., Jacobs F. 2008b. 10 Jaar honinganalyses in Vlaanderen (1998-2007). Themanummer HONING. Maandblad van de Vlaamse Imkersbond, Augustus 2008: 4-16.

Richter D., Bogdanov S., Edder P. 2005. Antibiotikarückstände – von der Gülle in der Honig? Schweiz. Bienen-Ztg. 128(2): 23-25. Seiler K., Kaufmann A. 2002. Kontamination von Honig mit Sulfathiazol durch Raüberei unter Bienen. Mitt. Geb. Lebensmittelunters. Hyg. 93: 437-446. Sheridan R., Policastro B., Thomas S., Rice D. 2008. Analysis and occurrence of 14 sulfonamide antibacterials and chloramphenicol in honey by Solid-Phase Extraction followed by LC/MS/MS analysis J. Agric. Food Chem. 56(10): 3509-3516. Shimanuki H., Knox D.A. 1994. Susceptibility of Bacillus larvae to Terramycin. Am. Bee J. 134: 125-126. Small A.V. 1950. Beecomb foundation. Application November 18,1947. Serial No. 786,791. Patented Jan. 17, 1950 No. 2,494,9047. United States Patent Office. Wallner K. 1999. Varroacides and their residues in bee products. Apidologie 30: 235-248. Wallner K. 2003. Sulfonamide-residues in German honey – The actual situation. Apidologie 34: 485.

5 Vation of the βeta-s.t.a.r. 1+1 139

Chapter 5 Validation of the βeta-s.t.a.r. 1+1

Adapted from:

Reybroeck W., Ooghe S., De Brabander H.F., Daeseleire E. 2010. Validation of the

βeta-s.t.a.r. 1+1 for rapid screening of residues of β-lactam antibiotics in milk. Food

Additives & Contaminants: Part A 27(8): 1084-1095.

5 Validation of the βeta-s.t.a.r. 1+1 148

Validation of βeta-s.t.a.r. 1+1 for rapid screening for residues of β-lactam antibiotics in milk

Abstract

The 2-min protocol (1+1) for the βeta-s.t.a.r. (manufactured by Neogen Corporation,

Lansing, MI) was validated at the Technology and Food Science Unit of the Institute

for Agricultural and Fisheries Research according to Commission Decision

2002/657/EC. The test is very selective for the group of β-lactam compounds: the

only interference found was by clavulanic acid at 2,500 µg kg-1 and above. The

modified protocol (βeta-s.t.a.r. 1+1) detected all β-lactams with a maximum residue

limit (MRL) in milk, but not all these compounds were detected at their respective

MRL. The detection of desfuroylceftiofur (detection capability = 6,000 µg kg-1; MRL =

100 µg kg-1), cefalexin (detection capability = 6,000 µg kg-1; MRL = 100 µg kg-1), and

penethamate (detection capability = 80 µg kg-1; MRL = 4 µg kg-1) was especially

poor, and also ceftiofur (MRL = 100 µg kg-1) was only detected from 500 µg kg-1.

Ampicillin, amoxicillin, nafcillin, cefquinome, cefazolin, and desacetylcephapirin were

also not detected at MRL. The repeatability of the reader and of the test was very

good. The test is very robust: test results were not significantly influenced by small

changes in the test protocol, by the milk composition, or by the type of milk. The test

was also suitable to test milk of animal species other than the cow. Favourable

results were obtained in testing monitoring samples, in two national ring trials, and in

an international proficiency test.

The βeta-s.t.a.r. 1+1 is a very fast, simple, and reliable test that could be used at the

farm level to prevent tanker milk contamination by β-lactams.


5.1 INTRODUCTION Penicillins and cephalosporins belong to the group of β-lactam antibiotics due to their

common β-lactam ring structure. Penicillins remain very important in human and

veterinary medicine. In 1997, 225 metric tonnes of penicillins were administered to

animals in the European Union (Anon., 1998).

Mastitis is the most expensive disease affecting dairy cattle worldwide and,

therefore, the single largest cause of antibiotic usage in dairy herds. Bacteria from

the genera Staphylococcus and Streptococcus, both Gram-positive, are the most

common causal agents of mastitis (Pyörälä, 1995). β-lactam antibiotics are the most

frequently administered drugs in parenteral and intra-mammary therapy. The drug of

choice in many countries is still penicillin, since the minimum inhibitory concentration

(MIC) and minimum bactericidal concentration (MBC) values for the most common

mastitis pathogens are very low. When mastitis pathogens show penicillin

resistance, a combination of penicillins and clavulanic acid or isoxazolyl penicillins

resistant to penicillinase are often used as alternative treatment (Pyörälä,1995).

Penicillins all have the same ring structure and are monobasic acids that readily form

salts and esters. The penicillin nucleus, 6-aminopenicillanic acid, consists of a fused

thiazolidine ring and a β-lactam ring with an amino group at the 6-position. The

cephalosporins are semi-synthetic antibacterials derived from cephalosporin C, a

natural antibiotic. The active nucleus is very closely related to the penicillin nucleus

and consists of a β-lactam ring fused with a six-membered dihydrothiazine ring and

having an acetoxymethyl group at position 7. Penicillins and cephalosporins are

bactericidal and act by inhibiting synthesis of the bacterial cell wall (Anon., 2005).

All antimicrobial drugs administered to cows enter the milk to some degree. Testing

for antimicrobial drug residues (i.e., the drug itself or a metabolite) in milk is therefore

necessary for ethical, health, and technological reasons (Honkanen-Buzalski and

Reybroeck, 1997). The presence of β-lactam residues in milk can have several

drawbacks: inhibition of dairy starter cultures used in the production of cheese and

yoghurt (Suhren, 1996; Grunwald, 2002), possible hypersensitivity reaction by the

consumer, and contribution to the development of antibiotic resistance.

In the European Union, maximum residue limits (MRLs) were fixed in bovine milk for

16 β-lactam compounds, ranging from 4 to 125 μg kg-1 (Regulation (EC) No

470/2009; Commission Regulation (EU) No 37/2010), and in many countries

inhibitory substances are screened in routine in farm milk samples as part of the


regulatory quality programme. In 2009, of the 1,374,801 farm milk samples in

Belgium analysed, 872 or 0.06% were found to be positive by the milk control

stations (Anon., 2010a & b). In most cases, residues of β-lactam substances were

the main reason for bulk tank milk failure. In an identification study performed at the

Technology and Food Science Unit of the Institute for Agricultural and Fisheries

Research (ILVO-T&V) on all positive Flemish farm milk samples leading to

penalization in the months May and June of 2003, 79% of the samples contained

non-synthetic penicillins, 8% synthetic penicillins, and 4% a combination of β-lactam

and non-β-lactam residues. In total, therefore, 91% of the samples contained β-

lactams (Reybroeck and Daeseleire, 2003). As given in the year report of 2005 of

Comité du Lait (Walloon region) the percentage of penalizations due to β-lactams

was 94.5% in 2003, 90.0% in 2004, and 83.9% in 2005 (Anon., 2006). In Germany,

β-lactams could, in 95% of cases, be identified in inhibitor-positive milk samples

(Kress et al., 2007). Benzylpenicillin was still the predominant antibiotic detected

(74.6%) during regulatory control, followed by ceftiofur (11%), ampicillin/amoxicillin

(6.3%), and isoxazolyl penicillins (3.2%).

It is worth noting that in routine testing mostly microbial inhibitor tests are used, often

with Geobacillus stearothermophilus var. calidolactis as the test organism, chosen

for its high sensitivity for penicillins (Suhren and Heeschen, 1996; Reybroeck, 2004).

The consequence of this is that milk is rigorously screened for the presence of β-

lactams, while residues of other antibacterial groups are not always detected at their

respective MRL. The percentage of β-lactams in the data above is, therefore, an

overestimation.

One of the potential issues for an assay for β-lactams in milk is their stability. Internal

standard samples, consisting of, respectively, benzylpenicillin and cloxacillin spiked

in raw milk and stored for 2 months below -20°C did not demonstrate any problems

with stability. Storing raw milk in the refrigerator (2-8°C), however, can result in

stability problems with β-lactams due to the possible formation of penicillinase by

certain milk bacteria (Guay et al., 1987).

Since the result of the routine testing of farm milk on antimicrobials by the milk

control stations is only known after the milk is processed, in some European

countries milk must be checked for the presence of β-lactam residues on entry to the

dairy plant or before production. This control is to ensure the technological safety of

the milk for the production of fermented dairy products and also to protect the


consumer, and several rapid screening tests are on the market for this purpose

(Neaves, 1999; Kroll, 2000; Kroll et al., 2000; Anon., 2002; Reybroeck, 2004 & 2008;

Reybroeck and Ooghe, 2004; Quandt, 2006; Ţvirdauskiené and Šalomskiené, 2007).

Further, non-commercial tests or biosensor and immunosensor-based tests are used

in some food laboratories (Gustavsson et al., 2002; Cacciatore et al., 2004; Knecht

et al., 2004; Lamar and Petz, 2007). Results of the testing can be obtained in less

than 10 minutes.

In some countries, rejected milk needs to be destroyed due to very strict legislation,

resulting in large costs being incurred in transport, incineration, and for the milk itself.

The dairy industry is therefore interested in testing at the farm before collection of the

milk, hence placing more responsibility on the farmer. However, in such a strategy, a

short test time is very important due to the number of tests involved.

The βeta-s.t.a.r. (Neogen Corporation, Lansing, MI) is a dipstick receptor assay,

using a selective receptor linked to gold particles for the detection of β-lactams in

milk (Reybroeck, 2000; Reybroeck and Ooghe, 2006) that originally involved a 5-min

protocol. The βeta-s.t.a.r. 1+1 is a faster version of the 5-min βeta-s.t.a.r. protocol

using identical reagents, but with 2 incubation steps of 1 minute each giving, overall,

a 2-min protocol. The present study describes how a validation of the βeta-s.t.a.r.

1+1 was performed at ILVO-T&V according to Commission Decision 2002/657/EC.

The specificity, detection capability, and test ruggedness of the assay were

demonstrated as meeting the criteria required by the European Commission

Decision.

Some of the results of this evaluation study were presented in 2008 at the

EuroResidue VI Conference on Residues of Veterinary Drugs in Food in Egmond

aan Zee, the Netherlands (Reybroeck and Ooghe, 2008).

5.2 MATERIALS AND METHODS 5.2.1 Reagents and standards Penicillin G or benzylpenicillin (PENNA), amoxicillin (A8523), oxacillin (O10002),

cloxacillin (C9393), dicloxacillin (D9016), nafcillin (N3269), cefazolin (C5020),

cephapirin (C8270), and cefoperazone (C4292) were all from Sigma-Aldrich

(Bornem, Belgium). Ampicillin (9930212) was from the WHO Collaborating Centre for


Chemical Reference Substances (Kungens Kurva, Sweden). Ceftiofur (34001) and

cefalexin (33989) were from Riedel-de Haën (Bornem, Belgium). Penethamate (PE-

0708004) was from Deltapharma s.a. (Barcelona, Spain); cefquinome (Batch 01-01)

from Intervet International GmbH (Unterschleiβheim, Germany); cefacetrile

(22020D000) from Novartis Animal Health Inc. (Basel, Switzerland); cefalonium

(2629) from Schering-Plough (Levallois-Perret, France); desacetylcephapirin from

ACS Dobfar S.p.a. (Tribiano, Italy); desfuroylceftiofur (D289980) from Toronto

Research Chemicals Inc. (Ontario, Canada); and clavulanic acid from DSM Anti-

Infectives (Delft, the Netherlands).

Comparisons were made for cefazolin (Cat. 1097 603) with reference material from

United States Pharmacopeia (Rockville, MD), and for penicillin G (9930226) and

cloxacillin (9930261) from the WHO Collaborating Centre for Chemical Reference

Substances (Kungens Kurva, Sweden).

Antibiotic standards were dissolved in water except for ceftiofur, cefalonium, and

cefazolin (acetonitrile/water, 1:1, v/v). Acetonitrile (01207802) was from Biosolve

B.V. (Valkenswaard, the Netherlands). Standard stock solutions of the antibiotic

standards of 100 mg l-1 were made in water and kept below 4°C for a maximum of 7

days. Dilutions of 1 and 0.1 mg l-1 were freshly prepared on a daily basis.

The βeta-s.t.a.r.-250 kits were from Neogen Corporation. In general, lot TH00616-

042405/4 (Exp. November 23, 2005) and 051607/2 (Exp. July 16, 2006) were used

for the evaluation study. For some parts, e.g. the study of batch-to-batch differences

and the stability of the reagents, lot 060409 (Exp. September 4, 2007) and lot

070331 (Exp. March 31, 2008) were also used. The reagents were stored in a cool

room at 4 ±2°C.

The Delvotest SP-NT 5-PACK kits were from DSM Food Specialties (Delft, the

Netherlands); the Charm MRL Beta-lactam tests were from Charm Sciences Inc.

(Lawrence, MA). A mixture of raw milk, aseptically collected from four individual

cows, was used as blank milk. The cows in mid-lactation were selected on the basis

of not being treated with veterinary drugs during the last months and giving milk with

a low number of somatic cells (<2 x 105 ml-1). The blank milk was always tested

before use with a Delvotest NT 5-PACK.


5.2.2 Material For the incubation of the glass vials, a dry-block heater Type BS25-230D (Aerne

Analytic, Pfaffenhofen, Germany) was used. For the reading of the dipsticks, a

reader system (Dipstick Reader, 77 Elektronika Kft, Budapest, Hungary) was used.

The reader system was checked daily with a blank calibration strip.

5.2.3 Test procedure and interpretation of the results For raw milk, no sample pre-treatment was required, while milk powder was

reconstituted with distilled water. A total of 200 µl of the milk sample were added to

the β-lactam receptor in the glass vial and the mixture was gently swirled after re-

closing the glass vial. The homogenous mixture was then incubated for 1 minute at

47.5 ±1°C in the block-heater. β-Lactam antibiotics in the milk form a stable non-

active complex with the selective β-lactam receptor. The dipstick of immuno-

chromatographic medium was placed into the glass vial and incubated at 47.5 ±1°C

for a further 1 min, during which incubation the liquid flowed vertically on the dipstick

and passed through the capture zone. The test line captured remaining active

receptor for β-lactams; the upper line or control line captured excess reagents. The

intensity of the colour that consequently developed at the selective test line and the

control line was inversely proportional to the amount of β-lactam residues and could

be interpreted both visually and instrumentally.

For instrumental reading the Dipstick Reader was used, which calculates the ratio of

colour, based on either the area or the amplitude, at the test line and the control line.

During measurement, the control line acts as a reference line. Milk with a ratio >1.00

is free of β-lactams („negative‟); milk with a ratio 1.00 is contaminated („positive‟).

Due to the very short incubation periods, it is possible that the control line is very

weak or virtually absent directly after finishing the test, and where a clear red test

line is already present the test can be interpreted as negative even before the control

line is fully developed. However, if the control and the test line are both weak, no

correct interpretation can be done until proper line development has occurred. In the

case where the control line was completely missing, the instrument indicated „invalid

reading‟. This happened a few times during the evaluation period, but the problem

was resolved by waiting until the control line developed. Only after appearance of the

control line a correct interpretation could be performed.


5.2.4 Test and reader repeatability

To calculate the repeatability of the Dipstick Reader, negative and positive strips

were measured twice. However, in the situation where the colour formation on the

dipsticks after the second incubation step was not fully finished, not exactly the same

situation was measured twice. Dipsticks were therefore allowed to become dry and

stable, then these dry dipsticks were measured ten times and the standard deviation

was calculated.

The repeatability of the test was calculated at different ratio levels by analysing and

measuring blank and positive milk samples in duplicate.

5.2.5 Test selectivity

The selectivity of the βeta-s.t.a.r. with the classic 5-min protocol test was previously

investigated by spiking blank milk with a relatively high concentration (10 x MRL in

milk) of a substance belonging to other groups of antibiotics or chemotherapeutics

(Reybroeck, 2000; Reybroeck and Ooghe, 2004) and testing in duplicate. One

substance was chosen from each of the most important groups: oxytetracycline

(tetracyclines), sulfadiazine (sulfonamides), enrofloxacin (quinolones), neomycin

(aminoglycosides), erythromycin (macrolides), lincomycin (lincosamides), clavulanic

acid (β-lactamase inhibitors), colistin (polymyxins), and trimethoprim (diamino-

pyrimidine derivatives). The forbidden compounds chloramphenicol and dapsone,

spiked at 3 and 50 μg kg-1, respectively, were also tested. With the compounds not

belonging to the group of β-lactam antibiotics, no interference was observed except

in the case of clavulanic acid (Reybroeck, 2000; Reybroeck and Ooghe, 2004).

The present study tested 100 blank milk samples free of antibiotics and the minimal

concentration of clavulanic acid in milk causing positive results for the βeta-s.t.a.r.

1+1. For this study, milk spiked with clavulanic acid at different concentrations was

tested.

5.2.6 Detection capability

The most important validation parameter is the detection capability (CCβ). This

parameter was determined for all β-lactams mentioned in the list of MRLs in milk

(Commission Regulation (EU) No 37/2010). Therefore, starting from the detection

capability concentrations of the classic βeta-s.t.a.r., blank milk was spiked with the β-

lactams investigated at different concentrations in various ranges in different


increments: in the range 1-10 μg kg-1 at 1 μg kg-1 increments; in the range 10-20 μg

kg-1 at 2 μg kg-1 increments; in the range 20-50 μg kg-1 at 5 μg kg-1 increments; in the

range 50-100 μg kg-1 at 10 μg kg-1 increments; in the range 100-250 μg kg-1 at 25 μg

kg-1 increments; in the range 250-500 μg kg-1 at 50 μg kg-1 increments; in the range

500-1,000 μg kg-1 at 100 μg kg-1 increments; in the range 1,000-5,000 μg kg-1 at 500

μg kg-1 increments; and in the range 5,000-10,000 μg kg-1 at 1,000 μg kg-1

increments. The spiked samples were blind-coded before analysis. The analysis was

performed within 4 hours after spiking. Each concentration was tested 20 times, in a

time period of at least 3 days. Each day a different blank milk was used. For each β-

lactam investigated, the CC or the lowest concentration giving 19 (low) positive test

results on 20 test results was determined, interpreting both visually and with the

Dipstick Reader.

5.2.7 Test robustness 5.2.7.1 Length of incubation

Since incubation time could possibly make the test less robust, other incubation

times were tested. The first incubation step was modified to 45 or 75 s while keeping

the second step at 1 min; the second incubation step was modified to 45 or 75 s

while keeping the first step at 1 min; and both incubation steps were modified (45 s

each, 75 s each, and the combinations of 45 and 75 s). Each situation was tested

with four blank milk samples and with four milk samples spiked with one of the

following three β-lactams: benzylpenicillin (4 μg kg-1), ampicillin (6 μg kg-1), or

cloxacillin (12 μg kg-1).

5.2.7.2 Influence of waiting time on reader results

Blank milk samples and samples spiked with one of the following three β-lactams

(benzylpenicillin, 4 μg kg-1; ampicillin, 6 μg kg-1; cloxacillin, 12 μg kg-1) were analysed

and the strips were read with the Dipstick Reader directly after the incubation and

after 0.5, 1, 3, and 10 min.


5.2.8 Milk influences

5.2.8.1 Milk quality and composition The impact of the milk quality (somatic cell count, total bacterial count) and

composition (fat and protein content, pH) was tested by comparing the test

performance of the βeta-s.t.a.r. 1+1 protocol for ten different blank milks with either

high somatic cell count (>106 ml-1, 34 samples) or high total bacterial count (> 5 x 105

cfu ml-1, 31 samples), and ten different spiked milk samples with a normal and an

abnormal composition. Milk of normal and abnormal composition was analysed with

and without spiking with one of three β-lactams (benzylpenicillin, 4 μg kg-1; ampicillin,

6 μg kg-1; cloxacillin, 12 μg kg-1). For each different milk type the average, the

highest, and the lowest ratio value was calculated.

Milk samples with a high number of somatic cells were selected at the milk control

station based on Fossomatic 5000 (FOSS, Hillerød, Denmark) measurements. Milk

samples with a high total bacterial count were obtained by keeping normal milk

samples for 4-6 h at room temperature. The final bacterial count was determined by

performing a spiral plate count (Eddy Jet, IUL sa, Barcelona, Spain) on plate count

agar plates after 3 days incubation at 30°C. Milk samples with a low fat content were

obtained by removal of the fat layer by centrifugation (3,050 g, 10 min, at 5°C). Milk

samples with a high fat content were obtained by addition of cream (50% fat, 6 g) to

milk (60 ml). The final fat content was measured by infrared with a MilcoScan 4000

(FOSS). Milk samples with a low (<2.5 g 100 ml-1) and a high protein content (>4 g

100 ml-1) were natural milk samples with extreme protein content that were selected

at the milk control station based on infrared spectroscopic results (MilcoScan 4000).

To gain samples with an abnormal pH, normal milk was initially adjusted to pH 6.0

and pH 7.5 with 1M HCl or 1M NaOH, respectively, then the pH was further adjusted

with the addition of either 0.1M HCl or 0.1M NaOH.

5.2.8.2 Type of milk and animal species

Ultra high temperature processing (UHT) milk, sterilized milk, reconstituted milk

powder, thawed milk, goats‟ milk, ewes‟ milk, and mares‟ milk were also tested to

determine if the βeta-s.t.a.r. 1+1 was a suitable test for these types of milk. Ten

different samples of each milk type were tested, with the exception that a higher

number of blank samples were tested for goats‟ milk (29 samples), ewes‟ milk (31

samples), and mares‟ milk (30 samples).


5.2.9 Test for false-positive/false-negative results

A total of 117 farm milk samples, 65 truck milk samples, 32 consumer milk samples,

and 18 milk powders were analysed with βeta-s.t.a.r. 1+1 as part of a monitoring

programme. The same samples were also tested by Delvotest SP-NT, Bacillus

cereus-test (Suhren and Heeschen, 1993), Escherichia coli-test (Suhren,1997), and

Charm MRL Beta-Lactam Test.

For testing the rate of false-negative results, 82 incurred milk samples originating

from 27 individual cows treated with a veterinary drug containing benzylpenicillin and

neomycin were analysed with the βeta-s.t.a.r. 1+1 and with other microbiological and

β-lactam receptor screening tests. Sampling started at the end of the withholding

period. The exact concentration of benzylpenicillin present in the milk samples was

determined by LC-MS/MS in an external laboratory.

5.2.10 Reagent influence (batch differences) To study the influence of different batches of reagents, blank and spiked milk

samples were analysed at the same time with 2 different batches of βeta-s.t.a.r.

reagents (Lot 70405, Exp. April 5, 2008, and Lot 70213, Exp. February 13, 2008),

and Lot 70205 (Exp. February 5, 2008). Besides spiking with benzylpenicillin (4 μg

kg-1), ampicillin (6 μg kg-1), or cloxacillin (12 μg kg-1), 20 milk samples were also

spiked with 28 μg kg-1 cephapirin to obtain ratios close to the cut-off value of 1.00. In

the area around the cut-off, any change in intensity of the test line can be quickly

noted.

The stability of reagents during shelf life was also checked. Blank and spiked milk

samples were tested with reagents of Lot 70405 shortly after the production date and

just one week before the expiry date.

5.2.11 Inter-laboratory testing

Twice a year, ILVO-T&V organizes a national ring trial for the Belgian dairy industry

regarding the detection of residues of antibiotics in milk by microbiological and rapid

tests. In the two ring trials of 2007, the βeta-s.t.a.r. 1+1 procedure was included.

ILVO-T&V participated with the βeta-s.t.a.r. 1+1 in the international proficiency study

for the analysis of -lactam residues in raw milk, organised in 2007 by Anses

Fougères, CRL for antimicrobial residues in food of animal origin.


5.2.12 Daily control samples

During the study, blank and control milk samples spiked separately with

benzylpenicillin (4 μg kg-1), ampicillin (6 μg kg-1), and cloxacillin (12 μg kg-1) were

analysed daily.

5.3 RESULTS AND DISCUSSION 5.3.1 Test and reader repeatability All repeatability results are shown in Table 1. The repeatability of the Dipstick

Reader was very good; very low standard deviations of repeatability (sr) were

obtained. The more positive the samples, the better the repeatability. It is worth

noting that, in general, with wet dipsticks a slightly lower ratio was obtained for the

second reading due to further colour formation on the strips when drying.

Table 1. Repeatability of the reader (dry and wet dipsticks) and repeatability of the βeta-s.t.a.r. 1+1 test at different ratios. Milk Compound;

concentration Number of samples

Mean ratio

a sr CV (%) b

Reader repeatability – wet dipsticks blank milk 30 4.08 0.30 0.52 positive milk benzylpenicillin; 4 μg kg-1 30 0.12 0.04 0.13 ampicillin; 6 μg kg-1 30 0.33 0.04 0.13 cloxacillin; 12 μg kg-1 30 0.05 0.02 0.04 Reader repeatability – dry dipsticks blank milk 10 2.40 0.02 0.06 positive milk benzylpenicillin; 1 μg kg-1 10 1.89 0.10 0.09 benzylpenicillin; 2 μg kg-1 10 1.94 0.06 0.05 benzylpenicillin; 2.5 μg kg-1 10 0.85 0.03 0.03 benzylpenicillin; 3 μg kg-1 10 0.35 0.03 0.03 benzylpenicillin; 4 μg kg-1 10 0.17 0.01 0.01 Test repeatability blank milk 20 4.35 0.33 0.55 positive milk benzylpenicillin; 4 μg kg-1 20 0.10 0.05 0.09 ampicillin; 6 μg kg-1 20 0.34 0.09 0.14 cloxacillin; 12 μg kg-1 20 0.05 0.03 0.04 Notes: a sr, standard deviation of repeatability. b CV, coefficient of variation.


The repeatability of the test was also very good; very low sr-values were again

obtained. It is noteworthy that the same sr- values for the test repeatability and

reader repeatability were obtained: this indicates that the test is very robust and that

for replicates of a sample the same binding, flow, and colour formation are

essentially obtained. The repeatability for positive samples is also better than that for

blank samples and is not influenced by the ratio level.

5.3.2 Test selectivity

All 100 blank milk samples tested negative. To test the minimum concentration of

clavulanic acid in milk that could cause positive results for the βeta-s.t.a.r. 1+1, milk

spiked with clavulanic acid at different concentrations was tested, and interference

by clavulanic acid was only obtained at 2,500 μg kg-1 and above. Therefore, the

βeta-s.t.a.r. 1+1 is very selective for the detection of β-lactams. Interference by the

β-lactamase inhibitor clavulanic acid could be expected since this molecule has a β-

lactam structure resembling that of the penicillin nucleus, except that the fused

thiazolidine ring of the penicillins is replaced by an oxazolidine ring (Anon., 2005).

Within the β-lactam group the test is not specific for any particular β-lactam, but

natural and aminopenicillins could be differentiated from the other penicillins and the

cephalosporins after pre-treatment of the milk with penase (data not shown).

5.3.3 Detection capability

A summary of the detection capability of the βeta-s.t.a.r. 1+1 is given in Table 2 and

Figure 1. Not all the β-lactam compounds were detected at their respective MRL. The

detection of desfuroylceftiofur, cefalexin, and penethamate was poor, and ceftiofur

was only detected from 500 μg kg-1. Ampicillin, amoxicillin, nafcillin, cefquinome,

cefazolin, and desacetylcephapirin were also not detected at MRL.

It should be noted, however, that from a practical perspective, the high detection

capability of 80 μg kg-1 for penethamate in relation to the MRL (4 μg kg-1) is of no

significance since the marker residue for penethamate is benzylpenicillin, and

penethamate is not stable in milk and is rapidly and completely hydrolysed to

benzylpenicillin and diethylaminoethanol. At 37°C and at pH 7.3 (reflecting the


physiological conditions of cows), the half-life time of penethamate in aqueous

solution is 23 min. In tissue homogenates at 32°C, 50% of the penethamate has

been shown to be hydrolysed within 2 h and 100% by 20 h (Anon., 2000). Table 2. Detection capability in raw cows’ milk of the βeta-s.t.a.r. 1+1 instrumental reading with a cut-off ratio of 1.00a in comparison to the classic βeta-s.t.a.r.. Group Compound MRLb

(μg kg-1) Detection capability (μg kg-1)a

βeta-s.t.a.r. 1+1 βeta-s.t.a.r.c,d penicillins benzylpenicillin 4 3 3 ampicillin 4 7 4 amoxicillin 4 8 4 oxacillin 30 11 6 cloxacillin 30 9 6 dicloxacillin 30 8 5 nafcillin 30 36 14 penethamate 4e 80 30 cephalosporins ceftiofur 100f 500 (6,000g) 110 (2,500g) cefquinome 20 28 10 cefazolin 50 175 60 cephapirin 60h 28 (125i) 12 (70i) cefacetrile 125 100 40 cefoperazone 50 7 6 cefalexin 100 6,000 >1,000 cefalonium 20 4 4 Notes: a Detection capability is defined as the lowest concentration tested giving a minimum

of 19 positive results out of 20. b MRL, maximum residue limit (Regulation (EC) No 470/2009; Commission

Regulation (EU) No 37/2010 and amendments as of October 12, 2010). c 3+2 classic protocol. d Reybroeck and Ooghe (2004) and unpublished data. e marker residue is benzylpenicillin. f sum of all residues retaining the -lactam structure expressed as desfuroylceftiofur. g desfuroylceftiofur. h sum of cephapirin and desacetylcephapirin. i desacetylcephapirin.


benzylpenicillin (4)ampicillin (4)

amoxicillin (4)

oxacillin (30)

cloxacillin (30)

dicloxacillin (30)

nafcillin (30)

penethamate (4)

ceftiofur (100)desfuroylceftiofur (100)

cefquinome (20)

cefazolin (50)

cephapirin (60)

desacetylcephapirin (60)

cefacetrile (125)

cefoperazone (50)

cefalexin (100)

cefalonium (20)

Figure 1. Detection pattern in raw cows’ milk of the βeta-s.t.a.r. 1+1, instrumental reading with a cut-off ratio of 1.00. Detection expressed relatively to the MRL (Commission Regulation (EU) No 37/2010 and amendments as of October 12, 2010). MRL (in µg kg-1) for each β-lactam compound mentioned in brackets. Inner circle = 2 MRL, circle 2 = MRL, circle 3 = 0.5 MRL, circle 4 = 0.25 MRL.

Since the analysis was performed not just immediately, but within 4 hours after

spiking, a negative influence of the protein binding effect on the test capabilities

could not be excluded. When performing the βeta-s.t.a.r. 1+1 protocol instead of the

classic 3+2-min protocol (Reybroeck and Ooghe, 2004), the test generally lost some

detection capability resulting in a reduced number of compounds detectable at the

MRL level.

5.3.4 Test robustness

5.3.4.1 Length of incubation

Performing the βeta-s.t.a.r. 1+1 protocol with the different incubation times tested

had no significant impact on the ratios obtained for blank milk (data not shown).

With a longer first incubation period, slightly lower ratio values were obtained with

positive milk samples, while a shorter first incubation step resulted in slightly higher


ratios (data not shown). The effect was limited for milk spiked with benzylpenicillin (4

µg kg-1) and cloxacillin (12 µg kg-1), but more visible for milk spiked with ampicillin (6

µg kg-1). This indicated that the time needed for a quantitative binding of the receptor

to ampicillin is longer in comparison with binding to benzylpenicillin or cloxacillin. The

data indicated that for the first incubation a minimum of 1 min is needed to be

respected. Even when the second incubation period differed from the standard

protocol, correct and acceptable results were obtained, proving that, within the limits

tested, strict adherence to timing for the second incubation was not a critical point.


If the reading of the dipsticks after incubation was delayed, the ratios decreased,

giving a tendency to more positive results. Nevertheless, all blank milk samples

remained clearly negative with 2.25 as the lowest ratio obtained for a delay of 10 min

after incubation before reading (data not shown). Therefore, delaying the reading

does not cause incorrect results but does improve the detection capability.

5.3.5 Milk influences 5.3.5.1 Milk quality and composition

With respect to testing the impact of the milk quality and composition (somatic cell

count, total bacterial count, fat and protein content, and pH), the mean, the highest

ratio, and the lowest ratio value for each milk type are given in Figures 2, 3, and 4.

The milk quality and composition had no influence on the performance of the βeta-

s.t.a.r. 1+1 when testing blank milk: all blank milk samples were clearly negative with

ratios all above 2.50, except for milk with a high pH for which the lowest ratio was

1.98. With positive milk samples spiked with benzylpenicillin or cloxacillin, only small

effects with abnormal milk quality or composition were noticed. In all spiked samples

the detection of the β-lactam was never completely hampered. In milk with a low pH

(6.0), sensitivity of the test was decreased, and this was most pronounced for milk

with 6 μg kg-1 ampicillin.

Further, the detection capability of the test was slightly diminished when testing milk

with a high bacterial load, which could be explained by the possible production of

penicillinase by certain bacteria. In such a case benzylpenicillin and ampicillin would

be expected to be more quickly cleaved than cloxacillin.


Figure 2. Ratios for normal and abnormal blank milk (, mean; , lowest; , highest) and normal and abnormal milks containing 12 μg kg-1 cloxacillin ( , mean; , lowest; , highest). Milks were of normal composition (1) or with: (2) a high somatic cell count; (3) a high bacterial count; (4) a low fat content; (5) a high fat content; (6) a low protein content; (7) a high protein content; (8) a low pH; and (9) a high pH. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result. Figure 3. Ratios for normal and abnormal milks containing 6 μg kg-1 ampicillin (, mean; , lowest; , highest). Milks were of normal composition (1) or with: (2) a high somatic cell count; (3) a high bacterial count; (4) a low fat content; (5) a high fat content; (6) a low protein content; (7) a high protein content; (8) a low pH; and (9) a high pH. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result.


Figure 4. Ratios for normal and abnormal milks containing 4 μg kg-1 benzylpenicillin (, mean; , lowest; , highest). Milks were of normal composition (1) or with: (2) a high somatic cell count; (3) a high bacterial count; (4) a low fat content; (5) a high fat content; (6) a low protein content; (7) a high protein content; (8) a low pH; and (9) a high pH. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result. It should be noted that the best detection was obtained in milk with low protein

content, which could be the result of decreased binding of the antibiotics to protein

material. However, in large volumes of commingled milk, such extreme values of

composition will not occur; for instance, while a high pH milk can occur in individual

cow milk due to damage of the blood/milk barrier by subclinical mastitis, it is unlikely

that an entire bulk collection of milk will be affected. Further, it must also be

recognized that the test is qualitative rather than quantitative and is used only to

discriminate between β-lactam residue-free milk and milk containing such residues.

In general, the test is very robust and not severely influenced by the milk

composition.


The results of the testing of UHT milk, sterilized milk, reconstituted milk powder,

thawed milk, goats‟ milk, ewes‟ milk, and mares‟ milk are presented in Figures 5, 6,

and 7. No significant differences were noticed in testing different types of milk. All

blank milk samples tested negative (all ratios >1.00), although for one out of 29 blank

goats‟ milk and for one out of 31 blank ewes‟ milk samples, a ratio below 2.00 was


obtained. This may have been caused by an abnormal flow of the milk on the

dipstick. Figure 5. Ratios for blank milk (, mean; , lowest; , highest) and different milks containing 12 μg kg-1 cloxacillin ( , mean; , lowest; , highest). Raw cows’ milk (1) compared with: (2) UHT milk; (3) sterilized milk; (4) reconstituted milk powder; (5) thawed milk; (6) goats’ milk; (7) ewes’ milk; and (8) mares’ milk. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result. Figure 6. Ratios for different milks containing 6 μg kg-1 ampicillin (, mean; , lowest; , highest). Raw cows’ milk (1) compared with: (2) UHT milk; (3) sterilized milk; (4) reconstituted milk powder; (5) thawed milk; (6) goats’ milk; (7) ewes’ milk; and (8) mares’ milk. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result.


Figure 7. Ratios for different milks containing 4 μg kg-1 benzylpenicillin (, mean; , lowest; , highest). Raw cows’ milk (1) compared with: (2) UHT milk; (3) sterilized milk; (4) reconstituted milk powder; (5) thawed milk; (6) goats’ milk; (7) ewes’ milk; and (8) mares’ milk. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result.

Cloxacillin (12 μg kg-1) and benzylpenicillin (4 μg kg-1) were always detected in quite

a uniform way in the different milk types and in the milk from animal species different

from the cow. The detection of 6 μg kg-1 ampicillin gives a higher variation in

detection and ratios. The level of detection of ampicillin in UHT milk, sterilized milk,

reconstituted milk powder, goats‟ milk, and for a lesser extent in ewes‟ milk, would be

higher than 7 µg kg-1 as determined in cows‟ milk (Table 2). It is worth noting that

the thawed milk samples were tested with reagents of Lot 70405, while the other milk

types were tested with Lot 70213. Lot 70405 was more sensitive, and the difference

in detection capability for ampicillin is especially notable. The biggest variation in

ratios was noticed for the detection of ampicillin in goats‟ milk. Of all milk types

spiked with ampicillin and tested with reagents of Lot 70213, the best detection was

obtained for mares‟ milk. The difference in detection for different milk types is smaller

than the difference in test capability by the use of different batches of reagents.

