pegah.irpegah.ir/uploads/Antibiotic 2009_1797.pdf · ________________________________________________________________________________________________ Page 2 TABLE OF CONTENTS 1 INTRODUCTIONPublished

Antibiotics in milk Actual situation & compilation of commercial available screening methods for the detection of inhibitors/antibiotics residues in milk

Jean-Marc Diserens, Andrea Beck Henzelin, Marie-Hélène Le Breton & Marie-Claude

Savoy Perroud Nestlé Research Center Quality & Safety Department 1000 Lausanne 26 (Switzerland) Tel.: 41/21 785 82 44 Fax.: 41/21 785 85 53 E-mail: jean-marc.diserens@rdls. nestle.com

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TABLE OF CONTENTS 1 INTRODUCTION .............................................................................................................. 5 2 METHODOLOGY AND TRIALS....................................................................................... 6

2.1 LACTATING COW TREATMENTS ................................................................................... 6 2.1.1 Mastitis ....................................................................................................................6 2.1.2 Drying off .................................................................................................................6

2.2 INTRAMAMMARY INJECTORS....................................................................................... 6 2.3 ANTIBIOTIC USE AND SALES........................................................................................ 7

2.3.1 United Kingdom .......................................................................................................7 2.3.2 Switzerland..............................................................................................................7 2.3.3 Most widely used antibiotics ....................................................................................7

2.4 COUNTRIES INVOLVED IN THE STUDY .......................................................................... 8 2.5 COMPOSITION OF THE INJECTORS .............................................................................. 8 2.6 FAMILIES OF ANTIBIOTICS CONTAINED IN THE INJECTORS............................................. 9 2.7 LIST OF ANTIBIOTICS AND THEIR RESPECTIVE FAMILIES................................................ 9 2.8 COMPOSITION OF THE INJECTORS ............................................................................ 11 2.9 PREDNISOLONE ....................................................................................................... 13 2.10 WITHDRAWAL PERIOD .............................................................................................. 13 2.11 MILK CONTAMINATION.............................................................................................. 14 2.12 MAXIMUM RESIDUE LIMITS (ENCLOSURE1) ................................................................ 16

3 RAPID METHODS FOR THE DETECTION OF ANTIBIOTIC RESIDUES .................... 17 4 CONCLUSIONS ............................................................................................................. 18 5 REFERENCES: .............................................................................................................. 19 6 COMPILATION OF COMMERCIALY AVAILABLE SCREENING METHODS FOR THE DETECTION OF INHIBITOR/ANTIBIOTIC RESIDUES IN MILK.......................................... 24

6.1 INFORMATIONS FROM THE KIT MANUFACTURERS ....................................................... 24 6.2 ANTIBIOTICS MENTIONED.......................................................................................... 24

7 BETASTAR®.................................................................................................................. 25 8 BETASTAR® COMBO ................................................................................................... 28 9 BRT INHIBITOR TEST - BRT MRL-SCREENING TEST ............................................... 31 10 BR-TEST AS SPECIAL.................................................................................................. 34 11 BR-TEST AS BRILLIANT............................................................................................... 37 12 CHARM II (NEOMYCIN TYPE) AMINOGLYCOSIDE TEST .......................................... 40 13 CHARM II (STREPTOMYCIN TYPE) AMINOGLYCOSIDE TEST ................................. 42 14 CHARM II AMPHENICOL TEST .................................................................................... 44 15 CHARM II BETA-LACTAM (SEQUENTIAL)TEST......................................................... 46 16 CHARM II CHLORAMPHENICOL TEST........................................................................ 49 17 CHARM II MACROLIDE TEST....................................................................................... 51 18 CHARM II NOVOBIOCIN TEST ..................................................................................... 53 19 CHARM II SULFA DRUG TEST..................................................................................... 55 20 CHARM II TETRACYCLINE TEST................................................................................. 58

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21 CHARM BLUE-YELLOW TEST ..................................................................................... 61 22 CHARM BSDA ............................................................................................................... 63 23 CHARM COWSIDE ........................................................................................................ 65 24 CHARM BLUE-YELLOW TEST ..................................................................................... 67 25 COPAN MILK TEST (CMT)............................................................................................ 69 26 DELVO-X-PRESS........................................................................................................... 73 27 DELVOTEST SP- NT...................................................................................................... 76 28 DELVOTEST SP............................................................................................................. 79 29 DELVOTEST P ............................................................................................................... 83 30 ECLIPSE FARM ............................................................................................................. 86 31 ECLIPSE 50.................................................................................................................... 88 32 ECLIPSE 100.................................................................................................................. 91 33 EIA CHLORAMPHENICOL ............................................................................................ 94 34 EIA ENROFLOXACIN .................................................................................................... 95 35 FLUMEQUINE EIA ......................................................................................................... 96 36 EIA FLUOROQUINOLONES.......................................................................................... 97 37 EIA GENTAMICIN .......................................................................................................... 99 38 EIA NEOMYCIN............................................................................................................ 100 39 EIA STREPTOMYCIN .................................................................................................. 101 40 EIA SULFAMETHAZINE .............................................................................................. 102 41 EUROCLONE CHLORAMPHENICOL ELISA.............................................................. 105 42 EUROCLONE KALIDOS TB ........................................................................................ 107 43 EUROCLONE TOTAL ANTIBIOTICS .......................................................................... 109 44 EUROCLONE KALIDOS TB ........................................................................................ 111 45 EUROCLONE KALIDOS MP........................................................................................ 113 46 PARALLUX .................................................................................................................. 115 47 PENZYM®100 .............................................................................................................. 117 48 PENZYM®100 S ........................................................................................................... 120 49 ROSA ENROFLOXACIN TEST.................................................................................... 123 50 ROSA MRL TEST FOR BETA-LACTAM..................................................................... 125 51 ROSA MRL3 TEST FOR BETA-LACTAM................................................................... 127 52 ROSA SDSM TEST ...................................................................................................... 129 53 ROSA SL3 TEST FOR BETA-LACTAM ...................................................................... 131 54 ROSA SLBL TEST FOR BETA-LACTAM ................................................................... 133 55 ROSA MRLBLTET ....................................................................................................... 135 56 ROSA 3 MINUTE MRL TEST FOR BETA-LACTAM AND TETRACYCLINE DRUGS 137 57 ROSA TETRACYCLINE TEST..................................................................................... 139 58 SCREENING PLUS ...................................................................................................... 141

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59 SNAP TEST KITS - IDEXX........................................................................................... 143 60 TECNA - I’SCREEN TYLOSIN..................................................................................... 146 61 TECNA - I’SCREEN GENTAMICIN.............................................................................. 148 62 TECNA - I’SCREEN NEOMYCIN ................................................................................. 150 63 TECNA - I’SCREEN CAP............................................................................................. 152 64 TECNA – SUPERSCREEN TETRA ............................................................................. 154 65 TETRASENSOR........................................................................................................... 156 66 TRANSIA PLATE CHLORAMPHENICOL ................................................................... 158 67 TWINSENSOR.............................................................................................................. 160 68 VALIO T 101 TEST....................................................................................................... 163 69 ALPHABETICAL LIST OF ANTIBIOTICS AND CORRESPONDING KITS ................ 167

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1 Introduction The use of antibiotics on lactating cows for dry cow therapy or to treat mastitis still presents a high risk of contamination of the milk supply. A wide study was performed on which antibiotics are used with the evaluation of 629 udder injectors from 23 countries. This article presents the results of the study, the antibiotics administered, some example of withdrawal times to be respected, the elimination of antibiotics after treatment, the legislation of some countries and the commercialy available screening methods. Looking at the composition of the intramammary injectors on the market, the main risk of milk contamination which could not be detected with an available screening test is very limited. Analysing the results of the composition of 629 intramammary injectors the actual situation of screening with rapid tests for beta-lactams and cephalosporins corresponds well to the drugs the most widely used.

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2 Methodology and Trials

2.1 LACTATING COW TREATMENTS Despite occasional treatment of cows with antibiotics to cure various diseases, the risk of milk contamination with antibiotics is mainly due to the treatment with intramammary drugs. Intramammary infusions applied for cure mastitis and drying off are certainely the main source of contamination worldwide but other source of contamination could contaminate the milk supply:

− Mis-application of injectables (example- ceftiofur (Excenel, Naxcel) is a 0 time withdrawal if injected in muscle but farmers mistakenly inject it into utter and when it is injected there it is a 96 hour withdrawal). Very common source of positives in US.

− Foot baths (tetracycline used for foot rot is licked afterwards by the cow) − Inter-uterine infusions and bolus- (sulfa drug and tetracycline used to flush placenta

after birth) − All kind of intramuscular and intravenous treatments

2.1.1 Mastitis Mastitis are microbial infections of the mammary gland, they are caused in >90 % of cases by pathogenic organisms that enter into the mammary gland through the teat end. Mastitis can be classified into two main types: - Subclinical: no visual symptoms, high somatic cell counts, occasionally elevated body

temperature

- Clinical: high somatic cell counts, fever, abnormal milk (off-colour, flaky, watery, clotted), udder is hot, hard, swollen and sensitive.

Both types of mastitis increase the somatic cell count and lower the milk production.

2.1.2 Drying off To have an optimal milk production, cows should calve once a year and milking must be stopped 50 to 60 days before the next calving. This dry period is necessary to regenerate the secretory cells of the mammary gland. Cows would produce ~ 25 % less milk if the dry period is omitted. After the last milking, the remaining milk may flocculate, the udder may get infected, etc. The cows udder is separated into four quarters which can be treated individually. Three approaches are available: - Treatment only if necessary and only the incriminated quarter.

- Systematic treatment of all quarters after the last milking of cows affected by mastitis in the previous lactation.

- Treatment of all cows in a herd.

2.2 INTRAMAMMARY INJECTORS We have studied only the use of intramammary injectors because they present the highest risk of milk contamination. Approximately 30 to 50 % of the antibiotic intramammary injected is elimintated with the first milking. The intravenous and intra-muscular treatments do not present such a high risk of contamination.

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2.3 ANTIBIOTIC USE AND SALES Not much information is available on the sales and use of intramammary injectors.

2.3.1 United Kingdom The total sales of antimicrobials used in veterinary medicine in 2006 in UK were of 405 tonnes. Out of those 405 tonnes, only three tonnes (0.74 %) were intramammary injector antibiotics. This data seems low but it represents the application of approx. 90 µg/kg of antibiotic per liter of milk produced in the country!

2.3.2 Switzerland In Switzerland in a study on the antibiotics used on lactating cows only, in 2003-2004, 74.6 % of the treatments were the application of intramammary infusions.