The βeta-s.t.a.r. 1+1 is therefore not only valid as a raw cows‟ milk test, but can also

be used to test UHT milk, sterilized milk, reconstituted milk powder and thawed milk,

and milk of non-bovine species (goat, ewe, and mare). However, for UHT milk,

sterilized milk, reconstituted milk powder, or thawed milk, the use of the classic 3+2

version with better detection capabilities for some compounds is recommended.


5.3.6 Test for false-positive/false-negative results

No false-positive or false-negative results were obtained when testing farm milk,

truck milk, consumption milk samples, and milk powders as part of a monitoring

programme. For testing the rate of false-negative results, incurred milk samples

originating from individual cows treated with a veterinary drug, containing

benzylpenicillin and neomycin, were analysed with the βeta-s.t.a.r. 1+1. All 59 milk

samples with a benzylpenicillin content ≤2.4 μg kg-1 tested negative on βeta-s.t.a.r.

1+1; all 23 incurred milk samples with a benzylpenicillin content ≥2.5 μg kg-1 were

testing positive, except for the sample containing 2.7 μg kg-1 benzylpenicillin. These

data confirm that the detection capability for benzylpenicillin in spiked milk (3 μg kg-1;

Table 2) is also valid for the detection of benzylpenicillin in incurred milk samples.

5.3.7 Reagent influence (batch differences) A summary of the results of the testing of spiked milk samples with two different

batches of βeta-s.t.a.r. reagents is given in Table 3. Table 3. Ratios obtained when testing the same positive (pos) and negative (neg) milk samples with βeta-s.t.a.r. reagents from different batches.

Tested Lot 70405 Lot 70213a Ratio Number Ratio Number mean minb maxb pos neg mean minb maxb pos neg

blank

3.67 3.20 4.04 0 20 3.06 2.63 3.70 0 20

benzylpenicillin at 4 μg kg-1

0.04 0.00 0.15 20 0 0.09 0.00 0.19 20 0

ampicillin at 6 μg kg-1

0.24 0.01 0.42 20 0 0.96 0.79 1.36 14 6

cloxacillin at 12 μg kg-1

0.15 0.10 0.25 20 0 0.35 0.29 0.42 20 0

cephapirin at 28 μg kg-1

0.80 0.41 1.06 19 1 1.03 0.77 1.44 11 9

Notes: a Except for cephapirin, which was tested using Lot 70205. b min, minimum; max, maximum. Differences in detection capability were found between batch Lot 70405 and Lots

70213 and 70205, the former giving lower ratios for the spiked milk samples than

Lots 70213 and 70205. The difference was most pronounced for the milk samples

spiked with 6 μg kg-1 ampicillin and 28 μg kg-1 cephapirin. Note that the detection


capability of ampicillin and cephapirin is 7 and 28 μg kg-1, respectively (Table 2).

Blank milk gave the same ratios with all the different lots tested.

The stability of reagents during shelf-life was also checked. Blank and spiked

standards were tested with reagents of Lot 70405 shortly after the production date

and one week before the expiry date. In general, comparable results were obtained,

confirming the stability of the reagents over the recommended shelf-life (mean ration

values were: blank = 3.23, benzylpenicillin at 4 μg kg-1= 0.03, ampicillin at 6 μg kg-1=

0.27, cloxacillin at 12 μg kg-1= 0.32).

5.3.8 Interlaboratory testing Twice a year, ILVO-T&V organizes a national ring trial for the Belgian dairy industry


tests. In the two ring trials of 2007, the βeta-s.t.a.r. 1+1 was also included as a rapid

test.

The results for the βeta-s.t.a.r. 1+1 in both ring trials were excellent: no false-positive

results were obtained and the spiked milk samples gave the expected positive

results with the exception that the sample spiked with penicillin at 3 μg kg-1 tested

negative (ratio = 1.28; detection capability = 3 μg kg-1). The sample spiked with

penicillin at 4 μg kg-1 gave a clear positive result (ratio = 0.03). Of all the samples

that tested positive with the classic βeta-s.t.a.r. 3+2 protocol, only three samples

tested negative with the rapid βeta-s.t.a.r. 1+1 protocol, i.e., samples spiked with 4

μg kg-1 ampicillin (detection capability = 7 μg kg-1), 20 μg kg-1 cefquinome (detection

capability = 28 μg kg-1) and 3 μg kg-1 benzylpenicillin (detection capability = 3 μg

kg-1). Details of the results are given in separate reports (Ooghe and Reybroeck,

2007a & 2007b).

In the international proficiency study organized by Anses Fougères, six blind coded

milk samples were distributed among the participating laboratories. The blank milk

and the five spiked milk samples, respectively, containing cefquinome at 50 μg kg-1,

cloxacillin at 40 μg kg-1, cefalonium at 20 μg kg-1, and benzylpenicillin at 6 μg kg-1 (in

duplicate), were all correctly analysed by βeta-s.t.a.r. 1+1 at ILVO-T&V (Fuselier et

al., 2008).



During the study, blank and control samples spiked with 4 μg kg-1 benzylpenicillin, 6

μg kg-1 ampicillin, or 12 μg kg-1 cloxacillin were analysed daily. The results are

shown in Figure 8.

Over 29 working days the control samples gave very constant ratios. It should be

noted that tests for days 1-26 used reagent Lot 70213, while from day 27 onwards

Lot 70405 was used, which was significantly better in detecting ampicillin.

Figure 8. Ratios obtained for 29 dairy control samples with blank milk (); and milk containing () 4 μg kg-1 benzylpenicillin; (), 6 μg kg-1 ampicillin; or () 12 μg kg-1 cloxacillin. The horizontal line at a ratio of 1.00 gives the cut-off between a negative and a positive result.

For the entire period, the following average ratios were obtained: blank milk = 3.38±

0.34, milk spiked with 4 μg kg-1 benzylpenicillin = 0.17 ±0.08, milk spiked with 6 μg

kg-1 ampicillin = 1.14 ±0.31, and milk spiked with 12 μg kg-1 cloxacillin = 0.40 ±0.10.

Since most of the time the 6 μg kg-1 ampicillin control sample was giving negative

results, its value for quality control could be discussed. However, small differences in

test capability of the reagents can be detected by the use of milk samples spiked

with ampicillin. This compound is therefore used by Neogen Corporation in their

quality control for product release.


5.4 CONCLUSIONS With a total test time of 2 min, the βeta-s.t.a.r. 1+1 is, presently, the fastest single

test on the market for the detection of β-lactam residues in milk meeting the criteria

required by Commission Decision 2002/657/EC. The short test time, the very easy

test protocol, and the possibility of visual interpretation of the test enables the use of

the test at the farm before collection. Shortening of the test protocol from 5 min to 2

min influences the detection capability for some β-lactam compounds, but does not

challenge test robustness. Further, the use of βeta-s.t.a.r. 1+1 at the farm level

instead of using the classic 5-min βeta-s.t.a.r. test at the entrance of the dairy plant

would resolve the issue of dilution “disguising” contaminated milk (since tanker milk

is bulked from an average of ten farms) and would lead to stronger on-farm

practices. On-farm checking would also reduce costs for the destruction of large

volumes of β-lactam-contaminated milk, since again it would be at the individual farm

level, rather than bulked milk from several farms. On the other hand, testing at the

farm means a larger number of determinations, higher costs for reagents, and more

work for the truck driver. It‟s the task of the responsible for the milk collection to

make a balance of pros and cons and to calculate the price differences between the

two strategies. If time is not the crucial factor (dairy entrance control) or no further

dilution of the milk is expected, the classic 5-min βeta-s.t.a.r. protocol could still be

preferred to obtain the best test sensitivity.

A limitation of the test with the modified test protocol is the detection capability for

some -lactams. Not all these compounds are detected at their respective MRL. The

detection of desfuroylceftiofur (detection capability = 6,000 µg kg-1; MRL = 100 µg

kg-1), cefalexin (detection capability = 6,000 µg kg-1; MRL = 100 µg kg-1),

penethamate (detection capability = 80 µg kg-1; MRL = 4 µg kg-1), and ceftiofur

(detection capability = 500 µg kg-1; MRL = 100 µg kg-1) is especially poor. Ampicillin,

amoxicillin, nafcillin, cefquinome, cefazolin, and desacetylcephapirin are also not

detected at MRL. But the new protocol was especially designed for testing on the

farm before collection. Theoretically, assuming a ten times dilution of the farm milk in

the tanker, the whole tanker milk can never contain -lactam residues above MRL

(except for desfuroylceftiofur, cefalexin, and penethamate) on condition that only milk

previously tested with eta-s.t.a.r. 1+1 is collected. Even more, the use of eta-


s.t.a.r. 1+1 at farm level instead of the classic 5 minute eta-s.t.a.r. at the entrance of

the dairy plant, is more severe for most of the compounds.

The βeta-s.t.a.r. 1+1 protocol is not only suitable for raw cows‟ milk, but could also

be used to test milk of species other than the cow (goat, ewe, and mare).

Acknowledgement The authors appreciate the valuable work performed by Veroniek De Paepe (ILVO-

T&V, Melle, Belgium) and Jorien Lambrecht (KaHo Sint-Lieven, Sint-Niklaas,

Belgium) and thank Neogen Corporation for kindly providing βeta-s.t.a.r. test

reagents, Tim Coolbear (Fronterra Co-operative Group Limited, Palmerston North,

New Zealand) for correction of the manuscript, Vzw Melkcontrolecentrum-

Vlaanderen for providing part of the raw cows‟ milk samples with a special

composition or quality and for the MilcoScan 4000 and Fossomatic 5000

measurements, Siegrid De Baere (Ghent University, Merelbeke, Belgium) for the

incurred milk samples used for testing of false-negative results and for the

determination of the benzylpenicillin content of these samples.

5.5 REFERENCES

Anonymous. 1998. Survey of antimicrobial usage in animal health in the EU. European Federation of Animal Health (Fedesa), Brussels, Belgium. Anonymous. 2000. Committee for Veterinary Medicinal Products: Penethamate (summary report) The European Agency for the Evaluation of Medicinal Plants, Veterinary Medicines Evaluation Unit, London, United Kingdom: 1-3. Anonymous. 2002. Rapid test kits for the detection of antibiotics and sulphonamides in milk. June 2002. Food Safety Authority of Ireland, Dublin, Ireland: 1-23. Anonymous. 2005. Martindale: The complete drug reference. Ed. Sweetman S. C. Royal Pharmaceutical Society of Great Britain, Pharmaceutical Press, London, United Kingdom, 34th Edition. Anonymous. 2006. Rapport des Activités 2005. Comité du Lait, Battice, Belgium: 1-82. Anonymous. 2010a. Jaarverslag 2009. Vzw Melkcontrolecentrum-Vlaanderen, Lier, Belgium: 1-52. Anonymous. 2010b. Rapport des Activités 2009. Comité du Lait, Battice, Belgium: 1-70. Cacciatore G., Petz M., Rachid S., Hakenbeck R., Bergwerff A.A. 2004. Anal. Chim. Acta, 520(1-2): 105-115.


Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Comm. 2002 L221: 8-36. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010 L15: 1-72. Grunwald L. 2002. Untersuchungen zur Analytik und zum Einfluss technologischer Prozesse auf Penicillinrückstände in Milch. Dissertation. Bergische Universität Wuppertal, Fachbereich Chemie, Wuppertal, Germany. Fuselier R., Gaudin V., Dubreil E., Verdon E., Sanders P. 2008. Proficiency study for the analysis of beta-lactam residues in raw milk. Final report. AFSSA, Fougères, France. Guay R., Cardinal P., Bourassa C., Brassard N. 1987. Decrease of penicillin G residue incidence in milk: A fact or an artefact? Int. J. Food Microbiol. 4(3): 187-196. Gustavsson, E., Bjurling, P., Sternesjö, Å. 2002. Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity. Anal. Chim. Acta 468(1): 153-159. Honkanen-Buzalski T., Reybroeck W. 1997. Antimicrobials. In: „Monograph on residues and contaminants in milk and milk products.‟ I.D.F. (International Dairy Federation), Brussels, Belgium, ISBN 92 9098 025 8: 28-34. Knecht B.G., Strasser A., Dietrich R., Märtlbauer E., Niessner R., Weller M.G. 2004. Automated microarray system for the simultaneous detection of antibiotics in milk. Anal. Chem. 73(3): 646-654. Kress C., Seidler C., Kerp B., Schneider E., Usleber E. 2007. Experiences with an identification and quantification program for inhibitor-positive milk samples. Anal. Chim. Acta 586(1-2): 275-279. Kroll S. 2000. Suitability of rapid test methods for the detection of residues of beta-lactam antibiotics in milk. Dissertation, University of Munchen, Germany. Kroll S., Usleber E., Zaadhof K.-J., Schneider E., Märtlbauer E. 2000. Evaluation of commercial rapid tests for β-lactam antibiotics in raw milk. Proceedings of the Euroresidue IV Conference on Residues of Veterinary Drugs in Food, Veldhoven, the Netherlands, May 8-10, 2000: 693-697. Lamar J., Petz M. 2007. Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices. Anal. Chim. Acta 586(1-2): 296-303. Neaves P. 1999. Monitoring antibiotics in milk - the changing world of test methods. British Mastitis Conference, Stoneleigh, England, October 13, 1999: 15-23. Ooghe S., Reybroeck W. 2007a. Rapport ringonderzoek antibiotica 24 mei 2007: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-23. Ooghe S., Reybroeck W. 2007b Rapport ringonderzoek antibiotica 18 oktober 2007: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-17.


Pyörälä S. 1995. Staphylococcal and streptococcal mastitis. In „The bovine udder and mastitis.‟ Ed. Sandholm M., Honkanen-Buzalski T., Kaartinen L., Pyörälä S. University of Helsinki, Faculty of Veterinary Medicine, ISBN 951-834-047-1: 143-148. Quandt B. 2006. Identifizierung van antimikrobiellen Rückständen in Milch mittels Schnelltestsystemen. Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität, München, Germany. Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Union 2009 L152: 11-22. Reybroeck W. 2000. Evaluation of the Beta s.t.a.r. for the detection of -lactam antibiotics. Proceedings 2nd International FoodSENSE Workshop, Zeven, Germany, March 30 - April 1, 2000 (abstract + poster). Reybroeck, W. 2004. Résidus d‟antibiotiques dans le lait. Utilisation des kits de dépistage des inhibiteurs. Point Vét. 242: 52-57. Reybroeck W. 2008. The use of microbiological, immunological and receptor tests for monitoring of residues of antimicrobials in milk: the Belgian approach. In „Bulletin of the IDF 424/2008. Advances in Analytical Technology.„, ISSN 0250-5118: 13-16. Reybroeck W., Daeseleire E. 2003. Invloed bewaring op antibioticaresiduen in melk. Heranalyse positieve stalen en identificatie van remstoffen verantwoordelijk voor een ongunstige resultaat bij de officiële kwaliteitsbepaling van melk. Resultaten. ILVO-T&V, Melle, Belgium: 1-20. Reybroeck W., Ooghe S. 2004. Rapid screening for residues of antibiotics in milk at the factory. In A Farm-to-table Approach for Emerging and Developed Dairy Countries, Proceedings IDF/FAO International Symposium on Dairy Safety and Hygiene, Cape Town, Republic of South Africa, March 2-5, 2004. ISSN 1810-0732: 157-161. Reybroeck W., Ooghe S. 2006. Gebruik van sneltesten als bevestigingstest bij de opsporing van bacteriegroeiremmende stoffen in melk in het kader van de officiële kwaliteitsbepaling van rauwe melk. Rapport voor het Wetenschappelijk Comité van het FAVV, 2006: 1-22. Reybroeck W., Ooghe S. 2008. Validation of the βeta-s.t.a.r. 1+1 for fast screening of raw milk on the presence of β-lactam antibiotics. Proceedings of the Euroresidue VI Conference on Residues of Veterinary Drugs in Food, Egmond aan Zee, the Netherlands, May 19-21, 2008: 793-797. Suhren G. 1996. Influence of residues of antimicrobials in milk on commercially applied starter cultures-model trials. Kieler Milchwirtschaftl. Forschungsber. 48(2): 131-149. Suhren G. 1997. Mikrobiologischer Hemmstofftest mit E. coli zum Nachweis von Chinolon-Rückständen in Milch. 38.Arbeitstagung des Arbeitsgebiet Lebensmittelhygiene der DGV, Garmisch-Partenkirchen, Germany. Teil II: 659-664.


Suhren G., Heeschen W. 1993. Detection of tetracyclines in milk by a Bacillus cereus microtitre test with indicator. Milchwissenschaft 48: 259-263. Suhren G., Heeschen W. 1996. Detection of inhibitors in milk by microbial tests. A review. Food / Nahrung 40(1): 1-7. Ţvirdauskiené R., Šalomskiené J. 2007. An evaluation of different microbial and rapid tests for determining inhibitors in milk. Food Control 18(5): 541-547.

Validation of the Charm MRL

Chapter 6 Validation of the Charm MRL-3

Adapted from:

Reybroeck W., Ooghe S., De Brabander H.F., Daeseleire E. 2011. Validation of the

Charm MRL-3 for fast screening of β-lactam antibiotics in raw milk. Journal of AOAC

International 94(2): page numbers not yet assigned.

6 Validation of the Charm MRL-3 176

Validation of the Charm MRL-3 for fast screening for β-lactam antibiotics in raw milk

Abstract

The biochemicals utilized in the Charm MRL Beta-Lactam Test (8 min test) were

applied to faster flowing lateral components to create a new 3 min, one-step β-

lactam test called Charm MRL-3 (Charm Sciences Inc., Lawrence, MA). This new

test was validated at ILVO-T&V according to Commission Decision 2002/657/EC.

In the validation study the test showed to be very specific as a real interference was

only caused by clavulanic acid at 175 μg kg-1 and above. The repeatability of the

reader was good; however regarding the test repeatability some problems for

negative milk samples were noticed. Throughout the evaluation study, false-positive

results were obtained when testing blank raw milk. Hence, it is recommended to

retest initial positive samples as indicated by the kit manufacturer.

The Charm MRL-3 detects all -lactams with a MRL in milk at their respective MRL

excepted for nafcillin and penethamate which were in 95% of the cases detected at

90 and 200 µg kg-1 and above, respectively. The test showed to be robust for

changes in the test protocol. The milk quality and composition had some influence

on the performance of the Charm MRL-3 when testing blank and spiked milk. Charm

MRL-3 is not only a raw milk test for the dairy industry, but it could also be used to

test UHT-milk, sterilized milk, reconstituted milk powder, or thawed milk under

condition that positive results are further tested with a different antibiotic test. The

Charm MRL-3 is not a suitable test to screen milk from animal species different from

the cow (goat, ewe, or mare).

The short test time and the very easy one-step test protocol enable the use of the

test at the farm before collection in order to prevent tanker milk contamination with β-

lactam antibiotics. A drawback is the recommendation of the use of a reader system

for the interpretation of the colour formation on the dipsticks.


6.1 INTRODUCTION The group of β-lactam antibiotics consists of penicillins and cephalosporins because

of their common β-lactam ring structure. β-Lactam antibiotics act bactericidal by

inhibiting the synthesis of the bacterial cell wall (Anon., 2005). β-Lactam antibiotics

are the most frequently administered drugs in parenteral and intra-mammary therapy

in dairy cattle, in most cases to treat mastitis (Pyörälä, 1995). All antimicrobial drugs

administered to cows enter the milk to some degree. A residue can be the drug itself

or its metabolite. Testing for antimicrobial drug residues in milk is necessary for

ethical, health, and technological reasons (Honkanen-Buzalski and Reybroeck,

1997). The dairy industry is screening milk for antimicrobials in order to prevent

inhibition of dairy starter cultures used in the production of cheese and yoghurt

(Suhren, 1996; Grunwald, 2002). Antimicrobial residues could also mean a risk for

consumer health through toxicological effects, allergies, or antimicrobial resistance of

pathogenic bacteria. Therefore, in the European Union maximum residue limits

(MRLs) were fixed in bovine milk for 16 β-lactam compounds, ranging from 4 to 125

µg kg-1 (Regulation (EC) No 470/2009; Commission Regulation (EU) No 37/2010).

Inhibitory substances are screened routinely in farm milk samples as part of the

regulatory quality programme. A positive result normally leads to a penalty for the

responsible farmer. In 2009 in Belgium, 872 (0.06%) out of 1,374,801 analysed farm

milk samples were found positive by the milk control stations (Anon., 2010a & b). In

most cases, residues of β-lactam substances were the main reason for bulk tank

milk failure. Since the result of the routine testing of farm milk for antimicrobials by

the milk control stations is only known after the milk is processed, the dairy industry

performs additional tests in order to prevent technological problems in the production

of fermented dairy products and to avoid problems with non-compliant consumption

milk. In most cases, milk is checked for the presence of β-lactam residues at the

entrance of the dairy plant by a rapid test. Several rapid screening tests are on the

market for this purpose (Kroll et al., 2000; Reybroeck, 2004 & 2008; Quandt, 2006;

Ţvirdauskiené and Šalomskiené, 2007). Results of the testing can be obtained in

less than 10 min. Most rapid tests are designed for a group-specific detection of β-

lactam residues, but recently tests for a simultaneous detection of β-lactams and

tetracyclines also became available. Instead of entrance control, some dairy


companies check the milk in the production tank by means of a broad spectrum

microbiological inhibitor test before starting production.

Due to very strict legislation, in most countries rejected milk needs to be destroyed.

This results in large costs for the transport, incineration, and the milk itself. The dairy

industry is, therefore, interested in testing at the farm before collection of the milk,

hence placing more responsibility on the farmer. However, in such a strategy, a short

test time is very important due to the number of tests involved.

Regarding fast tests for antimicrobial residue testing in milk, there is a tendency to

shorten test times or to test more groups of compounds in a single run. The

biochemicals utilized in the Charm MRL -Lactam Test (8-min test) (Charm Sciences

Inc., Lawrence, MA) were applied to faster flowing lateral components to create a

new 3-min, one-step β-lactam test called Charm MRL-3 (Charm Sciences Inc.). This

new test was validated at ILVO-T&V according to Commission Decision

2002/657/EC. The specificity, detection capability, and test ruggedness of the assay

were demonstrated to meet the criteria required by the EU Commission Decision.

Some of the results of this evaluation study were presented in 2008 at the

EuroResidue VI Conference on Residues of Veterinary Drugs in Food in Egmond

aan Zee, the Netherlands (Reybroeck and Ooghe, 2008).

6.2 MATERIALS AND METHODS 6.2.1 Reagents and standards Amoxicillin (A8523), cefazolin (C 5020), cefoperazone (C4292), cephapirin (C8270),

cloxacillin (C9393), dicloxacillin (D9016), oxacillin (O10002), nafcillin (N3269), and

penicillin G (benzylpenicillin, PENNA) were all from Sigma-Aldrich (Bornem,

Belgium). Ampicillin (9930212) was from the WHO Collaborating Centre for

Chemical Reference Substances (Kungens Kurva, Sweden). Cefalexin (33989) and

ceftiofur (34001) were from Riedel-de Haën (Bornem, Belgium). Cefacetrile

(22020D000) was from Novartis Animal Health Inc. (Basel, Switzerland); cefalonium

(2629) from Schering-Plough (Levallois-Perret, France); cefquinome (Batch 01-01)

from Intervet International GmbH (Unterschleiβheim, Germany); clavulanic acid from

DSM Anti-Infectives (Delft, the Netherlands); desacetylcephapirin from ACS Dobfar

S.p.a. (Tribiano, Italy); desfuroylceftiofur (D289980) from Toronto Research


Chemicals Inc. (Ontario, Canada); and penethamate (PE-0708004) from

Deltapharma s.a. (Barcelona, Spain).

The antibiotic standards were dissolved in water except for ceftiofur, cefalonium, and

cefazolin (acetonitile-water 50+50, v/v). Acetonitrile (01207802) was from Biosolve

B.V. (Valkenswaard, the Netherlands). Standard stock solutions of the antibiotic

standards (100 mg l-1) were made in water and kept below 4°C for a maximum of 1

week. Dilutions of 1 and 0.1 mg l-1 were freshly prepared on a daily basis.

To differentiate natural and aminopenicillins from the other -lactams, 25 µl penase

solution (1.2 x 105 units ml-1; BD Difco Penase Concentrate; Becton, Dickinson and

Company, Sparks, MD) was added to 1 ml of milk and incubated for 10 min at 37°C.

The Charm MRL-3 kits were from Charm Sciences Inc. (Lawrence, MA). All

sensitivity tests were performed with Lot 009001 (Expiration July 2007) (= Lot 009A

(Expiration September 2007, same lot packed on a different day)). For the study of

batch-to-batch differences, Lot 008003 was used. The reagents were stored in a cool

room at 4 ±2°C.

The eta s.t.a.r. kits were from Neogen Corporation (Lansing, MI); the Charm MRL

-Lactam kits from Charm Sciences Inc.; and the Delvotest SP-NT 5-PACK kits from

DSM Food Specialties (Delft, the Netherlands).

To check both the reader and reagents, a reconstituted Charm standard (Charm

Sciences Inc.) was used. A tablet was dissolved in 5 ml of blank raw milk to obtain a

milk solution containing cloxacillin 30 µg kg-1 and benzylpenicillin 4 µg kg-1.

A mixture of raw milk, aseptically collected from four individual cows, was used as

blank milk. Cows in mid-lactation were selected on the basis of not being treated with

veterinary drugs during the last months and giving milk with a low number of somatic

cells (<2 x 105 ml-1). The blank milk was always tested before use with a Delvotest

SP-NT 5-PACK.


6.2.2 Material For the incubation of the strips, a dry-block heater type „ROSA‟ with an integrated

timer (Charm Sciences Inc.) was used. For the interpretation of the colour formation

on the Charm MRL-3 strips, a three line reader system (ROSA Pearl Reader, Charm

Sciences Inc.) was used.

A low and a high calibration strip (Charm Sciences Inc.) were used to check the

performance of the reader system.

6.2.3 Test procedure and interpretation of the results

For raw milk no sample pretreatment was required, while milk powder was

reconstituted with distilled water. Charm MRL-3 test strips were placed in a 3-min

timed ROSA incubator at 56±1°C with the flat side facing up. The tape was peeled

back, 300 µl of milk was pipetted into either side well of the sample pad

compartment, and the strips were resealed. The lid of the incubator was closed; a

solid timer was automatically started. After 3 min incubation, the strips were removed

from the incubator, and the results were read on the ROSA Pearl Reader within 3

min.

As milk flows through the device, a line is formed in the X (cloxacillin) and T (test)

position when the sample contains no -lactams. A weaker intensity X or T line is

formed when -lactam antibiotics are present in the sample. The X and T lines are

compared to the C (control) line (Figure 1). If both the X and T lines are darker than

or equal to the C line, the sample is free of -lactams („negative‟). If either or both the

X and T line are lighter than the C line, or the X and/or T line does not form, the milk

is contaminated („positive‟). If the C line does not form, the test is invalid and must be

repeated.

The reader measured the colour formation at both test lines and the control line

position and converted the line comparison into a reading. Milk giving a reader value

0 was considered as free from -lactam antibiotics („negative‟), while milk giving a

reader value >0 was considered as suspect on the presence of -lactam antibiotics

(„positive‟).


C: control line T: -lactam X: cloxacillin

Figure 1. Configuration and interpretation of Charm MRL-3 dipsticks.

If the control line is missing or smeared or the colour is unevenly developed, or if

sample is obscuring either the C, T, or X lines, the reader will indicate „INVALID‟, and

the sample must be retested.

The performance of the reader system is checked daily by a low and a high

calibration strip and by testing a negative (residue-free raw milk) and a positive

control standard prior to testing samples.

Visual interpretation is not easy since most of the time three lines are present, and

the colour differences are not always very pronounced. So the use of a reader

system able to read strips at three positions is recommended.

6.2.4 Test and reader repeatability To calculate the repeatability of the ROSA Pearl Reader, negative and positive strips

were measured twice. The repeatability of the reader was calculated for different

reader value levels. Blank and spiked milk samples were analysed and the strips

were each time measured twice with the ROSA Pearl Reader.


The repeatability of the test was calculated at different reader value levels by

analysing and measuring blank and positive milk samples in duplicate.

6.2.5 Test selectivity The selectivity of the Charm MRL-3 was investigated by spiking residue-free raw

milk with a substance belonging to other groups of antibiotics or chemotherapeutics

at 10x MRL and testing in duplicate. One representative substance was chosen for

each of the most important groups: oxytetracycline (tetracyclines), sulfadiazine

(sulfonamides), enrofloxacin (quinolones), neomycin (aminoglycosides),

erythromycin (macrolides), lincomycin (lincosamides), clavulanic acid (β-lactamase

inhibitors), colistin (polymyxins), and trimethoprim (diaminopyrimidine derivatives).

The forbidden compounds chloramphenicol and dapsone, spiked at 3 and 50 μg kg-1,

respectively, were also tested. Non--lactam compounds testing positive were

spiked in different concentrations in milk to determine the minimal concentration

causing positive results.

6.2.6 Detection capability The detection capability (CCβ) was determined for all β-lactams mentioned in the list

of MRLs in milk (Commission Regulation (EU) No 37/2010). Therefore, starting from

the CCβ concentrations indicated by the kit manufacturer, blank milk was spiked with

the β-lactams investigated at different concentrations in various ranges in different

increments: in the range 1-10 μg kg-1 at 1 μg kg-1 increments; in the range 10-20 μg

kg-1 at 2 μg kg-1 increments; in the range 20-50 μg kg-1 at 5 μg kg-1 increments; in the

range 50-100 μg kg-1 at 10 μg kg-1 increments; and in the range 100-250 μg kg-1 at

25 μg kg-1 increments.

The spiked samples were blind coded before analysis. Each concentration was

tested 20 times, in a time period of at least 3 days. For each β-lactam investigated,

the lowest concentration giving 19 positive results out of 20 total test results was

determined, interpreting with the ROSA Pearl Reader.



The impact of the length of incubation on the test result was studied. The incubation

time was modified to 150 s and 4 min. Each situation was tested with four blank milk

samples and with four milk samples spiked with benzylpenicillin (3 μg kg-1) or

cloxacillin (30 μg kg-1).


Blank milk samples and samples spiked with benzylpenicillin (3 μg kg-1) or cloxacillin

(30 μg kg-1) were analysed, and the strips were read with the ROSA Pearl Reader

directly after the incubation and after 0.5, 1, and 3 min.


The impact of the milk quality (somatic cell count, total bacterial count) and

composition (fat and protein content, pH) was tested by comparing the test

performance of the Charm MRL-3 for milk with a normal quality and composition with

milk with a high somatic cell count (34 samples) or a high total bacterial count (36

samples). A comparison of the test performance was also executed on 10 different

spiked milk samples with a normal and an abnormal composition. Milk of normal and

abnormal composition was analysed with and without spiking with benzylpenicillin (3

μg kg-1) or cloxacillin (16 μg kg-1). For each different milk type, the average, the

highest, and the lowest reader value was calculated.

Milk samples with a high number of somatic cells (>106 ml-1) were selected at the

milk control station based on Fossomatic 5000 (FOSS, Hillerød, Denmark)

measurements. Milk samples with a high total bacterial count (>5 x 105 cfu ml-1) were

obtained by keeping normal milk samples for 4-6 h at room temperature. The final

bacterial count was determined by performing a spiral plate count (Eddy Jet, IUL sa,

Barcelona, Spain) on plate count agar plates after 3 days incubation at 30°C. Milk

samples with a low fat content (<2 g 100 ml-1) were obtained by removal of the fat

layer by centrifugation (3,050 g, 10 min, at 5°C). Milk samples with a high fat content

(>6 g 100 ml-1) , a low (<2.5 g 100 ml-1) and a high protein content (>4 g 100 ml-1)

were natural milk samples with extreme fat or protein content that were selected at

the milk control station based on IR spectroscopic results (MilcoScan 4000, FOSS).


To prepare samples with an abnormal pH, normal milk was initially adjusted to pH

6.0 and pH 7.5 with 1 M HCl or 1 M NaOH, respectively, then the pH was further

adjusted with the addition of either 0.1 M HCl or 0.1 M NaOH.


UHT milk, sterilized milk, reconstituted milk powder, thawed milk, goats‟ milk, ewes‟

milk, and mares‟ milk were also tested to determine if the Charm MRL-3 was a

suitable test for these types of milk. Ten different samples of each milk type were

tested, with the exception that only three samples were tested for thawed milk. The

aim was not only to investigate if certain milk types interfere and cause false-positive

results but also to test if the detection capability was or was not hampered.

Therefore, benzylpenicillin (3 µg kg-1) and cloxacillin (30 µg kg-1) were spiked in raw

cows‟ milk, milk of other types, or milk from animal species different from the cow.

6.2.9 Test for false-positive/false-negative results Twenty-two farm milk samples, 22 truck milk samples, 11 consumer milk samples,

and 8 milk powders were analysed with Charm MRL-3 as part of a monitoring

programme. The same samples were also tested by Delvotest SP-NT, Bacillus

cereus-test (Suhren and Heeschen, 1993), Escherichia coli-test (Suhren,1997), and

Charm MRL -Lactam Test. Also, special sampled milk samples were analysed with

Charm MRL-3 to verify the rate of false-positive results. The special sampling

concerned 41 individual cow milk samples, 300 farm milk samples, and 300 tanker

milk samples. Positive samples were further analysed with other microbiological and

immunological antimicrobial residue tests.

For testing the rate of false-negative results and to verify if the test capacity for

benzylpenicillin in incurred samples is comparable to the value determined in spiked

milk samples, 82 incurred milk samples originating from 27 individual cows treated

with a veterinary drug containing benzylpenicillin and neomycin were analysed with

Charm MRL-3 and with other microbiological and β-lactam receptor screening tests.

Sampling started at the end of the withholding period. The exact concentration of

benzylpenicillin present in the milk samples was determined by LC-MS/MS in an

external lab.


6.2.10 Reagent influence (batch differences) To study the differences of different batches of reagents, blank and spiked milk

samples were analysed at the same time with 2 different batches of Charm MRL-3

reagents (Lot 009001 (Expiration July 2007 = Lot 009A (Expiration Sept. 2007)) and

Lot 008003. Besides spiking with benzylpenicillin (3 μg kg-1) or cloxacillin (30 μg

kg-1), 20 milk samples were also spiked with 3 μg kg-1 cefalonium to obtain reader

values closer to the cut-off value of 0. In the area around the cut-off, any change in

intensity of the test line can be quickly noted.

The stability of reagents during shelf-life was also checked. Blank and spiked


and just before the expiration date.

6.2.11 Inter-laboratory testing Twice a year ILVO-T&V organizes a national ring trial for the Belgian dairy industry


tests. Since 2007, laboratories using Charm MRL-3 participated. In 2007, AFSSA

Fougères, Community Reference Laboratory for antimicrobial residues in food of

animal origin, organized an international proficiency study for the analysis of -

lactam residues in raw milk. ILVO-T&V participated with the Charm MRL-3 test.

6.2.12 Daily control samples During the study, the reader was checked daily with a low and a high calibration

strip. To check the reagents and the reader, blank milk and control samples were

analysed daily. Blank raw milk (once daily to check the reader and additionally four

times), a positive standard prepared by reconstitution of a Charm tablet containing

benzylpenicillin (3 μg kg-1) and cloxacillin (12 μg kg-1), raw milk spiked with

benzylpenicillin (3 μg kg-1, twice daily), and a reconstituted lyophilized Charm

standard of cloxacillin (30 μg kg-1, four times daily) were used as control samples.


6.3 RESULTS AND DISCUSSION 6.3.1 Test and reader repeatability All repeatability results are shown in Table 1. Table 1. Repeatability of the Charm ROSA Pearl Reader and repeatability of the Charm MRL-3 test at different levels.

Milk Compound; concentration

Number of samples

Mean level Repeatability

asr CV (%)b Reader repeatability blank milk -- 10 -1336 25 1.9 positive milk benzylpenicillin; 3 μg kg-1 10 1133 24 2.1 benzylpenicillin; 5 μg kg-1 10 1477 30 2.0 cloxacillin; 25 μg kg-1 10 1091 30 2.7 cloxacillin; 45 μg kg-1 10 1485 38 2.6 Test repeatability blank milk 30 -1008 507 50.3 positive milkc 30 1218 186 15.3 positive milkd 20 1830 109 6.0 Notes: a sr, standard deviation of repeatability. b CV, coefficient of variation. c reader level 0-1500. d reader level >1500.

The repeatability of the Charm ROSA Pearl Reader was very good; very low

standard deviations of repeatability (sr) values were obtained.

There is a difference in repeatability of the test in testing blank and positive samples.

A standard deviation of repeatability of 507 for negative milk samples is too high

(values up to 250-300 are acceptable). The high sr value for blank milk was mainly

caused by 5 samples tested in duplo giving a negative and a false-positive result.