Mastitis; 30.40%

Dry cow therapy; 44.20%

Teats; 1.50%

Calving; 10%

Locomotive system; 1.30%

Digestive track; 5.50%

Lungs; 1.30%

Other; 3.10%

Swiss study: repartition of the antimicrobials used on dairy cattle

Mastitis; 30.40%

Dry cow therapy; 44.20%

Teats; 1.50%

Calving; 10%

Locomotive system; 1.30%

Digestive track; 5.50%

Lungs; 1.30%

Other; 3.10%

Swiss study: repartition of the antimicrobials used on dairy cattle

2.3.3 Most widely used antibiotics Unfortunately, we have no information on which drug is the best-selling and the quantities sold. We only found informations on the best-selling intramammary treatments in UK. They are in the table as an example, note that they all contain at least one beta-lactam.

Best selling intramammary injectors in UK (2006) Name of the injector Composition Synulox LC Clavulanic acid, amoxicillin and prednisolone Tetra-Delta Novobiocin sodium, neomycin sulphate,

procaine penicillin, dihydrostreptomycin sulphate and prednisolone.

Leo Yellow Penethamate, dihydrostreptomycin, framycetin and prednisolone.

Leo Red Framycetin, penethamate and penicillin. Cepravin Cefalonium

In Switzerland 60% of the intramammary injectors used are a combination of penicillin and an aminoglycoside (often gentamicin). The other antibiotics the most frequently used are cloxacillin, cephalosporin and ampicillin.

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2.4 COUNTRIES INVOLVED IN THE STUDY We have studied the composition of 630 registered intramammary injectors from 23 countries: Argentina, Austria, Baltic countries, Belgium, Canada, Columbia, Croatia, Denmark, Finland, France, Germany, Greece, Iran, Ireland, Japan, Mexico, Portugal, Spain, Sweden, Switzerland, Thailand, United Kingdom and USA

2.5 COMPOSITION OF THE INJECTORS The majority of the injectors contain beta-lactams or cephalosporins.

83 %

17 %

Total number of injectorscontaining at least one ß-lactam

Total number of injectorswithout ß-lactam

% of injectors containing ß-lactams antibiotics

629 injectors analysed

83 %

17 %

Total number of injectorscontaining at least one ß-lactam

Total number of injectorswithout ß-lactam

% of injectors containing ß-lactams antibiotics

629 injectors analysed

Intramammary injectors contain between one and six antibiotics:

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225 243

47

6 1

5049

8

0 00

50

100

150

200

250

300

350

Injec

tors w

ith on

e anti

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Injec

tors w

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o anti

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Injec

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Without beta-lactamsWith beta-lactams

82 % 83

%

85 %

100 % 10

0 %

% of the various types of injectors containingß-lactam antibiotics

225 243

47

6 1

5049

8

0 00

50

100

150

200

250

300

350

Injec

tors w

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Injec

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Without beta-lactamsWith beta-lactams

82 % 83

%

85 %

100 % 10

0 %

% of the various types of injectors containingß-lactam antibiotics

2.6 FAMILIES OF ANTIBIOTICS CONTAINED IN THE INJECTORS

2.7 LIST OF ANTIBIOTICS AND THEIR RESPECTIVE FAMILIES

Antibiotic family Antibiotic Beta-lactams/cephalo Amoxicillin Ampicillin Cefacetril Cefalexin

Aminoglycosides Beta-lactams Cephalosporins DiaminopyrimidinesLincosaminide Macrolides Novobiocin Polymyxins Quinolones Rifamycins Sulfonamides Tetracyclines

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Antibiotic family Antibiotic Beta-lactams/cephalo Cefalonium Cefapirin Cefazolin Cefoperazon Cefquinom Ceftiofur Cefuroxime Cephalotine Cepharin Cloxacillin Dicloxacillin Nafcillin Oxacillin Penethamate Penicillin Beta-lactamase inhibithorClavulanic acid Tetracyclines Oxytetracycline Tetracycline Sulfonamides Sulfadiazine Sulfadiazole Sulfadimethoxine Sulfadimethylpyrimidine Sulfamethazine Sulfamethoxypyridine Sulfathiazole Aminoglycosides Dihydro-streptomycin Framycetin Gentamicin Kanamycin Neomycin Streptomycin Macrolides Erythromycin Spiramycin Tylosin Lincosamides Pirlimycin Lincomycin Ansamycin Rifaximin Polymyxins Colistin Novobiocin Novobiocin Diaminopyrimidines Trimethoprim Quinolones Norfloxacin Polypeptide Bacitracin

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2.8 COMPOSITION OF THE INJECTORS

Composition of the injectors

Number of injectors out of

629 Cloxacilin + ampicillin 88 Cloxacillin 81 Penicillin 29 Penicillin + neomycin 27 Neomycin + lincomycin 27 Cefalexin 19 Penicillin + dihydrostreptomycin 15 Cloxacilin + neomycin 15 Cefapirin 15 Cefoperazone 14 Penicillin + dihydrostreptomycin + nafcillin 13 Penicillin + novobiocin 12 Cefalonium 12 Cefquinom 12 Neomycin + spyramicin 11 Gentamicin 11 Amoxicillin + clavulanic acid 10 Cloxacilin + colistin 9 Cefacetril 8 Pirlymicin 8 Penicillin + gentamicin 7 Penicillin + penethamate + framycetin 6 Penethamate + dihydrostreptomycin + framycetin 6 Trimethoprim + sulfadiazine 6 Rifaximin 6 Cefazolin 6 Erythromycin 6 Penicillin + kanamycin 5 Cefuroxime 5 Penicillin + cloxacillin 4 Neomycin 4 Cloxacilin + gentamicin 4 Ampicillin 4 Ampicillin + dicloxacillin 4 Amoxicillin 4 Oxacillin 4 Rifaximin + cefacetril 4 Penicillin + penethamate + neomycin 3 Penicillin + neomycin + dihydrostreptomycin + novobiocin 3 Penicillin + framycetin 3

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629 Neomycin + tetracycline 3 Neomycin + tetracycline + bacitracin 3 Cloxacilin + amoxicillin 3 Colistin 3 Dihydrostreptomycin + erythromycin 3 Dihydrostreptomycin + cefalexin 3 Trimethoprim + sulfamethazine 3 Gentamicin + cefalexin 3 Cepharin 3 Ceftiofur 3 Penicillin + neomycin + streptomycin 2 Penicillin + nafcillin 2 Neomycin + cephalexin 2 Ampicillin + dicloxacillin gentamicin 2 Kanamycin + cefalexin 2 Trimethoprim + sulfamethazine + gentamicin 2 Spiramycin 2 Penethamate 2 Oxytetracycline 2 Novobiocin 2 Streptomycin + cefalexin 2 Sulfadiazine 2 Penicillin + penethamate 1 Penicillin + neomycin + cefalexin 1 Penicillin + neomycin + bacitracin 1 Penicillin + neomycin + tetracycline 1 Penicillin + neomycin + sulfamethazine 1 Penicillin + neomycin + streptomycin + novobiocin 1 Penicillin + neomycin + sulfamethazine + sulfathiazole 1 Penicillin + dihydrostreptomycin + neomycin + sulfamethazine + sulfadiazole + colistin 1 Penicillin + penethamate + dihydrostreptomycin + framycetin 1 Penicillin + cloxacillin + dihydrostreptomycin 1 Penicillin + dihydrostreptomycin + novobiocin 1 Penicillin + dihydrostreptomycin + gentamicin 1 Penicillin + oxytetracycline 1 Penicillin + nafcillin + streptomycin 1 Penicillin + streptomycin 1 Penicillin + sulfathiazole 1 Penicillin + bacitracin 1 Ampcillin + streptomycin + neomycin 1 Dihydrostreptomycin + neomycin + novobiocin 1 Dihydrostreptomycin + neomycin + colistin 1 Neomycin + gentamicin 1

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629 Cloxacilin + ampicillin + streptomycin 1 Cloxacilin + framycetin + sulfamethazine 1 Cloxacilin + nafcillin 1 Cloxacilin + kanamycin 1 Cloxacilin + streptomycin 1 Cloxacilin + trimetoprim + sulfamethazine 1 Ampicillin + colistin 1 Kanamycin + colistin 1 Kanamycin + colistin + cephalexin 1 Dihydrostreptomycin 1 Kanamycin + spiramycin 1 Kanamycin + sulfamethoxypyridine 1 Trimethoprim 1 Trimethoprim + gentamicin 1 Trimethoprim + gentamicin + sulfadimethoxine 1 Penethamate + streptomycin + framycetin 1 Gentamicin + norfloxacin 1 Sulfadimethylpyrimidine + tylosin 1 Sulfadiazole 1 Cephalotine 1 Tetracycline 1

2.9 PREDNISOLONE The prednisolone, a synthetic corticosteroid, is often added to the antibiotics present in the injectors. Prednisolone acts as an anti-inflammatory reducing swelling and associated pain. E.g.: Mastijet, Kanapen-P, Leo Yellow, etc.

2.10 WITHDRAWAL PERIOD

After antibiotic treatment a withdrawal period, during which the milk is eliminated, must be respected. This period is necessary to clear the udder from any remaining antibioitics and to ensure that the milk is free of residues. In the table, a few examples of withdrawal recommended periods. The withdrawal periods are defined by the governmental regulatory bodies.

Milk withdrawal time after intramammary treatment Name of the injector Active ingredients Withdrawal time Dry cow therapy Cloxacillin-TS-1000 Cloxacillin 8 weeks or negative to inhibition test Prevogent Penicillin + gentamicin 8 weeks or negative to inhibition test Prevolac Penicillin + bacitracin 8 weeks or negative to inhibition test Biclox DC Cloxacillin + ampicillin 56 days or negative to inhibition test Lactating cow therapy Mammacillin Penicillin 5 days Cobactan® LC Cefquinome 5 days Mastimyxin® Polymyxin B 5 days

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Miliopen Penicillin 7 days Monocillin® Penicillin 5 days Peracef® Cefoperazon 4 days Cloxa-coli Cloxacillin + colistin 5 days Medisan Neopen Penicillin + neomycin 5 days Synulox LC Plus Amoxicillin + clavulanic ac. 5 days

2.11 MILK CONTAMINATION If the withdrawal period is not respected, the contaminated milk will be diluted with the “clean” milk of the farm and could contaminate very high volumes of milk. The withdrawal of some intramammary injectors have been studied and some of their results are in the table below. The graphics are based on the results published by Jean-Pierre Moretain et al.

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Withdrawal of antibiotics after intramammary injection

Compositions of the injectors used in the study

Procaine penicillin G 450 mg/6 gProcaine penicillin G 1000 mg/ 10 mLProcaine penicillin G 100 mg/10 mlSodium cloxacillin 200 mg/3 gSodium cloxacillin 200 mg/3 gSodium cloxacillin 200 mg/9 gSodium cloxacillin+ampicillin 200 mg + 200 mg/10 mlSodium cloxacillin+ampicillin 200 mg + 75 mg/3 gSodium cloxacillin+ampicillin 200 mg + 200 mg/10 mlSodium cloxacillin+ampicillin 200 mg + 75 mg/3 gSodium oxacillin 1000 mg/7.5 gCephalexin monohydrate 1000 mg/10 mLTetracycline HCl 150 mg/5 gTetracycline HCl 200 mg/10 gTetracycline HCl 80 mg/5 gChlortetracycline HCl 130 mg/6.5 gChlortetracycline HCl 200 mg/5 g

Withdrawal of penicillin

1

10

100

1'000

10'000

100'000

1'000'000

0 2 4 6 8 10

Number of milkings

µg/k

g 450 mg/6 g1'000 mg/10 ml100 mg/10 mlCodex MRL

Withdrawal of cloxacillin

1

10

100

1'000

10'000

100'000

0 2 4 6 8 10

Number of milkings

µg/k

g

200 mg/ 3 g200 mg/ 3 g200 mg/9 g200 mg/10 ml200 mg/3 gEU MRL

Withdrawal of ampicillin

1

10

100

1'000

10'000

100'000

0 1 2 3 4 5 6 7 8

Number of milkings

µg/k

g 200 mg/10 ml75 mg/3 gEU MRL

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Withdrawal of tetracycline HCl

1

10

100

1000

10000

100000

0 2 4 6 8Number of milkings

µg/k

g

150 mg/5 g200 mg/10 g80 mg/5 gCodex MRL

Withdrawal of chlortetracycline HCl

1

10

100

1000

10000

100000

0 5 10 15 20 25 30Number of milkings

µg/k

g 130 mg/6.5 g200 mg/5 gCodex MRL

The results of the studies confirm that if the recommended withdrawal time is respected, in general five days apart for one injector, which means ten milkings in the studies, there is no more residues, or at a lower level than the MRL, in the milk. Only one treatment with chlortetracycline HCl presented residues even after 27 milkings! The authors do not explain the reason of such a long withdrawal period. The only explanation could be the non-miscibility of the cream contained in the injector.

2.12 MAXIMUM RESIDUE LIMITS (ENCLOSURE1) Maximum Residue Limits (MRLs) have been established for antibiotic residues in milk. Enclosure 1 lists the MRL’s of various countries or intergovernmental organisations.

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3 Rapid methods for the detection of antibiotic residues Many screening and confirmatory methods are available for the detection or the determination of antibiotic residues in milk. Confirmatory methods are chromatography methods, HPLC or HPLC-MS/MS. Screening methods are commercially available test kits and are listed in the second part of this article. They are well described by their respective manufacturers.

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4 Conclusions Looking at the composition of the intramammary injectors on the market, the main risk of milk contamination which could not be detected with an available screening test is very limited. Numerous screening tests are available to detect all kinds of antibiotics. The selection of test depends on the time at disposal and which antibiotics are used in the area of the milk production.

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5 References: Confédération suisse, Station de recherche Agroscope Liebefeld-Posieux ALP, Antibiotiques utilisés en production laitière en 2003-2004, W. SCHAEREN, Agroscope Liebefeld-Posieux ALP, Schwarzenburgstrasse 161, 3003 Berne Veterinary Medicine Directorate, SALES OF ANTIMICROBIAL PRODUCTS AUTHORISED FOR USE AS VETERINARY MEDICINES, ANTIPROTOZOALS, ANTIFUNGALS, GROWTH PROMOTERS AND COCCIDIOSTATS, IN THE UK IN 2006. AFSSA, Agence Française de Securite Sanitaire des Aliments, suivi des ventes de médicaments vétérinaires contenant des antibiotiques en France en 2006, A. Chevance et G. Moulin, AFSSA-ANMV, Fougères. AnimalPharm reports, Antibacterials in the Animal Health Industry: Current markets and future prospects, Publication Date: 05/04/2005 Elimination des residus d’antibiotiques dans le lait: les tetracyclines, J-P Moretain, S. Loussouarn et J. Boisseau, Recueil de Medecine Veterinaire, mai 1983, pages 473 - 480. Excretion of penicillins and cephalexin in bovine milk following intramammary administration, J-P Moretain and J. Boisseau, Food Additive and Contaminants, 1989, vol 6, no 1, pages 79 – 90. Food Standards, Australia New Zealand, 22 March 2006 Attachment 2, A Risk Profile of Dairy Products in Australia, Appendices 7-14, DRAFT ASSESSMENT REPORT PROPOSAL P296 PRIMARY PRODUCTION AND PROCESSING, STANDARD FOR DAIRY. The New Zealand Food Safety Authority, New Zealand (Maximum Residue Limits of Agricultural Compounds) Food Standards 2005 (No.2) http://www.nzfsa.govt.nz/policy-law/legislation/food-standards/nz-food-standards-2005-mrl-no-2.pdf European Medicine Agency, Veterinary Medicines - Maximum Residue Limits (MRL), http://www.emea.europa.eu/htms/vet/mrls/a.htm US Food and Drug Administration, September 27, 2005, Milk Safety References National Conference on Interstate Milk Shipments (NCIMS), Tolerance and/or Safe Levels of Animal Drug Residues in Milk, (Replaces M-I-03-9 (June 30, 2003) and identifies it as "INACTIVE") and also identifies M-I-92-1 as "INACTIVE" http://www.cfsan.fda.gov/~ear/mi-05-5.html Taiwan, Department of health, 22.Veterinary Drug Residue Limits in Foods http://www.doh.gov.tw/ufile/Doc/Veterinary.pdf South African Government Information, GOVERNMENT GAZETTE, 6 No. 28584, 10 MARCH 2006, DEPARTMENT OF HEALTH, No. R. 215, FOODSTUFFS, COSMETICS AND DISINFECTANTS ACT, 1972 (ACT NO.54 OF 1972) REGULATIONS GOVERNING THE MAXIMUM LIMITS FOR VETERINARY MEDICINE AND STOCK REMEDY RESIDUES THAT MAY BE PRESENT IN FOODSTUFFS http://www.info.gov.za/gazette/regulation/2006/28584b.pdf

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Confederation suisse, Ordonnance du DFI du 26 juin 1995 sur les substances étrangères et les composants dans les denrées alimentaires (Ordonnance sur les substances étrangères et les composants, OSEC) du 26 juin 1995 (Etat le 10 octobre 2006), 817.021.23 http://www.admin.ch/ch/f/rs/c817_021_23.html The Japan Food Chemical Research Foundation, The Japanese Positive List System for Agricultural Chemical Residues in Foods, (Enforcement on May 29, 2006) Maximum Residue Limits (MRLs) of Agricultural Chemicals in Foods http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/MRLs-p MERCOSUR/GMC /RES. Nº 54/00 REGLAMENTO TÉCNICO MERCOSUR METODOLOGÍAS ANALÍTICAS, INGESTA DIARIA ADMISIBLE Y LÍMITES MÁXIMOS DE RESIDUOS PARA MEDICAMENTOS VETERINARIOS EN ALIMENTOS DE ORIGEN ANIMAL. VISTO: El Tratado de Asunción, el Protocolo de Ouro Preto, las Resoluciones N° 91/93, 75/94, 152/96 y 38/98 del Grupo Mercado Común y la Recomendación Nº 28 /98 del SGT N° 3 “Reglamentos Técnicos y Evaluación de la Conformidad”. http://www.sagpya.gov.ar/new/0-0/programas/negociaciones/Mercosur/resoluciones/2000/R54-2000.htm Health Canada, Drugs and Health Products, Administrative Maximum Residue Limits (AMRLS) and Maximum Residue Limits (MRLS) set by Canada. http://www.hc-sc.gc.ca/dhp-mps/vet/mrl-lmr/mrl-lmr_versus_new-nouveau_e.html

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Antibiotic residues in bovine milk Legislation from various countries

All data in µg/kg

Substance (-group) Mercosur Canada Japan Australia South

Africa New

Zealand USA Switzerland Taiwan MRLCodex

MRL EU

ß-Lactams Penicillin 4 6 4 1.5 4 2 02/51 4 4 4 4 Penethamat 4 Ampicillin 10 20 10 4 102 4 10 4 Amoxicillin 8 10 4 102 4 10 4 Nafcillin 5 30 30 Clavulanic acid 100 10 200 Cloxacillin 30 20 10 30 102 30 10 30 Dicloxacillin 10 30 30 30 Oxacillin 30 30 30 30 30 Cefacetril 100 125 125 Cefalexin 100 100 Cefalonium 10 20 20 Cefoperazon 50 50 Ceftiofur 100 100 100 100 100 1002 100 100 100 100 Cefquinome 20 30 20 20 Cefuroxime 20 100 Cephapirin 20 30 10 10 202 10 10 60 Cephazolin 50 50 Tetracyclines 100 100 100 3002 100 100 100 Chlortetracycline 100 100 Doxycycline Oxytetracycline 100 100 100 Tetracycline 100 100 100 Sulfonamides 100 100 100 100 Sulfabenzamide 10 10

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Africa New


MRL EU

Sulfamethazine 100 10 25 - 25 101 25 Sulfadimethoxine 100 10 20 102 Sulfadoxine 10 60 100 Sulfaguanidine 10 10 Sulfamerazine 101 Sulfathiazole 100 10 90 101 Sulfadiazine 70 100 101 Sulfamethizole 101 Sulfachloropyridazine 10 101

Sulfanilamide 10 10 101 Sulfapyridine 10 10 101 Sulfaquinoxaline 100 10 10 101 Sulfatroxazole 100 100 Sulfacetamide 10 10 Sulfabromomethazine 10

Sulfaethoxypyridazine 10

Macrolides Erythromycin 40 50 40 40 02/501 40 40 40 Lincomycin 150 20 150 150 150 150 150 Spiramycin 200 200 200 200 200 200 Tilmicosin 50 50 Tylosin 50 50 502 50 50 50 Tulathromycin nd Oleandomycin 50 Aminoglycosides Gentamicin 200 200 301 100 200 200 100 Neomycin 500 500 1500 1500 1500 1502 1500 500 1500 1500 Spectinomycin 200 200 200 200 200 DH/Streptomycin 200 125 200 200 200 200 1252 200 200 200

________________________________________________________________________________________________

Page 23


Africa New


MRL EU

Kanamycin 400 150 Quinolones Enrofloxacin 20 100 100 Danofloxacin 50 30 30 Difloxacin nd Flumequin 100 50 Marbofloxacin 80 75 75 75 Various Bacitracin 400 500 5002 500 100 Baquiloprim 30 30 30 Chloramphenicol nd nd 1 nd Colistin 50 50 Dapsone nd Novobiocin 80 100 1002 100 50 Rifaximin 60 Thiamphenicol 50 50 50 Trimethoprim 50 50 Pirlimycin 400 100 4002 100 Virginiamycin 100 Polymyxin B 4 u/ml

1 Safe level, 2 Tolerance Remark: in EU, USA and several other countries there is a list of pharmacologically active substances for which no maximum levels can be fixed. In EU they are in the Annex IV of the legislation: Aristolochia spp. and preparations thereof, chloramphenicol, chloroform, chlorpromazine, colchicine, dapsone, dimetridazole, metronidazole, nitrofurans (including furazolidone) and ronidazole.