For these 5 samples, the difference between the first and the second test result

ranged between 1294 and 1964.

The repeatability for positive samples was better and is acceptable. The best

repeatability was found for the samples with the highest reader result level.

6.3.2 Test selectivity The Charm MRL-3 is very selective for the detection of penicillins and

cephalosporins. A real interference was only caused by clavulanic acid, a -

lactamase inhibitor, at 175 μg kg-1 and above.


benzylpenicillin (4)ampicillin (4)

amoxicillin (4)

oxacillin (30)

cloxacillin (30)

dicloxacillin

nafcillin (30)

penethamate (4)

ceftiofur (100)desfuroylceftiofur

(100)

cefquinome (20)

cefazolin (50)

cephapirin (60)

desacetylcephapirin (60)

cefacetrile (125)

cefoperazone (50)

cefalexin (100)

cefalonium (20)

Positive results for enrofloxacin (quinolones) and colistin (polymyxins) were also

obtained. A larger number of replicates showed that these positive results were

false-positive results rather than a cross-reactivity of the test for these compounds.

Despite a special test line for cloxacillin and related compounds, the test is not able

to differentiate between cloxacillin and the other β-lactams. Also, within the β-lactam

group, the test is not specific for any particular β-lactam, but natural penicillins

(benzylpenicillin) and aminopenicillins (ampicillin and amoxicillin) could be

differentiated from the group of isoxazolyl penicillins and cephalosporins after a pre-

treatment of the milk with penase (data not shown).

6.3.3 Detection capability A summary of the detection capabilities is given in Figure 2 and Table 2.

Figure 2. Detection pattern in raw cows’ milk of the Charm MRL-3, instrumental reading with a cut-off ratio of 0. Detection expressed relatively to the MRL (Commission Regulation (EU) No 37/2010 and amendments as of October 12, 2010). MRL (in µg kg-1) for each β-lactam compound mentioned in brackets. Inner circle = 2 MRL, circle 2 = MRL, circle 3 = 0.5 MRL, circle 4 = 0.25 MRL.


Table 2. Detection capability in raw cows’ milk of the Charm MRL-3 instrumental reading with a cut-off ratio of 0a in comparison to the Charm MRL (8-min test). Group Compound MRLb

(μg kg-1) Detection capability (μg kg-1)

Charm MRL-3 Charm MRLc penicillins benzylpenicillin 4 3 2 ampicillin 4 4 3 amoxicillin 4 4 3 oxacillin 30 18 30 cloxacillin 30 14 25 dicloxacillin 30 12 25 nafcillin 30 90 45 penethamate 4d 200 100 cephalosporins ceftiofur 100e 4 (6f) 6 (8f) cefquinome 20 14 14 cefazolin 50 16 10 cephapirin 60g 3 (1h) 5 (7h) cefacetrile 125 9 6 cefoperazone 50 4 4 cefalexin 100 10 15 cefalonium 20 3 3 Notes: a Detection capability is defined as the lowest concentration tested giving a minimum

of 19 positive results out of 20. b MRL, maximum residue limit (Regulation (EC) No 470/2009; Commission

Regulation (EU) No 37/2010 and amendments as of October 12, 2010). c Reybroeck and Ooghe (2004) and unpublished data. d marker residue is benzylpenicillin. e sum of all residues retaining the -lactam structure expressed as desfuroylceftiofur. f desfuroylceftiofur. g sum of cephapirin and desacetylcephapirin. h desacetylcephapirin.

The Charm MRL-3 detected all -lactams with a MRL in milk (Commission

Regulation (EU) No 37/2010 and amendments as of October 12, 2010) at their

respective MRL excepted for nafcillin (MRL = 30 µg kg-1) and penethamate (MRL = 4

µg kg-1), which were detected at 90 and 200 µg kg-1 and above.

From a practical perspective the high detection limit of 200 μg kg-1 for penethamate

in relation to the MRL is of no significance since penethamate is not stable in milk

and is rapidly and completely hydrolysed to benzylpenicillin and diethylaminoethanol

(Anon., 2000). Furthermore, the marker residue for penethamate is benzylpenicillin

(Commission Regulation (EU) No 37/2010).


Most cephalosporins were detected very sensitively; concentrations far below the

respective MRL values caused a positive result. Concentrations like 12 and 14 µg

kg-1 cloxacillin sometimes gave a higher colour intensity for the cloxacillin test line

compared to the intensity of the control line. Nevertheless, the ROSA Pearl Reader

converted the line comparison to a positive reading.

When performing the Charm MRL-3 instead of the classic Charm MRL Test (8 min)

(Reybroeck and Ooghe, 2004) nearly the same detection capabilities were obtained

for the group of natural penicillins, aminopenicillins, and cephalosporins. The Charm

MRL-3 was more sensitive for cloxacillin and the other isoxazolyl penicillins due to a

separate test line for cloxacillin on Charm MRL-3 strips. Only for nafcillin there was a

real loss in sensitivity: the detection capability shifted from 45 to 90 µg kg-1.


All data obtained when changing the length of incubation are summarized in Table 3.

Table 3. Values obtained when testing blank and spiked milk samples (4 replicates) after incubations of different lengths of time. Reader Incubation times

3 min 2 min 30 s 4 min Blank milk mean value -1564 -1320 -1330 lowest value -1942 -1531 -1442 highest value -1418 -1012 -1258

Milk spiked with benzylpenicillin at 3 μg kg-1 mean value 1259 1153 1125 lowest value 1131 1126 937 highest value 1306 1175 1413

Milk spiked with cloxacillin at 30 μg kg-1 mean value 1159 1019 1209 lowest value 840 828 1033 highest value 1420 1281 1419


Performing the Charm MRL-3 protocol with the different incubation times tested had

no significant impact on the reader values for blank milk or positive spiked milk

samples.

Even when the incubation differed from the standard 3 min, within the limits tested,

correct and acceptable results were still obtained, proving that a strict adherence to

timing was not a critical point.


All data obtained when delaying the reading of the strips are summarized in Table 4.

If the reading of the strips after incubation was delayed, the reader values did not

change for blank milk samples, while positive reader values had the tendency to

increase. So delaying the reading did not cause incorrect results but slightly

improved the detection capability.

Table 4. Values obtained when measuring 4 strips directly after incubation and after a delay of 0.5, 1, and 3 min.

Reader

Delay after incubation before reading (min) 0 0.5 1 3

Blank milk mean value -1564 -1484 -1546 -1526 lowest value -1942 -1640 -1638 -1678 highest value -1418 -1399 -1459 -1361 Milk spiked with benzylpenicillin at 3 μg kg-1 mean value 1060 1102 1127 1230 lowest value 1026 1016 1062 1177 highest value 1122 1150 1164 1321 Milk spiked with cloxacillin at 30 μg kg-1 mean value 1091 1117 1181 1298 lowest value 840 819 872 978 highest value 1420 1510 1632 1694


With respect to testing the impact of the milk quality and composition (somatic cell

count, total bacterial count, fat and protein content, and pH), the mean, the highest


reader value, and the lowest reader value for each milk type are given in Figures 3

and 4.

Figure 3. Reader values for normal and abnormal blank milk (, mean; , lowest; , highest) and normal and abnormal milks containing 3 μg kg-1 benzylpenicillin ( , mean; , lowest; , highest). Milks were of normal composition (1) or with: (2) high somatic cell count; (3) high bacterial count; (4) low fat content; (5) high fat content; (6) low protein content; (7) high protein content; (8) low pH; or (9) high pH. The horizontal line at a value of 0 gives the cut-off between a negative and a positive result.

The milk quality and composition had some influence on the performance of the

Charm MRL-3 when testing blank milk; most blank milk samples were clearly

negative with reader values below 0. However, positive reader values were obtained

for blank milk with a high somatic cell count (1 out of 34), for milk with a high protein

content (3 out 10), and for milk with a low pH (4 out of 10). Also, some influence of

milk quality and composition on the Charm MRL-3 results was noticed when testing

spiked milk samples. In milk with a high pH, the detection of benzylpenicillin was

hampered; 7 out of 10 samples tested negative. In 5 out of 10 milk samples with a

high fat content, 16 µg kg-1 of cloxacillin tested negative. The same result was

obtained for 2 out of 10 milk samples with a high somatic cell count and for 5 out of

12 milk samples with a high bacterial load. Also, one negative result was obtained for

milk with a high bacterial load spiked with benzylpenicillin at 3 µg kg-1. The

production of bacterial -lactamase in some milk samples with a high bacterial load

Type of milk0 1 2 3 4 5 6 7 8 9

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could not be excluded. Lower reader values were obtained when testing milk with a

high fat content spiked with cloxacillin at 16 µg kg-1. A hampered flow of milk with a

high fat content on the strip could be the reason for the low results.

Figure 4. Reader values for normal and abnormal milks containing 16 μg kg-1 cloxacillin (, mean; , lowest; , highest). Milks were of normal composition (1) or with: (2) high somatic cell count; (3) high bacterial count; (4) low fat content; (5) high fat content; (6) low protein content; (7) high protein content; (8) low pH; or (9) high pH. The horizontal line at a value of 0 gives the cut-off between a negative and a positive result.

A high pH can occur in milk of individual cows due to the presence of subclinical

mastitis, but it is unlikely that an entire bulk collection of milk will be affected. Further,

it must also be recognized that the test is qualitative rather than quantitative and is

used only to discriminate between β-lactam residue-free milk and milk containing

such residues.


The results of the testing of UHT milk, sterilized milk, reconstituted milk powder,

thawed milk, goats‟ milk, ewes‟ milk, and mares‟ milk are presented in Figures 5 and

6.

Type of milk

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Figure 5. Reader values for blank milk (, mean; , lowest; , highest) and different milks containing 3 μg kg-1 benzylpenicillin ( , mean; , lowest; , highest). Raw cows’ milk (1) compared with: (2) UHT milk; (3) sterilized milk; (4) reconstituted milk powder; (5) thawed milk; (6) goats’ milk; (7) ewes’ milk; and (8) mares’ milk. The horizontal line at a value of 0.0 gives the cut-off between a negative and a positive result.

Figure 6. Reader values for different milks containing 30 (situation 1 to 5) or 16 (situation 8) μg kg-1 cloxacillin (, mean; , lowest; , highest). Raw cows’ milk (1) compared with: (2) UHT milk; (3) sterilized milk; (4) reconstituted milk powder; (5) thawed milk; and (8) mares’ milk. The horizontal line at a value of 0.0 gives the cut-off between a negative and a positive result.

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Charm Sciences Inc. is only claiming the Charm MRL-3 as a test for raw cows‟ milk.

Dairy companies are mainly interested in testing raw milk from incoming tankers or

at the farm before collection. When testing blank UHT milk, sterilized milk, and

reconstituted milk powder, false-positive results (respectively 2 out of 20, 2 out of 15,

and 2 out of 18) were obtained. No significant differences were noticed in testing

different milk types spiked with benzylpenicillin at 3 µg kg-1 or cloxacillin at 30 µg kg-

1. So Charm MRL-3 is not only a raw milk test for the dairy industry, but it could also

be used by other laboratories to test UHT-milk, sterilized milk, reconstituted milk

powder, or thawed milk (monitoring samples are often kept frozen during transport

and storage) on the condition that positive results are further tested with a different

antibiotic test.

When testing blank goats‟ and ewes‟ milk, false-positive results (respectively 6 out of

8 and 10 out of 12) were obtained. Since such a high percentage of false-positive

results were obtained for blank milk from these animal species, no testing of spiked

samples was performed. Blank mares‟ milk gave 3 invalid readings out of 10, while

spiked mares‟ milk gave less positive results and even false-negative results. The

Charm MRL-3 is, therefore, not a suitable test to screen milk from animal species

different from the cow (goat, ewe, or mare).

6.3.6 Test for false-positive/false-negative results Throughout the evaluation study, false-positive results were obtained when testing

blank raw milk. Out of the special sampling of 41 individual cows‟ milk samples, 300

farm milk samples, and 300 tanker milk samples, the percentage of false-positive

results can be estimated as 2.4, 0.7, and 2.7%, respectively. Samples giving false-

positive results were retested (5 replicates). The replicates always gave a negative

Charm MRL-3 result. So it is recommended to retest a positive sample to confirm the

presence of -lactam antibiotics.

In legislation for screening methods there is only a criterion of <5% (-error) for the

false compliant rate at the level of interest (Commission Decision 2002/657/EC).

There is no criterion for the rate of false-positive results; for logistic and economic

reasons, this rate should be as low as possible. In the same legislation, it is

stipulated that, in the case of a suspected non-compliant result, this result shall be

confirmed by a confirmatory method.


No false-negative results were obtained when testing farm milk, truck milk,

consumption milk, and milk powders as part of a monitoring programme. However,

false-positive Charm MRL-3 results were also obtained in this monitoring

programme.

For testing the rate of false-negative results, incurred milk samples originating from

individual cows treated with a veterinary drug containing benzylpenicillin and

neomycin, were analysed with the Charm MRL-3. From a benzylpenicillin content

≥1.2 μg kg-1, all 32 incurred milk samples tested positive. These data confirm that the

detection capability for benzylpenicillin in spiked milk (3 μg kg-1, Table 2) is also valid

for the detection of benzylpenicillin in incurred milk samples. In this study of incurred

milk samples originating from individual cows, false-positive Charm MRL-3 results

were also obtained. Twenty-four out of 50 samples with a benzylpenicillin content

≤1.0 μg kg-1 tested positive on Charm MRL-3. If just the group of milk samples with a

benzylpenicillin content below the CC from the LC-MS/MS determination (0.3 µg

kg-1) is considered, still 12 out of 33 samples gave a positive Charm MRL-3 result.

It‟s difficult to indicate the reason for this high rate of false non-compliant results. Milk

of individual cows is more likely to have an anomalous fat or protein content,

although this was not the case in the depletion study. Some milk samples causing

false-positive results were centrifuged and decanted. On the bottom of some test

tubes debris was present, microscopically identified as parts of hair and dust. Such

small particles could possibly hamper the lateral flow of milk on the dipstick. The

presence of small particles in the milk samples of the depletion study is more likely,

since this milk was unfiltered; milk is normally passed through a filter fitted in the milk

tubes before entering in the milk silo at the farm.

6.3.7 Reagent influence (batch differences) A summary of the results of the testing of spiked milk samples with two different

batches of Charm MRL-3 reagents is given in Table 5.

Only small differences in test capability were found between different batches of

reagents of Charm MRL-3. Blank milk gave essentially the same reader values.

Batch 008003 was somewhat less sensitive when detecting the residues in the

spiked samples containing benzylpenicillin at 3 μg kg-1, cloxacillin at 12 μg kg-1, or


cefalonium at 3 μg kg-1, but the difference is of no importance when using the kit for

the discrimination of positive from blank milk.

Table 5. Values obtained and number of positive and negative milk samples when testing the same blank and the same spiked milk samples with Charm MRL-3 reagents from different batches or with reagents of a different age.

Tested Lot 009001 just after production Lot 008003

Ratio Number Ratio Number mean mina maxa posa nega mean mina maxa posa nega

blank milk -1207 -1753 -627 0 20 -1116 -1661 -530 0 20

milk spiked with benzylpenicillin at 3 μg kg-1 1173 785 1576 24 0 986 -853 1627 23 1

milk spiked with cloxacillin at 12 μg kg-1 1421 1239 1681 20 0 939 -190 1386 19 1

milk spiked with cefalonium at 3 μg kg-1 905 -688 1364 19 1 238 -1271 1220 14 6

Tested Lot 009001 just after production Lot 009001 just before expiry

Ratio Number Ratio Number mean mina maxa posa nega mean mina maxa posa nega

blank milk -1207 -1753 - 627 0 20 -230 1597 838 10 10

milk spiked with benzylpenicillin at 3 μg kg-1 1173 785 1576 24 0 1412 1252 1604 24 0

milk spiked with cloxacillin at 12 μg kg-1 1421 1239 1681 20 0 1444 1124 1939 20 0

milk spiked with cefalonium at 3 μg kg-1 905 -688 1364 19 1 1106 898 1379 20 0

Notes: a min, minimum; max, maximum; pos, positive; neg, negative.

The stability of reagents during shelf life was also checked. Blank and spiked


and again shortly before the expiration date. In general comparable results were

obtained for the spiked milk samples, but 10 out of 20 blank milk samples tested

positive (false-positive results) with the reagents just before the expiration date.

6.3.8 Interlaboratory testing

Twice a year ILVO-T&V organizes a national ring trial for the Belgian dairy industry


tests. Since 2007, laboratories could also participate with the Charm MRL-3. Each

time 8 blind-coded milk samples were distributed to the laboratories.

In the 5 ring trials organized since spring 2007, Charm MRL-results were reported by

2 laboratories. No false-negative results were obtained; however, in 4 ring trials 7


(out of 20) false-positive results were generated. Details of the results are given in

separate reports (Ooghe and Reybroeck, 2007a, 2007b, 2008a, 2008b & 2009).

In the international proficiency study organized by AFSSA Fougères, 6 milk samples

were distributed among the participating laboratories (Fuselier et al., 2008). The

blank milk was analysed as positive by ILVO-T&V with Charm MRL-3 (reader value

761), while the 5 spiked milk samples respectively containing cefquinome at 50 μg

kg-1, cloxacillin at 40 μg kg-1, cefalonium at 20 μg kg-1, and benzylpenicillin at 6 μg

kg-1 (in duplicate) all tested positive.


During the study, a low and a high calibration strip, a negative control sample (blank

raw milk), a positive standard sample (reconstituted Charm tablet containing benzyl-

penicillin (3 μg kg-1) and cloxacillin (12 μg kg-1)), four blank raw milk samples, two

raw milk samples spiked with benzylpenicillin (3 μg kg-1), and four reconstituted

lyophilized Charm standards of cloxacillin (30 μg kg-1) were analysed daily. The

results are shown in Figures 7-9.

Figure 7. Reader values obtained for 32 daily checks of the reader with a low () and a high calibration strip (). The horizontal line at a value of 0 gives the cut-off between a negative and a positive result.

Calibration strips

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Figure 8. Reader values obtained for 32 daily checks of the reader with a negative () and a positive control (). The horizontal line at a value of 0 gives the cut-off between a negative and a positive result. Figure 9. Reader values obtained for 128 control samples with blank raw milk (), 60 control samples containing 3 μg kg-1 benzylpenicillin (), and 128 control samples containing 30 μg kg-1 cloxacillin (). The horizontal line at a value of 0 gives the cut-off between a negative and a positive result.

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Control samples

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Over 32 working days, the control samples gave very constant reader values. For

the entire period, following average reader values were obtained: blank raw milk:

-1087 482; milk spiked with 3 µg kg-1 benzylpenicillin: 1279 195; and milk spiked

with 30 µg kg-1 cloxacillin: 1377 254. It is worth noting the influence by some false-

positive results for some blank milk samples on the standard deviation.

6.4 CONCLUSIONS With a total test time of 3 min, the Charm MRL-3 is presently one of the fastest tests

on the market for the detection of β-lactam residues in cows‟ milk.


test at the farm before collection in order to prevent tanker milk contamination. A

drawback is the recommendation of the use of a reader system for the interpretation

of the colour formation on the dipsticks. The same reagents and test protocol could

also be used at the entrance of the dairy plant to check the incoming milk on the

presence of β-lactams.

Of all -lactams with a MRL in milk, only nafcillin (detection capability = 90 µg kg-1;

MRL = 30 µg kg-1) and penethamate (detection capability = 200 µg kg-1; MRL = 4 µg

kg-1) are not detected at their respective MRL.

It is recommended to retest initial positive samples as indicated by the kit

manufacturer since false-positive results could occur (2.4% for individual cows‟ milk,

0.7% for farm milk, and 2.7% for tanker milk, respectively).

The Charm MRL-3 is not a suitable test to screen milk from animal species different

from the cow (goat, ewe, or mare). Charm Sciences Inc. is only claiming the Charm

MRL-3 as a test for raw cows‟ milk.

Acknowledgement We appreciate the valuable work performed by Veronique Ottoy and Katleen Vander

Straeten and also thank Charm Sciences Inc. for kindly providing Charm MRL-3 test

reagents, Vzw Melkcontrolecentrum-Vlaanderen for providing part of the raw cows‟

milk samples with a special composition or quality and for the MilcoScan 4000 and

Fossomatic 5000 measurements, and Siegrid De Baere (UGent) for the incurred milk

samples used for testing of false-negative results and for the determination of

benzylpenicillin content of these samples.


6.5 REFERENCES

Anonymous. 2000. Committee for Veterinary Medicinal Products: Penethamate (summary report) The European Agency for the Evaluation of Medicinal Plants, Veterinary Medicines Evaluation Unit, London, United Kingdom: 1-3. Anonymous. 2005. Martindale: The complete drug reference. Ed. Sweetman S. C. Royal Pharmaceutical Society of Great Britain, Pharmaceutical Press, London, United Kingdom, 34th Edition. Anonymous. 2010a. Jaarverslag 2009. Vzw Melkcontrolecentrum-Vlaanderen, Lier, Belgium : 1-52. Anonymous. 2010b. Rapport des Activités 2009. Comité du Lait, Battice, Belgium: 1-70. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Comm. 2002 L221: 8-36. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010 L15: 1-72. Fuselier R., Gaudin V., Dubreil E., Verdon E., Sanders P. 2008. Proficiency study for the analysis of beta-lactam residues in raw milk. Final report. AFSSA, Fougères, France. Grunwald L. 2002. Untersuchungen zur Analytik und zum Einfluss technologischer Prozesse auf Penicillinrückstände in Milch. Dissertation. Bergische Universität Wuppertal, Fachbereich Chemie, Wuppertal, Germany. Honkanen-Buzalski T., Reybroeck W. 1997. Antimicrobials. In: „Monograph on residues and contaminants in milk and milk products.‟ I.D.F. (International Dairy Federation), Brussels, Belgium, ISBN 92 9098 025 8: 28-34. Kroll S., Usleber E., Zaadhof K.-J., Schneider E., Märtlbauer E. 2000. Evaluation of commercial rapid tests for β-lactam antibiotics in raw milk. Proceedings of the Euroresidue IV Conference on Residues of Veterinary Drugs in Food, Veldhoven, the Netherlands, May 8-10, 2000: 693-697. Ooghe S., Reybroeck W. 2007a. Rapport ringonderzoek antibiotica 24 mei 2007: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-23. Ooghe S., Reybroeck W. 2007b. Rapport ringonderzoek antibiotica 18 oktober 2007: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-17. Ooghe S., Reybroeck W. 2008a. Rapport ringonderzoek antibiotica 24 april 2008: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-21. Ooghe S., Reybroeck W. 2008b Rapport ringonderzoek antibiotica 23 oktober 2008: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-17. Ooghe S., Reybroeck W. 2009. Rapport ringonderzoek antibiotica 23 april 2009: microbiologische testen en sneltesten. ILVO-T&V, Melle, Belgium: 1-20.


Pyörälä S. 1995. Staphylococcal and streptococcal mastitis. In: „The bovine udder and mastitis‟ Ed. Sandholm M., Honkanen-Buzalski T., Kaartinen L., Pyörälä S. University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland. ISBN 951-834-047-1: 143-148. Quandt B. 2006. Identifizierung van antimikrobiellen Rückständen in Milch mittels Schnelltestsystemen. Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität, München, Germany. Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Union 2009 L152: 11-22. Reybroeck W. 2004. Résidus d‟antibiotiques dans le lait. Utilisation des kits de dépistage des inhibiteurs. Point Vét. 242: 52-57. Reybroeck W. 2008. The use of microbiological, immunological and receptor tests for monitoring of residues of antimicrobials in milk: the Belgian approach. In Bulletin of the IDF 424/2008 “Advances in Analytical Technology” , ISSN 0250-5118: 13-16. Reybroeck W., Ooghe S. 2004. Rapid screening for residues of antibiotics in milk at the factory. In A Farm-to-table Approach for Emerging and Developed Dairy Countries, Proceedings IDF/FAO International Symposium on Dairy Safety and Hygiene, Cape Town, Republic of South Africa, March 2-5, 2004. ISSN 1810-0732: 157-161. Reybroeck W., Ooghe S. 2008. Validation of the CHARM MRL-3 for fast screening of β-lactam antibiotics in raw milk. Proceedings of the Euroresidue VI Conference on Residues of Veterinary Drugs in Food, Egmond aan Zee, the Netherlands, May 19-21, 2008: 787-792. Suhren G. 1996. Influence of residues of antimicrobials in milk on commercially applied starter cultures-model trials. Kieler Milchwirtschaftl. Forschungsber. 48(2): 131-149. Suhren G. 1997. Mikrobiologischer Hemmstofftest mit E. coli zum Nachweis von Chinolon-Rückständen in Milch. 38.Arbeitstagung des Arbeitsgebiet Lebensmittelhygiene der DGV, Garmisch-Partenkirchen, Germany. Teil II: 659-664. Suhren G., Heeschen W. 1993. Detection of tetracyclines in milk by a Bacillus cereus microtitre test with indicator. Milchwissenschaft 48: 259-263. Ţvirdauskiené R., Šalomskiené J. 2007. An evaluation of different microbial and rapid tests for determining inhibitors in milk. Food Control 18(5): 541-547.

ory

Chapter 7 Inhibitory Effects by Pseudomonas spp. in Raw Milk on Microbiological Inhibitor Assays

Adapted from:

Reybroeck W., Marchand S., De Block J., Coudijzer K., Heylen K., Daeseleire E., De

Brabander H.F., Heyndrickx M. 2010. Inhibitory effects by Pseudomonas spp. in raw

milk causing false-positive results in microbiological inhibitor assays for the detection

of antibiotic residues. Journal of Dairy Science, to be submitted.

7 Inhibitory Effects by Pseudomonas spp. in Raw Milk on Microbiological Inhibitor Assays 204

Inhibitory effects by Pseudomonas spp. in raw milk causing false-positive results in microbiological inhibitor assays for the detection of antibiotic residues

Abstract Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii,

were isolated from milk of a farm with frequent problems of false-positive Delvotest

results as part of the regulatory quality programme. Growth at 5 to 7°C of the isolates

in milk resulted in high lipolysis and the production of bacterial inhibitors. These

bacterial inhibitors with a molecular weight <1 kDa showed to be heat-tolerant and

inhibitory to Geobacillus stearothermophilus var. calidolactis, the test strain of most of

the commercially available microbiological inhibitor tests for milk. The bacterial

inhibitors also showed antimicrobial activity against Staphylococcus aureus, Bacillus

cereus, and B. subtilis and interfered in yoghurt production.

The bacterial inhibitors were not yet identified but the results of the characterization

assays could rule out that the inhibition was caused by the elevated level of free fatty

acids and indicate in the direction of cyclic lipodepsipeptides, toxins with antimicrobial

properties.

Our findings indicate a new challenge for the dairy industry. By extending the

refrigerated storage of raw milk, the keeping quality of milk is influenced by growth

and metabolic activities of psychrotrophic bacteria at low temperatures. This could

besides a possible spoilage of long-life milk also result in false-positive microbial

inhibitor test results.


7.1 INTRODUCTION Antibiotic residues in milk are of great concern to dairy farmers, milk processors,

authorities, and consumers because of public health and industrial implications. In

European countries, inhibitory substances are routinely screened in farm milk

samples as part of a regulatory quality programme as required in Regulation (EC) No

853/2004 and its Corrigendum. Microbiological inhibitory tests are widely used for

screening for inhibitors such as antibiotics in the milk delivered to dairies. The

principle of microbial inhibition assays is traditionally based on the detection of

growth inhibition, indicated by clear inhibition zones in disc assays or by a colour

change of the pH-indicator in the test medium. Major drawbacks with microbiological

methods are the fact that no indications of the identity of the inhibitory substance(s)

are given and the possible interference by natural inhibitors.

Inhibitions without any reasonable explanation occur occasionally. Inhibitory

substances other than antibiotics have been reported in milk (Wolin and Kosikowski,

1958; Kosikowski and O‟Leary, 1963; Duthie et al., 1976; Andrew et al., 1977;

Okada, 1986; Carlsson, 1991; Cullor et al., 1992; Halbert et al., 1996). Colostrum

and mastitic milk are known to cause false-positive results in microbiological assays

for antibiotic residues (Goudswaard et al., 1978; Kitchen, 1981; Egan and Meaney,

1984; Schiffmann et al., 1992; Suhren 1995). Lactoferrin and lysozyme, two natural

antibacterial substances in milk, have been indicated to have separately and

synergistically an inhibitory effect on Geobacillus stearothermophilus var. calidolactis,

the most commonly used test organism in microbiological inhibitor assays (Carlsson

and Björck, 1987; Carlsson et al., 1989; Pan et al., 2007). The synergistic effect was

explained by complex formation between the two proteins. Apparently, the protein

complex could diffuse more easily through the agar and be more inhibitory in the

Delvotest than each protein separately (Carlsson et al., 1989). Lactoferrin is an iron-

binding glycoprotein found in larger concentrations in colostrums and mastitic milk.

The antimicrobial activity of lactoferrin and its derivatives has been attributed mainly

to three mechanisms: a) binding with iron present in the medium leading to inhibition

of bacterial growth since iron is indispensable for many bacteria; b) direct binding of

lactoferrin to the microbial membrane, especially to lipopolysaccharides in Gram-

negative bacteria, causing fatal structural damages to outer membranes; and c)

prevention of microbial attachment to epithelial cells or enterocytes (Korhonen,


2009). Other authors suggest that lactoferricin represents the antimicrobial domain of

lactoferrin or that lactoferrin acts as a precursor molecule releasing lactoferricin

(Bellamy et al., 1992; Jones et al., 1994). Lysozyme catalyses hydrolysis of the

bacterial wall peptidoglycan, notably of Gram-positive bacteria like Bacillus (Salton,

1966). The concentrations of lysozyme and lactoferrin in bovine milk are low during

lactation. The lactoferrin content increases during involution of the udder. Early

colostrums contain high concentrations of lactoferrin and slightly elevated lysozyme

levels. In mastitic milk the concentrations of both lactoferrin and lysozyme increase.

The antibacterial effect of the lactoperoxidase/SCN-/H2O2 system and

immunoglobulins is also known (Carlsson, 1991; Mullan, 2003a). Lactoperoxidase is

a normal constituent of bovine milk. Hydrogen peroxide and thiocyanate are required

in addition to lactoperoxidase in order to get inhibition of acid production of starter

cultures. It appears that the antimicrobial effects are likely due to oxyacids of

thiocyanate e.g. OSCN- . The inhibitor(s) are heat-labile and are inactivated by

sulphur containing reducing agents. Lactoperoxidase is relatively heat-resistant but a

total inactivation can be obtained by heating at 80°C for 5 minutes (Mullan, 2003a).

The ability of some vitamin binding proteins to inhibit the growth of bacterial species

was suggested by Mullan (2003b). Feed complemented with minerals, oligo-

elements, and vitamins appeared to be a factor that may give rise to false-positive

results in the determination of antibiotics in milk with Delvotest SP (Romnée, 1999).

Along with the biochemical changes, the physical properties of mastitic milk change.

As a consequence of strong inflammation, the pH of milk increases from 6.6 to more

than 7. This reaction is mainly due to the transfer of bicarbonate ions from blood to

milk (Korhonen and Kaartinen, 1995). This pH increase can cause false-positive

results in microbiological assays based on acid production as criterion for bacterial

growth. High concentrations of alkaline disinfecting products or detergents, due to

inadequate rinsing and draining, could also induce false-positive screening results

(Schiffmann et al., 1992; Reybroeck, 1997; Zvirdauskiené and Salomskiené, 2007).

False-positive outcomes can also be induced by high levels of antiparasitic agents or

anti-inflammatory products.

Several reports have suggested that the rate of false outcomes increases with

increasing somatic cell count (SCC) (Van Eenennaam et al., 1993; Hillerton et al.,


1999; Kang and Kondo, 2001; Reybroeck and Ooghe, 2010). Non-proteinaceous

inhibitors may be present in early lactation and high SCC milk.

In 1963, Stadhouders associated the inhibitory principle in raw milk against starter

culture Streptococcus cremoris with the fat globules. Following Anders and Jago

(1964), some fatty acids like caprylic (C8:0), capric (C10:0), lauric (C12:0), and myristic

(C14:0) acid show inhibition for certain lactic acid bacteria in milk. Milk samples may

contain high concentrations of fatty acids, which may inhibit microbial inhibitor tests

(Mäyrä Mäkinen, 1990; Carlsson and Björck, 1992) by their ability to kill or to inhibit

the growth of bacteria (Deeth and Fitz-Gerald, 1983; Desbois and Smith, 2010). Fatty

acids predominantly affect Gram-positive organisms, and the most active saturated

and unsaturated fatty acids were lauric and palmitoleic (C16:1) acid (Kabara, 1983).

Following Boyaval et al. (1995), linoleic (C18:2), lauric, myristic, and oleic acid (C18:1)

inhibit the growth and acid production of Propionibacterium freudenreichii subsp.

Shermani. Mullan (2003b) indicated that for the inhibition of the starter culture

Lactococcus lactis subsp. cremoris, relatively high levels (0.1%) of butyric (C4:0),

capric, caproic (C6:0), or oleic acid were required. Such high concentrations of fatty

acids do normally not occur in modern hygienically produced milk that has been

stored at correct temperatures.

The typical concentration of fatty acids in bovine milk and their nomenclature are

given in Table 1. The concentration of free fatty acids in raw milk may increase due

to the activity of indigenous milk lipase or bacterial lipases. Milk is susceptible to

induced lipolysis, which can be initiated by cold mechanical disruption of the milk fat

globule membrane so that the indigenous milk lipase can easily access the fat

fraction of the milk. This can happen either mechanically, due to agitation, pumping,

stirring, and freezing/thawing of milk, or by enzymatic means, such as

phospholipases or glycosidases (De Jonghe, 2010).

Pseudomonas spp. which are the most important spoilers of raw milk are also

capable to produce heat-stable lipases which on their turn can lead to high

concentrations of free fatty acids (Mullan, 2003b). A biologically interesting lipid

group in milk fat are the polar lipids, which are mainly located in the milk fat globule

membrane. In particular, sphingolipids and their derivatives are considered highly

bioactive components possessing antibacterial activities (Rombaut and Dewettinck,

2006).


Table 1. Nomenclature and typical concentration of fatty acids occurring in bovine milk (Calder and Burdge, 2004).

Systematic name Trivial name Shorthand notation

Fatty acid (g 100 g-1 total fatty acids)

butanoic butyric 4:0 a

hexanoic caproic 6:0 a

octanoic caprylic 8:0 a

decanoic capric 10:0 a

dodecanoic lauric 12:0 a

tetradecanoic myristic 14:0 12 hexadecanoic palmitic 16:0 26 octadecanoic stearic 18:0 11

cis 9-hexadecenoic palmitoleic 16:1n-7 3 cis 9-octadecenoic oleic 18:1n-9 28

cis 9, cis 12-octadecadienoic linoleic 18:2n-6 2 all-cis 9,12,15-

octadecatrienoic α-linolenic 18:3n-3 1

Note: a, sum of all ≤12:0 fatty acids = 13 g 100 g-1 of total fatty acids.

The diapedesis of neutrophils may lead to a leakage of serum components across

the mammary epithelium, which, in turn, could lead to lipolysis and inhibitory action

(Carlsson and Björk, 1992) and also to an increase in the electrical conductivity of the

milk due to an increase in sodium content (Hillerton et al., 1999). In a study

conducted by Carlsson and Björck (1992), Delvotest SP showed to be more robust

and less easily affected by high free fatty acids in comparison to both the Arla

microtest and the Valio T101. The difference could be explained by the fact that the

Delvotest SP-NT is, in contrast to the two other assays, a test based on the agar

diffusion principle and uses Geobacillus stearothermophilus as test organism. The

Arla microtest and the T101 test are agar-free and consist of freeze-dried cultures of

Bacillus subtilis and Streptococcus salivarius ssp. thermophilus, respectively

(Carlsson and Björck, 1992). Pekkanen and Soback (1977) found heat-stable

inhibitors of Geobacillus stearothermophilus produced in two raw milk samples out of

20, which had been kept at room temperature until the pH fell to 6.2-6.3, without

giving any explanation or indication of type of inhibitor.

High concentrations of milk protein and milk fat can adversely affect antimicrobial

residue test performance, but the degree depends upon the analytical method of the

screening test (Andrew, 2000). Higher concentrations of immunoglobulins and milk


protein can also cause false-positives with screening tests used on samples from

recently freshened heifers or cows (Andrew, 2001).

Fat content of milk was positively related to an increase in false-positive rates for the

Delvotest Accelerator and Eclipse 50 (Reybroeck and Ooghe, 2010). Also a low

protein content could cause false-positive results, what possibly could be explained

by the fact that a minimum protein content is essential for normal growth of the test

organism (Reybroeck and Ooghe, 2010).