24

6 Compilation of commercialy available screening methods for the detection of inhibitor/antibiotic residues in milk

6.1 INFORMATIONS FROM THE KIT MANUFACTURERS All the informations contained in the following tables are from the test kit manufacturers. They were collected in 2007 and early 2008.

6.2 ANTIBIOTICS MENTIONED

List of antibiotics mentioned in the compendium Amoxicillin Dapsone Rifampicin Ampicillin Dicloxacillin Rifamycin Bacitracin Dihydrostreptomycin Spectinomycin Benzylpenicillin Doxycycline Spiramicin Cefacetrile Enoxacin Streptomycin Cefadroxil Erythromycin Sulfacetamide Cefalexin Florfenicol Sulfachlorpyridazine Cefalonium Flumequine Sulfadiazine Cefapirin Gentamicin Sulfadimethoxine Cefazolin Hetacillin Sulfadoxine Cefoperazone Kanamycin Sulfamerazine Cefotaxime Lincomycin Sulfamethazine Cefquinome Lomefloxacin Sulfamethizole Ceftiofur Macrolides Sulfamethoxazole Cefuroxime Methacycline Sulfamethoxypyridazine Cephacetrile Methicillin Sulfamonomethoxine Cephalexin Nadifloxacin Sulfanilamide Cephalonium Nafcillin Sulfapyridine Cephaloridine Neomycin Sulfaquinoxaline Cephalothin Norfloxacin Sulfathiazole Cephalothin Novobiocin Sulfisoxazole Cephapirin Oxacillin Sulfonamides Cephoxazole Oxytetracycline Tetracycline Cephradine PABA Thiamphenicol Chloramphenicol Pefloxacin Ticarcillin Chlortetracycline Penethamate Tilmicosin Ciprofloxacin Penicillin Trimethoprim Cloxacilin Piperacillin Tylmicosin Danofloxacin Pirlimycin Tylomycin Dapsone Piromidic acid Tylosin

25

7 BetaStar®

Name of the test: BetaStar® Test for Beta-Lactam Drugs in Cow Milk

Presentation: BetaStar®100 – 2 vials (enough for 100 tests) and 100 test strips BetaStar®25 – 25 vials and 25 test strips BetaStar®250 – 250 vials and 250 test strips

Group of drugs detected: Beta-lactams

Type of reaction: Receptor based lateral flow assay utilizing dipsticks in vials

Time per analysis: 5 minutes

Number of samples at a time: Up to 6

Detection limits: See table

Equipment: Dry block incubator 47.5°±1.0°C

Lorry incubator availability: Yes

Special equipment: BetaStar reader (optional)

Special reagents: None

Reference milk availability with known level of antibiotics:

Available at 5ppb Penicillin

Applicable at: Lorry: Yes

Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer: Neogen Corporation 620 Lesher Place Lansing, Michigan, 48912 United States of America (517) 372-9200

Contact person E-Mail:

Gary White [email protected]

Internet site: www.neogen.com; www.chr-hansen.com

Reference: Validation by AFNOR CHR/3-03/03

26

GENERAL DESCRIPTION BetaStar® is a receptor-based lateral flow assay for detection of beta-lactam antibiotic residues in raw, commingled cow milk. A receptor-gold particle conjugate is incubated with the milk sample. Beta-lactam antibiotic present in the milk sample will bind to the receptor-gold conjugate. A membrane-based dipstick with beta-lactam antibiotic bound to the test line is then added to the sample. As the sample flows up the membrane, a red line forms with intensity inversely proportional to the amount of beta-lactam antibiotic present in the milk sample. Additional labelled reagents present in the conjugate preparation react at the control line to form a second red line. Visual interpretation of the lines will indicate whether the sample is positive or negative. The relative intensity of the test and control lines may also be determined using a BetaStar Reader. A ratio of greater than 1.0 indicates a negative test result, while a ratio of less than or equal to 1.0 indicates a positive test result. GENERAL TEST FORMAT/PRESENTATION The test kit for BetaStar 100 includes: 2 vials with the lyophilized receptor and 4 vials of dipsticks. The vials of lyophilized receptor are reconstituted and 25 µl aliquots are delivered to conical Eppendorf vials for use. A milk sample of 100µl is added to the Eppendorf vial and incubated at 47.5°±1.0°C for 3 minutes. The dipstick is placed in the vial and further incubated at 47.5°±1.0°C for 2 minutes. The test is then visually interpreted. The optional BetaStar reader can be utilized for interpretation of results.

27

TEST BETASTAR®

LIMITS OF DETECTION IN MILK Results in µg/kg

Antibiotic Detection limit Penicillin 2-4 Ampicillin 2-5 Amoxicillin 2-4 Cloxacillin 5-10 Dicloxacillin 5-10 Oxacillin 5-10 Nafcillin 8-20 Ceftiofur 75-150 Cefquinome <20 Cefapirin 8-16 Cefoperazone 5-8 Cefalonium 7-15 Cefazolin 40-60

28

8 BetaStar® Combo

Name of the test: BetaStar® ComboTest for Beta-Lactam and Tetracycline Drugs in Cow Milk

Presentation: BetaStar® Combo 25 – 25 vials and 25 test strips

Group of drugs detected: Beta-lactams and Tetracyclines

Type of reaction: Receptor based lateral flow assay utilizing dipsticks in vials




Equipment: Dry block incubator 47.5°±1.0°C


Special equipment: Accuscan III reader (optional)



Available at 5ppb Penicillin


Farm: Yes

Milk reception: Yes

Laboratory: Yes




Internet site: www.neogen.com; www.chr-hansen.com

Reference:

29

GENERAL DESCRIPTION BetaStar® Combo is a binding protein-based lateral flow assay for detection of beta-lactam and tetracycline antibiotic residues in raw, commingled cow milk. A binding protein-gold particle conjugate is incubated with the milk sample. Beta-lactam and tetracycline antibiotic present in the milk sample will bind to the binding protein-gold conjugate. A membrane-based dipstick with beta-lactam antibiotic bound to one test line and tetracycline antibiotic bound to a second test line is then added to the sample. As the sample flows up the membrane, a red line forms with intensity inversely proportional to the amount of beta-lactam and tetracycline antibiotic present in the milk sample. Additional labeled reagents present in the conjugate preparation react at the control line to form a third red line. Visual interpretation of the lines will indicate whether the sample is positive or negative. The relative intensity of the test and control lines may also be determined using a Accuscan III Reader. A ratio of greater than 1.0 indicates a negative test result, while a ratio of less than or equal to 1.0 indicates a positive test result. GENERAL TEST FORMAT/PRESENTATION The test kit for BetaStar Combo includes: 25 vials with the lyophilized binding protein and 1 tube of dipsticks. A milk sample of 200µl is added to the vial and incubated at 47.5°±1.0°C for 2 minutes. The dipstick is placed in the vial and further incubated at 47.5°±1.0°C for 3 minutes. The test is then visually interpreted. The optional Accuscan III reader can be utilized for interpretation of results.

30

TEST BETASTAR® COMBO


Antibiotic Detection limit Penicillin 2-4 Ampicillin 2-3 Amoxicillin 2-3 Cloxacillin 5-10 Dicloxacillin 5-10 Oxacillin 5-10 Nafcillin 8-20 Ceftiofur 75-100 Cefquinome <20 Cefapirin 8-16 Cefoperazone 5-8 Cefalonium 7-15 Cefazolin 40-60 Tetracycline 50 Oxytetracycline 25-50 Chlortetracycline 25-50

31

9 BRT Inhibitor Test - BRT MRL-Screening Test

Name of the test:

BRT Inhibitor Test BRT MRL-Screening Test

Presentation: Tubes for single sample Strips divisible in 96 single samples Plate for 96 samples

Group of antibiotics detected: Wide range of antibiotics and sulfonamides

Type of reaction: Bacterial growth inhibition

Time per analysis: 2hrs to 2hrs 30min

Number of samples at a time: Not limited

Detection limits: See tables

Equipment: Water bath or thermo-block

Lorry incubator availability : Yes

Special equipment: ELISA reader



Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer AiM Analytik in Milch Produktions- und Vertriebs-GmbH Kaiser-Ludwig-Platz 2 80336 München Germany Tel.: +49 89 53075120 Fax: +49 89 53075122


Dr. Birgit Kreis [email protected]

Internet site: www.aimbavaria.com

Reference:

32

GENERAL DESCRIPTION Microbiological inhibitor tests operate by making use of the sensitivity of specific micro-organisms - so-called test bacteria - to antibiotics, sulfonamides and other inhibitors. With the BRT, the test medium is contained in cavities, or wells, in microtiter test plates or ampoules. This test medium is a mixture of nutrients, test bacteria (Geobacillus stearothermophilus var. calidolactis C953), brilliant black, and other supplements which help improve detection sensitivity towards chosen inhibitors. The milk samples are pipetted into the wells. Any inhibitors present can then diffuse throughout the test medium. During incubation the growing test bacteria shift the redox indicator (brilliant black) to its yellow or colorless reduction stage through the division of double azocompounds, and the test medium changes from blue to yellow. If inhibitors are present in the sample, growth will be minimal or non-existent. There will then be no reduction of the coloring agent, or to only a very small degree, and the test medium will remain blue. The BRT Inhibitor Test was developed especially for the detection of inhibitors in milk, and distinguishes itself by its particularly high sensitivity to betalactam antibiotic residues. The maximum residue limits (MRLs) laid down by the EU for betalactam antibiotics are reliably encompassed by the BRT Inhibitor Test; other antibiotics and sulfonamides present will also be detected. The BRT Inhibitor Test is especially suitable for examining raw milk and heat treated milk, it is particularly designed to fulfill the demands of milk quality regulation in Germany, but is also used successfully in other countries in quality evaluation and payment for tanker milk. In notice of our evidences the testing of milk products and preserved samples is also quite possible. The BRT MRL-Screening Test is a modified BRT which is distinguished by an increased sensitivity towards certain antibiotics and sulfonamides. Besides the statutory maximums (MRLs, laid down by the EU) for betalactam antibiotics, the test also encompasses substances from the sulfonamide group, macrolide group and aminoglycoside group to MRL level. Other substance groups are also detected with greater sensitivity than in the BRT Inhibitor Test. BRT MRL-Screening Test is especially suitable for examining raw milk and heat treated milk and is particularly suitable for quality assurance as well as for the examination of dairy milk and milk for the production of fermented milk products. PRESENTATION Tubes for single sample: glass tubes with screw cap, packing size 25 or 100 tubes, shelf life 9 month from production Strips divisible in 96 single samples: polystyrene test plates, 96 wells, crystal clear, 12 x 8 strips breakable up to single sample, shelf life 3 month (version ESL) from production Plate for 96 samples: polystyrene tests plates, 96 wells, crystal clear, shelf life 3 month or 6 month (version ESL) from production