Evaluating the performance of screening tests, requirements are stipulated for the

rate of false compliant results. Following Commission Decision 2002/657/EC this rate

should be <5% (-error) at the level of interest. In the same Commission Decision, as

a general requirement for specificity it is stated that a method shall be able to

distinguish between the analyte (antibiotic residue) and the other substances under

the experimental conditions and that it is of prime importance that interference, which

might arise from matrix components, is investigated. But in the European residue

legislation there is no maximum norm fixed for screening tests for the rate of false

non-compliant results. In Commission Decision 2002/657/EC is indicated for

screening tests that in the case a suspected non-compliant result is obtained, this

result shall be confirmed by a confirmatory method. The American Food and Drug

Administration has established that, for commingled milk, the acceptable specificity

rate for antibiotic residue screening tests is ≥0.9 with a 95% confidence interval

(Anon., 1994).

False-positive results can have serious consequences. Good milk will be discarded

and in regulatory testing programme a financial penalty will be given to the

responsible farmer.

This paper describes the induction of false-positive screening results on

microbiological inhibitor tests for the detection of antibiotic residues in milk by

bacterial inhibitors produced by Pseudomonas bacteria, isolated from raw milk and

identified as closely related to P. tolaasii.


7.2 MATERIALS AND METHODS

7.2.1 Milk sampling Raw milk samples (500 ml) for laboratory analysis and a larger quantity (5 l) for

yoghurt production were aseptically collected from the farm cooling tank of two

Belgian farms with frequent problems of occurrence of inhibitors in the milk.

In a second sampling at farm 1, milk was aseptically collected from each individual

cow (n=11) and the farm silo (in duplicate).

A mixture of raw milk was aseptically collected from four individual cows of a farm in

the neighbourhood of ILVO-T&V to be used as blank reference milk. The cows in

mid-lactation were selected on the basis of not being treated with veterinary drugs

during the last months and giving milk with a low number of somatic cells (<2 x 105

ml-1). The blank milk was always tested before use with Delvotest SP-NT 5-PACK. In

some experiments commercial full-cream UHT consumption milk was used.

7.2.2 Antimicrobial testing by microbiological inhibitor tests and receptor assays For the detection of residues of antimicrobials in milk, following antibiotic test kits

were used: Delvotest SP-NT 5-PACK and Delvotest MCS from DSM Food

Specialties (Delft, the Netherlands); Copan Milk Test microplates from Copan Italia

S.p.A. (Brescia, Italy) and DSM Food Specialties (since February 2009); PremiTest

from DSM Nutritional Products (Geleen, the Netherlands); Charm MRL Beta-lactam

test, Charm II Sulfonamides Milk, Charm II Tetracyclines Milk, Charm II

Aminoglycosides Milk, and Charm II Macrolides Milk were from Charm Sciences Inc.

(Lawrence, MA). The reagents were stored in a cool room at 4 2°C, except for the

microbiological inhibitor tests that were stored between 6 and 15°C.

Plates of Delvotest SP-NT, Delvotest MCS, Copan Milk test, and ampoules of

Premitest were incubated in a covered waterbath (Type 19 + MP thermostat from

Julabo Labor-technic GmbH (Seelbach, Germany)) at 64.0 1°C. Plates of

Geobacillus stearothermophilus disc assay were incubated in an incubator BD240

with natural convection from Binder GmbH (Tittlingen, Germany) at 55.0 1°C.

The colour interpretation of Delvotest SP-NT and Delvotest MCS plates was done by

means of a flatbed scanner (HP Scanjet 7400C, Hewlett-Packard Company, Palo


Alto, CA) connected to DelvoScan software, version 3.05 (DSM Food Specialties).

The cut-off was set at a Z-value = -3.00. For the reading of Copan Milk Test plates a

HP GRLYB-0307 flatbed scanner (Hewlett-Packard Company) connected to CScan

software, version 1.32 (Copan Italia S.p.A.) was used. The cut-off was set at a CIF-

value = 4.5. The colour of PremiTest ampoules was interpreted by means of a flatbed

scanner (HP Scanjet 7400C, Hewlett-Packard Company) connected to PremiScan

software, version 1.02 (DSM Premitest B.V., Heerlen, the Netherlands). The cut-off

was set at a Z-value = -4.50.

Reagents of Charm MRL Beta-lactam test were incubated in a ROSA Incubator at

56°C (Charm Sciences Inc.). The strips were interpreted by means of a ROSA

Reader (Charm Sciences Inc.) with a reader value = 0 as cut-off. Charm II reagents

were incubated in a Charm Inctronic 2 Dual Incubator (Charm Sciences Inc.). The

amount of bound radioactive tracer was read with a liquid scintillation counter 1409

(Wallac, Waltham, MA). The cut-off cpm for each type of test was set according to

the protocols provided by the reagents manufacturer.

All commercial microbial inhibitor tests were used following the instructions of the kit

manufacturers. The disc assay was prepared and performed as described by Ginn et

al. (1982). For the disc assay an inhibition zone around the filter disc of 2.0 mm is

considered as positive.

In every run of each inhibitor test, blank reference milk and antibiotic standards were

included. Antibiotic standards were dissolved in water. Standard stock solutions of

the antibiotic standards of 100 mg l-1 were made in water and kept below 4°C for

maximum 14 days. Dilutions of 1 and 0.1 mg l-1 were freshly prepared on a daily

basis. Cloxacillin (C9393), oxytetracycline (O5875), penicillin G (benzylpenicillin,

PENNA), sulfadiazine (S8626), and para-aminobenzoic acid (A9878) were all from

Sigma-Aldrich (Bornem, Belgium). Penicillinase (L037) was from Genzyme (West

Malling, UK).

The interference of free fatty acids on Delvotest SP-NT and Copan Milk Test was

tested by spiking blank raw milk with 0.15% (w/v) of the following fatty acids: butyric

(B103500), capric (C1875), caproic (153745), caprylic (C2875), elaidic (E4637),

lauric (292168), linoleic (L1376), α-linolenic acid (L2376), myristic (M3128), oleic acid

(O1008), palmitic (P0500), palmitoleic acid (P9417), stearic (S4751), and vaccenic


acid (O1008). All fatty acids used were from Sigma-Aldrich. Some fatty acids were

also spiked in milk at 0.075% (w/v).

7.2.3 Assessment of milk quality 7.2.3.1 Composition parameters and pH

The content of the milk samples (fat, protein, and lactose) was determined by

Milcoscan 4000 (FOSS, Hillerød, Denmark) and the somatic cell count (SCC) by

Fossomatic 5000 (FOSS) at Vzw Melkcontrolecentrum-Vlaanderen (Lier, Belgium). A

SevenMulti pH meter was used from Mettler-Toledo Inc. (Columbus, OH) for pH-

measurements. In Europe, the norm for somatic cells is ≤4 105 ml-1; the criterion is

applied on rolling geometric averages (Corrigendum to Regulation (EC) No

853/2004).

7.2.3.2 Total bacterial count, enumeration of psychrotrophic and lipolytic bacteria

The total bacterial count and enumeration of the psychrotrophic and fat splitting

bacteria were performed on Plate Count Agar (PCA, 3d at 30°C), Violet Red Bile

Agar (VRBA, 1d at 30°C), PCA (7d at 7°C), and Tributyrin Agar (TBA, 3d at 30°C),

respectively, following ISO 4833 (Anon., 2003), ISO 4832 (Anon., 2006), ISO

6730/IDF101 (Anon., 2005), and Method 3.32 (Anon., 1999), respectively. PCA

(CM0325), VRBA (CM0107), and TBA (PM0004) were from Oxoid Limited

(Basingstoke, UK). In Europe, the norm for raw cows‟ milk for total bacterial count is

≤105 cfu ml-1; the criterion is applied on rolling geometric averages (Corrigendum to

Regulation (EC) No 853/2004).

7.2.3.3 Determination of lipolysis, fat oxidation and proteolysis

The lipolysis in milk was estimated as described by Driessen et al. (1977). Therefore

the milk fat was extracted according to IDF Bulletin 265 (Anon., 1991) using the

Bureau of Dairy Industries (BDI)-reagent. Afterwards the free fatty acids were titrated

with NaOH and phenolphthalein as indicator according to IDF Standard 6B (Anon.,

1989). The free fatty acids content is expressed as % oleic acid 100 g-1 fat. The

peroxide value was determined following AOAC Official Method 965.93 (Anon., 1997)

and expressed in meq O2 kg-1 fat. The proteolysis in milk was monitored following an

adaptation of the method described by Polychroniadou (1988).


7.2.4 Further bacteriological testing

7.2.4.1 Isolation, characterization and identifications of strains

Two strains, Pseudomonas P866 and P867, were isolated from the raw milk

samples, purified, identified and conserved at -80°C in Protect vials (International

Medical Products, Brussels, Belgium).

Single colonies were streaked out on different selective media to visualise enzymatic

activities: Tributyrin Agar for lipolysis (Anon., 1999) and PCA + 2% UHT milk for the

detection of proteolysis (FNZ Method 53.27 (Anon., 1986)). The haemolytic activity

was determined on Blood Agar (BA) plates with addition of 5% of sheep blood as

described in ISO Standard 7932 (Anon., 2004). Display of a clear halo around the

colony was considered as positive. Blood Agar Base No 2 (BA, CM0271) and Sheep

blood (SR0051) were from Oxoid Limited. The ability to use lactose was checked by

the isopropyl-β-D-thio-galactoside-test (IPTG).

Identification of the strains was done with API 20NE (20050) from bioMérieux France

(Craponne, France) and rpoB sequencing. The rpoB gene was amplified as

described previously by Ait Tayeb et al. (2005). The PCR-amplified rpoB gene

products were purified using the Nucleofast® 96 PCR system (Millipore, Billerica,

MA). For each sequence reaction a mixture was made using 3 µl purified and

concentrated PCR product, 1 µl of BigDye™ Termination RR mix version 3.1

(Applied Biosystems, Carlsbad, CA), 1.5 µl of BigDye™ buffer (5x), 1.5 µl sterile

milliQ water, and 3 µl (20 ng µl-1) of one of the sequencing primers. The amplification

primers were used as sequencing primers. The temperature-time profile was as

follows: 30 cycles of denaturation for 15 s at 96°C, primer annealing for 1 s at 35°C

and extension for 4 min at 60°C. The sequencing products were cleaned up as

described previously (Naser et al., 2005). Sequence analysis of the rpoB gene was

performed using a 3100 DNA Sequencer (Applied Biosystems) according to

protocols provided by the manufacturer.

7.2.4.2 Phylogenetic analysis

Forward and reverse strands of rpoB were assembled with the BioNumerics 4.6

software (Applied Maths, Sint-Martens-Latem, Belgium) and were aligned with

sequences retrieved from the EMBL database using Clustalx (Thompson et al.,


1994). Phylogenetic analyses were performed with Treecon (Van de Peer and De

Wachter, 1994). Trees were constructed with the neighbour joining algorithm without

corrections. Statistical evaluation of the tree topologies was performed by bootstrap

analysis with 1000 resamplings.

7.2.4.3 Growth and bacterial inhibitor production

Growth and bacterial inhibitor production was followed in raw milk, full-cream UHT

milk, and Brain Heart Infusion Broth (BHI, CM1135, Oxoid Limited). After inoculation

with Pseudomonas P866 or P867, the medium was incubated at 5-7 and 30°C with

daily sampling for enumeration of total bacterial count while the bacterial inhibitor

production was followed by Delvotest SP-NT, PremiTest, or on the disc assay with

Geobacillus stearothermophilius var. calidolactis. Bacterial inhibitor production of P.

tolaasii LMG2342 in full-cream UHT milk was checked on Delvotest.

The production of bacterial inhibitor was also followed for pure cultures of

Pseudomonas P866 and P867, for P866 and P867 in competition with raw milk flora,

and for P867 in competition with a mixture of 10 psychrotrophic Pseudomonas strains

isolated from Belgian raw milk samples. The list of strains is given in Table 2.

Table 2. List of Pseudomonas strains used as competitors in growth and bacterial inhibitor production experiment (De Jonghe, 2010).

Cluster Isolate R-number Tentative ID B S1-2-RM8-CFC27 R-37257 Pseudomonas sp. 1

D2 S1-2-RM11-CFC17 R-37194 P. fluorescens-like 1 E1 S3-1-RM8-CFC24 R-38757 P. fluorescens-like 2 H2 S1-2-RM1-CFC4 R-37283 P. fluorescens-like 3 I S3-1-RM8-CFC14 R-38748 P. gessardii

J2 S2-1-RM14-CFC26 R-38242 P. gessardii-like1 O4 S2-2-RM14-CFC19 R-38438 P. gessardii-like 2 R2 S3-1-RM11-PCMA10 R-38618 P. fragi V2 S3-2-RM8-CFC3 R-38766 P. lundensis W1 S2-2-RM14-CFC28 R-38253 P. fragi-like

7.2.4.4 Bacterial inhibitor characterization assays

7.2.4.4.1 Estimation of the molecular weight

The molecular weight of the bacterial inhibitor was tested by means of regenerated

cellulose dialysis membrane with a molecular weight cut-off (MWCO) of 1 kDa


(Spectra/Por Dialysis membrane 6 (132638) from Spectrum Laboratories, Inc.

(Rancho Dominguez, CA, USA)). The membrane was conditioned by soaking in

warm water for 30 minutes at 50°C, followed by rinsing with distilled water. Full-

cream UHT milk was inoculated with Pseudomonas strain P867 and incubated for 24

h at 30°C. In a first experiment a dialysis membrane tube was filled with 2 ml of

positive UHT milk due to growth of Pseudomonas P867 and surrounded by 35 ml of

blank raw milk. In a second experiment the inside of the membrane tube was filled

with 2 ml of blank raw milk, with 35 ml of positive UHT milk on the outer side of the

membrane. Milk from the in- and outside was sampled after 24 hours at 4°C to be

tested on Delvotest SP-NT.

Similar dialysis experiments were conducted with the dialysis membrane filled with 2

ml of milk containing 0.15% (w/v) of a fatty acid, surrounded by 35 ml of blank raw

milk. Following fatty acids were tested: caprylic, capric, lauric, palmitoleic, and α-

linolenic acid.

Finally, two similar dialysis experiments were conducted with 125 mM NaCl solution

against full-cream milk before and after growth of Pseudomonas P867. NaCl (06404)

was from Merck KGaA (Darmstadt, Germany).

7.2.4.4.2 Heat tolerance of bacterial inhibitor and fatty acids

The heat tolerance of the bacterial inhibitor produced by Pseudomonas P866 and

P867 was tested by heating 5 ml of the skimmed fraction of full-cream UHT milk after

growth of the bacteria in a glass test tube in a waterbath at different temperatures.

After heating at 80, 90, or 100°C for 10 min, the milk samples were rapidly cooled to

20°C with cold water and tested on Delvotest SP-NT. Besides Pseudomonas positive

milk, also raw milk spiked with 2 and 4 µg kg-1 benzylpenicillin, 20 µg kg-1 cloxacillin,

30 µg kg-1 cloxacillin, 300 µg kg-1 oxytetracycline or 100 µg kg-1 sulfadiazine was

tested.

7.2.4.4.3 Inhibition spectrum of the bacterial inhibitor

BHI broth was inoculated with Pseudomonas strain P867, incubated for 24 h at 30°C

and filtered through a Millipak 0.22 µm Filter Unit (Millipore Corporate (Billerica, MA)).

The inhibition spectrum of the bacterial inhibitor produced was tested by placing 12.7

mm diameter filter discs (Antibiotic Test Discs, FN0905A00005, Novolab,

Geraardsbergen, Belgium) impregnated with 80 µl of filtrate on PCA plates, filled with


10 ml of agar and inseminated with different bacterial strains. The plates were

incubated for 24 hours at 30°C, except for the plates with Geobacillus

stearothermophilus var. calidolactis, which were incubated for 24 hours at 55°C. The

antimicrobial activity was tested against following bacterial species: Bacillus cereus,

Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus var. calidolactis

C953, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella Enteritidis,

and Staphylococcus aureus.

7.2.4.4.4 Impact of test medium on the inhibition

The inhibitory action of Pseudomonas P867 bacterial inhibitor on different culture

media was determined by wetting Antibiotic Test Discs with 80 µl of positive UHT

milk. The filter discs were placed on the agar of different culture media (10 ml in 90

mm petri dishes), inseminated with 5.2 x 103 Geobacillus stearothermophilus var.

calidolactis. After an incubation for 24 h at 55°C, the plates were checked on the

presence of clear inhibition zones. If present, the diameters of the inhibition zones

were measured by means of a calliper. Following culture media were tested: PCA,

Milk Plate Count Agar (MPCA, CM0681), Nutrient Agar (NA, CM0004), Tryptone

Soya Agar (TSA, CM0131), and Brain Heart Infusion Agar (BHIA, CM1136). All

culture media were from Oxoid Limited.

7.3 RESULTS

7.3.1 Screening of milk for antibiotic residues Both farm silo milk samples from a special sampling on two farms with frequent

penalizations for inhibitory substances in the silo milk, tested (low) positive on

Delvotest MCS, but negative on Copan Milk Test, but with higher CIF values than for

blank milk samples. After pre-heatment at 80°C for 10 min, the inhibition on the

Delvotest SP-NT remained (data not shown). The pH of both milk samples was 6.4

and 6.6, respectively. After removal of the fat by centrifugation (10 min at 3,067 g at

4°C), the skimmed milk fraction was still causing positive Delvotest results. After

addition of penicillinase or para-aminobenzoic acid still positive Delvotest results

were obtained. To exclude combinations of antibiotic residues, the milk samples were

further tested on Charm MRL Beta-lactam test and Charm II Sulfonamides Milk with

negative results. Both milk samples tested also negative on Charm II Tetracyclines

Milk, Charm II Aminoglycosides Milk, and Charm II Macrolides Milk.


The results of a second sampling of milk from each individual cow (n=11) and the

farm silo (in duplicate) at farm 1 are presented in Table 3. All individual cow milk

samples tested negative on Delvotest MCS the day after sampling except for cow 3,

giving a borderline positive Z-value (-0.24). This positive result could be explained by

the higher pH of the milk (7.06), characteristic for subclinical mastitis, since the

growth of Geobacillus in the Delvotest is followed with bromocresol purple, a pH

indicator. The farm silo milk, sampled in duplicate, tested negative that day, but the

Z-values (-3.32 and 3.47) were close to the cut-off of -3.00 indicating some inhibition

of the Delvotest test organism Geobacillus stearothermophilus var. calidolactis.

Retesting the farm silo and individual cow milk samples, stored at 4°C, three days

after sampling, resulted in positive Delvotest MCS results for the farm silo milk

samples and the milk of cow 3 and 9. For the milk of cows 4 and 7, borderline

negative results were obtained. All milk samples tested negative on Copan Milk Test

(data not shown).

7.3.2 Assessment of milk quality The farm silo milk samples were also analysed on composition and quality. The

somatic cell counts, 300,000 and 273,000 ml-1, respectively, were below the norm of

400,000 ml-1 but slightly above the country average of 190,000 ml-1. The pH values

were slightly lower than the average value for raw milk. The content of free fatty acids

was 1.8 and 3% of the fat (≈ 0.15% of the milk), respectively. These free fatty acid

concentrations were high for raw milk and may be indicative for the activity of

bacterial lipases. Both silo milk samples showed a normal proteolysis, and a normal

fat oxidation.

Analytical results of the second sampling of milk from each individual cow (n=11) and

the farm silo (in duplicate) at farm 1 are presented in Table 3. High SCC above the

norm of 4 x 105 ml-1 were measured in 6 out 11 individual cow milk samples. A high

level of free fatty acids was present in the milk of cow 9 and in both farm silo milk

samples.


Table 3. Composition, pH, bacteriological parameters, Delvotest MCS (SP-NT) results (1 and 3 days after sampling), fat oxidation (3.5 days after sampling), and free fatty acids (3.5 days after sampling) of reference blank milk, and of 11 individual cow milk samples, and 2 samples of the farm silo milk, from a Flemish farm frequently penalized for antimicrobials in the farm silo milk. Delvotest cut-off Z-value= -3.00.

Milk Fat (g per 100 g)

Protein (g per 100 g)

Somatic cell count (x103 ml-1)

pH Total bacterial

count (cfu ml-1)

Psychro-trophic bacteria (cfu ml-1)

Lipo- phylic

bacteria (cfu ml-1)

Delvotest MCS (d+1)a

(Z-value)

Delvotest MCS (d+3)a

(Z-value)

Fat oxidation (d+3.5)a (meq O2 kg-1 fat)

Free fatty acids

(d+3.5)a (% oleic

acid 100 g-1 fat)

cow 1 1.71 3.42 409 6.71 1,100 780 2,900 -7.90 -4.14 0.09 0.0251 cow 2 2.14 3.38 20 6.52 350 10 70 -9.66 -6.18 0.08 0.0385 cow 3 2.28 3.55 959 7.06 8,300 10 400 -0.24c 2.61c 0.12 0.0291 cow 4 4.57 3.99 314 6.86 40 <10 <10 -5.90 -3.87 0.09 0.0359 cow 5 1.52 3.31 251 6.70 16,000 <10 300 -5.15 -4.08 0.06 0.0426 cow 6 0.73 3.21 12 6.60 4,900 2,000 4,100 -7.98 -6.39 0.08 0.0267 cow 7 2.13 3.45 626 6.86 6,200 <10 110 -7.34 -3.77 0.12 0.0332 cow 8 5.28 5.95 1304 6.81 4,900 540 <10 -7.22 -4.61 0.12 nab cow 9 4.43 4.48 1407 6.76 7,900 260 100 -7.56 -2.67c 0.13 0.1329

cow 10 3.76 3.81 36 6.62 70 <10 <10 -7.59 -4.67 0.07 0.0971 cow 11 2.02 3.61 643 6.69 6,800 <10 10 -6.97 -4.75 0.10 0.0335

silo 3.92 3.58 168 6.64 1,510 170 220 -3.32 -0.10c 1.24 0.1415 silo 3.92 3.58 179 6.65 1,290 110 180 -3.47 0.21c 1.24 0.1415

blank milk 4.30 3.53 159 6.80 na na na -8.96 -6.87 0.09 0.0665 Notes: a d+, days after sampling. b na, no data available. c, positive result.


Regarding the microbiological quality of the individual cow milk samples, no extreme

values were found for the total bacterial count (40-16,000 cfu ml-1), the number of

coliforms (<1-29 cfu ml-1, data not shown), psychrotrophic (<10-2,000 cfu ml-1), and

fat splitting bacteria (<10-4,100 cfu ml-1). The composition parameters fat, protein,

and lactose (data not shown) of the silo milk were normal.

7.3.3 Isolation, characterization and identification of strains On VRBA plates, coliforms characteristically form dark red colonies usually

surrounded by a reddish zone with a granular appearance by precipitation of bile

salts. However, on some plates some bacteria gave atypical results with a clear halo

between the colony and the reddish zone. P866 and P867 (R-36630 and R-36631,

respectively), isolated as atypical bacterial strains from the VRBA plates with milk of

cow 3 and 4 were purified and identified as 99.9% Pseudomonas fluorescens by

using API 20NE. Since API identification is limited in comparison to sequence-based

identification (Marchand et al., 2009), rpoB sequence analysis was performed. A

phylogenetic clustering with all public available rpoB sequences from members of

the Pseudomonas genus indicated P. tolaasii LMG2342 as the closest relative for

both strains (Figure 1).

The strains were able to grow at 7°C (psychrophilic) and did not show any lipase

activity using tributyrin as substrate. Both strains showed a very strong haemolytic

activity on blood agar plates; also a proteolytic activity was indicated. The P. tolaasii

LMG2342 strain did not show a haemolytic activity, a finding in contradiction with

literature data.

The bacteria were not able to split lactose. The pH of raw milk inoculated with

Pseudomonas P867 was lowered from 6.70 to 6.53 after 48 hours incubation at

30°C. Also fat oxidation was observed in the inoculated milk incubated for 1 day at

30°C or for 4 days at 5°C (inoculated milk: 0.46 (at 30°C) and 1.08 (at 5°C) meq O2

kg-1 fat; reference milk: 0.15 meq O2 kg-1 fat).

7.3.4 Growth and bacterial inhibitor production The growth of Pseudomonas strains P866 and P867 inoculated in full-cream UHT

milk and incubated at 5-7 and 30°C was several times followed, together with the

production of bacterial inhibitor.


Figure 1. Phylogenetic analysis of the Pseudomonas isolates P866 and P867 on the basis of rpoB sequences. Unrooted neighbour joining tree was based on partial rpoB sequences (1030 bp). Bootstrap values were generated from 1000 replicates of neighbor joining. Bootstrap values higher than 50% are given.


As could expected, both parameters were strongly influenced by the number of

bacteria in the inoculum. But also for a similar number of bacteria inoculated, the

growth and the bacterial inhibitor production were not constant. An example for the

growth of Pseudomonas strain P867 incubated at 7°C is shown in Table 4.

Table 4. Growth and bacterial inhibitor production by Pseudomonas strain P867 in full-cream UHT milk at 7°C. Delvotest cut-off Z-value= -3.00.

Time (days)

Bacterial count (cfu ml-1)

Delvotest SP-NT Z-value result

t= 0 7.0 x 102 -10.24 neg t=1 5.6 x 105 -10.10 neg t=2 1.6 x 107 -10.32 neg t=3 4.5 x 107 -9.19 neg t=4 1.3 x 108 -4.93 neg t=5 6.0 x 107 2.18 pos

Notes: pos, positive; neg, negative.

Milk inoculated with P866 or P867 and incubated at 7°C, needed 5 days and a high

bacterial count to become positive on Delvotest. Also in growth experiments at 30°C,

a high number of bacteria (>107 ml-1) were needed before positive Delvotest results

could be generated (data not shown).

In most growth experiments the highest production of bacterial inhibitor production

was obtained in the stationary growth phase.

The interaction between bacterial cultures on the production of bacterial inhibitor was

also followed. The results are summarized in Table 5.

Table 5. Delvotest SP-NT results (Z-values) for reference blank raw milk and for UHT and raw milk inoculated with Pseudomonas strain P866 and P867 (5.7 and 3.5 x 103 cfu ml-1, respectively), after 24 hours incubation at 30°C or after 3 days incubation at 5°C. Delvotest cut-off = -3.00. Milk Inoculation Delvotest SP-NT

1 day at 30°C 3 days at 5°C Z-value result Z-value result

UHT milk P866 6.94 positive 9.01 positive P867 8.25 positive 13.72 positive

P866 +P867 10.02 positive 13.37 positive raw milk P866 0.97 positive 11.34 positive

P867 0.12 positive 12.92 positive P866 +P867 1.25 positive 12.19 positive

UHT milk --- -11.14 negative -10.86 negative raw milk --- -8.04 negative -7.29 negative


The production of inhibitory substances at 30°C was smaller in raw milk than in UHT

milk, indicating a possible hampering by background milk flora. After an incubation of

3 days at 5°C, all inoculated milk samples were highly positive in Delvotest with Z-

values exceeding 9.01. At that temperature, there were no significant differences in

bacterial inhibitor production between raw and UHT milk.

The experiment with growth of P867 in the presence of other psychrotrophic

Pseudomonas strains isolated from Belgian raw milk samples, resulted in lower

bacterial inhibitor production (data not shown). The presence of the background flora

used in our experiment hampered the production of bacterial inhibitor and possibly

the growth of P867.

Pseudomonas P867 was also cultured in BHI broth and the bacterial inhibitor

production was followed by testing the inhibitory effect in PremiTest, a variation of

the Delvotest developed for monitoring of meat. PremiTest was used instead of

Delvotest since blank BHI broth itself resulted in a positive Delvotest. This effect is

due to the fact that the test organism in the Delvotest needs certain milk nutrients for

a normal acid production. Positive PremiTest results were obtained for the BHI broth

after 2 days of growth at 6°C. The mean Z-value (n=4) of blank BHI was -7.19

(negative); the Z-values (n=4) of BHI inoculated with P966 and P867 after incubation

were 7.98 and 9.33, respectively.

7.3.5 Bacterial inhibitor characterization assays 7.3.5.1 Dialysis experiments

The results of dialysis of full-cream UHT milk, testing positive for Delvotest SP-NT

due to growth of Pseudomonas strain P867, against blank raw milk and of the

inverse dialysis are summarized in Table 6.

The results for the inhibitory action on Geobacillus disc assay of a 125 mM NaCl

solution after dialysis against full-cream UHT milk before and after growth of

Pseudomonas P867 are presented in Table 7.


Table 6. Inhibition (Z-value on Delvotest SP-NT) of positive full-cream UHT milk + bacterial inhibitors by growth of Pseudomonas P867, before and after dialysis (1 kDa membrane) against blank milk for 24 hours at 4°C, and results for the inverse dialysis. Delvotest cut-off Z-value = -3.00.

Milk Delvotest SP-NT (Z-value) before dialysis after dialysis

Milk + bacterial inhibitors by growth of P867 inside dialysis membrane Milk + bacterial inhibitors 10.77 -5.60 Blank raw milk -6.68 -6.45 Blank milk inside dialysis membrane Blank raw milk -6.68 8.08 Milk + bacterial inhibitors 10.77 8.34

Table 7. Inhibition of 125 mM NaCl solution on Geobacillus disc assay after dialysis (1 kDa membrane) for 24 hours at 4°C against UHT milk, before and after growth of Pseudomonas P867. Diameter filter discs = 12.7 mm.

Milk Geobacillus disc assay

(diameter inhibition zone in mm) before dialysis after dialysis

NaCl solution inside dialysis membrane Salt solution ---a ---a Blank UHT milk ---a ---a NaCl solution inside dialysis membrane Salt solution ---a 23.4 UHT milk after growth of P867 24.9 24.7 Note: a ---, no inhibition.

The results of both experiments show that the bacterial inhibitor is able to permeate

the 1 kDa dialysis membrane and that the bacterial inhibitor in a nearly pure salt

solution was still inhibiting Geobacillus with an activity equal to its activity when

present in milk. The salt solution that is giving an inhibition zone of 23.4 mm was

only containing a very limited amount of 4 fatty acids: butyric (9.24 µg ml-1), caproic

(4.73 µg ml-1), caprylic (2.17 µg ml-1), and capric acid (1.97 µg ml-1). All other fatty

acids with a longer chain length were not present in the salt buffer. The

concentration of caprylic and capric acid is more than 345 times smaller than the

concentration of 0.075%, needed to get (low) positive Delvotest results.


7.3.5.2 Heat tolerance

The impact of a heat-treatment of 10 minutes at 80, 90, or 100°C on the antimicrobial

activity of the bacterial inhibitor is shown in Table 8.

Table 8. Effect of heat-treatment on the antimicrobial activity of raw milk with bacterial inhibitors produced by strain Pseudomonas P867, on raw milk spiked with veterinary drugs, and on blank raw milk. Results (Z-values) of Delvotest SP-NT tests, Delvotest cut-off = -3.00.

Sample Delvotest SP-NT (Z-value)

no heat-treatment

10 min at 80°C

10 min at 90°C

10 min at 100°C

milk + bacterial inhibitors by Pseudomonas growth 7.96 6.34 7.25 5.86

milk + 2 µg kg-1 penicillin G 8.10 8.76 10.50 9.79 milk + 4 µg kg-1 penicillin G 12.71 12.13 13.80 12.40 milk + 20 µg kg-1 cloxacillin 6.54 7.24 6.61 7.15 milk + 30 µg kg-1 cloxacillin 11.49 12.01 9.32 10.98 milk + 300 µg kg-1 oxytetracycline 7.76 8.61 6.76 4.22 milk + 100 µg kg-1 sulfadiazine 5.86 7.02 6.60 6.24 blank raw milk -8.48 -7.43 -9.04 -8.52

Heating of milk (5 min at 82°C or 10 min at 80°C) inactivates natural inhibitors and is

often applied to prove false-positive results in microbial growth inhibition assays

(Kang et al., 2005). The bacterial inhibitors produced by Pseudomonas P866 (data

not shown) and P867 showed to be heat-stable at 80°C for 10 min. Even 10 min at

100°C was not sufficient to inactivate the bacterial inhibitors by Pseudomonas

growth, while this heat-treatment already had negative effects on the activity of the

heat-labile oxytetracycline in the Delvotest.

7.3.5.3 Inhibitory spectrum

On disc assay plates, the culture of Pseudomonas strain P867 in milk caused

inhibition of Bacillus cereus, Geobacillus stearothermophilus var. calidolactis,

Bacillus subtilis, and Staphylococcus aureus. No inhibition zones were found on the

plates inseminated with Listeria monocytogenes, Escherichia coli, Pseudomonas

fluorescens, or Salmonella Enteritidis.


7.3.5.4 Impact of test medium on the inhibition

The diameters (mean of 3 replicates) measured around the filter disks (diameter 12.7

mm) wetted with 80 µl of the skimmed fraction of milk, positive on Delvotest due to

growth of Pseudomonas P867, on plates filled with PCA, MPCA, NA, or TSA and

inoculated with Geobacillus stearothermophilus var. calidolactis were 20.6, 23.4,

21.4, and 15.4 mm, respectively. There was no inhibition on the plates with BHIA as

test medium.

7.3.5.5 Technological significance

The dairy industry is not just testing milk to make sure that the milk is not containing

residues of antibiotics above MRL, but also to safeguard the production of fermented

products like cheese and yoghurt. In an experiment of yoghurt production, the

acidification during the fermentation of the milk of both farms to yoghurt was slower

in comparison with the acidification obtained for blank control milk. After 5 hours

fermentation at 43°C the pHs of the abnormal milks were 5.92 (farm 1) and 5.89

(farm 2), respectively, while the pH of the blank control milk was 4.29. The yoghurt

samples were further incubated overnight at 30°C. After 19 hours of incubation at

30°C, the end pHs of the yoghurts were 4.08 (farm 1) and 4.12 (farm 2), respectively,

and 3.82 for the control. Also the texture of the end product was not as expected;

there was no whey secretion and the smell of the yoghurt was rancid.

7.3.6 Experiments with fatty acids

The link between pseudomonads and the lipolytic degradation of milk is known and

an inhibitory activity by some fatty acids is described in literature. Since high concen-

trations of free fatty acids were measured in the „problem‟ farm milk samples,

experiments with fatty acids were set up to clear out if the matrix effects could be

caused by lipolysis.

7.3.6.1 Effect of fatty acids on microbiological inhibitor assays

The results of inhibitory effect of fatty acids on Delvotest MCS and Copan Milk Test

are shown in Table 9. Besides the most occurring fatty acids in milk, as mentioned

in Table 1, also elaidic and vaccenic acid were tested. Of all fatty acids tested, a

positive Delvotest result was only obtained for blank raw milk fortified with 0.15%

(w/v) of caprylic, capric, lauric, palmitoleic, and α-linolenic acid (borderline). The Z-


values for raw milk with 0.15% of myristic or linoleic acid were higher compared with

the value for blank raw milk, indicating partly inhibition of the test organism.

Table 9. Inhibitory effects of fatty acids spiked at 0.15% and 0.075% in blank raw milk on Delvotest (n=2; cut-off = -3.00) and Copan Milk Test (n=2; cut-off = 4.5).

Fatty acid Delvotest MCS Copan Milk Test Z-value result CIF result

Milk spiked at 0.15% (w/v) butyric -6.54 neg 1.3 neg caproic -7.26 neg 0.4 neg caprylic -0.65 pos 1.0 neg capric 6.27 pos 5.5 pos lauric 1.83 pos 0.7 neg myristic -4.62 neg 0.1 neg palmitic -8.07 neg 0.2 neg stearic -8.57 neg 0.2 neg palmitoleic -2.12 pos 1.7 neg oleic -6.66 neg 0.3 neg linoleic -4.91 neg 0.6 neg α-linolenic -3.00 pos 1.4 neg elaidic -7.44 neg nt nt vaccenic -7.23 neg nt nt Milk spiked at 0.075% (w/v) caprylic -2.71 pos 0.5 neg capric 2.71 pos 3.7 neg lauric -1.26 pos 0.3 neg palmitoleic -5.34 neg 0.7 neg α-linolenic -6.01 neg 0.3 neg Blank raw milk --- -7.67 neg 0.1 neg Notes: pos, positive; neg, negative; nt, not tested. None of the in milk most abundant fatty acids (myristic, palmitic, stearic, and oleic

acid), spiked in milk at 0.15% was causing a positive Delvotest SP-NT result. There

was also no interference by 0.15% of butyric, caproic, elaidic, or vaccenic acid on

Delvotest. A concentration of 0.075% of caprylic, capric, or lauric acid in milk still

resulted in positive Delvotest results, while 0.075% of palmitoleic or α-linolenic acid

tested negative.

The fatty acids were also tested on Copan Milk Test. The only positive Copan Milk

Test result was obtained for milk spiked with capric acid at 0.15 % but not at 0.075%.


No inhibitory effect by any fatty acid (caprylic, capric, lauric, palmitoleic, or α-linoleic

acid) spiked at 0.15% in raw milk was noticed on the Geobacillus disc assay despite

the fact that in Delvotest and Copan Milk Test also Geobacillus stearothermophilus is

used.

7.3.6.2 Dialysis experiments

In Table 10 the results of the dialysis of milk spiked (0.15%, w/v) with caprylic,

capric, lauric, palmitoleic, or α-linolenic acid against blank raw milk are presented.

Table 10. Inhibition (Z-value on Delvotest SP-NT) of milk spiked with 0.15% (w/v) of fatty acids, before and after dialysis (1 kDa membrane) for 24 hours at 4°C. Delvotest cut-off Z-value = -3.00.

Milk Delvotest SP-NT (Z-value) before dialysis after dialysis

Milk + 0.15% caprylic acid inside dialysis membrane Milk + 0.15% caprylic acid -1.37 -7.69 Blank raw milk -9.59 -7.02

Milk + 0.15% capric acid inside dialysis membrane Milk + 0.15% capric acid 7.41 -7.40 Blank raw milk -9.39 -7.69

Milk + 0.15% lauric acid inside dialysis membrane Milk + 0.15% lauric acid 1.44 3.07 Blank raw milk -9.59 -7.69

Milk + 0.15% palmitoleic acid inside dialysis membrane Milk + 0.15% palmitoleic acid -2.33 -2.11 Blank raw milk -10.76 -11.76

Milk + 0.15% α-linolenic acid inside dialysis membrane Milk + 0.15% α-linolenic acid -1.76 -0.33 Blank raw milk -11.64 -9.80 Of the fatty acids with inhibitory activity against Geobacillus, the two fatty acids with

the shortest chain, caprylic and capric acid, were able to permeate the 1 kDa dialysis

membrane.

7.3.6.3 Effect of heat-treatment

The impact of heat-tratment on the inhibitory effect of fatty acids spiked in raw milk is

shown in Table 11.

The five fatty acids tested showed to be very heat-tolerant. The inhibitory activity in

Delvotest was not influenced by a heat-treatment for 10 min at 100°C.


Table 11. Effect of heat-treatment on the antimicrobial activity of raw milk spiked with 0.15% (w/v) of fatty acids, and on blank raw milk. Results of Delvotest SP-NT tests (Z-values), Delvotest cut-off = -3.00.

Sample Delvotest SP-NT (Z-value)

no heat-treatment

10 min at 80°C

10 min at 90°C

10 min at 100°C

milk + 0.15% caprylic acid -1.37 -1.06 -2.26 -1.88 milk + 0.15% capric acid 7.41 5.90 6.86 7.06 milk + 0.15% lauric acid 1.71 2.98 3.04 3.30 milk + 0.15% palmitoleic acid -2.12 -1.24 -2.45 -1.23 milk + 0.15% α-linolenic acid -3.00 -2.68 -3.49 -4.77 blank raw milk -8.48 -7.43 -9.04 -8.52 7.4 DISCUSSION Silo milk from two Belgian dairy producers, fined on several occasions because their

milk tested positive for the presence of antimicrobials in the Delvotest SP-NT at the

milk control station, was further studied. The dairy- and fieldmen of the two milk

producers could not explain the reason for the bulk tank failures since no cows were

treated during the last month and no fresh cows had been added to the milking herd

within a month of the positive bulk tank samples. The purpose of the investigation

was to examine any true presence of antibiotic residues in the positive samples and

to indicate the reason of inhibition by the silo milk. The results of the antimicrobial testing confirmed the opinion of the dairy- and

fieldmen that the inhibition in the Delvotest was not caused by the presence of

residues of antibiotics or chemotherapeutics.

In most of the positive milk samples a high lipolysis was found. This could be

expected since high SCC were present in the milk of 6 out of 11 cows. A high SCC

could implicate an increased lipase activity, both endogenic as bacterial, together

with a higher concentration of short chain free fatty acids in this milk.

From the VRBA plates with milk of cows 3 and 4, bacterial strains, surrounded by a

clear halo were isolated, purified, and codified as P866 and P867. Both bacteria

were identified as 99.9% Pseudomonas fluorescens by API 20NE. P. tolaasii

LMG2342 was indicated as the closest relative by phylogenetic clustering. The


isolation of Pseudomonas strains is not surprising. The current practices for the

collection and storage of the raw milk and the use of milking robots favor the growth

of psychrotrophic bacteria, able to grow below 7°C (Munsch-Alatossava and

Alatossava, 2006; De Jonghe, 2010). Pseudomonas spp. are the most common

organisms in raw or pasteurized milk at the time of spoilage (Sørhaug and

Stepaniak, 1997); they constitute the predominant micro-organisms limiting the shelf-

life of processed fluid milk at 4°C. In a Finnish study, 88% of the isolates from raw

milk samples were psychrotrophs. The majority of the isolates were representatives

of the Pseudomonas genus (Munsch-Alatossava and Alatossava, 2006). Marchand

et al. (2009) found P. lundensis and P. fragi as predominant milk spoilers isolated

from Belgian raw milk samples. In a recent Dutch study (Rademaker et al, 2009)

mainly pseudomonads and Staphylococcus aureus were detected and quantified in

farm tank milk of poor quality, based on parameters such as high total bacterial

counts (TBC) and high somatic cell counts (SCC). It concerned a large diversity of

Pseudomonas spp., including members of the P. fluorescens and P. putida group as

well as P. lundensis, P. fragi, and P. aeruginosa. Significant contaminations by

pseudomonads occur due to inadequately sanitized surfaces of milking, storage, and

transporting equipments. Besides their rapid growth ability in refrigerated milk,

psychrotrophs produce heat-stable extracellular proteases, lipases, and

phospholipases. Pseudomonas spp. are the primary concern with regard to lipolytic

degradation of milk fat (McPhee and Griffiths, 2002).

Pseudomonas spp. are known to produce cyclic lipopeptides (CLPs). CLPs are

composed of a fatty acid tail linked to a short oligopeptide, which is cyclised to form a

lactone (depsi) ring between two amino acids in the peptide chain (Raaijmakers et al.

2006). CLPs are very diverse both structurally and in terms of their biological activity

(Raaijmakers et al., 2010). CLPs produced by Pseudomonas spp. play a key role in

antimicrobial activity against a range of other micro-organisms (Ron and Rosenberg,

2001; Nielsen et al., 2002). Activity against Bacillus megaterium was shown for

corpeptins, syringopeptins, and tolaasin (Emanuele et al., 1998; Lavermicocca et al.,

1997; Soler-Rivas et al., 1999) and for pseudodesmin A and B, two new cyclic

lipodepsipeptides from Pseudomonas bacteria (Sinnaeve et al., 2009). Besides an

antimicrobial activity, the lipodepsipeptide cormycin A (Scaloni et al., 2004) and

tolaasin showed erythrocyte haemolytic properties.


Both strains, P866 and P867, showed to be psychrophilic, haemolytic, and

proteolytic. Inoculated milk incubated at 30 or 5-7°C showed fat oxidation and tested

positive on Delvotest. In comparison, P. tolaasii LMG2342 showed no haemolytic

activity and a lower inhibitory effect.

In all growth experiments, a high number of bacteria (>107 ml-1) was needed before

positive Delvotest results could be generated. These experimental findings are

contradictory to the initial false-positive Delvotest MCS results at the milk control

stations, generated by farm silo milk with a normal bacterial count. Literature (Lewis

Sauer et al., 2002) is suggesting that the presence of a bacterial biofilm could have

an influence, or quorum sensing is regulating the production of inhibitory substances.

For example, the opportunistic bacterium Pseudomonas aeruginosa uses quorum

sensing to coordinate the formation of biofilms, exopolysaccharide production, and

cell aggregation (Rossignol et al., 2009). Perhaps the bacterial flora in the milk was

inhibited by the production of antibacterial inhibitor. But also influences by a typical

flora present at the farm cannot be excluded. Preliminary experiments with

incubation of Pseudomonas inoculated in milk in the presence of other

pseudomonads or the normal flora of blank raw milk showed no enhancement of

bacterial inhibitor production but more likely the opposite. Possibly Pseudomonas

P867 strain do not produce toxins against other Pseudomonas strains.

The Pseudomonas strains P866 and P867 do not need milk for bacterial inhibitor

production. A culture of Pseudomonas P866 and P867 grown in BHI broth tested

positive on PremiTest.

The permeation of the bacterial inhibitor through the membrane in the dialysis

experiments indicated that the molecular weight of the bacterial inhibitor is below

1,000 Da. The molecular weight of tolaasin A is 980 Da (Bassarello et al., 2004);

most other lipodepsipeptides have a higher molecular weight.

The different heat-treatments had no inactivating effect on the activity of the bacterial

inhibitor in Delvotest. The extracellular lipodepsipeptide toxin, tolaasin, produced by

P. tolaasii, is described as heat-stable (Peng 1986, Rainey et al., 1991, Nutkins et

al., 1991).


The bacterial inhibitor by growth of Pseudomonas P867 in milk was not only

inhibitory to Geobacillus stearothermophilus var. calidolactis but also to other Gram-

positives (B. cereus, B. subtilis, and Staphylococcus aureus) while the Gram-

negatives tested were not inhibited. Tolaasins also showed antimicrobial activity

against Gram-positive bacteria. Bassarello and co-workers (2004) assayed the

antimicrobial activity of tolaasins A-E in comparison with tolaasin I and II against the

Gram-positive bacteria Bacillus megaterium and Rhodococcus fascians,

respectively, and the Gram-negative bacteria Escherichia coli and Erwinia amylovora

subsp. carotovora. All the analogues, except tolaasin C, inhibited the growth of the

tested Gram-positive bacteria, although differences among their specific activities

were observed, but none of the tested Gram-negative bacteria. The minimal

inhibitory quantity of tolaasin A to inhibit Bacillus megaterium was 1.28 µg.

The antimicrobial activity on the disc assay of a fixed amount of bacterial inhibitor

against Geobacillus stearothermophilus var. calidolactis was not constant for

different types of test media. The highest inhibition was measured on the plates filled

with MPCA, followed by NA and PCA. The inhibition was significantly smaller on

TSA, while no inhibition was noticed on BHIA plates. This may be explained by the

fact that of all media tested, BHIA is the only buffered medium, namely with di-

sodium phosphate 2.5 g l-1. Further experiments (results not shown) revealed that pH

was the reason of the effects: an increase of pH of the medium in disc assays

resulted in smaller inhibition zones. Perhaps the pH has a regulating role on the

permeation of the bacterial inhibitor through the bacterial cell wall.

An impact by the bacterial inhibitor on yoghurt production was noticed and an impact

on cheese making and the cheese quality could not be excluded. These results are

of importance especially for farmers with home production of fermented products. On

the other side, it‟s very unlikely that the manufacturing in larger dairies of cheese and

yoghurt with abnormal milk commingled with normal milk of other production

holdings, would be hampered.

Besides the possibility that Pseudomonas P867 or P866 are producing biological

active toxins like lipodepsipeptides interfering in the microbiological inhibitor essays,

another reason for interference cannot be excluded.

Many authors reported about inhibitory properties of free fatty acids as described in

the introduction. In our experiments a positive Delvotest result was obtained for

blank raw milk fortified with 0.075% of caprylic, capric, or lauric acid and for milk


spiked with 0.15% of palmitoleic or α-linolenic acid (borderline). But the most in milk

abundant fatty acids (myristic, palmitic, stearic, and oleic acid) spiked at 0.15% were

not inhibiting the Delvotest. The results are in line with Anders and Jago (1964), who

stated an inhibiting effect of caprylic, capric, and lauric acid on lactic acid bacteria,

and the publication of Boyaval et al. (1995) who described inhibitory properties of

linoleic acid. The results also proof that the inclusion of agar to microbial inhibitor

tests like the Delvotest is not prohibiting the incidence of interferences by fatty acids

or by natural antimicrobial factors in the milk, as suggested by certain authors

(Carlsson, 1991; Carlsson and Björck, 1992).

A positive Copan Milk Test result was only obtained for milk spiked with capric acid

at 0.15 % but not at 0.075%. There is no direct explanation why less positive results

were obtained in the Copan Milk Test compared to the Delvotest because both tests

are based on the same test principle and test organism. Perhaps the test organism is

less stressed in the Copan Milk Test, or the agar used in both tests has different

rheological properties, or the test medium of the Copan Milk Test is more buffered.

None of the fatty acids spiked in milk at 0.15% was inhibiting Geobacillus on the disc

assay, while large inhibition zones were obtained the filter discs impregnated with

milk after Pseudomonas growth or salt solution dialysed against positive milk after

Pseudomonas growth. These results suggest that the fat destruction and the release

of fatty acids are not the reason for the matrix effects noticed on Delvotest.

Of the fatty acids with inhibitory activity against Geobacillus, only caprylic and capric

acid were able to permeate the 1 kDa dialysis membrane. The inhibitory effect by

fatty acids tested showed not to be influenced by a heat-treatment.

Considering all the results, there are serious indications that the false-positive

Delvotest results are caused by antimicrobial toxins like lipodepsipeptides, produced

by the Pseudomonas strains. However, further research is needed to identify the

inhibitor.

7.5 FINAL CONCLUSIONS To our knowledge this paper is the first statement of interference of microbial

inhibitor tests for antibiotic residues in milk by bacterial inhibitors produced by

Pseudomonas strains. The strains, isolated from milk of a farm with frequent

problems of false-positive Delvotest results were identified as closely related to


Pseudomonas tolaasii. Growth of the isolates in milk was not only resulting in high

lipolysis of the raw milk, but also in the production of bacterial inhibitors. These

bacterial inhibitors with a molecular weight <1 kDa showed to be heat-tolerant and

inhibitory to Geobacillus stearothermophilus var. calidolactis, the test strain used in

most of the commercially available microbiological inhibitor tests. The bacterial

inhibitors also showed antimicrobial activity against Staphylococcus aureus, B.

cereus, and B. subtilis. These two Bacillus strains are often used in microbiological

tests for screening for antibiotic residues. The bacterial inhibitor in culture filtrate

caused false-positive results on Delvotest MCS, Delvotest SP-NT, and PremiTest.

The acid production of Copan Milk Test was also hampered, but not to the same

extent, resulting in no false positive test results.

So far, we were not able to identify the microbial inhibitor but the results of the

microbial inhibitor characterization assays, and the identification of the isolates

indicate in the direction of cyclic lipodepsipeptides, toxins with antimicrobial

properties. Further research on extracted and purified toxin by LC-MS or MALDI-

TOF MS is needed to confirm this suggestion.

Our findings have a serious consequence for regulatory quality programmes. In such

programmes, in most cases, a confirmation of initial positive screening results is

foreseen, including a heat-treatment of the milk to exclude influence by natural

inhibitors. However, the bacterial inhibitors produced by Pseudomonas P866 and

P867 are heat-stable and will in most test schemes lead to false-positive results. In

some countries, even special measures like higher financial penalties or a

suspension of the milk collection, are contributed to recidivists. The production of

bacterial inhibitors is very likely to be a persistant problem on certain farms.

Pseudomonads are known to be well adapted to survival in milk processing

environment. They are able to adhere strongly to the surface of milk processing

equipment and they are capable to colonize in the milking equipment and storage

tank at the farm. We speculate that they can produce from time to time enough

inhibitors to cause positive inhibitor test results.

It remains difficult to give a rate of occurrence of this phenomenon. It‟s obvious that

in most cases positive inhibitor tests are caused by the presence of residues of

antibiotics, mainly belonging to the family of -lactams.


Nevertheless, our data show that results of microbiological inhibitor tests should be

interpreted with care, especially when the outcome of such tests is not confirmed by

a confirmatory method, providing full or complementary information enabling the

substance to be unequivocally identified.

Our findings indicate a new challenge for the dairy industry. By extending the

refrigerated storage of milk, the keeping quality of milk is influenced by growth and

metabolic activities of psychrotrophic bacteria at low temperatures. This is not only

resulting in possible spoilage of long-life milk but also in false-positive microbial

inhibitor tests.

Acknowledgement We express our gratitude to all technicians involved at ILVO-T&V, to Ann Van de

Walle and Katleen Vander Straeten in special, and to Vzw Melkcontrolecentrum-

Vlaanderen (Lier, Belgium) and Comité du Lait (Battice, Belgium) for indicating the

farms, milk sampling, and Milcoscan and Fossomatic measurements.

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iscussio

General Discussion

General Discussion 242

General Discussion To ensure enough food production to feed the enlarging world population, agriculture

is pushed towards a more intensive production. Intensification of animal production

can lead to an increase of bacterial infections and consequently a higher therapeutic

and even prophylactic use of antimicrobials. The administration of antibiotics or

chemotherapeutics may result in the presence of residues of these substances or

their metabolites in food from animal origin like milk and honey. In order to protect

the consumer, Maximum Residue Limits (MRLs) of veterinary medicinal products in

foodstuffs of animal origin, were set in the European Union, based on the basis of

scientific assessment of the safety of those substances. Pharmacologically active

substances not listed in Table 1 of the Annex of Commission Regulation (EU) No

37/2010 are not approved to be administered to food-producing animals. The

specifically prohibited substances are listed in Table 2 of that Annex.

The European Union was also harmonizing the monitoring of certain substances and

residues thereof in certain animal products by indicating for each commodity (honey,

milk, eggs, etc.) the groups of substances that must be monitored, fixing the levels

and frequencies of sampling, laying down detailed rules on official sampling and

setting performance criteria and other requirements for analytical methods and the

interpretation of results. Under this legislation, Member States are required to submit

national residue control plans for approval by the European Commission on an

annual basis. Besides the official controls, also checks are carried out by food

business operators under HACCP based control programmes or quality assurance

programmes in order to meet the requirements of food law.

Therefore, many samples need to be analysed for unauthorized substances and

registered veterinary drugs. For the determination of residues of antibiotics in food,

screening methods are widely used thanks to their cost-effectiveness. By preference

screening methods show the following characteristics: high throughput, short

analysis time, ease of use, low cost, cheap instrumentation, minimal sample pre-

treatment, possibility of automation, detection of the analyte or family of analytes at

the level of interest (no false-negative (false-compliant) results), and a low rate of

false-positive results. False-compliant results would mean in the case of milk that the

processing of yoghurt and cheese could be hampered or in general that food with


residues surpassing the regulatory tolerance could enter the market for human

consumption and present a potential health risk to the consumer.

False-positive results, mostly caused by matrix effects, often result in extra

confirmatory analyses to be performed and in an increase of costs and efforts for the

destruction of the product.

In some cases, especially in milk testing, the decision is taken only on the basis of a

screening result. This is the case in regulatory quality systems where farmers

receive a financial penalty for the presence of antimicrobials in the ex-farm milk

detected by a microbiological inhibitor test. Also tanker milk is rejected and

destructed in consequence of a positive screening result of a rapid test, without any

knowledge about the identity or the concentration of the residue. Therefore, the

development of reliable screening methods is a challenge for kit manufacturers. On

the other side it is a responsability of the authorities to work out protocols which

prevent the destruction of safe food or the punishment of food producers respecting

the rules.

The decision to send a milk producer an invoice for the destruction of a complete

tanker milk load should by preference be based on a physico-chemical analysis

(qualitative & quantitative) proving that the marker residue in the ex-farm milk is

corresponding to the residue found in the tanker milk and at the same time that the

concentration of residues in the ex-farm milk sample divided by the dilution factor

(ratio total load of the tanker / amount of farm milk delivered) is in line with the

concentration of residues in the tanker milk.

Most regulatory systems are based on a microbiological test referring to the

detection of „inhibitory substances‟ instead of „antibiotic residues‟. In that view, the

dairy industry is claiming that, in case an inhibition is noticed, no false-positive

results could be generated. Moreover, to their opinion the financial punishment is

justified because with such milk no yoghurt or cheese can be produced. On the other

hand, more and more milk producers would favour a protocol with the inclusion of a

physico-chemical analysis. In such a regulatory system, a penalty would only be

assigned providing that the presence of residues of antibiotics or chemotherapeutics

is confirmed and the concentration of residues is exceeding the regulatory limit

(MRL). The extra costs involved could be paid by a fund of all the money gathered

from financial penalties for the presence of inhibitory substances in ex-farm milk.

Nowadays, in most cases the penalties are cashed by the dairies even when the


tanker milk, tested as negative on entry at the dairy plant, was accepted and being

processed. Some farmer associations also insist on in duplo sampling enabling a

counter analysis in a laboratory of choice.

In some cases, the antibiotic residue present in a positively screened sample is not

confirmed by the confirmatory method due to the fact that the substance is not

included in the detection method used. The CCα of the confirmatory method can also

be higher than the CC of the screening method. When this is the case, despite the

presence of residues, the positive screening result will be interpreted as false-

positive. If more than one antimicrobial is occurring in a sample, or the parent drug is

present together with its metabolite(s), the antimicrobials can have a cumulative or

even a synergistic interaction on the screening method and hence traces can lead to

a positive screening result.

Another problem of screening methods are false-violative results due to a detection

capability (far) below MRL for allowed substances. In these cases the consumer

protection with regard to food safety is respected but false-violative results

erroneously call for the withholding of milk from being used for food. This also results

in an economic loss for the producer (financial penalty) or the food business operator

and to an increase of the general carbon footprint of the milk production. Therefore, it

is a real challenge for method developers to bring the detection capability of

screening methods close to the level of interest, in other words for allowed

substances close to the MRLs. This is of less importance for official monitoring

samples where suspect results have to be confirmed by a confirmatory method or for

honey where a zero-tolerance is applied. Here again, a quantitative confirmatory

result could solve the problem. But due to the short shelf-life of raw milk, the

inclusion of a physico-chemical confirmatory step to take the decision to dispose of

the contaminated (tanker) milk is practically not feasible.

Some people suggest to dilute the milk before testing but if the identity of the

inhibitor is not known, the correct dilution factor cannot be calculated and applied. In

some systems the correction factor is based on the test capability for

benzylpenicillin, the most occurring residue in raw milk.


The use of third- and fourth-generation cephalosporins in dairy production is

increasing (Anon., 2010b). Several cephalosporins can be detected by microbial

inhibitor and rapid tests at levels far below their MRL, leading to positive results for

tests performed on milk of treated cows collected after the prescribed withdrawal

period. Due to false-violative results in the milk control stations, penalties for the milk

producer delivering positive milk containing residues at concentrations below MRL

cannot be excluded. From time to time, compliant tanker milk, containing low

concentrations of cephalosporins but indicated as „positive‟ by the screening test, is

destroyed. This is leading to conflicts and a poor trust in the system and in the

indicated withdrawal times. On the other side, the European MRL legislation is not

giving a 100% guarantee for technical food safety. Some compounds, e.g. several

cephalosporins, inhibit the production of fermented products like yoghurt and cheese

at concentrations below MRL.

Another issue is the fact that European legislation is rather vague where the MRL

needs to be applied: on the milk per treated quarter leaving the udder, on the milk of

each individual cow, or on the commingled milk in the farm silo? In Corrigendum to

Regulation (EC) No 853/2004, it is stipulated that raw milk collected from milk

production holdings must be in compliance with the MRL legislation regarding

antibiotic residues. But the MRL is used in the calculation for the setting of

withholding times, valid for individual cows. The European legislation is also not

indicating if MRLs for residues of veterinary products in milk powder should be

applied on reconstituted milk or on powder basis.

A related problem is the lack of harmonization of legislation as not every country is

applying the same regulatory limits. Depending on the country, MRLs, safety levels,

Codex MRLs, or tolerances are established, what could be a barrier for international

trade. Despite promises made after a curd cheese scandal in 2006 where Bowland

Dairy Products Limited (Barrowford, United Kingdom) produced curd cheese out of

raw milk containing antibiotic residues, detergents, and dyes (Commission Decision

2006/694/EC), there are still no clear European Union criteria and protocols for the

testing of milk for residues of antibiotics in the HACCP based control programmes or

quality assurance programmes performed by the food business operators. The


European legislation is also not clear if screening for -lactam residues in milk on

entry at the dairy plant is sufficient.

Despite an animal by-products regulation (Regulation (EC) No 1069/2009) Member

States still apply different provisions in case of positive milk.

Till present, no single rapid screening method is able to detect all -lactams at their

respective MRL. Even more, no single microbiological test can screen milk for all

antibiotics and chemotherapeutics with a MRL at the regulatory level. To ensure a

100% food safety of milk for antibiotic residues, combinations of screening tests are

needed, or even screening tests may be supplemented by other types of methods

(e.g. LC-MS/MS) in order to cover all EU-MRLs. On the other side, the likelihood of

an acute toxicity from residues by the consumption of milk is extremely low since no

other food matrix is so intensively checked for antimicrobials as milk.

The difference in detection capability between different screening tests is also

leading to misunderstandings and conflicts. The differences between tests can be of

such a magnitude that the tanker milk is testing positive on a rapid test while all

corresponding individual ex-farm milk samples are testing negative at the milk

control station. The bigger the size of the dairy farms, the higher the chance this will

happen because of the decreasing dilution factor and the smaller gap in residue

concentration between ex-farm and tanker milk. In such a case, no producer

responsible can be indicated and, hence, the dairy company cannot get

compensated by any farmer for the costs of the destruction of the contaminated

tanker load. Here, a fund as suggested above, could also intervene and pay all

related costs.

By checking the milk at the farm before collection, the costs for destruction of a

whole tanker load could be avoided. Nowadays, test kits with a test time of three

minutes or less are commercially available (e.g. eta-s.t.a.r. 1+1, Charm MRL-3, and

TwinExpress Milk) but dairies still hesitate to implement such tests in routine at the

farm.

The problem of false-positive Charm MRL-3 results can partly be solved by retesting

initial positive samples as requested by the kit manufacturer. However, since this is

not only a problem of repeatability, the retesting should by preference be performed


with a different test. This is feasible at the dairy plant (screening of incoming tanker

loads) but is much more difficult at the farm (screening of farm milk before

collection).

It is difficult to estimate the number of false-positive microbiological test results

caused by interference of bacterial inhibitors produced by certain Pseudomonas

strains. In Belgium, the Copan Milk Test was introduced instead of the Delvotest

MCS to limit the number of wrong penalties. In September 2010, national

acceptance criteria for screening tests used in the framework of the screening for

antimicrobials as part of a regulatory quality programme were set (Anon., 2010c).

Since the Copan Milk Test is not fulfilling these criteria, it can be expected that in the

near future in Belgium the Copan Milk Test will be replaced by another test with

better detection capabilities. This would implicate not only a higher number of

positive results but also an increase of false-positive results. In that view, it is

advisable to adapt the test protocol to avoid that in such cases penalties will be

attributed. By extending the intervals between two milk collections so that

Pseudomonas spp. could grow and metabolize, the dairy industry is partly

responsible for the problem and the situation can be expected to get worse the

longer the raw milk is kept. Special attention should also be given to the disinfecting

of milking equipment to avoid the formation of biofilms.

In the last decade some trends in milk testing could be observed. The most changes

happened in the field of rapid milk tests. Due to the simple test protocols, testing of

milk is more and more performed on-site by truck drivers. The actual test protocols

are much shorter and rapid testing is no longer restricted to compounds of the family

of -lactam antibiotics. Presently, compounds of other families (tetracyclines,

sulfonamides, and quinolones) can be tested simultaneously. The results of several

tests can be interpreted visually and a recent test (SNAP Beta-Lactam ST) does no

longer require incubation equipment.

The improvement of microbiological tests is a much more difficult task. Nevertheless,

recently some kit manufacturers were able to optimize the detection capability of

Geobacillus stearothermophilus-based tests. For example with Eclipse 3G and

Charm Blue-Yellow II, all tetracyclines can be screened in milk at MRL. The


subjective colour reading of plates or ampoules could be replaced by a reflectometric

scanner reading. The colour change of Delvotest Accelerator plates is monitored

during incubation enabling to shorten the incubation time.

Currently, ultra-performance liquid chromatography combined with time-of-flight

mass spectrometry (UPLC-ToF-MS) or Orbitrap mass analyser technology (UPLC-

Orbitrap) can be used for screening and quantification of more than 100 veterinary

drugs in milk (Stolker et al., 2008; Kaufmann et al., 2010; Malik et al., 2010). The

available technology permits very high separation (accuracy) and mass resolution,

together with a high selectivity and sensitivity. By maximizing the number of analytes

that may be determined by a single procedure, the cost-effectiveness was

significantly improved. Nevertheless, the widely used, simple, and cheap

microbiological and bioassay techniques will not be completely replaced by this

technology.

Validation of test methods is an important task in order to prove that the analytical

method is fulfilling the relevant requirements established in Commission Decision

2002/657/EC, e.g. a false compliant rate of <5% (-error). For screening methods, a

determination of the detection capabilities and other performance characteristics like

test applicability, ruggedness, and stability, and an investigation of interference,

which might arise from matrix components, are required. A guideline for the

validation of screening methods for residues of veterinary medicines was worked out

by the Community Reference Laboratories for residues (Anon., 2010a). In the final

version, the number of samples spiked at the screening target concentration for the

determination of the detection capability CC in an initial validation was set at 60 for

a screening target concentration close (≥90%) to the regulatory limit

(MRL/MRPL/action limit). If the screening target concentration is set between 50 and

90% or ≤ 50% of the regulatory limit, the number of replicates is at least 40 and 20,

respectively. This enlarges the work and consequently the costs for validation

studies. The guideline is also not indicating the number of replicates needed for the

determination of the detection capability for substances without a MRL/MRPL/action

limit. This is often due to the fact that an application for the establishment of a MRL

for these substances in respect of the foodstuff or species concerned never has


been requested, e.g. several antibiotics in honey. For such substances an indication

of the level of interest is recommended.

A problem when validating analytical methods for the detection of antibiotics and

chemotherapeutics is the availability of standard reference material. Some

substances or their metabolites are not commercially available and, if directly

obtained from the pharmaceutical company, the certificate of analysis with all

information regarding purity, moisture content, residual solvents, or activity is

sometimes not available. In some cases, when calculating the amount of active

compound to be weighed for the preparation of the stock solution, a disagreement

exists between the result based on the activity of the pharmacologically active

substance mentioned on the certificate of analysis and the calculation based on

corrections for impurities, moisture, and salt content. Even taking into account

corrections for impurities, still a different activity can be obtained for standard

reference material originating from different companies.

The situation regarding residues of antimicrobials in honey is rather complicated. In

the European Union, no MRLs have been established for antibiotics and

sulfonamides in honey, resulting in a „zero tolerance‟ for residues of anti-infectious

agents in honey. The EU has a honey deficit and is relying for about half of the total

honey consumption on import of honey from regions where the use of antimicrobials

in apiculture against infectious bacterial brood diseases is allowed or applied. Zero

tolerance depends in most countries on the decision limit (CCα) of the confirmatory

method which is resulting in an uneasiness for the honey importers. Every laboratory

involved in honey residue analysis has his own CCα-value and hence the same

honey sample may be declared compliant or non-compliant depending upon the CCα

of the particular testing laboratory.

Residues or their metabolites remain stable for a long period in honey since

antimicrobials are not actively metabolized by the honeybees and elimination of the

residues in the hive can only happen by consumption by the bees or removal of the

contaminated food by the beekeeper. Hence, high numbers of rapid alert

notifications for residues of antimicrobials in honey are not surprising. But residues of

antimicrobials also occurred in honey of local production. High residue levels in

honey indicate an illegal administration but external sources have to be considered

in the case of small residue concentrations. Other sources of contamination could be


the robbery by bees of contaminated honey from hives of other apiaries, the

migration of residues from contaminated wax foundations to honey, the collection by

bees of contaminated surface water or medicated drinking water from farms, or the

collection of contaminated nectar from fruit trees treated with streptomycin or from

other agricultural crops treated with the herbicide asulam (sulfanilamide). Bad

beekeeping practices like the feeding of contaminated honey to bee colonies or the

mixing of privately produced honey with external honey of dubious quality could also

be the reason of honey contamination. Some honey contaminations by external

sources can be avoided by the beekeeper. But the beekeeper has little impact on the

contaminations by certain agricultural practices.

In beekeeping the setting of a withdrawal time to be respected after the last usage of

antimicrobials is not an easy task. In honey, there is no elimination of residues as a

result of pharmacokinetics. In practical studies, large variations in residue

concentration (i.e. high %CVs) were observed between honey sampled from

different hives within an apiary and even between honey collected from different

frames within a hive. This is caused by the fact that the honey flow has the largest

impact on the level of residues in the honey. According the new MRL regulation

(Regulation (EC) No 470/2009), if the metabolism and depletion of the substance

cannot be assessed, the scientific risk assessment may take into account monitoring

data or exposure data.

The concern about potential build-up of antibiotic resistant organisms in humans is

raising. In the Netherlands, in E.coli isolates from mastitis samples, resistance

against fourth-generation cephalosporins was found, which is indicative for the

presence of Extended Spectrum Beta-Lactamase (ESBL) (Anon., 2010b). Therefore

the nearly systematic dry cow treatment in all four quarters commonly used in our

regions is more and more questioned. The link between residues in food of animal origin and the problem of resistance is

limited to the use of antimicrobials in food-animal production. The problem of

presence of residues in food above the regulatory level could be prevented by

setting and respecting a correct withholding time. For the problem of increasing

resistance in bacteria of human and animal origin, every application of antibiotics has

to be considered.


References Anonymous. 2010a. Guidelines for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). Community Reference Laboratories Residues (CRLs). 20/01/2010: 1-18.

Anonymous. 2010b. MARAN-2008. Monitoring of antimicrobial resistance and antibiotic usage in animals in the Netherlands in 2008. Corrected version July 2010. Veterinary antibiotic usage and resistance surveillance working group (VANTURES). Anonymous. 2010c. Criteria for the approval of an inhibitory substance test for screening antibiotics and sulfonamids in raw milk. 15/10/2010. http://www.favv-afsca.fgov.be/laboratories/ approvedlaboratories/generalinformation/ Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Comm. 2002 L221: 8-36. Commission Decision 2006/694/EC of 13 October 2006 prohibiting the placing on the market of curd cheese manufactured in a dairy establishment in the United Kingdom. Off. J. Eur. Union 2006 L283: 59-61. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Union 2010 L15: 1-72. Corrigendum to Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin. Off. J. Eur. Union 2004 L226: 22-82. Kaufmann A., Butcher P., Maden K., Widmer W., Walker S. 2010. Possibilities and limitations of current UPLC-Orbitrap technology for multi-residue methods. Abstract book of the 6th International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent, Belgium, June 1-4, 2010: 7. Malik A.K., Blasco C., Pico Y. 2010. Liquid chromatography-mass spectrometry in food safety. Review. J. Chromatogr. A 1217: 4018-4040. Regulation (EC) No 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Union 2009 L152: 11-22. Regulation (EC) No 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No 1774/2002 (Animal by-products Regulation). Off. J. Eur. Union 2009 L300: 1-33.


Stolker A.A.M., Rutgers P., Oosterlinck E., Lasaroms J.J.P., Peters R.J.B., van Rhijn J.A., Nielen M.W.F. Comprehensive screening and quantification of veterinary drugs in milk using UPLC-ToF-MS. Anal. Bioanal. Chem. 391: 2309-2322.

9 S

Summary

Summary 254

Summary In the general introduction of this doctoral thesis, theoretical aspects and background

information on residues of antibiotics and chemotherapeutics in honey and milk are

emphasized (Chapter 1).

The European Union was working out many Regulations, Directives, and Decisions,

establishing Maximum Residue Limits (MRLs), laying down specific hygiene rules for

food of animal origin and harmonized standards for the testing for certain residues,

fixing the levels and frequencies of sampling, and providing performance criteria for

analytical methods and the interpretation of results. Food needs to be analysed to

determine its compliance with legislation. Screening methods are playing an

important role in the analysis of a large number of samples by food business

operators in the framework of their autocontrol programmes and by laboratories for

official control.

Qualitative screening methods have to be validated to demonstrate their reliability.

The parameters which are needed to be thoroughly investigated are

selectivity/specificity, detection capability (CC), and applicability/ruggedness/stabili-

ty.

The main bee diseases that could be treated with antimicrobials (American and

European foulbrood, and nosemosis) are shortly discussed. The antibiotics and

chemotherapeutics of interest in apiculture are tetracyclines, streptomycin, sulfo-

namides, tylosin, erythromycin, lincomycin, chloramphenicol, nitrofurans, nitroimida-

zoles, fluoroquinolones, and fumagillin. Most of these drugs are very stable in honey

and remain for a long time in the beehive after application since honeybees do not

actively metabolize these antimicrobials. Despite the fact that in the European Union

no MRLs are fixed for antimicrobials in honey, in some Member States antibiotics or

sulfa drugs could be used in apiculture, based on the cascade system for veterinary

medicines under Minor Uses, Minor Species. After application, very long withdrawal

periods need to be considered in view of the zero-tolerance for residues of

antimicrobials in honey. Despite honey is considered as a natural healthy product,

the occurrence of residues in honey in the period 2000-2005 was very high. Even the

prohibited compound chloramphenicol was regularly detected in honey and royal

jelly on the European market, indicating blending with import from China.