33

AiM BRT Testsystems LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic BRT MRL-Screening Test

BRT Inhibitor Test

Penicillines Amoxicillin 2 – 3 2 – 3 Ampicillin 2 – 3 2 – 3 Benzylpenicillin 1,5 – 2 2 – 3 Cloxacillin 10 – 20 20 – 30 Dicloxacillin 5 – 10 10 – 20 Nafcillin 5 – 10 10 – 15 Oxacillin 5 – 10 10 – 20 Piperacillin 5 – 10 10 – 15 Cephalosporines Cefapirin 4 – 5 4 – 5 Cefazolin 10 – 25 10 – 25 Cefoperazone 20 – 30 25 – 50 Cephalexin 100 – 200 200 – 300 Cefalonium 10 – 15 10 – 20 Cefquinome 80 – 100 100 – 200 Ceftiofur 50 – 100 50 – 100 Cefuroxime 100 – 200 200 – 300 Macrolides Erythromycin 40 – 60 40 – 60 Spiramycin 200 – 400 400 – 600 Tylosin 25 – 50 25 – 50 Tetracyclines Oxytetracycline 250 – 500 500 – 750 Tetracycline 100 – 200 200 – 400 Sulfonamides Sulfadiazine 100 500 – 750 Sulfadimethoxine 100 500 – 750 Sulfamethazine 100 – 300 500 – 750 Sulfathiazole 100 200 – 400 Aminoglycosides Dihydrostreptomycin

400 – 600 1000 – 1500

Gentamicin 100 – 200 200 – 300 Neomycin 500 – 750 500 – 750 Others Lincomycin 75 – 150 150 – 300 Chloramphenicol 2500 – 5000 5000 – 7500

Evaluation 07/2008: reading with ELISA Reader measuring wavelength 450 nm reference wavelength 620 nm

34

10 BR-Test AS Special

Name of the test: BR-Test® AS Special

Presentation: BR-Test® AS Special: plates

Group of drugs detected: Wide range of antibiotics and sulfonamides


Time per analysis: Control time: starting from 2 hrs

Number of samples at a time: waterbath no limit


Equipment: Incubator (MCI-12) or waterbath

Lorry incubator availability : Yes (24V incubator available)

Special equipment: -

Special reagents: No

Reference milk availability with known level of antibiotics Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer

DSM Dairy Ingredients P.O. Box 1 2600 MA Delft The Netherlands


Tineke Hummelen [email protected]

Internet site: www.dsm-dairy.com www.delvotest.com

Reference: DSM

35

GENERAL DESCRIPTION BR-Test® AS Special is a brilliant black reduction test for the detection of antibiotic and sulfonamide residues in milk. The test consists of an agar medium with nutrients seeded with a standardised number of spores of Bacillus stearothermophilus, an antifolate and is coloured purple by the redox indicator brilliant black. Milk samples that are free from antibiotic and/or sulfonamide residues, or milk samples containing inhibitory substances below the specified sensitivity levels, added to the test in an amount of 0.1 ml will upon incubation at 64°C, allow germination and growth of the bacteria leading to a change in colour of the indicator from purple to yellow. When the milk sample is containing antibiotic residues at or above the test sensitivity, growth is inhibited and by this the colour remains purple. GENERAL TEST FORMAT/PRESENTATION Polypropylene plate consisting of 6 blocks of 16 test wells each. The blocks may be separated depending on the number of tests to be executed. Adhesive covers for sealing of the plates during incubation are included.

36

BR-Test® AS-Special


Antibiotic Sensitivity at control timeß-lactams Penicillin 1-2 Ampicillin 2-3 Amoxicillin 2-3 Cephalexin 20-30 Cefquinome 90 Tetracyclines Oxytetracycline 100 - 300 Chlortetracycline 100 - 300 Macrolides Erythromycin 100 Spiramycin 400 - 800 Tylosin 20 Aminoglycosides Gentamicin 200 - 400 Neomycin 400 - 800 Sulfonamides Sulfadiazine 50-150 Sulfamethazine 100-200

The data indicated in the table are based upon readings judged as being yellow/purple to purple and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found. Data in the table may be used for validation purposes. Definitions Drugs: Active ingredient of cattle medication such as antibiotics and Sulfonamides. Spectrum: An indication of the number of different active ingredients that can be detected by

a given method at or below the required detection level. Detection level: The lowest concentration at which a drug is still detected by the test. Control time: Time at which the test agar containing the negative a control sample has just

changed to yellow. At this time the best sensitivity for the test system is obtained.

37

11 BR-Test AS Brilliant

Name of the test: BR-Test® AS Brilliant

Presentation: BR-Test® AS Brilliant: 100 ampoules BR-Test® AS Brilliant: 1* 96, 12*8 and 96*1 plates



Time per analysis: Reading time: 2hours and 45 minutes Control time: starting from 1 hr 45 min

Number of samples at a time: 10 per incubator/waterbath no limit




Special equipment: -


Reference milk availability with known level of antibiotics Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer

DSM Dairy Ingredients P.O. Box 1 2600 MA Delft The Netherlands




Reference: DSM

38

GENERAL DESCRIPTION BR-Test® AS Brilliant is a brilliant black reduction test for the detection of antibiotic and sulphonamide residues in milk. The test consists of an agar medium with nutrients seeded with a standardised number of spores of Bacillus stearothermophilus, an antifolate and is coloured blue by the redox indicator brilliant black. Milk samples that are free from antibiotic and/or sulphonamide residues, or milk samples containing inhibitory substances below the specified sensitivity levels, added to the test in an amount of 0.1 ml will upon incubation at 64°C, allow germination and growth of the bacteria leading to a change in colour of the indicator from blue to yellow. When the milk sample is containing antibiotic residues at or above the test sensitivity, growth is inhibited and the colour stays blue. GENERAL TEST FORMAT/PRESENTATION Ampoules: Polypropylene ampoules in a 100 test kit presentation. The ampoules can be separated into single test ampoules dependent on the number of tests to be executed. Disposable pipettes for taking samples are included. Plates: Polystyrene multi-well presentation; - 1x96 ; multiplate consisting of 1x96 test wells. - 12x8 ; multiplate consisting of 12 strips of 8 test wells. The strips may be separated depending on the number of tests to be executed. - 96x1 ; multiplate consisting of 96 individual test wells. The test wells may be separated depending on the number of tests to be executed. Adhesive covers for sealing of the plates during incubation are included.

39

BR-Test® AS-Brilliant LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Sensitivity at control time

ß-lactams Penicillin 3 - 4 Ampicillin 3 - 4 Amoxicillin 3 - 4 Cephalexin 25 - 50 Cefquinome 100 Tetracyclines Oxytetracycline 100 - 400 Chlortetracycline 100 - 300 Macrolides Erythromycin 100 Spiramycin 400 - 1000 Tylomycin 30 - 40 Aminoblycosides Gentamicin 200 - 400 Neomycin 400 - 800 Sulfonamides Sulfadiazine 25 - 100 Sulfamethazine 50 - 200

The data indicated in the table are based upon readings judged as being yellow/blue to blue and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found. Data in the table may be used for validation purposes. Definitions Drugs: Active ingredient of cattle medication such as antibiotics and Sulfonamides. Spectrum: An indication of the number of different active ingredients that can be detected by a given method at or below the required detection level. Detection level: The lowest concentration at which a drug is still detected by the test. Control time: Time at which the test agar containing the negative a control sample has just changed to yellow. At this time the best sensitivity for the test system is obtained.

40

12 Charm II (Neomycin Type) Aminoglycoside Test

Name of the test: Charm II Aminoglycoside Test for Gentamicin and Neomycin

Presentation: GTBL-100K: 100 test for T/SL GTBL-500K: 500 tests for T/SL GMRL-100K: 100 tests for MRL GMRL-500K: 500 tests for MRL

Group of antibiotics detected: Gentamicin and Neomycin

Type of reaction: Rapid microbial receptor assay

Time per analysis: 12-16 minutes, plus 1 minute per sample

Number of samples at a time: Up to 12 samples per assay

Detection limits: See Table

Equipment: Test Tubes,13x100mm Corks, 13mm Centrifuge Mixer Incubator, Inctronic II Cotton Swabs

Lorry incubator availability : No

Special equipment: Charm 6600 analyzer

Special reagents: Scintillation Fluid, Optifluor Zero Control Standard Positive Control Standard, Multi-Antimicrobial

Applicable at: Lorry: No

Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc. 659 Andover St. Lawrence, MA 01843-1032 USA Tel +1 978 687 9200


Charm Sciences Inc Technical Support [email protected]

Internet site: www.charm.com

Reference: Technical information from Charm Sciences, Inc. Charm et al. J.AOACVol.71,N.2 1988

GENERAL DESCRIPTION

41

Charm II Aminoglycoside Test for Gentamicin and Neomycin is a rapid microbial receptor assay that detects gentamicin and neomycin in milk. The test consists of a white microbial receptor tablet and a yellow [3H] labeled gentamicin tablet. The Charm II Aminoglycoside Test uses bacteria with specific receptor sites that bind gentamicin and neomycin type aminoglycosides. The bacteria are added to a skimmed 5 ml milk sample along with [3H] labeled gentamicin. Any aminoglycoside already in the milk, upon incubation at 35°C, competes with this labeled gentamicin for the binding sites. The amount of [3H]-gentamicin that binds to the receptor sites is measured and compared to a previously determined control point or to a standard curve. The greater the amount of [3H]-gentamicin measured, the lower the aminoglycoside concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Foil-backed plastic blister card, with 10 tests (10 receptor tablets and 10 [3H] labeled gentamicin tablets) per card, and each tablet in a separate blister. Cards can be separated into single tests dependent on the number of tests to be run.

CHARM II Aminoglycoside Test LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit (MRL)

Detection limit (T/SL)

Gentamicin 96 24 Neomycin 40 10

42

13 Charm II (Streptomycin Type) Aminoglycoside Test

Name of the test: Charm II Aminoglycoside Test for Gentamicin, Streptomycin, and Dihydrostreptomycin (Competitive Assay) for Raw, Commingled Bovine Milk

Presentation: STTBL-100 or STMRL-100: 100 Tracer and binder tablets with zero and Strep positive controls for US or MRL levels. STTBL-500 or STMRL-500: 500 Tracer and binder tablets with zero and Strep positive controls for US or MRL levels.

Group of antibiotics detected: Streptomycin Type Aminoglycoside Drugs



Number of samples at a time: 6 samples per assay


Equipment: 300uL pipette 5 mL pipette 13x100mm borosilicate Test Tubes Corks Vortex 35°C Inctronic II Timer Centrifuge Cotton Swabs



Special reagents: Multi-Antimicrobial Positive Control Standard (Contains 30ppb Gentamicin) Positive standard and Zero Control Standard powdered milk supplied with kits. Optifluor scintillation fluid


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc.


Charm Sciences Inc. Technical Support [email protected]


43

Reference: Tech. Info. from Charm Sciences Inc. Charm et al. J.AOACVol.71,N.2 1988

GENERAL DESCRIPTION The Charm II Aminoglycoside Drug Test uses bacteria with specific receptor sites that bind all streptomycin type aminoglycoside drugs. The bacteria are added to a pre-spun milk sample along with an exempt amount of [3H] labeled streptomycin. Any streptomycin type aminoglycoside already in the milk competes for the binding sites with this labeled streptomycin. The amount of [3H]-streptomycin that binds to the receptor sites is measured and compared to a previously determined control point or to a standard curve. The greater the amount of [3H]-streptomycin measured, the lower the aminoglycoside drug concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during incubation. Binder tablet is centrifuged and rehydrated with Scintillation fluid. 3H CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative.

CHARM II AMINOGLYCOSIDE TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic MRL Protocol US Protocol Gentamicin 50 25 Streptomycin 40 20 Dihydrostreptomycin 150 75

44

14 Charm II Amphenicol Test

Name of the test: Charm II Test for Chloramphenicol and other Amphenicols (Competitive Assay) for Raw and Pasteurized, Cow Milk

Presentation: CAP-100K: 100 test tablets and negative and positive controls

Group of antibiotics detected: Chloramphenicol and other Amphenicols

Type of reaction: Rapid Radioimmunoassay




Equipment: 300ul pipette 5 mL pipette 13x100mm borosilicate test tubes Corks Vortex Ice bath Thermometer Centrifuge



Special reagents: Optifluor Scintillation Fluid Zero Control Standard and 1 ppb Chloramphenicol Positive Control Standard supplied with kits


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Tech. Info. from Charm Sciences Inc. Screening Methods for use under Appendix N of PMO- http://www.cfsan.fda.gov/~ear/mi92-11.html

GENERAL DESCRIPTION:

45

The Charm II Test for Chloramphenicol and other Amphenicols is a rapid radioimmunoassay that detects amphenicols in milk. The test uses antibody with specific sites that bind amphenicol drugs. The antibody are added to milk along with an exempt amount of [3H] labelled chloramphenicol. Amphenicols that are present in the milk then compete with the [3H]-chloramphenicol for the binding sites. The unbound [3H]-chloramphenicol is removed with charcoal absorption, so that the bound anti-body is measured and compared to a previously determined control point or to a standard curve. The greater the amount of [3H]-chloramphenicol measured, the lower the amphenicol drug concentration in the sample. This method has been performance tested by an independent laboratory. The control point to determine negative or suspect positive is set to detect chloramphenicol at or below the established US action level (1ppb). This test is designed for use by milk, intake, laboratory, field and regulatory personnel. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during 4ºC incubation. Charcoal tablet is centrifuged and 300ul supernatant rehydrated with Scintillation fluid. 3H CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative.

CHARM II AMPHENICOL TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Chloramphenicol 1 Florfenicol 40 Thiamphenicol 50 Chloramphenicol glucuronide 1

46

15 Charm II Beta-lactam (Sequential)Test

Name of the test: Charm II Beta-lactam Test for Amoxicillin, Ampicillin, Ceftiofur, Cephapirin and Penicillin (Sequential Assay) for Commingled Raw and Pasteurized Cow and Goat Milk

Presentation: TBL8-100 or TBL8MRL-100: 100 Tracer and binder tablets with zero and PenG positive controls for US or MRL levels. TBL8-500 or TBL8MRL- 500: 500 Tracer and binder tablets with zero and PenG positive controls for US or MRL levels.

Group of antibiotics detected: Beta-lactams.


Time per analysis: Approximately 12 minutes

Number of samples at a time: 6

Detection limits: Beta-lactams at MRL or US Safe Level- See Table

Equipment: 300uL pipette 5 mL pipette 100mm borosilicate Test Tubes Charm II 6600 analyzer Charm Inctronic 2 Vortex Cotton Swabs Centrifuge.

Lorry incubator availability: No

Special equipment: Charm II 6600 analyzer, Charm Inctronic 2

Special reagents: 0.0080 IU/ml Penicillin Standard with TBL8 kits and 4ppb Pencillin G standard with TBL8MRL kits. Positive standard and Zero Control Standard powdered milk supplied with kits. Optifluor scintillation fluid


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm sciences, inc.



47


Reference: Tech. Info. from Charm Sciences Inc. Charm et al. J.AOACVol.71,N.2 1988 Beta-Lactam Methods for use under Appendix N of Past. Milk Ordinance- http://www.cfsan.fda.gov/~ear/m-a-85.html D.D. Muir - Milk Quality Forum, 19 Cornwall Terrace, London, NW1 4QP B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005 Zeng et al. J. Food Prot. 61-3 1998 Contreras et al. J. Dairy Sci. V.80 1997

GENERAL DESCRIPTION The Charm II Beta-lactam Test uses bacteria with specific receptor sites that bind all beta-lactam drugs. The bacteria are added to a milk sample along with an exempt amount of [14C] labeled Penicillin. Any beta-lactam already in the milk competes for the binding sites with this labeled Penicillin. The amount of [14C]-Penicillin that binds to the receptor sites is measured compared to a previously determined control point or to a standard curve. The greater the amount of [14C]-Penicillin measured, the lower the beta-lactam concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during incubation. Binder tablet is centrifuged and rehydrated with Scintillation fluid. 14C CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative.

48

CHARM II BETA-LACTAM TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic MRL Protocol US SL Protocol Amoxicillin 4-5 8.1 Ampicillin 3-4 6.6 Ceftiofur (total Metabolite) 20-40 40 Cephapirin 3-4 4.1 Penicillin 2-3 3.4 Cefazolin 10-15 ND Cefquinomee 15-20 ND Cloxacillin 25-35 ND

Dicloxacillin 15-20 ND

Nafcillin 20-30 ND

Oxacillin 25-35 ND

Penethamate 2-3 ND ND= Not Determined

49

16 Charm II Chloramphenicol Test

Name of the test: Charm II Test for Chloramphenicol for Raw Commingled, Cow Milk

Presentation: AIIHM-100K: 100 test tablets and negative and positive controls

Group of antibiotics detected: Chloramphenicol specific

Type of reaction: Rapid Radioimmunoassay




Equipment: 300ul pipette 5 mL pipette 13x100mm borosilicate test tubes Corks Vortex Incubator 50°C Thermometer Centrifuge



Special reagents: Optifluor Scintillation Fluid Zero Control Standard and Multi-standard containing 0.3 ppb Chloramphenicol supplied with kits


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: GENERAL DESCRIPTION: The Charm II Test for Chloramphenicol is a rapid radioimmunoassay that detects chloramphenicol in milk. The test uses antibody with specific sites that bind chloramphenicol. The antibody are added to milk along with an exempt amount of [3H] labelled chloramphenicol. Chloramphenicol present in the milk then compete with the [3H]-chloramphenicol for the binding sites. The bound [3H]-chloramphenicol is removed with centrifugation and analyzed. The greater the amount of [3H]-chloramphenicol measured, the lower the chloramphenicol drug concentration in the sample. The control point to determine negative or suspect positive is set to detect chloramphenicol at MRL

50

performance levels (0.1ppb). This test is designed for use by milk, intake, laboratory, field and regulatory personnel. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during 50ºC incubation. The binding tablet is centrifuged and 300ul supernatant rehydrated with Scintillation fluid. 3H CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative.

CHARM II CHLORAMPHENICOL TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Chloramphenicol 0.1

51

17 Charm II Macrolide Test

Name of the test: Charm II Macrolide Test for Macrolides at US Safe Level/Tolerance and MRL

Presentation:

ETBL-100 or MMRL-100: 100 test kit including positive and negative control ETBL-500 or MMRL-500: 500 test kit including positive and negative control

Group of antibiotics detected: Macrolides

Type of reaction: Bacterial receptor radio-label assay-sequential assay

Time per analysis: 10-15m


Detection limits: US Safe Levels and MRL, See Table

Equipment: Charm Inctronic Incubator 1-5mL pipette Tablet punch


Special equipment: Charm 6600 system.

Special reagents:

Multi-antibiotic positive contains 50 ppb Erythomycin with ETBL kits and 100 ppb spiramycin with MMRL kits. Positive standard and Zero Control Standard powdered milk supplied with kits. Optiflour scintillation fluid


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc


Charm Sciences Technical Support [email protected]


Reference: Tech. info. from Charm Sciences Inc. Charm et al. J.AOACVol.71,N.2 1988

GENERAL DESCRIPTION The Charm II Macrolide Test uses bacteria with specific receptor sites that bind all macrolide drugs. The bacteria are added to a milk sample along with an exempt amount of [14C] labeled

52

erythromycin. Any macrolide or lincomycin already in the milk competes for the binding sites with this labeled erythromycin.

The amount of [14C]-erythromycin that binds to the receptor sites is measured compared to a previously determined control point or to a standard curve. The greater the amount of [14C]-erythromycin measured, the lower the macrolide concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during incubation. Binder tablet is centrifuged and rehydrated with Scintillation fluid. 14C CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative.

TEST CHARM II MACROLIDE LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Erythromycin 40 Lincomycin 100 Pirlimycin 80 Spiramycin 50 Tilmicosin 20 Tylosin 50

53

18 Charm II Novobiocin Test

Name of the test: Charm II Test for Novobiocin

Presentation: NTBL-100K: NTBL-500K:

Group of antibiotics detected: Novobiocin






Lorry incubator availability: No


Special reagents: Optifluor Scintillation Fluid Zero Control Standard 100 ppb Novobiocin Positive Control Standard


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Technical Information from CSI Charm et al. J.AOACVol.71,N.2 1988

GENERAL DESCRIPTION

54

The Charm II Test for Novobiocin is a rapid microbial receptor assay that detects novobiocin in milk. The control point to determine negative or suspect positive is set to detect novobiocin at or below the U.S. tolerance/safe level (T/SL) of 100ppb. This test is designed for use by dairy, veterinary, laboratory, field and regulatory personnel. GENERAL TEST FORMAT/PRESENTATION Available in 100 count or 500 count kits. One test incorporates one “binder” tablet paired with one “tracer” tablet. Tablets are packaged in blister-pack foil in sets of 10, perforated by test. White tablets contain novobiocin-specific bacterial receptors. Each blue tablet contains less than 1.85 kilobecquerels (kBq) [3H]-novobiocin. These tablets are also indicated by printing on the packaging “N->”.