Summary 255

In milk production, the main reasons for the use of anti-infectious agents are the

control of bovine mastitis and dry cow therapy. In total, 82 different veterinary drugs

(brand/trade names) with an anti-infectious agent as active substance are registered

in Belgium for use in milk producing cows. More than half of these drugs contain a -

lactam antibiotic. Benzylpenicillin, occurring in nine brand names, is the most

common active substance. Most veterinary drugs are registered for intramuscular

use, followed by intra-mammary and intravenous route of administration. The

pharmacokinetics of antimicrobials in lactating cows is mainly related to the route of

administration. Only intra-mammary application is resulting in a high concentration of

residues in the milk, especially in the first milking after treatment. So large volumes

of milk can only get contaminated above MRL by intra-mammary use of veterinary

drugs. The dairy sector could be exemplary for other food sectors with its in routine

monitoring and low occurrence of antibiotic residues. Antibiotics could pose a hazard

to human health, not only by their toxicological properties in the narrow sense such

as teratogenic, mutagenic, or carcinogenic effects but also by the possibility of

allergic reactions. In addition, antimicrobial resistance of zoonotic bacteria isolated

from foodstuffs is an increasing public health concern. Evidence shows that the food-

borne route is the major transmission pathway for resistant bacteria from food-

producing animals to humans. Antimicrobial residues could also cause technological

problems by inhibiting dairy starter cultures used for the manufacturing of yoghurt

and cheese.

The objectives of this thesis are shortly given in Chapter 2. The first objective was

the validation of three screening tests, namely the Tetrasensor Honey test kit for

screening for tetracyclines in honey and two dipstick rapid tests, the eta-s.t.a.r. 1+1

and the Charm MRL-3, for detection of -lactam residues in milk. The second

objective was the determination of migration of sulfonamides from contaminated

beeswax foundations to honey. The last objective was the study of milk originating

from farms with regular problems of false-positive Delvotest results in order to

indicate the interfering inhibitory substances.

The validation of Tetrasensor Honey is described in Chapter 3. With this receptor-

based assay using dipsticks, honey can be screened for tetracyclines in 30 min. The

Summary 256

test was validated at ILVO-T&V according to Commission Decision 2002/657/EC.

The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in

honey in a specific way at concentrations below 10 µg kg-1 (dry dipstick reading).

The test procedure is very simple and the test is rugged as no influence on the test

capability was noticed with regard to the geographical or botanical origin or by

physical parameters. Only small problems were encountered with a Spanish

honeydew honey. No false-negative and no false-positive results were obtained

during two international proficiency tests and in a study of 100 table honey samples.

It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test

kit that even can be performed by the beekeeper at the production site since no

special equipment (incubator, reader, etc.) is required.

Some Flemish honey samples showed to be contaminated with a low concentration

of sulfamethazine not caused by the use of sulfa drugs by the beekeeper. A

migration test was set up to check if sulfa-containing beeswax foundations could

lead to contamination of honey. The results of the experiment are given in Chapter 4.

Beeswax foundations made out of wax doped with sulfamethazine at three levels

were given to three different hives to let the honeybees draw out the spiked wax

foundations to honeycombs. Once filled with capped honey, the honeycombs were

sampled and further incubated in the laboratory to follow the migration of the

sulfamethazine. The higher the concentration of sulfamethazine doped in the wax,

the higher the concentration of sulfamethazine found in the honey. The maximum

transfers of the initial amount spiked in the wax foundation to the honey were 15.6,

56.9, and 29.5%, respectively.

In a second experiment, the percentage of sulfamethazine migrating from medicated

winter feed to beeswax in relation to the concentration in the syrup and the contact

time was studied. The maximum transfer of sulfamethazine from medicated sucrose

syrup to beeswax was 3.1%.

The results of both experiments indicate that after the use of sulfonamides at

medicated concentration in the hive, residues remain in the wax of the combs which

can contaminate the honey during the next honey season, with serious implications

for the beekeeper in countries without tolerance levels for sulfa drugs in honey. The

results also indicate serious implications regarding the recycling of beeswax. This

Summary 257

publication is the first to report that contaminated beeswax could be the vector of

honey contamination with antimicrobial residues.

Chapters 5 and 6 are describing the validation of two new rapid tests for screening

for -lactams in milk, βeta-s.t.a.r. 1+1 and Charm MRL-3, respectively. The validation

studies were performed at ILVO-T&V according to Commission Decision

2002/657/EC.

The βeta-s.t.a.r. 1+1 with a 2-min protocol (1+1) is very selective for the group of β-

lactam compounds. The test was only interfered by clavulanic acid, a -lactamase

inhibitor, at 2,500 µg kg-1 and above. All β-lactams with a MRL in milk were detected,

but not all at their respective MRL. The detection of desfuroylceftiofur, cefalexin,

penethamate, and ceftiofur was poor and ampicillin, amoxicillin, nafcillin,

cefquinome, cefazolin, and desacetylcephapirin were also not detected at MRL. The

repeatabilities of both reader and test were very good. The test was very robust as

test results were not significantly influenced by small changes in the test protocol,

milk composition, or type of milk. The test was also applicable on milk of animal

species other than the cow (goat, ewe, or mare). Favourable results were obtained in

testing monitoring samples, in two national ring trials, and in an international

proficiency test.

With a total test time of two min, the βeta-s.t.a.r. 1+1 is, presently, the fastest single

test on the market for the detection of β-lactam residues in milk. The short test time,

the very easy test protocol, and the possibility of visual interpretation of the test

enables the use of the test at the farm before collection. The use of βeta-s.t.a.r. 1+1

at farm level instead of the classic 5-min βeta-s.t.a.r. test at the entrance of the dairy

plant would resolve the issue of dilution “disguising” contaminated milk and would

lead to stronger on-farm practices. On-farm checking would also reduce costs for the

destruction of large volumes of β-lactam-contaminated milk. If time is not the crucial

factor (dairy entrance control) or no further dilution of the milk is expected, the

classic 5-min βeta-s.t.a.r. protocol could still be preferred to obtain the best test

sensitivity.

The one-step 3-min assay, Charm MRL-3, is a new and faster version of the Charm

MRL Beta-Lactam Test for the detection of β-lactam residues in cows‟ milk. In the

Summary 258

validation study the test showed to be very specific as a real interference was only

caused by clavulanic acid at 175 μg kg-1 and above. The repeatability of the reader

was good; however regarding the test repeatability some problems for negative milk

samples were noticed. Throughout the evaluation study, false-positive results were

obtained when testing blank raw milk. Hence, it is recommended to retest initial

positive samples as indicated by the kit manufacturer.

The Charm MRL-3 detects all -lactams with a MRL in milk at their respective MRL

excepted for nafcillin and penethamate which were in 95% of the cases detected at

90 and 200 µg kg-1 and above, respectively. The test showed to be robust for

changes in the test protocol. The milk quality and composition had some influence

on the performance of the Charm MRL-3 when testing blank and spiked milk. Charm

MRL-3 is not only a raw milk test for the dairy industry, but it could also be used to

test UHT-milk, sterilized milk, reconstituted milk powder, or thawed milk under

condition that positive results are further tested with a different antibiotic test. The

Charm MRL-3 is not a suitable test to screen milk from animal species different from

the cow (goat, ewe, or mare).


test at the farm before collection in order to prevent tanker milk contamination with β-

lactam antibiotics. A drawback is the recommendation of the use of a reader system

for the interpretation of the colour formation on the dipsticks.

Microbiological inhibitor tests which are widely used for the screening for inhibitors,

such like antibiotics in the farm milk delivered to dairies, are known to produce false-

positive results by interference of natural inhibitors in the milk. At two farms with

frequent problems of false-positive Delvotest results as part of the regulatory quality

programme, milk was sampled for antibiotic detection at ILVO-T&V. As described in

Chapter 7, positive Delvotest results were noticed despite no residues of antibiotics

and chemotherapeutics were found. Two Pseudomonas strains, identified as closely

related to Pseudomonas tolaasii, were isolated from milk of one of these farms.

Growth at 5 to 7°C of the isolates in milk resulted in high lipolysis and the production

of bacterial inhibitors. These bacterial inhibitors with a molecular weight <1 kDa

showed to be heat-tolerant and inhibitory to Geobacillus stearothermophilus var.

calidolactis, the test strain of most of the commercially available microbiological

Summary 259

inhibitor tests for milk. The bacterial inhibitors also showed antimicrobial activity

against other Gram-positives and interfered in yoghurt production.

The bacterial inhibitors are not yet identified but the results of the characterization

assays could rule out that the inhibition was caused by the elevated level of free fatty

acids and indicate in the direction of cyclic lipodepsipeptides, toxins with

antimicrobial properties.

To our knowledge, these results are the first statement of interference of microbial

inhibitor tests for antibiotic residues in milk by bacterial inhibitors produced by

Pseudomonas strains. Our findings show that extended refrigerated storage of raw

milk could not only result in possible spoilage of long-life milk but also in false-

positive microbial inhibitor tests.

With the validation of three screening tests, a contribution was given to the quality

assurance of milk and honey. In addition, the problem of low sulfa-contamination of

honey and the occurrence of false-positive results caused by certain milk flora were

studied in this thesis. In both cases, where no antibiotics were used by the food

producers (beekeepers or dairy farmers), new elements were found that could help

to explain the product contamination.

envattin

Samenvatting

Samenvatting 262

Samenvatting In de algemene inleiding van dit proefschrift worden theoretische aspecten en

achtergrondinformatie omtrent residuen van antibiotica en chemotherapeutica in

honing en melk toegelicht (Hoofdstuk 1).

De Europese Unie heeft tal van verordeningen, richtlijnen en beschikkingen

uitgevaardigd, maximumwaarden voor residuen (MRLs) vastgesteld, specifieke

hygiënevoorschriften voor voeding van dierlijke oorsprong en geharmoniseerde

standaarden voor het testen van bepaalde substanties uitgewerkt, de niveaus en de

frequenties van staalname bepaald, en performantiecriteria voor analytische

methoden en de interpretatie van resultaten vastgelegd. Voedsel dient geanalyseerd

te worden om na te gaan of aan de wettelijke bepalingen wordt voldaan.

Screeningsmethoden spelen een belangrijke rol bij de analyse van een groot aantal

stalen door voedingsoperatoren in het kader van een autocontroleprogramma en bij

de officiële controleonderzoeken uitgevoerd in laboratoria.

Kwalitatieve screeningsmethoden dienen gevalideerd te worden om hun betrouw-

baarheid aan te tonen. De parameters die uitvoerig onderzocht moeten worden zijn

de selectiviteit/specificiteit, detectiecapaciteit (CC) en de toepasbaarheid

/robuustheid/stabiliteit.

De belangrijkste bijenziekten (Amerikaans en Europees vuilbroed, en nosemose) die

met infectiewerende stoffen kunnen worden behandeld worden kort besproken. De

antibiotica en chemotherapeutica van belang voor de bijenteelt zijn tetracyclines,

streptomycine, sulfonamiden, tylosine, erythromycine, lincomycine, chloor-

amphenicol, nitrofuranen, nitro-imidazolen, fluoroquinolones en fumagilline. De

meeste van deze geneesmiddelen zijn zeer stabiel in honing en blijven na toediening

lange tijd in de bijenkast aangezien de bijen deze substanties niet actief

metaboliseren. Ondanks het feit dat in de Europese Unie geen MRLs in honing zijn

vastgesteld, mogen in bepaalde lidstaten antibiotica en sulfonamiden toch in de

imkerij toegepast worden op basis van het cascadesysteem voor diergenees-

middelen onder het beleid voor weinig voor-komende toepassingen en diersoorten

(Minor Uses and Minor Species, MUMS). Na toediening dienen evenwel zeer lange

wachttijden te worden gerespecteerd, gezien de nultolerantie voor residuen van

infectiewerende stoffen in honing. Ondanks het feit dat honing algemeen beschouwd

wordt als een natuurlijk gezond product, werden er in de periode 2000-2005 frequent

Samenvatting 263

residuen in aangetroffen. Zelfs de verboden substantie chlooramphenicol werd

geregeld gedetecteerd in honing en koninginnenbrij op de Europese markt wat wijst

om menging met import uit China.

In de melkproductie zijn de voornaamste redenen voor het gebruik van

infectiewerende stoffen de beheersing van mastitis en droogstandstherapie. In totaal

zijn er 82 verschillende diergeneesmiddelen met een infectiewerende stof als actieve

substantie geregistreerd in België voor gebruik bij melkvee. Meer dan de helft van

deze dier-geneesmiddelen bevat een -lactamantibioticum. Benzylpenicilline,

voorkomend in negen handelsproducten, is de meest courante substantie. De

meeste diergenees-middelen zijn geregistreerd voor intramusculair gebruik, gevolgd

door intramammaire en intraveneuze wijze van toediening. De farmacokinetiek van

infectiewerende stoffen in lacterende dieren is hoofdzakelijk gerelateerd met de

toedieningswijze. Enkel intramammaire toedieningen resulteren in hoge

concentraties van residuen in de melk, en dit voornamelijk bij de eerste melkbeurt na

toediening. Bijgevolg kunnen grote volumes melk enkel gecontamineerd geraken met

residugehaltes boven de maximum-waarde (MRL) door intramammair gebruik van

diergeneesmiddelen. De zuivelsector, met zijn routinematige monitoring en lage

prevalentie van antibioticaresiduen, kan als voorbeeld dienen voor andere

voedingssectoren.

Antibiotica kunnen een gevaar inhouden voor de volksgezondheid omwille van hun

toxicologische eigenschappen in de enge betekenis van het woord, zoals teratogene,

mutagene of carcinogene effecten, en mogelijke allergische reacties. Daarbij komt

een toenemende bezorgdheid omtrent de antimicrobiële resistentie van zoönotische

bacteriën geïsoleerd uit levensmiddelen. Er zijn immers aanwijzingen dat de

belangrijkste wijze van transmissie van resistente bacteriën vanuit

voedselproducerende dieren naar de mens toe via levensmiddelen geschiedt.

Antibioticaresiduen kunnen bovendien technologische problemen veroorzaken door

het remmen van de starterculturen bij de productie van yoghurt of kaas.

In Hoofdstuk 2 zijn de doelstellingen van het proefschrift kort weergegeven. De

eerste doelstelling van deze thesis was het valideren van drie screeningstesten. Het

betreft de Tetrasensor Honey test kit voor de screening van tetracyclines in honing

en twee dipstick sneltesten, de eta-s.t.a.r. 1+1 en de Charm MRL-3, voor de

Samenvatting 264

detectie van -lactamresiduen in melk. De tweede doelstelling was een onderzoek

naar de migratie van sulfonamiden vanuit gecontamineerde bijenwas naar honing.

De laatste doelstelling betreft een studie van melk afkomstig van hoeven waar

regelmatig problemen van valspositieve Delvotest-resultaten werden vastgesteld,

teneinde de interfererende bacteriegroeiremmende stoffen te kunnen achterhalen.

De validatie van de Tetrasensor Honey is beschreven in Hoofdstuk 3. Met deze

receptortest, die gebruik maakt van dipsticks, kan honing gescreend worden op

tetracyclines in 30 minuten. De test werd op het ILVO-T&V gevalideerd volgens

Beschikking 2002/657/EG van de Commissie. De test detecteert heel specifiek

tetracycline, oxytetracycline, chloortetracycline en doxycycline in honing in

concentraties lager dan 10 µg kg-1 (aflezing van droge dipsticks). De testprocedure is

zeer eenvoudig en de test is robuust vermits er op de testcapaciteit geen invloeden

door verschillen inzake geografische of botanische herkomst van de honing, noch

door fysische parameters werden vastgesteld. Noch valsnegatieve, noch

valspositieve resultaten werden bekomen in twee internationale ringonderzoeken en

in een studie van 100 stalen tafelhoning. Bijgevolg kan worden gesteld dat de

Tetrasensor Honey een eenvoudige en betrouwbare test is, die zelfs door de imker in

het slingerlokaal kan worden uitgevoerd daar de uitvoering geen speciale apparatuur

(incubator, afleestoestel,…) behoeft.

Enkele Vlaamse honingstalen bleken gecontamineerd met lage concentraties

sulfamethazine, zonder dat door de imker sulfa‟s waren aangewend. Een migratietest

werd opgezet om na te gaan of sulfa-gecontamineerde bijenwas kon leiden tot

besmetting van honing. De resultaten van het experiment zijn weergegeven in

Hoofdstuk 4. Waswafels gemaakt van bijenwas gedopeerd op drie niveaus

sulfamethazine werden in drie aparte bijenkasten gehangen om door de bijen

uitgebouwd te worden tot wasraten. Eenmaal gevuld met honing en verzegeld,

werden de honingraten bemonsterd en verder in het laboratorium geïncubeerd om de

migratie van sulfamethazine te volgen. Hoe hoger de concentratie aan

sulfamethazine gedopeerd in de was, hoe hoger de concentratie van sulfamethazine

teruggevonden in de honing. De maximale overdracht van het initieel gehalte in de

waswafel naar de honing was respectievelijk 15,6; 56,9 en 29,5%.

Samenvatting 265

In een tweede experiment werd het percentage sulfamethazine dat migreert van

gemedicineerd wintervoedsel naar bijenwas bepaald en dit in relatie tot de

concentratie in de siroop en de contacttijd. De maximale overdracht van

sulfamethazine vanuit de gemedicineerde suikersiroop naar de was bedroeg 3,1%.

De resultaten van beide experimenten tonen aan dat er na gebruik van sulfonamiden

in een bijenvolk residuen achterblijven in de was van de honingraten die de

honingoogst van het volgende seizoen kunnen verontreinigen, met zware gevolgen

voor imkers uit landen met een nultolerantie voor residuen in honing. De resultaten

hebben ook implicaties betreffende de recyclage van bijenwas. In deze publicatie

wordt voor het eerst gerapporteerd dat gecontamineerde bijenwas de vector kan zijn

van besmetting van honing met residuen van antimicrobiële stoffen.

In de Hoofdstukken 5 en 6 wordt de validatie beschreven van twee nieuwe sneltesten

voor de screening van -lactam antibiotica in melk, namelijk de βeta-s.t.a.r. 1+1 en

de Charm MRL-3. De validatiestudies werden op het ILVO-T&V uitgevoerd volgens

Beschikking 2002/657/EG van de Commissie.

De βeta-s.t.a.r. 1+1 met zijn 2-minuten protocol (1+1) is zeer selectief voor de groep

van β-lactamantibiotica. Er werd enkel een interferentie vastgesteld voor clavulaan-

zuur, een -lactamaseremmer, bij concentraties vanaf 2500 µg kg-1. Alle β-lactam-

antibiotica met een MRL in melk werden gedetecteerd, doch niet allen op hun

respectievelijke MRL. De detectie van desfuroylceftiofur, cefalexine, penethamaat en

ceftiofur was zwak; en ampicilline, amoxicilline, nafcilline, cefquinome, cefazoline en

desacetylcephapirine werden ook niet op MRL gedetecteerd. De herhaalbaarheid

van het afleestoestel en van de test was zeer goed. De test was zeer robuust: de

testresultaten werden niet significant beïnvloed door kleine wijzigingen in het

testprotocol, de melksamenstelling of het type melk. De test kon ook aangewend

worden op melk van andere diersoorten (geit, schaap of paard). Bij de monitoring

van praktijkstalen, en tevens in twee nationale ringonderzoeken en in een

internationale geschiktheidsbeproeving werden gunstige resultaten bekomen.

Met een totale testduur van twee minuten is de βeta-s.t.a.r. 1+1 momenteel de

snelste individuele test op de markt voor de detectie van β-lactamresiduen in melk.

De korte testtijd, het zeer eenvoudig testprotocol en de mogelijkheid tot visuele

aflezing zorgen ervoor dat deze test inzetbaar is bij de melkophaling op de hoeve.

Het gebruik van de βeta-s.t.a.r. 1+1 op hoeveniveau, in plaats van de klassieke 5-

Samenvatting 266

minuten test eta-s.t.a.r. bij de ingangscontrole van het zuivelbedrijf, lost het

probleem van het maskeren van gecontamineerde melk door verdunning op, en leidt

tot meer waakzaamheid op de hoeve. Het testen op de hoeve vόόr het opzuigen van

de melk zou tevens de kosten drukken voor de vernietiging van grote volumes met β-

lactam-gecontamineerde melk. Evenwel, als de tijd geen cruciale factor speelt

(ingangscontrole op het zuivelbedrijf) of indien de melk nadien niet meer wordt

verdund, is het aangewezen om de klassieke 5-minuten βeta-s.t.a.r. in te zetten, om

zo een betere testgevoeligheid te bekomen.

De éénstapstest Charm MRL-3 met een testprotocol van drie minuten is een nieuwe

en snellere versie van de Charm MRL Beta-Lactam Test voor het opsporen van β-

lactamresiduen in koemelk. In een validatiestudie bewees de test zeer specifiek te

zijn vermits enkel een echte interferentie voor clavulaanzuur werd vastgesteld en dit

vanaf een concentratie van 175 μg kg-1. De herhaalbaarheid van de reader was

goed; wel werden er enkele problemen vastgesteld inzake de herhaalbaarheid van

de test voor negatieve melkstalen. Over de gehele evaluatieperiode werden

valspositieve resultaten bekomen bij het testen van remstofvrije rauwe melk.

Bijgevolg is het aangewezen om de aanbeveling van de kitproducent op te volgen

door initieel positieve resultaten te hertesten.

De Charm MRL-3 detecteert alle -lactamantibiotica met een MRL in melk op hun

respectievelijke norm met uitzondering van nafcillin en penethamaat die

respectievelijk in 95% van de gevallen werden gedetecteerd vanaf 90 en 200 µg kg-1.

De test was bestand tegen wijzigingen in het testprotocol. De melkkwaliteit en

-samenstelling hadden invloed op de prestatie van de Charm MRL-3 bij het testen

van blanco en gedopeerde melk. De Charm MRL-3 is niet alleen een rauwe melktest

voor de zuivelindustrie, maar kan eveneens worden ingezet voor het testen van

UHT-melk, gesteriliseerde melk, gereconstituteerde melkpoeder of melk na

ontdooiing, op voorwaarde dat positieve resultaten verder getest worden met een

andere antibioticatest. De Charm MRL-3 is niet geschikt voor het screenen van melk

van andere diersoorten dan de koe (geit, schaap of paard).

De korte testduur en het eenvoudig éénstapsprotocol maken het gebruik van de test

op de hoeve mogelijk, om op die manier een contaminatie van de ophaalwagenmelk

met β-lactamantibiotica te voorkomen. Een nadeel vormt de aanbeveling tot het

Samenvatting 267

gebruik van een readersysteem voor de interpretatie van de kleurvorming op de

dipsticks.

Het is bekend dat microbiologische inhibitietesten, die op grote schaal worden

ingezet voor het screenen van remstoffen zoals antibiotica in de melk geleverd aan

de zuivelbedrijven, valspositieve resultaten kunnen geven die te wijten zijn aan een

interferentie door natuurlijke bacteriegroeiremmende stoffen in de melk. Op twee

hoeves waar frequent problemen van valspositieve Delvotest-resultaten werden

bekomen in het kader van de uitbetaling van de melk, werd melk bemonsterd voor

onderzoek op antibioticaresiduen op het ILVO-T&V. Zoals beschreven in Hoofdstuk

7, werden positieve Delvotest-resultaten bekomen zonder dat residuen van

antibiotica of chemotherapeutica in de melk werden aangetroffen. Twee

Pseudomonas-stammen, geïdentificeerd als sterk verwant met Pseudomonas

tolaasii, werden geïsoleerd uit de melk van één van de hoeves. Groei van de isolaten

bij 5 tot 7°C in melk resulteerde in een hoge lipolyse en de productie van

bacteriegroeiremmende stoffen. Deze stoffen met een moleculair gewicht <1 kDa

waren hitte-tolerant en remden Geobacillus stearothermophilus var. calidolactis, het

testorganisme van de meeste commercieel beschikbare microbiologische

inhibitietesten voor melk. De bacteriegroeiremmende stoffen vertoonden eveneens

een antimicrobiële activiteit tegen andere Gram-positieven en verstoorden de

yoghurtproductie.

De bacteriegroeiremmende stoffen zijn nog niet geïdentificeerd maar de resultaten

van de karakterisatietesten sluiten uit dat de groeiremming veroorzaakt wordt door

een verhoogd gehalte aan vrije vetzuren en wijzen in de richting van cyclische

lipodepsipeptiden, toxines met antimicrobiële eigenschappen.

Voor zover ons bekend, zijn deze resultaten de eerste vaststelling van interferentie

bij microbiologische inhibitietesten door bacteriële groeiremmende stoffen

geproduceerd in melk door Pseudomonas-bacteriën. Onze bevindingen tonen aan

dat een langdurige gekoelde bewaring van rauwe melk, naast een mogelijks bederf,

ook aanleiding kan geven tot valspositieve resultaten van remstoftesten.

Met de validatie van drie screeningstesten werd een bijdrage geleverd aan de

kwaliteitsgarantie van melk en honing. Daarnaast werden in dit proefschrift het

probleem van lage sulfa-contaminatie van honing en het voorkomen van

Samenvatting 268

valspositieve resultaten bij screening van melk door een bepaalde melkflora

bestudeerd. In beide gevallen, waarbij door de producenten (imkers en

melkveehouders) geen antibiotica werden verstrekt, werden nieuwe elementen

gevonden die de contaminaties zouden kunnen verklaren.

Curriculum Vitae

Curriculum Vitae

Curriculum Vitae 270

Wim Reybroeck werd geboren op 17 december 1960 te Gent. Na zijn studies

middelbaar onderwijs, richting Latijn-Wetenschappen, aan het Sint-Barbaracollege te

Gent, behaalde hij in 1983 met onderscheiding het diploma van master in de

biowetenschappen: landbouwkunde (industrieel ingenieur) aan de Hogeschool Gent.

In 1984 behaalde hij met onderscheiding het diploma van licentiaat in de

milieusanering aan de Universiteit Gent.

Na zijn legerdienst trad hij in 1985 in dienst bij ILVO-T&V (toenmalig

Rijkszuivelstation) in de bacteriologische afdeling. In 1994 ontwikkelde hij een ATP-

methode voor de snelle kiemgetalbepaling van rauwe melk, gecommercialiseerd

door Celsis als „Raw Milk Microbial Kit‟. Sinds 1994 is hij laboverantwoordelijke voor

het onderzoek en de dienstverlening inzake screening van antibioticaresiduen in

levensmiddelen van dierlijke oorsprong en kwaliteitsaspecten van rauwe melk en

honing.

Hij is lid van de International Honey Commission en meerdere Standing Committees

van de International Dairy Federation. Hij is tevens beëdigd assistent bijenziekten en

lesgever bij het PCLT vzw te Roeselare en de K. O. I. B. vzw.

A PUBLICATIES A.1 Artikels A.1.1 In peer-reviewed tijdschriften

Van Crombrugge J., Waes G., Reybroeck W. 1989. The ATP-F test for estimation of bacteriological quality of raw milk. Neth. Milk Dairy J. 43: 347-354. Reybroeck W., Schram E. 1995. Improved filtration method to assess bacteriological quality of raw milk based on bioluminescence of adenosine triphosphate. Neth. Milk Dairy J.49: 1-14. D‟Haese E., Nelis H.J., Reybroeck W. 1997. Inhibition of -galactosidase biosynthesis in Escherichia coli by tetracycline residues in milk. Appl. Environ. Microbiol. 63(10): 4116-4119. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Chemiluminometric -galactosidase detection as a basis for a tetracycline screening test in milk. J. Biolumin. Chemilumin., 13: 279-284. D‟Haese E., Nelis H.J., Reybroeck W., De Ruyck H. 1999. Evaluation of a modified enzymatic test for the detection of tetracyclines in milk. J. Food Prot. 62(6): 632-636. D‟Haese E., Nelis H.J., Reybroeck W. 2001. Determination of somatic cells in milk by solid phase cytometry as a new tool. J. Dairy Res. 68: 9-14.


Dobbelaere W., Jacobs F.J., Reybroeck W., Desmedt E., Peeters J.E., de Graaf D.C. 2001. Disinfection of wooden structures contaminated with Paenibacillus larvae subsp. larvae spores. J. Appl. Microbiol. 91: 212-216. Impens S., Reybroeck W., Vercammen J., Courtheyn D., Ooghe S., De Wasch K., Smedts W., De Brabander H. 2003. Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC-MS2 and LC-MS2. Anal. Chim. Acta 483: 153-163. Reybroeck W. 2004. Résidus d‟antibiotiques dans le lait. Utilisation des kits de dépistage des inhibiteurs. Point Vét. 242: 52-57. Van Coillie E., De Block J., Reybroeck W. 2004. Development of an Indirect Competitive ELISA for Flumequine Residues in Raw Milk Using Chicken Egg Yolk Antibodies. J. Agric. Food Chem. 52: 4975-4978. Van Hoof N., De Wasch K., Okerman L., Reybroeck W., Poelmans S., Noppe H., De Brabander H. 2005. Validation of a liquid chromatography-tandem mass spectrometric method for the quantification of eight quinolones in bovine muscle, milk and aquacultured products. Anal Chim Acta, 529: 265-272. Reybroeck W., Ooghe S., De Brabander H., Daeseleire E. 2007. Validation of the Tetrasensor Honey Test Kit for the Screening of Tetracyclines in Honey. J. Agric. Food Chem. 55: 8359-8366. De Brabander H.F., Noppe H., Verheyden K., Vandenbussche J., Wille K., Okerman L., Vanhaecke L., Reybroeck W., Ooghe S., Croubels S. 2009. Review. Residue analysis: Future trends from a historical perspective. J. Chromatogr. A 1216: 7964-7976. Reybroeck W., Jacobs F.J., De Brabander H.F., Daeseleire E. 2010.Transfer of sulfamethazine from contaminated beeswax to honey. J. Agric. Food Chem. 58: 7258-7265. Reybroeck W., Ooghe S., De Brabander H., Daeseleire E. 2010. Validation of the eta-s.t.a.r. 1+1 for rapid screening of residues of -lactam antibiotics in milk. Food Addit. Contam.: Part A, 27(8): 1084-1095, DOI: 10.1080/19440041003724871. Reybroeck W., Ooghe S., De Brabander H., Daeseleire E. 2011. Validation of the Charm MRL-3 for fast screening of -lactam antibiotics in raw milk. J. AOAC Int. 94(2): page numbers not yet assigned. A.1.2 In wetenschappelijke tijdschriften Van Heddeghem A., Reybroeck W. 1990. De bepaling van het totaal kiemgetal en het aantal coliforme bacteriën in rauwe melk met de Petrifilm-methode. Landbouwtijd. - Rev. Agr. 43(5): 839-848. Van Heddeghem A., Reybroeck W., Van Crombrugge J. 1990. De bruikbaarheid van de Bactoscan 2/87 bij de bepaling van het kiemgetal van rauwe melk. Landbouwtijd. - Rev. Agr. 43(6): 1039-1051. Reybroeck W., Van Heddeghem A. 1991. Bruikbaarheid van de Partec CA-II voor de bepaling van het celgetal van melk. Landbouwtijd. - Rev. Agr. 44(6): 1257-1268. Ninane V., De Reu K., Oger R., Reybroeck W., Guyot A. 2000. Evaluation du BactoScan FC pour la numération des bactéries du lait cru. Le Lait 80: 527-538.


Reybroeck W. 2003. Residues of antibiotics and sulphonamides in honey on the Belgian market. Apiacta 38: 23-30. Reybroeck W. 2004. Role of the Farmer in Preventing Residues of Antibiotics in Farm Milk. Bulletin of the IDF 386: Brussels, Belgium: 8-9. Reybroeck W. 2008. The use of microbiological, immunological and receptor tests for monitoring of residues of antimicrobials in milk: the Belgian approach. In Bulletin of the IDF 424/2008 “Advances in Analytical Technology”, ISSN 0250-5118. Brussels, Belgium: 13-16. A.1.3 In overige tijdschriften en vulgaristische artikels Schotsaert P., Reybroeck W., Verstraeten B., Jacobs F.J. 1982. Het oogsten van stuifmeel II. Resultaten en bespreking. Maandblad van de Vlaamse Imkersbond 68(10): 360-365. Reybroeck W. 1994. Bioluminescentie en enkele toepassingen. Chemie Magazine, 20(19): 36-37. De Ville W., Reybroeck W. 1994. Reiniging en desinfectie bij de melkwinning. De Boer & Tuinder 100(14): 13-14. Reybroeck W. 1999. Kwaliteit van honing. Honinganalyses. Proceedings studienamiddag met als thema "Nectar- van bloem tot honing" georganiseerd door het CLO, Departement Gewasbescherming en het Vlaams Vulgarisatiecentrum voor Bijenteelt in het kader van Agriflora. Merelbeke , 9 januari 1999: 1-6. Van Hoorde A., Reybroeck W., Jacobs F.J. 1999. Honinganalyses in Vlaanderen anno 1998. Maandblad van de Vlaamse Imkersbond 85(6): 237-242. Reybroeck W., Van Hoorde A., Jacobs F.J. 2000. Honinganalyses in Vlaanderen anno 1999. Maandblad van de Vlaamse Imkersbond 86(7/8): 21-25. Schotsaert P., Reybroeck W., Van Hoorde A., Jacobs F.J. 2000. Mierenzuur, thymol. Alternatief tegen Varroase? Maandblad van de Vlaamse Imkersbond 86(11): 26-31. Reybroeck W. 2001. Mogelijkheden en beperkingen van antibioticaresidutesten. Melkveebedrijf 1(4): 189-191. Reybroeck W., Van Hoorde A., Jacobs F.J. 2001. Honinganalyses in Vlaanderen anno 2000. Maandblad van de Vlaamse Imkersbond, 87(11): 309-312. Reybroeck W. 2002. Honingonderzoek op het DVK-CLO. DVK Nieuwsbrief 5: 2-3. Reybroeck W. 2002. Honingonderzoek op het DVK-CLO. Maandblad van de Vlaamse Imkersbond 88(7-8): 51-5. Reybroeck W., Simoens C., Jacobs F.J. 2002. Honinganalyses in Vlaanderen anno 2001. Maandblad van de Vlaamse Imkersbond 88(12): 222-224. Reybroeck W. 2003. Ingangscontrole van melk op antibioticaresiduen verscherpt. Melkveebedrijf 3(1): 16-17. Reybroeck W., De Block J., Van Hoorde A., Jacobs F.J. 2003. Honinganalyses in Vlaanderen anno 2002. Maandblad van de Vlaamse Imkersbond 89(12): 311-313.


Reybroeck W., Ooghe S. 2004. Wetenschappelijke begeleiding inzake productie en controle van biologische honing. Overzicht van het onderzoek biologische landbouw 2003 in Vlaanderen. Uitgave van L. Delanote, I. Vuylsteke, F. Temmerman, M. Demeulemeester en A. Calus, PCTB (Interprovinciaal Proefcentrum voor de Biologische Teelt v.z.w.: 106-107. Reybroeck W., De Block J., Van Hoorde A., Jacobs F.J. 2004. Honinganalyses in Vlaanderen anno 2003. Maandblad van de Vlaamse Imkersbond 90(12): 17-20. Reybroeck W., Ooghe S., Van Hoorde A., Jacobs F.J. 2005. Honinganalyses in Vlaanderen anno 2004. Vlaamse Imkersblad 35(11): 245-253. Reybroeck W., Ooghe S., Van Hoorde A., Jacobs F.J. 2005. Honinganalyses in Vlaanderen anno 2004. Federaal Imkersblad 8(6): 10-15. Reybroeck W., Ooghe S., Van Hoorde A., Jacobs F.J. 2005. Honinganalyses in Vlaanderen anno 2004. Maandblad van de Vlaamse Imkersbond 91(12): 23-26. Reybroeck W., Ooghe S., Van Hoorde A., Jacobs F.J. 2006. Honinganalyses in Vlaanderen anno 2005. Vlaams Imkersblad 36(9): 305-312. Reybroeck W., Ooghe S., Van Hoorde A., Jacobs F.J. 2006. Honinganalyses 2005. Maandblad van de Vlaamse Imkersbond 92(7): 29-32. De Boosere I., Reybroeck W. 2007. Vragen en antwoorden over het gebruik van antibioticatesten. Landbouw & Techniek 4: 16-18. De Boosere I., Reybroeck W. 2007. Antibioticatesten: vraag en antwoord. Drietand-magazine 9: 8-10 Reybroeck W., Ooghe S., Rotthier B., Jacobs F.J. 2007. Honinganalyses 2006. Maandblad van de Vlaamse Imkersbond 93(9): 15-19. Reybroeck W. 2007. Apiculture, version Australie. Abeilles & Cie 121(6): 31-32. Reybroeck W. 2008. Honinganalyses in Vlaanderen anno 2006. De Vlaamse Imker. Tijdschrift voor imkers 12(2): 27-30. Jacobs F.J., Rotthier B., Beeuwsaert K., De Keukelaere K., Reybroeck W. 2008. Analyse van honing in Vlaanderen, reeds 11 jaar lang. Maandblad van de Vlaamse Imkersbond 94(4): 34-35. Reybroeck W. 2008. Eindredactie themanummer „HONING‟. Uitgave van de Vlaamse Imkersbond, Augustus 2008: 1-40. Reybroeck W., Ooghe S. ,Jacobs F. 2008. 10 Jaar honinganalyses in Vlaanderen (1998-2007). Themanummer „HONING‟ Augustus 2008, K.V.I.B.: 4-16. Reybroeck W., Ooghe S., Jacobs F. 2008. Kristallisatie van honing. Themanummer „HONING‟ Augustus 2008, K.V.I.B.: 23-33. Reybroeck W., Ooghe S. ,Jacobs F. 2008. Etikettering van honing. Themanummer „HONING‟ Augustus 2008, K.V.I.B.: 37-40.