CHARM II NOVOBIOCIN LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Novobiocin 100

55

19 Charm II Sulfa Drug Test

Name of the test: Charm II Sulfa Drug Test for Sulfadiazine, Sulfadimethoxine, Sulfamethazine, and Sulfathiazole (Competitive Assay) for Raw, Commingled Bovine Milk

Presentation: SULFA-100 or SULFAMRL-100: 100 Tracer and binder tablets with zero and SMZ positive controls for US or MRL levels. SULFA-500 or SULFAMRL-500: 500 Tracer and binder tablets with zero and SMZ positive controls for US or MRL levels.

Group of antibiotics detected: Sulfonamide and Disulfone Drugs




Detection limits: US or MRL levels, see table

Equipment:

300uL pipette 5 mL pipette 13x100mm borosilicate Test Tubes Corks Vortex 85°C Inctronic II Timer Centrifuge Cotton Swabs



Special reagents: 10ppb Sulfamethazine Positive Control Standard Positive standard and Zero Control Standard powdered milk supplied with kits. Optifluor scintillation fluid


Farm: No

Milk reception: Yes

Laboratory: Yes




56


Reference: Tech. Info. from Charm Sciences Inc. Charm et al. J.AOACVol.71,N.2 1988 Charm et al. J.Food Prot. 51-12 1988 Methods for use under Appendix N of Past. Milk Ordinance- http://www.cfsan.fda.gov/~ear/m-a-85.html AOAC Referee- Volume 19 no.8 1995 B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005

57

GENERAL DESCRIPTION The Charm II Sulfa Drug Test uses bacteria with specific receptor sites that bind all sulfa drugs. The bacteria are added to a milk sample along with an exempt amount of [3H] labeled sulfamethazine. Any sulfa drug already in the milk competes for the binding sites with this labeled sulfamethazine. The amount of [3H]-sulfamethazine that binds to the receptor sites is measured and compared to a previously determined control point or to a standard curve. The greater the amount of [3H]-sulfamethazine measured, the lower the sulfa drug concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Reagents are tableted and packaged in blister packaging. Tablets are pushed into test tubes where they are rehydrated and reacted with milk during incubation. Binder tablet is centrifuged and rehydrated with Scintillation fluid. 3H CPM detection with a Charm II analyzer is compared to a control point determined with reference positive control for determination of positive or negative. MRL positive levels confirmed by dilution and retesting.

CHARM II SULFA DRUG TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic MRL Procedure US Procedure Sulfadiazine 49 4.9 Sulfadimethoxine 40 4.0 Sulfamethazine 94 9.4 Sulfathiazole 73 7.3 Dapsone 20 2 PABA 30 3 Sulfacetamide 500 50 Sulfachlorpyridazine 50 5 Sulfadoxine 70 7 Sulfamerazine 50 5 Sulfamethizole 150 15 Sulfamethoxazole 30 3 Sulfamethoxypyridazine 50 5 Sulfapyridine 100 10 Sulfaquinoxaline 30 3 Sulfisoxazole 60 6

58

20 Charm II Tetracycline Test

Name of the test: Charm II Tetracycline Test for Chlortetracycline, Oxytetracycline and Tetracycline (Competitive Assay) Operator's Manual for Raw, Commingled, Bovine Milk

Presentation: TET2-100-DK: 100 Binder and Tracer Tablets with zero and positive controls and W/Dilution Standards for MRL limits. TET2-100K: 100 Binder and Tracer Tablets with zero and positive controls TET2-500DK: 500 Binder and Tracer Tablets with zero and positive controls and W/Dilution Standards for MRL limits. TET2-500K: 100 Binder and Tracer Tablets with zero and positive controls.

Group of antibiotics detected: Tetracycline Drugs


Time per analysis: 12 Minutes

Number of samples at a time: 6 Samples Per Assay

Detection limits: U.S or MRL levels See Table




Special reagents: Optifluor Scintillation Fluid Zero Control Standard Multi-Antimicrobial Positive Control Standard


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences Inc. 659 Andover St. Lawrence, MA 01843

59

Contact person/Email Charm Sciences Inc. Technical Support [email protected]


Reference: Tech. Info. from Charm Sciences Inc. Charm et al. J.AOACVol.71,N.2 1988 Methods for use under Appendix N of Past. Milk Ordinance- http://www.cfsan.fda.gov/~ear/m-a-85.html AOAC Referee- Volume 20 no.1 1996 B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005

60

GENERAL DESCRIPTION The Charm II Tetracycline Drug Test uses antibodies that bind all tetracycline drugs. The antibodies are added to a milk sample along with an exempt amount of (3H) labeled tetracycline. Any tetracycline drug already in the milk competes for the antibodies with this labeled tetracycline. The amount of (3H) tetracycline that binds to the antibodies is measured and compared to a previously determined control point. The greater the amount of (3H) tetracycline measured, the lower the tetracycline drug concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Available in 100 count or 500 count kits. One test incorporates one “binder” tablet paired with one “tracer” tablet. Tablets are packaged in blister-pack foil in sets of 10, perforated by test. White tablets contain Tetracycline-specific bacterial receptors. Each orange tablet contains less than 1.85 kilo Becquerel’s (kBq) [3H]-tetracycline.

CHARM II TETRACYCLINE TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Chlortetracycline 28 Oxytetracycline 19 Tetracycline 5

61

21 Charm Blue-Yellow Test

Name of the test: Charm Blue-Yellow Test Inhibition test for milk

Presentation: FARM-BY-192K: 2 plates, 192 tests

Group of antibiotics detected: Five Families of Antibiotics

Type of reaction: Microbial inhibition in multiwell format

Time per analysis: Approx 2hr 45minutes

Number of samples at a time: 4 sample for each cowside incubator. Unlimited samples with air incubator.


Equipment: Charm cowside incubator or 64+1°C air Incubator 50uL pipette forceps

Lorry incubator availability : Yes- 24V Cowside incubator

Special equipment: NA

Special reagents: 4 ppb PenG Positive Control Standard


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: Technical information from CSI D.D. Muir – (FARM test) Milk Quality Forum, 19 Cornwall Terrace, London, NW1 4QP

62

GENERAL DESCRIPTION Charm Blue Yellow test consists of a multiwell plate with agar medium seeded with a standardized number of B. Stearothermophilus spores. Bacteria, cultured in a microplate well with milk, generate acid that is indicated by color using a pH indicator dye. GENERAL TEST FORMAT/PRESENTATION Plates consisting of 96 Polypropylene wells may be tested in strips of 8 samples or may be further divided and incubated/tested individually. 50ul milk added to test. Test is sealed with plastic strip and incubated for specified time and the color observed to determine positive or negative.

CHARM BLUE-YELLOW TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 3-4 Amoxicillin 5-6 Ampicillin 5 Ceftiofur 50-100 Cloxacillin 30-50 Cephapirin 10 Oxytetracycline 300 Sulfadimethoxine 50 Gentamicin 300-400 Tylosin 75-100 Drugs listed are commonly used drugs in 5 antibiotic families. Other drugs in those families will also be detected. Sensitivity to other drugs such as chloramphenicol, enrofloxacin, streptomycin and tiamulin exceeds 1000ppb.

63

22 Charm BSDA

Name of the test: Charm (BSDA) B. Stearothermophilus Disc Assay - Inhibition test for milk

Presentation: BSDA-1000K: Reagents and supplies to perform 1000 tests. BSDA-3000K: Reagents and supplies to perform 3000 tests.

Group of antibiotics detected: Beta-lactam and other antibiotics/inhibitors

Type of reaction: Seeded lawn with microbial inhibition around a paper disc absorbed with sample


Number of samples at a time: 7 ½” discs per 100mm plate. Unlimited samples with air incubator.

Detection limits: US safe levels of beta-lactam, see Table

Equipment: 64+1°C air Incubator 90uL pipette Forceps Hot plate


Special equipment: Calipers for measuring zone size.

Special reagents (supplied in kits): 5 ppb PenG Positive Control Standard B. Stearothermophilus spore 100,000,000/tablets PM indicator agar 100mm Petri dishes ½” paper discs


Farm: No

Milk reception: No

Laboratory: Yes





Reference: Tech. info. from Charm Sciences Inc. Beta-Lactam Methods for use under Appendix N of Past. Milk Ordinance- http://www.cfsan.fda.gov/~ear/m-a-85.html Charm et al. IDF Analytical week-Toronto

64

1995 Zeng et al. J. Food Prot. 61-3 1998 Contreras et al. J. Dairy Sci. V.80 1997

GENERAL DESCRIPTION Charm BSDA test consists of a B. Stearothermophilus spore tablets, PM indicator agar, multiwell plate with agar medium seeded with a standardized number of B. Stearothermophilus spores. Bacteria generate acid as they grow and change pH indicator from purple to yellow. Purple zones of inhibition are generated around paper discs absorbed with antibiotic contaminated sample. This is the traditional antibiotic test referenced in IDF document 057, inhibitors in dairy products. GENERAL TEST FORMAT/PRESENTATION Plates are prepared by heating PM agar to 100C, cooling and seeding with spore tablet, 6ml pipetted into 100 mm disc. 90ul milk added to paper disc and applied to plate. Test incubated for specified time and the color/zone of inhibition is observed to determine positive or negative. Area of zone may be correlated to concentration when identity of drug causing zone is known.

CHARM TEST BSDA LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 3.8 Amoxicillin 7.5 Ampicillin 6.7 Ceftiofur (parent) 150 Cloxacillin 50 Cephapirin 11.7 Oxytetracycline 400 Gentamicin 500 Tylosin 200 Beta-lactam Drugs listed are 90% positive levels

with 95% confidence. Other drugs are representative drugs from other antibiotic

families. Other drugs in those families will also be detected. Sensitivity to drugs in other families

such as sulfa drugs, chloramphenicol, enrofloxacin, streptomycin and tiamulin exceed

1000ppb.

65

23 Charm Cowside

Name of the test: Charm Cowside Inhibitor Test

Presentation: Cowside-20 20 Tests Cowside-80 80 Tests

Group of antibiotics detected: Broad Spectrum

Type of reaction: Microbial Inhibition

Time per analysis: Approximately 2 Hrs 30 Min

Number of samples at a time: 4-8 Samples for each charm incubator. Supported by other dry incubators. Multiple samples with air incubator/water bath.


Equipment: Charm cowside incubator or 64+1° air incubator or water bath 100uL pipette

Lorry incubator availability : Yes (24v unit available)


Special reagents: Positive and Negative Control Standards sold separately


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: Technical Information from CSI D.D. Muir – (FARM-V)Milk Quality Forum, 19 Cornwall Terrace, London, NW1 4QP

66

GENERAL DESCRIPTION Charm Blue Yellow test consists of an agar medium seeded with a standardized number of Bacterial spores. Bacteria, cultured in a microplate well with milk, generate acid that is indicated by color using a pH indicator dye. If color fails to change, sample is interpreted as containing inhibitor/antibiotics. GENERAL TEST FORMAT/PRESENTATION Vials filled with seeded agar may be tested individually. Sample is added through pierced foil seal and vial is incubated for specified time. Color is observed to determine positive or negative.