Jacobs F.J., Rotthier B., Beeuwsaert K., De Keukelaere K., Reybroeck W. 2009. Analyse van honing in Vlaanderen, reeds 12 jaar lang. Maandblad van de Vlaamse Imkersbond 95(4): 29-30. Reybroeck W., Jacobs F., Lequeux R., Bruneau E. 2009. Gids voor goede Bijenteeltpraktijken. Informatiecentrum voor Bijenteelt, Gent, D/2009/5360/01: 1-80. Lequeux R., Bruneau E., Reybroeck W., Jacobs F. 2009. Guide de bonnes pratiques Apicoles. CARI, Louvain-la-Neuve: 1-80. Reybroeck W., Ooghe S., Rotthier B., Jacobs F.J. 2010. Honinganalyses 2008 – Deel 1. Maandblad van de Vlaamse Imkersbond, 96(1): 26-28. Reybroeck W., Ooghe S., Rotthier B., Jacobs F.J. 2010. Honinganalyses 2008 – Deel 2. Maandblad van de Vlaamse Imkersbond, 96(2): 20-22. Reybroeck W. 2010. Controle van antibioticaresiduen in melk. Geïntegreerd systeem. Labinfo. Informatieblad voor de erkende laboratoria voedselveiligheid, nr 4, juni 2010: 22-25. Lequeux R., Bruneau E., Reybroeck W., Jacobs F. 2010. Leitlinien für eine Gute Imkerliche Praxis. CARI, Louvain-la-Neuve: 1-80. A.2 Hoofdstukken in boeken Waes G., Van Crombrugge J., Reybroeck W. 1989. The ATP-F test for estimation of the bacteriological quality of raw milk. Modern Microbiological Methods for Dairy Products, I.D.F. Special Issue 8901, I.D.F. (International Dairy Federation), Brussels: 279-286. Reybroeck W. 1997. ATP Monitoring for microbes and antibiotics in milk. In: "A practical guide to industrial uses of ATP-luminescence in rapid microbiology." Eds. Stanley PE, Smither R. and Simpson W.J. Cara Technology Ltd, Lingfield, Surrey, UK: 87-94. Honkanen-Buzalski T., Reybroeck W. 1997. Antimicrobials. In: "Monograph on residues and contaminants in milk and milk products." I.D.F. (International Dairy Federation), Brussels, Belgium, ISBN 92 9098 025 8: 28-34. Reybroeck W. 1997. Detergents and disinfectants. In: "Monograph on residues and contaminants in milk and milk products." I.D.F. (International Dairy Federation), Brussels, Belgium, ISBN 92 9098 025 8: 112-123. Reybroeck W. 1999. ATP BIOLUMINESCENCE: (b) Application in dairy industry. In: "Encyclopedia of Food Microbiology", Eds. Robinson R, Batt C and Patel P. Academic Press Ltd., London, England: 88-94. A.3 Andere publicaties: proceedings van wetenschappelijke congressen Waes G., Van Crombrugge J., Reybroeck W. 1989. El ensayo del ATP-F para la evaluacion de la calidad bacteriologica de la leche cruda.Proceedings of International Seminar in Santander, Spain, May 1989: 286-287. Reybroeck W. 1989. The ATP-F test for estimation of bacteriological quality of raw milk. Proceedings Dairy Workshop No 2, Lumac, Landgraaf, Nederland: 29-38.


Waes G., Reybroeck W. 1990. Raw milk ATP-F test. Brief communications of XXIII International Dairy Congress, Montreal, Canada, October 8-12, Vol I: 244. Reybroeck W., Schram E. 1992. Study of parameters involved in the assessment of the bacteriological quality of raw milk by two bioluminescent ATP assays (abstract). "A Symposium on ATP Rapid Microbiology for the Food and Beverage Industries", Cambridge, England, 17 juni 1992. J. Biolumin. Chemilumin. 7: 259. Reybroeck W., Waes G. 1992. Comparison of two ATP test kits for the assessment of the bacteriological quality of raw milk (abstract). "A Symposium on ATP Rapid Microbiology for the Food and Beverage Industries", Cambridge, England, 17 juni 1992. J. Biolumin. Chemilumin. 7: 259. Reybroeck W., Schram E. 1994. Integrated filtration method for testing the bacteriological quality of raw milk by ATP bioluminescence. In: Bioluminescence and Chemiluminescence; Fundamentals and Applied Aspects; Proc. 8th Int. Biolum. and Chemilum. Symposium, Cambridge, England, 5-8 september 1994. Eds. A.K. Campbell, L.J. Kricka and P.E. Stanley, John Wiley & Sons, Chichester, England: 482-485. Reybroeck W., Schram E. 1994. New improved ATP filtration method for the assessment of the bacteriological quality of raw milk (abstract). 8th International Symposium on Biolumi-nescence and Chemiluminescence, Cambridge, England, 5-8 september 1994. J. Biolumin. Chemilumin. 9(5): 343. Reybroeck W. 1994. Comparison of two ATP bioluminescence methods for the bacterio-logical assay of raw milk (abstract). 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge, England, 5-8 september 1994. J. Biolumin. Chemilumin. 9(5): 343. Reybroeck W. 1995. Evaluation of screening tests for the detection of antimicrobial residues in milk. Proceedings "I.D.F.-Symposium on Residues of Antimicrobial Drugs and other Inhibitors in Milk”, Kiel, Germany, 28-31 August, IDF, Brussels: 182-186. Reybroeck W. 1995. Sensitivity and selectivity of screening tests for the detection of antimi-crobial residues in milk. Proceedings "I.D.F.-Symposium on Residues of Antimicrobial Drugs and other Inhibitors in Milk”, Kiel, Germany, 28-31 August, IDF, Brussels: 216-217. Reybroeck W. 1995. Field test of screening tests for the detection of antimicrobial residues in milk. Proceedings "I.D.F.- Symposium on Residues of Antimicrobial Drugs and other Inhibitors in Milk”, Kiel, Germany, 28-31 August, IDF, Brussels: 218-219. Reybroeck W. 1996. Modern methods for the bacteriological quality control of raw milk. Proceedings "I.D.F.-Symposium on Bacteriological Quality of raw milk, Wolfpassing, Austria", IDF, Brussels: 131-140. Reybroeck W., Schram E. 1994. New improved ATP filtration method for the assessment of the bacteriological quality of raw milk (abstract). "Brief Communications and Abstracts of Posters and Invited Papers - 24th International Dairy Congress", Melbourne, Australia,18-22 September 1994: 129. Reybroeck W. 1997. Determination of ceftiofur in milk after intramuscular administration of Excenel to lactating cows. Proceedings "World Congress on Food Hygiene. World Association of Veterinary Food Hygienists, the Hague, the Netherlands", 24-29 augustus 1997, Wageningen Pers, Wageningen, Nederland: 94.


Reybroeck W. 1997. Influence of residues of ceftiofur in milk on the production of yoghurt and cheese, after application of Excenel to lactating cows. Proceedings "World Congress on Food Hygiene. World Association of Veterinary Food Hygienists, the Hague, the Netherlands", 24-29 augustus 1997, Wageningen Pers, Wageningen, Nederland: 95. D‟Haese E., Nelis H.J., Reybroeck W. 1997. Development and evaluation of a chemilu-minescence screening assay for the detection of tetracycline residues in milk (abstract). Abstracts "Symposium on Luminescence Assays for Industry, Cambridge, UK, 21-22 september 1997", J. Biolumin. Chemilumin. 12: 97. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Detection of antibiotic residues in milk by solid phase cytometry (abstract). Abstracts "Third international symposium on hormone and veterinary drug residue analysis, Brugge, 2-5 juni 1998": 15. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Specificity of a chemiluminometric test for the detection of tetracycline residues in milk (abstract). Abstracts 112th AOAC Annual Meeting, Montréal, Canada, 13-17 september 1998: 97. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Detection of membrane disrupting antibiotics in milk and feeds by solid phase cytometry (ChemScan)(abstract). Abstracts 112th AOAC Annual Meeting, Montréal, Canada, 13-17 september 1998: 104-105. Herman L., Reybroeck W., D‟Haese E., Zorman T., Nelis H.J. 1999. Detection and enumeration of Mycobacterium avium subsp. paratuberculosis in milk. Proceedings of the Sixth International Colloquium on Paratuberculosis, Eds. E. Manning and M. Collins, Int. Ass. Paratuberculosis, Madison, WI: 543-552. Herman L., Reybroeck W., D‟Haese E., Zorman T., Nelis H.J. 1999. Comparison of different detection methods for Mycobacterium paratuberculosis in milk. Abstract op the IDF Brainstorming Session on Mycobacterium paratuberculosis, Brussel, 5-6 mei 1999: 17-19 (invited). Reybroeck W. 1999. Immunologische en microbiële screening van diergeneesmiddelen. Proceedings KVCV-studiedag “(Bio)chemische sensoren”, Tervuren, 24 september 1999: 1-7. Reybroeck W. 2000. Alternatieve technieken voor zuivelcontrole. Proceedings KVCV studiedag “Snellere methoden voor kwaliteitsbepaling van levensmiddelen: microbiologische methoden”, Melle, 2 maart 2000: 1-9. Reybroeck W. 2000. Evaluation of the Parallux test for the detection of -lactam antibiotics and tetracyclines. Proceedings 2nd International FoodSENSE Workshop, Zeven, Duitsland, 30 maart - 1 april 2000. Reybroeck W. 2000. Evaluation of the Beta s.t.a.r. for the detection of -lactam antibiotics. Proceedings 2nd International FoodSENSE Workshop, Zeven, Duitsland, 30 maart - 1 april 2000. Reybroeck W. 2000. Milk residues of lincomycin and neomycin after a 5-fold administration of Lincocin Intramammaire to lactating cows: their technological significance. Proceedings EuroResidue IV, Veldhoven, Nederland, 8-10 mei 2000: 903-908. Reybroeck W. 2000. Performance of the PremiTest using naturally contaminated meat. Proceedings EuroResidue IV, Veldhoven, Nederland, 8-10 mei 2000: 909-912.


Reybroeck W. 2000. Detection of residues of antibiotics in foodstuffs with microbiological tests using Bacillus (abstract). Bacillus2000 symposium, Brugge, 31 augustus 2000. Reybroeck W., Van Hoorde A., Jacobs F.J. 2001. Quality control of Flemish honey: results 1998-2000. Arbeitsgemeinschaft der Institute für Bienenforschung e.V, 48. Jahrestagung, Bad Neuenahr / Ahrweiler, Duitsland, 27-29 maart 2001 (abstract). Van Hoorde A., Reybroeck W., Jacobs F.J. 2001. Quality control of honey. A critical analysis of useful parameters. Arbeitsgemeinschaft der Institute für Bienenforschung e.V, 48. Jahrestagung, Bad Neuenahr / Ahrweiler, Duitsland, 27-29 maart 2001 (abstract). Reybroeck W. 2001. Kwaliteitsbepaling van honing binnen de huidige wetgeving. Proceedings K.V.I.B. Symposium “Vlaamse Honing”, Gent, 23 juni 2001: 1-7. Reybroeck W. 2001. Detection of residues of antibiotics in foodstuffs with microbiological tests using Bacillus.Proceedings Epidemiology & Economics (VEE), Melle, 25 oktober 2001: 90-93. Reybroeck W. 2001. Screening van residuen van diergeneesmiddelen in melk. Proceedings Post Universitair Onderwijs Diergeneeskunde “Residuen in de melk, oorzaken en gevolgen”, Merelbeke, 21 december 2001: 1-18. Reybroeck W., Ooghe S. 2002. Evaluation of the applicability of fast screening tests for the detection of residues of antibiotics in milk. Proceedings 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, 4-7 juni 2002: 10. Impens S., Reybroeck W., Courtheyn D., De Wasch K., Ooghe S., Smedts W., De Brabander H.F. 2002. Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC-MS2 and LC-MS2. Proceedings 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, 4-7 juni 2002: 113. Reybroeck W. 2002. Residues of antibiotics and sulphonamides in honey on the Belgian market. Proceedings Apimondia Symposium on Preventing Residues in Honey, Celle, Duitsland, 10-11 oktober 2002: 1-6. Reybroeck W. 2003. Van nectar tot honing, het kristallisatieproces. Proceedings voordrachtenreeks “Diverse Honingaspecten” georganiseerd door de Sint Ambrosius Bijengilde Mortsel en Omstreken. Provinciaal Antwerps Verbond van Imkersverenigingen – PAVI v.z.w.. Mortsel, 25 januari 2003: 1-12. Van Coillie E., Reybroeck W., De Block J., Van Renterghem R., 2003. Production of chicken egg yolk antibodies for the development of an immunoassay for the detection of flumequine in milk (abstract). “Pre-summit Symposium on Innovative Research in Dairy Science and Technology”, IDF World Dairy Summit & Centenary, Brugge, 7-12 september 2003: 93-94.

Van Coillie E., Reybroeck W., De Block J., Van Renterghem R., 2003. Production of chicken egg yolk antibodies for the development of an immunoassay for the detection of flumequine in milk (abstract). Abstracts book of the VIIth International Conference on Agri-Food Antibodies, Uppsala, Zweden, 10-13 september 2003. Reybroeck W. 2003. Role of the Farmer in Preventing Residues of Antibiotics in Farm Milk. Conference paper “Conference on Quality management at farm level”, IDF World Dairy Summit & Centenary, Brugge, 7-12 september 2003: 239-240.


Reybroeck W., Ooghe S. 2003. Results of a multilaboratory trial regarding rapid tests for the control of milk on -lactam antibiotics. Proceedings Euro Food Chem XII, “Stategies for Safe Food”, Brugge, 23-26 september 2003, Proceedings Volume 2: 523-526. Reybroeck W., Ooghe S. 2004. Rapid screening for residues of antibiotics in milk at the factory. Abstract UBISI 2004, IDF/FAO/OIE International Symposium on Dairy Safety and Hygiene. A Farm-to-table Approach for Emerging and Developed Dairy Countries, Cape Town, Zuid-Afrika, 2-5 maart 2004. Reybroeck W., Daeseleire E. ,Ooghe S., Jacobs F. 2004. Sulpha drugs in honey and other bee products (abstract). Apimondia symposium 2004 “Prevention of Residues in Honey 2”, Celle, Duitsland, 27-28 april 2004. Van Hoof N., De Wasch, K., Okerman L., Reybroeck W., Poelmans S., Noppe H., De Brabander H.F. 2004. Quantification of 8 quinolones in bovine muscle using LC-MS2. Proceedings EuroResidue V, Conference on Residues of Veterinary Drugs in Food, Noordwijkerhout, Nederland, 10-12 mei 2004, Proceedings Vol.1: 538-543. Reybroeck W., Ooghe S. 2004. Validation of the Copan Milk Test for the detection of residues of antibiotics and sulphonamides in milk and milk products (abstract). EuroResidue V, Conference on Residues of Veterinary Drugs in Food, Noordwijkerhout, Nederland, 10-12 mei 2004. Reybroeck W. 2004. Belang van het Voedselagentschap en de honingkwaliteit. Proceedings 16de Vlaams Imkerscongres, Gent, 11 september 2004: 1-4. Reybroeck W., Ooghe S. 2004. Rapid screening for residues of antibiotics in milk at the factory. In A Farm-to-table Approach for Emerging and Developed Dairy Countries, Proceedings IDF/FAO International Symposium on Dairy Safety and Hygiene, Cape Town, Rep. of South Africa, 2-5 March 2004. ISSN 1810-0732: 157-161. Reybroeck W., Ooghe S. 2005. Validation of the Tetrasensor Honey for the Screening of Tetracyclines in Honey (abstract), Proceedings 39th Apimondia International Apicultural Congress” te Dublin (Ireland) 21-26 August 2005: p 34. Beaune P., Diserens J.M., Reybroeck W. 2005. Proficiency Testing of Charm II Tests for Residue Control of Honey (abstract), Proceedings 39th Apimondia International Apicultural Congress” te Dublin (Ireland) 21-26 August 2005: p 33-34. Daeseleire E., Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS (abstract). Participants Book /Abstracts At the forefront of agri-food innovations, 2006 CIFST/AAFC Joint Conference, Montreal, Canada, 28-30 May 2006: p. 185 (poster 62). Reybroeck W., Ooghe S. 2006. Validation of the TwinSensor Milk for rapid screening of -lactams and tetracyclines in milk (abstract). Abstract Book 5th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, May 16-19 2006: 26. Daeseleire E., Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS (abstract). Abstract Book 5th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, May 16-19 2006: 79. Reybroeck W., Ooghe S., Daeseleire E. 2006. Presence of antibiotics and sulfonamides in honey and royal jelly on the European market (abstract). Proceedings of the Second European Conference of Apidology EurBee 2006, Prague, Czech Republic, 10-14


September 2006. Edited by Vesely V., Vorechovska M. and Titĕra D. ISBN 80-903442-5-9: 117. Daeseleire E., Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS (abstract). Proceedings of the Second European Conference of Apidology EurBee 2006, Prague, Czech Republic, 10-14 September 2006. Edited by Vesely V., Vorechovska M. and Titĕra D. ISBN 80-903442-5-9: 118-119. Reybroeck W. 2007.The rapid detection of antibiotic residues in food products. Proceedings Rapid methods Europe 2007, Noordwijkerhout (the Netherlands), 29-30 januari 2007: 63-65. Reybroeck W. 2007.The control of milk on the presence of antimicrobials: the value of screening tests and the Belgian approach. Proceedings 5th Fresenius conference „Contaminants and residues in food‟ at Cologne (Germany): May 3rd 2007.

Reybroeck W., Daeseleire E., Jacobs F. 2007.Residue formation of sulfonamides in honey by migration from contaminated beeswax (abstract), Apimondia Programme & Abstracts, 40th Apimondia International Apicultural Congress” te Melbourne (Australië) 9-13 September 2007: 206. Reybroeck W., Daeseleire E., Jacobs F. 2008. Can sulfa-contaminated beeswax lead to residues in honey? Residues of Veterinary Drugs in Food, Proceedings of the EuroResidue VI Conference, Egmond aan Zee (the Netherlands) edited by Van Ginkel, L.A. & Bergwerff, A.A., ISBN 978-90-804925-3-0: 43-45. Reybroeck W., Ooghe S. 2008. Validation of the CHARM MRL-3 for fast screening of β-lactam antibiotics in raw milk. Residues of Veterinary Drugs in Food, Proceedings of the EuroResidue VI Conference, Egmond aan Zee (the Netherlands) edited by Van Ginkel, L.A. & Bergwerff, A.A., ISBN 978-90-804925-3-0: 787-792. Reybroeck W., Ooghe S. 2008. Validation of the BETA-S.T.A.R. 1+1 for fast screening of raw milk on the presence of β-lactam antibiotics. Residues of Veterinary Drugs in Food, Proceedings of the EuroResidue VI Conference, Egmond aan Zee (the Netherlands) edited by Van Ginkel, L.A. & Bergwerff, A.A., ISBN 978-90-804925-3-0: 793-797. De Reu K., Renders K., Maertens G., Messens W., Reybroeck W., Ooghe S., Herman L., Daeseleire E. 2009. A market study on the quality of eggs from different housing systems. XIXth European Symposium on the Quality of Poultry Meat & XIIIth Symposium on the Quality of Eggs and Egg Products, Turku, Finland, 21-25 June 2009, Book of abstracts: 65. Full paper op XIXth European Symposium on the Quality of Poultry Meat & XIIIth Symposium on the Quality of Eggs and Egg Products, Turku, Finland, 21-25 June 2009, Proceedings Eggmeat 2009 – category Posters: 7p. Reybroeck W., Ooghe S. 2008. Validation of Eclipse 50 and Delvotest Accelerator for screening of inhibitors in milk. Abstract book 6th International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent, Belgium, June 1-4, 2010: 22. Reybroeck W., Ooghe S. 2008. Validation of TwinSensor Milk (BT00660+) and TwinExpress Milk (BT00760) for rapid screening of -lactams and tetracyclines in milk. Abstract book 6th International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent, Belgium, June 1-4, 2010: 195. Reybroeck W. Screening antibioticaresiduen: mogelijkheden en beperkingen (abstract). AOAC Low Lands Symposium „Antibiotica en alternatieven: zijn we op de goede weg….?, Breda, Nederland, 25 maart 2010.


Reybroeck W., Ooghe S. 2010. Presence of antibiotics and chemotherapeutics in honey on the European market: situation in 2009. Proceedings of the 4th European Conference of Apidology. Kence M; (ed.), EurBee 2010, September, 7-9, 2010, Ankara, Turkey: 148-149. Reybroeck W., Ooghe S. 2010. Detection of sulfa drugs in honey by Sulfa-Sensor Honey and Charm II Sulfa Test Honey: a comparison. Proceedings of the 4th European Conference of Apidology. Kence M; (ed.), EurBee 2010, September, 7-9, 2010, Ankara, Turkey: 148. Reybroeck W., Jacobs F., Daeseleire E. 2010. Migration of residues of chloramphenicol from contaminated beeswax foundations to honey. Proceedings of the 4th European Conference of Apidology. Kence M; (ed.), EurBee 2010, September, 7-9, 2010, Ankara, Turkey: 78-79. B WETENSCHAPPELIJKE ACTIVITEITEN B.1 Organisatie van een internationaal symposium Organisatie van het Symposium BijenBenelux te Melle op 21 december 2005. B.2 Internationale congressen en symposia B.2.1 Voordrachten op internationale congressen en symposia op uitnodiging van de organisatoren "De ATP-F test voor de kiemgetalbepaling in rauwe melk" te Antwerpen op 18 mei 1989 op een studiedag "Rapid Microbial Quality Control in the Dairy Industry" ingericht door Lumac B.V. (Nederland). "Toepassingen van ATP-bepalingen" te Wageningen (Nederland) op 23 juni 1993 op een symposium omtrent toepassingen van snelle methoden, georganiseerd door EFFI (Training, Research and Control in Food-Industry). "Rapid estimation of the bacteriological quality of raw milk: comparison of the BactoFoss and an improved filtration ATP-method" te Leatherhead (Engeland) op 8 maart 1994 op een studiedag ingericht op het Leatherhead Food Research Association. "Estimation rapide de la qualité bactériologique du lait cru par une nouvelle méthode de dosage de l'ATP (Lumac Raw Milk Microbial kit)" te Parijs (Frankrijk) op 31 maart 1994 op een studiedag omtrent "Contrôle rapide d'hygiène en industries laitières" op het E.N.S.I.A.A.. "Raw milk testing with ATP measurement" op 22 juni 1995 te Oud-Turnhout op een Benelux Dairy Seminar georganiseerd door Perstorp Analytical (Nederland). "Modern methods for bacteriological quality control of raw milk", introductory paper (invited) te Wolfpassing (Oostenrijk) op 15 maart 1996 op het I.D.F.-Symposium "Bacteriological Quality of Raw Milk". "ATP monitoring for microbes and antibiotics in milk", main dairy presentation (invited) te Londen (Engeland) op 25 juni 1996 op "ATP96: An International Symposium on Industrial Applications of Bioluminescence in Microbiology", georganiseerd door Cara Technology Ltd. “Role of the Farmer in Preventing Residues of Antibiotics in Farm Milk”, conference on Quality management at farm level”, IDF World Dairy Summit & Centenary te Brugge op 11 september 2003.


“Rapid screening for residues of antibiotics in milk at the factory”, tijdens UBISI 2004, IDF/FAO/OIE International Symposium on Dairy Safety and Hygiene. A Farm-to-table Approach for Emerging and Developed Dairy Countries te Cape Town (Zuid-Afrika) op 5 maart 2004. “Validation of the CMT-Copan Milk Test for the detection of residues of antibiotics and sulphonamides in milk” voor het I.D.F. Joint Action Team “Veterinary Residues” op een workshop i.v.m. de CMT test te Brescia (Italië) op 23 april 2004. “Validation of the CMT-Copan Milk Test and the C-Scan for the screening of residues of antibiotics in milk” tijdens IDF-ISO Analytical Week te Magaliesburg (Zuid-Afrika) op 22 mei 2005. “Detection of residues of antibiotics in foodstuffs of animal origin: the Belgian approach (milk, meat, eggs, honey)” op The Academic Intercourse and Symposium on Food Antibiotic Residues Detection of China and EU te Beijing (China) op 30 november 2005. “Possibilities and limitations of screening tests for the detection of antimicrobials in milk – case study „Milk‟ ” op de Workshop: veterinary drug residues and farm to fork approach. Platform for Scientific Concertation: Food Safety at Marloie, November 24th 2006. “The rapid detection of antibiotic residues in food products” Rapid methods Europe 2007 te Noordwijkerhout (Nederland) op 30 januari 2007. “The control of milk on the presence of antimicrobials: the value of screening tests and the Belgian approach”, 5th Fresenius conference „Contaminants and residues in food‟ at Cologne (Germany), May 3rd 2007. “The use of microbiological, immunological and receptor tests for monitoring of residues of antimicrobials in milk: the Belgian approach “ (invited), IDF-ISO symposium on „Advances in Analytical Technology‟ during IDF/ISO Analytical Week at Münich (Germany), May 23rd 2007. “Residuen in honing” op een studiedag voor honingkeurmeesters te Eerbeek, Nederland op 27 september 2008. “Screening antibioticaresiduen: mogelijkheden en beperkingen” AOAC Low Lands Symposium „Antibiotica en alternatieven: zijn we op de goede weg….?, Breda, Nederland, 25 maart 2010. “EU Residue controls (general aspects, MRLs, National Residue Control Plans)” Workshop on EU residue controls (veterinary medicines) Organised by European Commission (TAIEX) in co-operation with the Ministry of Agriculture. Tbilisi, Georgia, November 5, 2010. “Case study – residue control for honey” Workshop on EU residue controls (veterinary medicines) Organised by European Commission (TAIEX) in co-operation with the Ministry of Agriculture. Tbilisi, Georgia, November 5, 2010. B.2.2 Voordrachten op internationale congressen en symposia "Evaluation of screening tests for the detection of antimicrobial residues in milk" te Kiel (Duitsland) op 30 augustus 1995 op het I.D.F.- Symposium "Residues of Antimicrobial Drugs and other Inhibitors in milk".


“Evaluation of the applicability of fast screening tests for the detection of residues of antibiotics in milk”, 4th International Symposium on Hormone and Veterinary Drug Residue Analysis te Antwerpen op 5 juni 2002. “Residues of antibiotics and sulphonamides in honey on the Belgian market” te Celle (Duitsland) op 10 oktober 2002 op Apimondia Symposium “Preventing Residues in Honey”. “Sulpha drugs in honey and other bee products” op het Apimondia symposium 2004 “Prevention of Residues in Honey 2” te Celle (Duitsland) op 27 april 2004. “Residuen van antibiotica en sulfonamiden in honing op de Belgische markt” op BijenBenelux te Wageningen (Nederland) op 25 november 2004. “Validation of the Tetrasensor Honey for the screening of tetracyclines in honey” op het 39th Apimondia International Apicultural Congress” te Dublin (Ierland) op 22 augustus 2005. “Kwaliteitsbepaling van Vlaamse honing” op Symposium BijenBenelux te Melle op 21 december 2005. “Validatie van de TetraSensor Honey voor de screening van tetracyclines in honing” op Symposium BijenBenelux te Melle op 21 december 2005. “Recent residuonderzoek” op Symposium BijenBenelux te Melle op 21 december 2005. “Validation of the TwinSensor Milk for rapid screening of -lactams and tetracyclines in milk”, 5th International Symposium on Hormone and Veterinary Drug Residue Analysis te Antwerpen op 19 mei 2006. “Presence of antibiotics and sulfonamides in honey and royal jelly on the European market”, EurBee 2006, Second European Conference of Apidology te Praag, Tsjechië, 12 september 2006. “Levels of residues in consumption honey samples in 5 different European countries. Lezing voor de International Honey Commission te Praag, Tsjechië, 15 september 2006. “Residuen van antibiotica en sulfonamiden in honing op de markt in 5 Europese landen” op het BijenBenelux symposium te Gorsem op 17 januari 2007. “Carry-over naar honing van sulfonamiden uit gecontamineerde bijenwas” op het BijenBenelux symposium te Gorsem (PC Fruit vzw) op 17 januari 2007. “Residue formation of sulfonamides in honey by migration from contaminated beeswax”, 40th Apimondia International Apicultural Congress at Melbourne (Australia), September 11, 2007. “Migration of sulfonamides: from contaminated beeswax to honey & from winter feed to beeswax” op Bee Benelux 2008, KUL te Leuven op 8 januari 2008. “Can sulfa-contaminated beeswax lead to residues in honey?”, EuroResidue VI at Egmond aan Zee (the Netherlands), May 19th 2008. “Validation of Eclipse 50 and Delvotest Accelerator for screening of inhibitors in milk”, 6th International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent, Belgium, June 3rd 2010.


B.2.3 Voordrachten op internationale workshops en bijeenkomsten "Fast detection of antibiotics in milk. Results of the evaluation of LacTek Milk Screening kits" te Antwerpen op 14 november 1990 op een informatienamiddag van Novo Nordisk (Denemarken) i.v.m. "LacTek for fast detection of antibiotics in milk" (op uitnodiging). "The Lumac Raw Milk Microbial Kit for fast screening of the bacteriological quality of raw milk" te Bradford-on-Avon, Wiltshire (Engeland) op 15 november 1994 op een technische studiedag ingericht door St.Ivel Dairy Group (op uitnodiging). “Control of milk on the presence of residues of antibiotics and chemotherapeutics” te Amsterdam op 15 mei 2002 op een bijeenkomst van de Europese vertegenwoordigers, georganiseerd door Idexx Europe B.V. (op uitnodiging). “Toepasbaarheid van snelle screeningstesten voor de detectie van antibioticaresiduen in melk”, Workshop voor de Beneluxvertegenwoordigers van Pharmacia Animal Health te Melle op 2 juli 2002 (op uitnodiging). “Control of milk on the presence of residues of antibiotics and chemotherapeutics”, press meeting met landbouwpers uit het Verenigd Koninkrijk en Ierland, georganiseerd door Garnett Keeler (Surrey, UK) en Pharmacia Animal Health te Melle op 8 november 2002 (op uitnodiging). “Control of milk on the presence of residues of antibiotics and chemotherapeutics”, press meeting met landbouwpers uit Frankrijk, georganiseerd door Garnett Keeler (Surrey, UK) en Pharmacia Animal Health te Melle op 14 november 2002 (op uitnodiging). “Control of milk on the presence of residues of antibiotics and chemotherapeutics”, press meeting met landbouwpers uit Italië, georganiseerd door Garnett Keeler (Surrey, UK) en Pharmacia Animal Health te Melle op 20 november 2002 (op uitnodiging). “Control of milk on the presence of residues of antibiotics and chemotherapeutics”, press meeting met landbouwpers uit de Benelux, georganiseerd door Garnett Keeler (Surrey, UK) en Pharmacia Animal Health te Melle op 21 november 2002 (op uitnodiging). “Screening of milk on antimicrobials: methods available and the Belgian approach”, meeting georganiseerd door Pharmacia Animal Health voor veeartsen uit Oost-Europa te Brugge op 18 maart 2003 (op uitnodiging). “Screening of meat and eggs on the presence of antimicrobials: the Belgian approach”, voordracht voor een groep veeartsen uit Brazilië gespecialiseerd in pluimvee te Melle op 12 mei 2003 (op uitnodiging). “Honey legislation”, lezing gehouden ter gelegenheid van het bezoek van dhr. Girma Birru, Minister van Handel en Industry en dhr. Brook Debebe, Deputy Chief of Mission van de Democratische Republiek van Ethiopië te Melle op 20 januari 2005 (op uitnodiging). “Screening of meat and eggs on the presence of antimicrobials – The Belgian approach”, lezingen op uitnodiging van Laboratórios Pfizer Ltda. (Sâo Paulo, Brazilië) bij de Braziliaanse vleesproducenten Seara Cargill te Itajai (Santa Catarina) op 3 juli 2006, bij Perdigão Agroindustrial S.A. te Videira (Santa Catarina) op 4 juli 2006, bij Frangosul te Salvador Do Sul (Rio Grande do Sul) op 5 juli 2006, bij Grupo Avipal te Porto Alegre (Rio Grande do Sul)op 6 juli 2006 en bij Penasul Alimentos te Garibaldi (Rio Grande do Sul) op 7 juli 2006 (op uitnodiging).


“Quality control of Flemish honey, honey legislation & validation of the Tetrasensor Honey” in het kader van het bezoek van Universiteit Wageningen – Expertise Centre for Chain and Network Studies en een groep imkers uit Bosnië-Herzegovina in het kader van Supply Chain Course te Melle op 20 november 2006 (op uitnodiging). “European legislation regarding residues of veterinary drugs in honey”, International Honey Commission, Melbourne (Australia), September 10, 2007.

“The testing of antimicrobials in milk: legislation, analytical possibilities and the Belgian approach”, Dairy Antibiotics Training Seminar, Chr. Hansen & Neogen, October 11, 2007, Hørsholm, Denemark (op uitnodiging).

“Validation of the βeta-s.t.a.r. 1+1”, Dairy Antibiotics Training Seminar, Chr. Hansen & Neogen, October 11, 2007, Hørsholm, Denemark (op uitnodiging).

“Residuen van diergeneesmiddelen in honing”, op vraag van de Nederlandse Bijenhoudersvereniging-NBV te Bunnik ( Nederland) op 14 december 2007 (op uitnodiging). “The screening of antimicrobials in milk” op vraag van Schering-Plough te Parijs, Frankrijk op 24 april 2009 (op uitnodiging). “The detection of cefalonium in milk by different microbiological and receptor tests” op vraag van Schering-Plough te Parijs, Frankrijk op 24 april 2009 (op uitnodiging). “Residues of antibiotics and sulfonamides in honey”. Conffidence, Liège op 29 juni 2009 (invited). “Screening of antimicrobials in honey – new possibilities”, Technische workshop georganiseerd door Randox Laboratories tijdens 41st Apimondia Congress in Montpellier, France op 17 september 2009. “Validation of βeta-s.t.a.r. 1+1”, lezing voor kitontwikkelaars bij Neogen Corporation in Lansing, Michigan, USA op 12 oktober 2009 (op uitnodiging). “Residues of anti-infectious agents in food of animal origin”, lezing op Scientific Review Council Meeting bij Neogen Corporation in Lansing, Michigan, USA op 13 oktober 2009 (op uitnodiging). “Residues of antibiotics in dairy milk. Current situation and perspectives for the future”, lezing op workshop georganiseerd door Intervet-Schering-Plough in Santiago de Compostela, Spanje op 6 april 2010 (invited). “Residues of antibiotics in dairy milk. Current situation and perspectives for the future”, lezing op workshop georganiseerd door Intervet-Schering-Plough in Gijón, Spanje op 7 april 2010 (invited). “Residues of antibiotics in dairy milk. Analysis of the problem from the lab point of view”, lezing op workshop georganiseerd door Intervet-Schering-Plough in Madrid, Spanje op 8 april 2010 (invited).


B.2.4 Postermededelingen op internationale congressen en symposia Reybroeck W., Schram E. 1992. Study of parameters involved in the assessment of the bacteriological quality of raw milk by two bioluminescent ATP assays. "A Symposium on ATP Rapid Microbiology for the Food and Beverage Industries", Cambridge, England, 17 juni 1992. Reybroeck W., Waes G. 1992. Comparison of two ATP test kits for the assessment of the bacteriological quality of raw milk. "A Symposium on ATP Rapid Microbiology for the Food and Beverage Industries", Cambridge, England, 17 juni 1992. Reybroeck W., Schram E. 1993. New improved ATP filtration method for the assessment of the bacteriological quality of raw milk. "7th International Congress on Rapid Methods and Automation in Microbiology and Immunology", London, England, 12-15 september 1993. Reybroeck W., Schram E. 1994. New improved ATP filtration method for the assessment of the bacteriological quality of raw milk. 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge, England, 5-8 september 1994. Reybroeck W. 1994. Comparison of two ATP bioluminescence methods for the bacterio-logical assay of raw milk. 8th International Symposium on Bioluminescence and Chemiluminescence, Cambridge, England, 5-8 september 1994. Reybroeck W., Schram E. 1994. New improved ATP filtration method for the assessment of the bacteriological quality of raw milk. "24th International Dairy Congress", Melbourne, Australia, 18-22 september 1994. . Reybroeck W. 1994. Comparison of two ATP bioluminescence methods for the bacterio-logical assay of raw milk. "24th International Dairy Congress", Melbourne, Australia, 18-22 september 1994. Reybroeck W. 1995. Sensitivity and selectivity of screening tests for the detection of antimi-crobial residues in milk. "I.D.F.- Symposium on Residues of Antimicrobial Drugs and other Inhibitors in Milk", Kiel, Germany, 28-31 augustus 1995. Reybroeck W. 1995. Field test of screening tests for the detection of antimicrobial residues in milk. "I.D.F.- Symposium on Residues of Antimicrobial Drugs and other Inhibitors in Milk", Kiel, Germany, 28-31 augustus 1995. D‟Haese E., Nelis H.J., Reybroeck W. 1997. Screening for tetracycline residues in milk based on inhibition of -galactosidase induction in E. coli. "97th General Meeting of the American Society for Microbiology", Miami Beach, USA, 4-8 mei 1997. Reybroeck W. 1997. Determination of ceftiofur in milk after intramuscular administration of Excenel to lactating cows. "World Congress on Food Hygiene. World Association of Veterinary Food Hygienists", Den Haag, Nederland, 24-29 augustus 1997. Reybroeck W. 1997. Influence of residues of ceftiofur in milk on the production of yoghurt and cheese, after application of Excenel to lactating cows. "World Congress on Food Hygiene. World Association of Veterinary Food Hygienists", Den Haag, Nederland, 24-29 augustus 1997.


D‟Haese E., Nelis H.J., Reybroeck W. 1998. Specificity of a chemiluminometric test for the detection of tetracycline residues in milk. 112th AOAC Annual Meeting, Montréal, Canada, 13-17 september 1998. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Detection of membrane disrupting antibiotics in milk and feeds by solid phase cytometry (ChemScan). 112th AOAC Annual Meeting, Montréal, Canada, 13-17 september 1998. D‟Haese E., Nelis H.J., Reybroeck W. 1998. Solid phase cytometry (ChemScan detection) as a new tool for the detection of somatic cells and antibiotic residues in milk. 25th International Dairy Congress, Arhus, Denemarken, 21-23 september 1998. Reybroeck W. 2000. Evaluation of the Parallux test for the detection of -lactam antibiotics and tetracyclines. 2nd International FoodSENSE Workshop, Zeven, Duitsland, 30 maart - 1 april 2000. Reybroeck W. 2000. Evaluation of the Beta s.t.a.r. for the detection of -lactam antibiotics. 2nd International FoodSENSE Workshop, Zeven, Duitsland, 30 maart - 1 april 2000. Reybroeck W. 2000. Milk residues of lincomycin and neomycin after a 5-fold administration of Lincocin Intramammaire to lactating cows: their technological significance. EuroResidue IV, Veldhoven, Nederland, 8-10 mei 2000. Reybroeck W. 2000. Performance of the PremiTest using naturally contaminated meat.EuroResidue IV, Veldhoven, Nederland, 8-10 mei 2000. Reybroeck W. 2000. Detection of residues of antibiotics in foodstuffs with microbiological tests using Bacillus. Bacillus2000 symposium, Brugge, 31 augustus 2000. Reybroeck W., Van Hoorde A., Jacobs F.J. 2001. Quality control of Flemish honey: results 1998-2000. Arbeitsgemeinschaft der Institute für Bienenforschung e.V, 48. Jahrestagung, Bad Neuenahr / Ahrweiler, Duitsland, 27-29 maart 2001. Van Hoorde A., Reybroeck W., Jacobs F.J. 2001. Quality control of honey. A critical analysis of useful parameters. Arbeitsgemeinschaft der Institute für Bienenforschung e.V, 48. Jahrestagung, Bad Neuenahr / Ahrweiler, Duitsland, 27-29 maart 2001. Reybroeck W. 2001. Determination of ceftiofur in milk after intramuscular administration of Excenel to lactating cows. Symposium “Kansen in Kwaliteit", Groep Geneeskunde van het Rund van de Koninklijke Nederlandse Maatschappij voor Diergeneeskunde, KNMvD, Ede, Nederland, 14 december 2001. Reybroeck W. 2001. Influence of residues of ceftiofur in milk on the production of yoghurt and cheese, after application of Excenel to lactating cows. Symposium “Kansen in Kwaliteit", Groep Geneeskunde van het Rund van de KNMvD, Ede, Nederland, 14 december 2001. Impens S., Reybroeck W., Courtheyn D., De Wasch K., Ooghe S., Smedts W., De Brabander H.F. 2002. Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC-MS2 and LC-MS2. 4th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, 4-7 juni 2002. Van Coillie E., Reybroeck W., De Block J., Van Renterghem R. 2003. Production of chicken egg yolk antibodies for the development of an immunoassay for the detection of flumequine


in milk. “Pre-summit Symposium on Innovative Research in Dairy Science and Technology”, IDF World Dairy Summit & Centenary, Brugge, 7-12 september 2003.

Van Coillie E., Reybroeck W., De Block J., Van Renterghem R. 2003. Production of chicken egg yolk antibodies for the development of an immunoassay for the detection of flumequine in milk. VIIth International Conference on Agri-Food Antibodies, Uppsala, Zweden, 10-13 september 2003. Reybroeck W., Ooghe S. 2003. Results of a multilaboratory trial regarding rapid tests for the control of milk on -lactam antibiotics. Euro Food Chem XII, “Stategies for Safe Food”, Brugge, 23-26 september 2003. Reybroeck W., Van Hoorde A., Jacobs F.J. 2003. Quality control of Flemish honey. Results 1998-2002. European beekeeping Congress. What future for European beekeeping? UCL – Socrate Auditorium, Louvain-La-Neuve, 22-23 november 2003. Reybroeck W., Ooghe S., Grijspeerdt K. 2004. First results of the validation of the Copan Milk Test for the detection of residues of antibiotics and sulphonamides in milk. UBISI 2004, IDF/FAO/OIE International Symposium on Dairy Safety and Hygiene. A Farm-to-table Approach for Emerging and Developed Dairy Countries, Cape Town, Zuid-Afrika, 2-5 maart 2004. Reybroeck W., Ooghe S. 2004. Results of a multilaboratory trial regarding rapid tests for the control of milk on -lactam antibiotics. UBISI 2004, IDF/FAO/OIE International Symposium on Dairy Safety and Hygiene. A Farm-to-table Approach for Emerging and Developed Dairy Countries, Cape Town, Zuid-Afrika, 2-5 maart 2004. Reybroeck W.,, Van Hoorde A., Jacobs F.J. 2004. Residues of sulphonamides in Flemish Honey – Results 2003”. 51e Jahrestagung der Arbeitsgemeinschaft der Bieneninstitute, Haus Düsse, 23-25 maart 2004. Reybroeck W., Daeseleire E. ,Ooghe S., Jacobs F. 2004. Sulpha drugs in honey and other bee products. Apimondia symposium 2004 “Prevention of Residues in Honey 2”, Celle, Duitsland, 27-28 april 2004. Van Hoof N., De Wasch K., Okerman L., Reybroeck W., Poelmans S., Noppe H., De Brabander H.F. 2004. Quantification of 8 quinolones in bovine muscle using LC-MS2. EuroResidue V, Conference on Residues of Veterinary Drugs in Food, Noordwijkerhout, Nederland, 10-12 mei 2004. Reybroeck W., Ooghe S. 2004. Validation of the Copan Milk Test for the detection of residues of antibiotics and sulphonamides in milk and milk products. EuroResidue V, Conference on Residues of Veterinary Drugs in Food, Noordwijkerhout, Nederland, 10-12 mei 2004. Daeseleire E. , Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS. “At the forefront of agri-food innovations”, 2006 CIFST/AAFC Joint Conference, Montreal, Canada, 28-30 May 2006. Daeseleire E. , Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS. 5th International Symposium on Hormone and Veterinary Drug Residue Analysis, Antwerp, May 16-19, 2006.


Reybroeck W., Ooghe S., Daeseleire E. 2006. Presence of antibiotics and sulfonamides in honey and royal jelly on the European market. Second European Conference of Apidology EurBee 2006, Prague, Czech Republic, 10-14 September 2006. Daeseleire E., Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS., Second European Conference of Apidology EurBee 2006, Prague, Czech Republic, 10-14 September 2006. Reybroeck W., Ooghe S. 2008. Validation of the CHARM MRL-3 for fast screening of β-lactam antibiotics in raw milk. EuroResidue VI Conference, Egmond aan Zee, Nederland, 19-21 mei 2008. Reybroeck W., Ooghe S. 2008. Validation of the BETA-S.T.A.R. 1+1 for fast screening of raw milk on the presence of β-lactam antibiotics. EuroResidue VI Conference, Egmond aan Zee, Nederland, 19-21 mei 2008. Platteau C., Bridts C., Reybroeck W., De Loose M., Devreese B., Daeseleire E., Ebo D. 2008. Comparison of three different extraction methods for the isolation of hazelnut proteins. 3rd International Symposium on Molecular Allergology, Parma, Italy, 26-29 May 2008. De Reu K., Renders K., Maertens G., Messens W., Reybroeck W., Ooghe S., Herman L. en Daeseleire E. 2009. A market study on the quality of eggs from different housing systems. XIXth European Symposium on the Quality of Poultry Meat & XIIIth Symposium on the Quality of Eggs and Egg Products, Turku, Finland, 21-25 June 2009. Reybroeck W., Ooghe S. 2010. Validation of TwinSensor Milk (BT00660+) and TwinExpress Milk (BT00760) for rapid screening of -lactams and tetracyclines in milk. 6th International Symposium on Hormone and Veterinary Drug Residue Analysis, Ghent, Belgium, 1-4 June 2010. Reybroeck W., Ooghe S. 2010. Presence of antibiotics and chemotherapeutics in honey on the European market: situation in 2009. 4th European Conference of Apidology, EurBee 2010, September, 7-9, 2010, Ankara, Turkey. Reybroeck W., Ooghe S. 2010. Detection of sulfa drugs in honey by Sulfa-Sensor Honey and Charm II Sulfa Test Honey: a comparison. 4th European Conference of Apidology, EurBee 2010, September, 7-9, 2010, Ankara, Turkey. B.3 Nationale congressen, symposia en workshops B.3.1 Voordrachten op nationale congressen, symposia en workshops op uitnodiging van de organisatoren "Demonstratie ChemFlow methode voor snelle detectie van gisten en schimmels" in samenwerking met Chemunex (Nederland) te Melle op 25 oktober 1990. "Celgetalbepaling met het Fossomatic en het Somascopetoestel" te Gent op 24 januari 1994 op een informatievergadering ingericht op de Faculteit Diergeneeskunde in het kader van een I.W.O.N.L. - project. "Beter imkeren vanuit raszuivere bijen" te Gent op 10 september 1994 op het 11e Vlaams Imkerscongres, georganiseerd door de Koninklijke Vlaamse Imkersbond.


"Snelle microbiologische controlesystemen" te Gent op 25 november 1994 op de 29e Zuivelstudiedag met als thema: "Zuivelrichtlijn 92/46 en H.A.C.C.P.: waarborgen voor hygiënisch produceren", georganiseerd door het A.I.V.C. en Hogeschool Gent - C.T.L.. "Werking, instelling en borging van de BactoScan 8000" op 28 oktober 1996 te Lier op de Workshop BactoScan 8000. "Opsporing van residuen van antibiotica en chemotherapeutica in melk: een geïntegreerde benadering" te Merelbeke op 3 oktober 1997 op een studiemiddag georganiseerd door de Vlaamse Vereniging voor Buiatrie. "Residu- en resistentieproblematiek bij bijenziektebestrijding" te Gent op 18 oktober 1997 op een studienamiddag omtrent Varroa-bestrijding bij honingbijen, georganiseerd door Studiekring Oost-Vlaanderen, KOIV. "Kwaliteit van honing. Honinganalyses" te Merelbeke op 9 januari 1999 op een studienamiddag met als thema "Nectar- Van bloem tot honing" georganiseerd door het CLO, Departement Gewasbescherming en het Vlaams Vulgarisatiecentrum voor Bijenteelt in het kader van Agriflora. "Immunologische en microbiële screening van diergeneesmiddelen" te Tervuren op 24 september 1999 op een KVCV (Koninklijke Vlaamse Chemische Vereniging)-studiedag omtrent (bio)chemische sensoren. "Alternatieve technieken voor zuivelcontrole" te Melle op 2 maart 2000 op een KVCV (Koninklijke Vlaamse Chemische Vereniging-Sectie Voeding)-studiedag omtrent snellere methoden voor kwaliteitsbepaling van levensmiddelen: microbiologische methoden. “Resultaten honinganalyses (‟98 & ‟99) in het kader van het Vlaams honingproject” te Gent (RUG) op 6 juni 2000 op Colloquium “Honing”. “Kwaliteitsbepaling van honing binnen de huidige wetgeving” op het K.V.I.B. Symposium “Vlaamse Honing” te Gent op 23 juni 2001. “Analysemethoden voor het remstoffenonderzoek in de melk”, contactnamiddag ”Remstoffenonderzoek in de melk” georganiseerd door de Vereniging voor de Melkkwaliteit te Lier op 17 april 2002. “Screening van residuen van diergeneesmiddelen in eieren”, studienamiddag “Residu-problematiek in de pluimveevoeding” georganiseerd door The World Poultry Science Association –Belgium v.z.w. te Gontrode op 30 april 2002. “Residuen van antibiotica en chemotherapeutica in honing” op het “Symposium Honingkwaliteit” georganiseerd door het Informatiecentrum voor Bijenteelt te Gent op 13 december 2003. “Opsporen van antibioticaresiduen in vlees” studienamiddag georganiseerd door GOM Oost-Vlaanderen te Melle op 28 november 2003. “Belang van het Voedselagentschap en de honingkwaliteit”. 16de Vlaams Imkerscongres te Gent op 11 september 2004. “Voorstelling Sectorgids voor goede imkerspraktijken” op een mini-symposium te Gent op 29 januari 2006.


“Validatie van screeningstesten”, Studiedag ivm methodenvalidatie georganiseerd door het nationaal referentielaboratorium, Brussel, 9 juni 2006. “De sectorgids, leidraad tot het afleveren van kwaliteitshoning.” Symposium „Uitdagingen voor de imker in 2006‟ georganiseerd door de Koninklijke Oost-Vlaamse Imkersvereniging op 18 juni 2006 te Wetteren. “Screening of antimicrobials in milk and meat: survey of available tests”. Meeting of the laboratories in the framework of the 96/23 at FAVV in Brussels on December 1st 2006. “Kwaliteit van Vlaamse honing” op het symposium „Kwaliteitsaspecten van honing‟ te Gent op 29 juni 2008. “Kristallisatie van honing: hoe gebreken voorkomen?” op het symposium „Kwaliteitsaspecten van honing‟ te Gent op 29 juni 2008. “Legislation of veterinary drug residues”, workshop “The use of LC-MS/MS for the determination of residues of veterinary drugs in milk”, organisatie van de Nationale Referentie Laboratoria Melk en Melkproducten te Brussel op 5 mei 2009. “Validation of Eclipse 50 for screening of inhibitors in milk”, workshop “Validation of new microbiological tests for screening of antimicrobial residues in milk”, organisatie van de Nationale Referentie Laboratoria Melk en Melkproducten te Brussel op 22 juni 2010. “Validation of Delvotest Accelerator for screening of inhibitors in milk”, workshop “Validation of new microbiological tests for screening of antimicrobial residues in milk”, organisatie van de Nationale Referentie Laboratoria Melk en Melkproducten te Brussel op 22 juni 2010. “Kristallisatie van honing: praktische tips voor het bekomen van kwaliteitshoning” op het symposium „Kwaliteitshoning in 2010‟ georganiseerd door de Kon. Oost-Vlaamse Imkersvereniging‟ te Gent op 27 juni 2010. “Primaire productie bijenteelt: wat te verwachten bij controle door het voedselagentschap?” op het symposium „Kwaliteitshoning in 2010‟ georganiseerd door de Kon. Oost-Vlaamse Imkersvereniging‟ te Gent op 27 juni 2010. B.3.2 Voordrachten op nationale congressen, symposia en workshops “Uitvoering en borging kiemgetalbepaling van rauwe melk” op 22 februari 2001 te Melle op de Workshop “Totaal Kiemgetal”. “Snelle screeningstesten voor de detectie van antibioticaresiduen in melk”, Workshop voor zuivelbedrijven en ambtenaren van FAVV en FOD te Melle op 7 november 2002. “Remstoffenonderzoek van melk”, “SNAP Beta-Lactam Test” en “Delvotest SP” op de workshop “SNAP Beta-Lactam Test & Delvotest SP” georganiseerd in het kader van de wetenschappelijke begeleiding van de Belgische zuivelindustrie bij de autocontrole op antibioticaresiduen, te Melle op 18 december 2002. “Remstoffenonderzoek van melk”, “Parallux” en “Delvotest SP” op de workshop “SNAP Beta-Lactam Test & Delvotest SP” georganiseerd in het kader van de wetenschappelijke begeleiding van de Belgische zuivelindustrie bij de autocontrole op antibioticaresiduen, te Melle op 22 januari 2003.


“Remstoffenonderzoek van melk”, “eta s.t.a.r.”, “Charm MRL -Lactam Test (ROSA)” en “Delvotest SP” op de workshop “SNAP Beta-Lactam Test & Delvotest SP” georganiseerd in het kader van de wetenschappelijke begeleiding van de Belgische zuivelindustrie bij de autocontrole op antibioticaresiduen, te Melle op 27 januari 2003. “Remstoffenonderzoek van melk”, “Charm MRL -Lactam Test (ROSA)” en “Delvotest SP” op de workshop “SNAP Beta-Lactam Test & Delvotest SP” georganiseerd in het kader van de wetenschappelijke begeleiding van de Belgische zuivelindustrie bij de autocontrole op antibioticaresiduen, te Melle op 11 februari 2003. “Controle van antibiotica in hoevemelk: gisteren en vandaag”. Voordracht in het kader van opleiding door Nationale Referentie Laboratoria – Melk en Zuivelproducten in samenwerking met het FAVV te Brussel op 11 mei 2007. “Evaluatie van nieuwe sneltesten voor de detectie van antibiotica in melk” te Merelbeke op 22 juni 2007 in de sessie „Meettechniek en analysestrategieën‟ op de themadag Eenheid Technologie & Voeding ter gelegenheid van 75 jaar Overheidslandbouw- en visserijonderzoek. B.3.3 Postermededelingen op nationale congressen, symposia en workshops Reybroeck W. 2001. Detection of residues of antibiotics in foodstuffs with microbiological tests using Bacillus. Epidemiology & Economics (VEE), Melle, 25 oktober 2001: 90-93. Daeseleire E., Reybroeck W. 2006. Determination of sulpha drugs in honey by LC-MS/MS. Workshop: veterinary drug residues and farm to fork approach. Platform for Scientific Concertation: Food Safety at Marloie, 24 november 2006. Reybroeck W., Ooghe S., Daeseleire E. 2006. Presence of antibiotics and sulfonamides in honey and royal jelly on the European market. Workshop: veterinary drug residues and farm to fork approach. Platform for Scientific Concertation: Food Safety at Marloie, 24 november 2006. B.3.4 Lezingen aan universiteiten, wetenschappelijke en officiële instellingen “Screening van residuen van diergeneesmiddelen in melk. Welke verbindingen kunnen worden opgespoord? Welke testen zijn daartoe beschikbaar?” Lezing in het kader van het Post Universitair Onderwijs Diergeneeskunde omtrent “Residuen in de melk, oorzaken en gevolgen”, Ugent, Faculteit Diergeneeskunde, Merelbeke, 21 december 2001. “Detection des résidus d‟antibiotiques dans les denrées alimentaires d‟origine animale: l‟approche belge”, seminarie in het kader van DES - Sciences d‟Alimentation op de Faculté Vétérinaire de Université de Liège op 4 april 2006. Workshop „Biochemische en enzymatische toepassingen‟ te Melle voor studenten industrieel ingenieurs van de HoGent-BIOT op 21 april 2006. “Honey, quality of honey residues in honey. Pollen trapping.” Les aan UGent, Faculteit Wetenschappen, Gent in het kader van de cursus “Beekeeping for poverty alleviation” op 2 juni 2006, 19 juni 2007, 13 juni 2008, 22 april en 3 juli 2009, 26 april en 1 juli 2010.


Workshop “Quality control of honey” gegeven aan studenten van de cursus “Beekeeping for poverty alleviation” te Melle op 6 juni 2006, 20 juni 2007, 17 juni 2008, 29 en 30 april 2009, 10 en 16 juni 2010. “Screening en groepsspecificatie van antibioticaresiduen in melk”. Les voor studenten industrieel ingenieurs van de HoGent-BIOT te Melle op 4 december 2007. “Détection des résidus d‟antibiotiques dans le lait” Dag opleiding, gegeven te Luxembourg, Groot-Hertogdom Luxemburg op vraag van Ministère de la Function Publique et de la Réforme Administrative, Institut national d‟administration publique op 22 september 2009. C. MAATSCHAPPELIJKE DIENSTVERLENING C.1 Rapporten opgesteld voor de overheid, het Wetenschappelijk Comité (FAVV), het FAVV en andere instellingen Reybroeck W. 1994. Uitwerking en het uitschrijven van de analysemethode voor het opsporen van remstoffen en sulfonamiden in melk voor de officiële bepaling van de kwaliteit van melk geleverd aan kopers (Ministerieel Besluit van 14 oktober 1994, Belgisch Staatsblad van 2 december 1994: 29868-29876. Reybroeck W., Dierick K., Degroodt J., Herman L. 2001. Residuen van antibiotica en sulfonamiden in honing. Dossier opgemaakt in opdracht van het wetenschappelijk comité van het federaal agentschap voor de veiligheid van de voedselketen. Advies Wetenschappelijk Comité FAVV 2001/11. www.FAVV.be/nl/structure/01-12-19%20 WC honing.pdf: 1-14. Dehareng F., Genard O., Grijspeerdt K., Havelange M., Reybroeck W., Romnée J.-M., Van Crombrugge J.-M., Van Royen G., Veselko D. 2003. Rapport d‟utilisation du DelvoScan: Système de lecture automatique des plaques de détection des substances inhibitrices. Gembloux-Belgique, CRA-W. Rapport, (Fr-Nl): 1-12. Herman L., Reybroeck W. 2003. Streptomycineresiduen in honing door het gebruik van het product Fructocin op appelaars en perelaars. Advies Wetenschappelijk Comité FAVV 2003/07: 1-2. http://www.favv-afsca.fgov.be/portal/page?_pageid=34,65288&_dad=portal&_ schema=PORTAL#07. Van Royen G., Reybroeck W., Dehareng F., Romnée J.M. 2004. Opsporen van melkvreemde remstoffen in melk met de Delvotest MCS, Rapport voor het Wetenschappelijk Comité van het FAVV: 1-11. Reybroeck W., Ooghe S., Grijspeerdt K. 2005. Evaluatie van de Copan Milk Test voor de opsporing van bacteriegroeiremmende stoffen in melk. Rapport voor het Wetenschappelijk Comité van het FAVV: 1-17. Reybroeck W., Ooghe S. 2006. Gebruik van sneltesten als bevestigingstest bij de opsporing van bacteriegroeiremmende stoffen in melk in het kader van de officiële kwaliteitsbepaling van rauwe melk. Rapport voor het Wetenschappelijk Comité van het FAVV: 1-22. Reybroeck W. 2008. Final report on laboratory results (residues and contaminants). Taiex Mission on the Inspection of honey, 25/04/2008 – 02/05/2008, Nicosia, Cyprus, Rapport opgesteld voor de Europese Commissie, TAIEX: 1-11 (2008).

http://www.favv.be/nl/structure/01-12-19


Reybroeck W. 2008. IDF Observer‟s Report on Ad Hoc Intergovernmental Task Force on Antimicrobial Resistance.Intersession Working Group Meeting on Risk Management, 29 May and 30 May 2008, A. Borschette Conference Centre, Brussels, Belgium. Rapport opgesteld voor de I.D.F.: 1-10. Reybroeck W., Ooghe S. 2010. Validatie van Premi-test na solventextractie. Rapport voor het FAVV, dienst Laboratoria. Reybroeck W., Ooghe S. 2010. Evaluation of the Delvotest Accelerator. Rapport voor het FAVV, dienst Laboratoria: 1-25. Reybroeck W., Ooghe S. 2010. Evaluation of the ECLIPSE 50. Rapport voor het FAVV, dienst Laboratoria: 1-20. C.2 Voordrachten voor sectorverenigingen e.d. C.2.1 Werkgroep voor de Melkkwaliteit en de valorisatie van melk en melkbestanddelen, Voordrachten op contactgroepvergaderingen “Kwaliteit van de rauwe melk”. “De bepaling van het totaal kiemgetal en het aantal coliforme bacteriën in rauwe melk met de Petrifilm-methode.” Melle, 30 november 1990. “Opsporen van remstoffen in melk. Evaluatie van de Lactek-methode.” Melle, 30 november 1990. “Alternatieven voor de kiemgetalbepaling. Wetenschappelijke onderbouw ATP-F test. Evaluatie Biotrace MMAK.” Melle, 5 december 1991. “Opsporen van remstoffen in melk. Evaluatie Lumac rapid Antibiotic Test kit.” Melle, 5 december 1991. “Bruikbaarheid van Foss Autosampler 205 als monsternameapparaat voor RMO.” Melle, 5 december 1991. “Evaluatie ATP-F test en Biotrace MMAK.” Melle, 27 november 1992. “DEFT-methode voor celgetalbepaling.” Melle, 27 november 1992. “Alternatieven voor de kiemgetalbepaling. Evaluatie Bactofoss (voorlopige resultaten).” Melle, 27 november 1992. “Celgetalbepaling. Evaluatie Somascope (voorlopige resultaten).” Melle, 27 november 1992. “Bepaling remstoffen (stand van zaken).” Melle, 27 november 1992. “Vergelijking raw milk Microbial Kit en Bactofoss.” Melle, 8 april 1994. “Celgetalbepaling – Evaluatie Somascope.” Melle, 8 april 1994. “Bewaarproeven standaard celgetal.” Melle, 8 april 1994. “Opsporen remstoffen. Resultaten verbeterde agardiffusietest. Stand van zaken.” Melle, 8 april 1994.


C.2.2. Voordrachten op vergaderingen contactgroep “Melk en zuivelproducten” “Evaluatie sneltesten voor ingangscontrole antibioticaresiduen op de zuivelfabriek.” Melle, 10 december 1999. “De nieuwe Delvotest MCS.” Melle, 10 december 1999. “Spectrofotometrische aflezing van de Delvotest MCS-test.” Melle, 19 januari 2001. “Evaluatie van de Charm-MRL--lactamtest (ROSA).” Melle, 19 januari 2001. “Uitscheiding van lincomycine na toepassing van Lincomycin Intramammaire.” Melle, 19 januari 2001. “Automatisering van de kleuraflezing van microbiologische inhibitortesten d.m.v. reflectometrie.” Melle, 23 mei 2003. “Evaluatie van de Copan-test voor het opsporen van remstoffen in rauwe melk.” Melle, 23 mei 2003. “Organisatie van ringonderzoeken voor de zuivelindustrie in het kader van de ingangscontrole van rauwe melk op antibiotica. Automatisering van de kleuraflezing van microbiologische inhibitortesten d.m.v. reflectometrie.” Melle, 23 mei 2003. “Validatie van de Copan Milk Test met C-Scan aflezing.” Melle, 25 november 2005. C.2.3. Voordrachten op vergaderingen contactgroep “Pluimvee” “Huidige wetgeving i.v.m. residuen van diergeneesmiddelen.” Melle, 3 juni 1999. “Immunologische opsporing van tetracyclines in eieren en kippenvlees.” Melle, 3 juni 1999. “Screening van residuen van infectiewerende stoffen in eieren – resultaten 1998.” Melle, 3 juni 1999. “Evaluatie van de Premitest voor de opsporing van antibioticaresiduen in kippenvlees.” Melle, 28 juni 2000. “Screening van residuen van infectiewerende stoffen in eieren (resultaten 1999).” Melle, 28 juni 2000. “Demonstratie Premitest.” Melle, 28 juni 2000. “Screening van residen van infectiewerende stoffen in eieren (resultaten 2000 en 2001).“ Melle, 26 juni 2002. “Detectie van neomycine en sulfdiazine in eieren na toediening van respectievelijk Biosol 70% en Tucoprim aan legkippen.” Melle, 26 juni 2002. “Monitoring van eieren op de aanwezigheid van residuen van infectiewerende stoffen en coccidiostatica.” Melle, 23 september 2004. C.2.4. Voordrachten op contact(namid)dagen met de Belgische zuivelindustrie “Bespreking ringonderzoeken antibiotica.” Gontrode, 5 oktober 2004. “Bespreking resultaten ringonderzoeken antibiotica.” Gontrode, 24 november 2005. “Demonstratie TwinSensor Milk.” Gontrode, 24 november 2005. “Demonstratie instrumentele aflezing Copan Milk Test.” Gontrode, 24 november 2005.


“Bespreking resultaten ringonderzoeken antibiotica.” Gontrode, 28 november 2006. “Toelichting dossier „Gebruik sneltesten bij I.O.‟s.” Gontrode, 28 november 2006. “Bespreking resultaten ringonderzoeken antibiotica 2007.” Melle, 4 december 2007. “Antibioticatesten – vraag en antwoord”. Melle, 4 december 2007. “Bespreking resultaten ringonderzoeken antibiotica 2008.” Melle, 4 december 2008. “Nieuwe (snel)testen voor antibioticabepaling in melk”. Melle, 4 december 2008. “Bespreking resultaten ringonderzoeken antibiotica 2009”. Melle, 1 december 2009. “Validatie van de TwinExpress Bespreking resultaten ringonderzoeken antibiotica 2008”. Melle, 1 december 2009. C.2.5 Andere voordrachten en lessen Diverse voordrachten op studievergaderingen van de Boerenbond en voor diverse imkersverenigingen. Sinds 1984 diverse lessen in de cursus „Beginnend imker‟, „Gevorderd imker‟ en „Bijenproducten‟ op het PC.L.T. vzw te Roeselare. Lessen in de cursusen „Tweede Jaar Imker‟ georganiseerd door de K.O.I.B. op diverse locaties. C.3 Lidmaatschap van wetenschappelijke commissies Lid van IDF-FIL (International Dairy Federation): - Van maart 1994 tot aan de nieuwe structuur binnen IDF: lid van de Groep A4 "Residues and Contaminants in milk and milk products". Van mei 1995 ondervoorzitter van de FIL-IDF Groep A4. - Van september 1995 tot aan de nieuwe structuur binnen IDF: lid van de Groep A30 "Microbiological Quality and Safety of Raw Milk and Milk Products". Van februari 1998 ondervoorzitter van de FIL-IDF Groep A30. - Van mei 1997 tot aan de nieuwe structuur binnen IDF: lid van de Groep E503 "Antibiotics and Contaminants in milk and milk products". In de nieuwe structuur binnen IDF, lid van:

- Standing Committee Analytical Methods for Additives and Contaminants - Standing Committee on Residues and Chemical Contaminants - Standing Committee on Microbiological Hygiene - Action Team on Antimicrobial Resistance

Lid van IHC (International Honey Commission), sinds 2002. Onafhankelijk honingexpert (Europese Commissie), sinds augustus 2007. Expert voor Ex-Change vzw, Vlaams Uitzendplatform voor Experten. Beëdigd assistent bijenziekten, sinds 1986. Voorzitter Studiekring „Bijengezondheid‟, Koninklijke Oost-Vlaamse Imkersvereniging.

ACKNOWLEDGEMENTS

My parents Prof. Dr. Hubert F. De Brabander Promotor, UGent Dr. Apr. Els Daeseleire Promotor, ILVO-T&V Dr. Lieve Herman Promotor, ILVO-T&V Dr. ir. Lynn Vanhaecke Promotor, UGent ir. Sigrid Ooghe Lab „Screening Antibiotics‟, ILVO-T&V

Prof. Dr. Siska Croubels Member of the examination commission, UGent

Prof. Dr. Aart de Kruif Member of the examination commission, UGent

Dr. Philippe Delahaut Member of the examination commission, CER

Prof. Dr. Frans Jacobs Member of the examination commission, UGent

Dr. Matthew Sharman Member of the examination commission, FERA, United Kingdom

Prof. Dr. ir. Paul Van Assche Member of the examination commission, HoGent

Prof. Dr. ir. Erik Van Bockstaele Member of the examination commission, ILVO

Prof. Dr. Apr. Carlos Van Peteghem Member of the examination commission, UGent

Christa Boumon, Martine De Clercq, Veroniek De Paepe, Veronique Ottoy, Dominique Van Heeschvelde, Katleen Vander Straeten, Sofie Verheyden

Technicians lab „Screening Antibiotics‟, ILVO-T&V

Petra De Neve, Ann Van de Walle, Patricia Van Herreweghe †

Technicians chromatographic and microbio-logical lab, ILVO-T&V

Dr. Katleen Coudijzer, Dr. Jan De Block, Prof. Dr. Marc Heyndrickx, Dr. Sophie Marchand Co-authors of ILVO-T&V

All other colleagues of ILVO-T&V Mieke De Mits, Conny De Schepper, Kurt Hullebusch, Sabrina Vermeulen, Nurten Yigit

Ex-technicians lab „Screening Antibiotics‟, ILVO-T&V

ACKNOWLEDGEMENTS

Elie Cousens Housekeeper, ILVO-T&V

Tim Coolbear (Fronterra, New Zealand), Monique Duquet, Miriam Levenson (ILVO) Language correction

Jean Brasseur Administration des services vétérinaires, Luxembourg

Ansgar Adriany, Christian Baumgartner, Birgit Kreis

Analytik in Milch Produktions- und Vertriebs-GmbH

Anne-Claire Martel Anses, Laboratoire de Sophia Antipolis

Cor Arts Arts Projects Support

Renaat Debergh, Katrien D‟Hooghe, Kris Lambrechts, Delphine Sunnaert BCZ-CBL

Etienne Bruneau, Sabine Malfait CARI asbl

Steve Holmes, Wilbert Kokke, David Legg, Bob Markovsky, Julio Quintana-Rizzo, Bob Salter Charm Sciences Inc.

Ole Madsen, Jean-Louis Thétas Chr. Hansen

Michel Havelange, Emile Piraux, Didier Véselko Comité du Lait asbl

Daniela Brignocchi, Michela Ferrari, Stefania Novelli, Daniele Triva Copan Italia S.p.A.

Pierre Dardenne, Frédéric Dehareng, Véronique Ninane, Jean-Michel Romnée

CRA-W, Département Valorisa-tion des productions

Jan-Pieter Barendse, Mylène Caussette, Marion Desaunois, Stéphen Hennart, Tineke Hummelen DSM Food Specialties B.V.

Françoise De Goeijen, Angelique de Rijk DSM Nutritional Products B.V.

Ana-Maria Blass-Rico European Commission

Liberty Sibanda, Lucia Streppel, Piet van Wichen EuroProxima B.V.

Lionel Laurier FAMHP

Patricia Beaune, Monique Morlot Famille Michaud Apiculteurs

Luc Bollen, Dirk Courtheyn, Geert De Poorter, Walter Smedts, Rudi Vermeylen FASFC

ACKNOWLEDGEMENTS

Ivan Demeyer ForLab nv

Aurélie Dubois, Joerg Seifert, colleagues of different Standing Committees IDF

Laurent Depeige, Kristina Koch, Terance Fisher, Travis Waldron Idexx Laboratories, Inc.

Christiaan De Bruyne Indumed N.V.

Koen Beeuwsaert, Dirk de Graaf, Katrien De Keukelaere, Bernadette Rotthier Informatiecentrum Bijenteelt

Colleagues International Honey Commission

Chantal Asselman, Beni De Wever, Herman Locquefeer International Medical Products

Lutz Elflein, Martin Linkogel, Kurt-Peter Raezke Intertek Food Services GmbH

Sylvain Bareille, Manuel Cervino, Jorge Donate, Philippe Houffschmitt, Frederic Leboeuf, Bart Sustronck

Intervet Schering-Plough Animal Health

Luc De Meulemeester, Anne Gijsels, Jean-Marie Van Crombrugge

Melkcontrolecentrum-Vlaanderen vzw

Jef Joostens, Dirk Vandervelde M.P.M. De Block cvba

Bill Hoerner, Tony Maltese, Jennifer Rice, Gary White Neogen Corporation

Jean-Marc Diserens Nestlé Research Center

Sigrid Stoop, Hedwige Vanaken, Monique Van Goubergen Pfizer Animal Health

Damien McAleer, Aaron Tohill Randox Laboratories Ltd.

Ron Wolbert Tecna S.r.l.

Sigrid Lauryssen, Gwendolyn Maertens, Robert Remy Test-Aankoop – Tests Achats

Jacques Degelaen UCB Bioproducts sa

Siegrid De Baere, Soetkin De Wannemacker, Kim Heylen, Dries Laget, Inge Roman UGent

Tony Brisaert, Vincent Chabottaux, Benoît Granier, Benoît Lemmens Unisensor s.a.

Pedro Razquin, Sofia Andaluz ZEU INMUNOTEC S.L.

Family, friends, and all other nice contacts

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To Eva and Amber - Universiteit Gent