CHARM COWSIDE TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 3-4 Amoxicillin 6 Ampicillin 5 Ceftiofur 50-100 Cephapirin 10 Cloxacillin 30-50 Oxytetracycline 200-300 Sulfamethazine 100-200 Sulfadimethoxine 50 Gentamicin 300-400 Tylosin 75-100 Pirlimycin 100-200

Drugs listed are commonly used drugs in 5 antibiotic families. Other drugs in those families will also be detected. Sensitivity to other drugs

such as chloramphenicol, enrofloxacin, streptomycin and tiamulin exceeds 1000ppb.

67

24 Charm Blue-Yellow Test

Name of the test: Charm Blue-Yellow Test Inhibition test for milk

Presentation: FARM-BY-192K: 2 plates, 192 tests

Group of antibiotics detected: Five Families of Antibiotics

Type of reaction: Microbial inhibition in multiwell format


Number of samples at a time: 4 sample for each cowside incubator. Unlimited samples with air incubator.


Equipment: Charm cowside incubator or 64+1°C air Incubator 50uL pipette forceps

Lorry incubator availability : Yes- 24V Cowside incubator


Special reagents: 4 ppb PenG Positive Control Standard


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: Technical information from CSI D.D. Muir – (FARM test) Milk Quality Forum, 19 Cornwall Terrace, London, NW1 4QP

68

GENERAL DESCRIPTION Charm Blue Yellow test consists of a multiwell plate with agar medium seeded with a standardized number of B. Stearothermophilus spores. Bacteria, cultured in a microplate well with milk, generate acid that is indicated by color using a pH indicator dye. GENERAL TEST FORMAT/PRESENTATION Plates consisting of 96 Polypropylene wells may be tested in strips of 8 samples or may be further divided and incubated/tested individually. 50ul milk added to test. Test is sealed with plastic strip and incubated for specified time and the color observed to determine positive or negative.

CHARM BLUE-YELLOW TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 3-4 Amoxicillin 5-6 Ampicillin 5 Ceftiofur 50-100 Cloxacillin 30-50 Cephapirin 10 Oxytetracycline 300 Sulfadimethoxine 50 Gentamicin 300-400 Tylosin 75-100 Drugs listed are commonly used drugs in 5 antibiotic families. Other drugs in those families will also be detected. Sensitivity to other drugs such as chloramphenicol, enrofloxacin, streptomycin and tiamulin exceeds 1000ppb.

69

25 Copan Milk Test (CMT)

Name of the test: Copan Milk Test (CMT)

Presentation: CMT Single Test format CMT Microplate format

Group of drugs detected: Betalactams, Sulfonamides, Tetracyclines and others.

Type of reaction: CMT combines the principle of agar diffusion with the reduction of an indicator pigment by microbial inhibition.

Time per analysis: Fixed time of 3 hours

Number of samples at a time: 10 single tests per dry heat incubator No limitation when incubated in water bath

Detection limits: As per table below.

Equipment: Water bath or Dry Heat Block

Lorry incubator availability : Available

Special equipment: C-Scan automatic reading system both for Microplate and Single Test versions

Special reagents: Not necessary

Reference milk availability with known level of antibiotics

Lyophilized control samples available at: 4ppB PenG , 100ppB Sulfadiazine and 400ppB Oxytetracycline


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Copan Italia Spa Via Perotti, 10 25125 Brescia Italy Tel. +39 030 2687211 [email protected] www.copaninnovation.com


Daniela Brignocchi [email protected]

Internet site: www.copaninnovation.com

Reference:

70

GENERAL DESCRIPTION Copan Milk Test (CMT) is a microbiological ready to use test with large spectrum sensitivity. The test is applicable as screening method for the detection of antibiotic, Sulfonamides and tetracycline residues, at lower than MRL concentration, in milk samples. It is confirmed the suitability for milk from cow, ewe and goat (see Annexe as reference); substrate: raw milk, heat-treated milk, milk powder. CMT combines the principle of agar diffusion with the reduction of an indicator pigment. The absence of inhibitory substances in the milk sample is shown by a colour change, instead, there is no colour change when inhibitory substances are present in concentration above the limit of detection of the test. A micro-organism, particularly sensible to inhibition by penicillin the Bacillus Stearothermophilus var. Calidolactis, is trapped in spore form in an agar gel matrix, containing nutritive substances and a pH indicator. When the milk sample is added and the test is submitted to the optimal temperature, the spore can germinate, grow and duplicate. The normally growing cells produce acid that causes change in the pH of the gel and consequently the pH indicator colour changes from purple to yellow. Instead, if the growth is inhibited by the action of antimicrobials in the milk sample, the acid produced does not cause a significant change in the gel pH and as a result the pH indicator colour remains purple. GENERAL TEST FORMAT/PRESENTATION MICROPLATE FORMAT The transparent Polypropylene microplate preserves the test from dehydration enabling a long shelf-life of 12 months. The cartesian plane reproduced on the micro-plate borders, uniquely identifies each well by a combination of a number / letter label. CMT is ready to use, the nutrient are incorporated inside the media therefore no tablet addition or activation is necessary. Each microplate is breakable in sixteen wells strips, enabling the analysis of a number of samples less than 96. The kit also contains adhesive plastic films for closing the micro-plate before incubation in water bath. SINGLE TEST FORMAT The transparent Polypropylene vial preserves the test from dehydration enabling a long shelf-life of 12 months. CMT is ready to use, the nutrient are incorporated inside the media therefore no tablet addition or activation is necessary. Each vial represents one test and patented “no drop count” pipets included in the kit guarantee accurate milk sample inoculation. Foam floating racks are available for water bath incubation. Single test format is available either in box of 100 tests or 25 tests.

71

COPAN MILK TEST


Antimicrobial drugs

At control time

3 hours incubation

Beta-lactams Penicillin-Na salt 2 2-3 Ampicillin 2 4 Amoxicillin 2 3-4 Cloxacillin 15 25 Dicloxacillin 10 20 Oxacillin 5 10 Naficillin 5 10 Ceftiofur 50 50-70 Cefquinomee 75 100 Cefapirin 5-10 10 Cefoperazonee 40 60 Cephalexin 50 60-70 Cefalonium 8 8-10 Cephacetrile 20 25 Cefazolin 15 20-25 Tetracyclines Chlortetracycline 300 350-400 Oxytetracycline 250 300-350 Tetracycline2) 200 250-300 Sulfonamides Sulfathiazole 50 50-100 Sulfamethazine5) 100 100-150 Sulfadoxine 100 100-200 Sulfadimethoxine 50 50-100 Sulfadiazine 50 50-100 Sulfamethoxazole 50 50-100 Sulfamerazine 70 100 Sulfamonomethoxine 50 50-100 Sulfacetamide 100 100-150 Aminoglycosides Dihydrostreptomycin 1000 1000-2000 Streptomycin 1000 1000-2000 Gentamicin 250 250-350 Spectinomycin 3000 3000-4000 Neomycin 1000 1000-2000 Kanamycin 4000 4000-5000 Macrolides Erythromycin 250 250-350 Spiramycin 1000 1000-2000 Tylosin 50 50-100 Tilmicosin 75 75-100 Quinolones Flumequine >10000 >10000 Lincosamides Lincomycin 300 300-500 Other antibiotics Dapsone 2 2-4 Trimethoprim 50 50-100

72

Chloramphenicol 3000 3000-5000 Thiamphenicol 100 >100

1) Regulation 2377/90 ff EEC2) Mother compound; 3) Mother compound and metabolites; 4) Mother compound and 4-epimer; 5) Sulfadimidine; 6) Sum of all substances of this group; 7) Not allowed

73

26 Delvo-X-press

Name of the test: Delvo-X-press

Presentation: Tubes

Group of drugs detected: B-lactams

Type of reaction: Receptor enzyme assay


Number of samples at a time: 6 per incubator (7 including reference)


Equipment: IS(R)P workstation


Special equipment: No



Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer DSM Dairy Ingredients P.O. Box 1 2600 MA Delft The Netherlands



Internet site: www.dsm-dairy.com

Reference: DSM

74

GENERAL DESCRIPTION The Delvo-X-PRESS® ßL-II (ß-lactam) is a qualitative competitive receptor-enzyme assay designed to detect the presence of beta-lactam drug residues in commingled cow’s milk. A calibrated amount of TRACER is mixed with a fixed volume of milk sample in a COATED TUBE. The TRACER reacts with ß-lactams that may be present in the milk sample. Only free TRACER will bind to the coated tube. After the removal of the TRACER-ß-lactam complex by washing, a colour developer is added to detect residual TRACER bound to the coated tube. Colour development in the tube is inversely proportional to the ß-lactam concentration in the sample. The same procedure is carried out with a beta-lactam standard diluted in milk. By comparing the intensity of the colour in the sample tube with the standard tube in a reader, the presence or absence of ß-lactam residue in the milk sample can be determined. GENERAL TEST FORMAT/PRESENTATION A test kit Delvo-X-PRESS® ßL-II contains 96 test tubes and all necessary ready-for-use reagents; 96 Coated Tubes 1 bottles lyophilised Standard 1 bottles Standard diluent 1 bottle TRACER 1 bottle 50x concentrated Wash Solution 1 bottle Colour Developer The test is executed with ISR or ISRP-workstation ensuring correct incubation temperatures and times (3 minutes at 64°C and 3 minutes at room temperature) under constant shaking of test ampoules and tubes. The results are specified as Reader Units as measured by the ISR/ISRP-reader system.

75

DELVO-X-PRESS LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 2-4 Ampicillin 4-8 Amoxicillin 4-8 Cloxacillin 30-60 Cephapirin 4-8 Ceftiofur 4-8 Penicillin procaine 3-5 Hetacillin 6-10 Penicillin-V 3-5 Piperacillin 5-10 Cephalonium 3-4 Methicillin 10-20 Ticarcillin 30-100 Cefadroxil 5-25 Cefotaxime 4-5 Cefoperazonee 5-20 Cephalexin 25-50 Oxacillin 25-50 Dicloxacillin 25-50 Cephradine 25-50 Cefuroxime 4-20 Cephoxazole 75-100

The data indicated in the table are based upon readings by the ISR(P)-workstation as being < 00 Reader Units and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found

76

27 Delvotest SP- NT

Name of the test: Delvotest® SP- NT

Presentation: Delvotest SP-NT: 100 ampoules Delvotest SP-NT: 25 ampoules (mini) Delvotest SP-NT: 5 pack multi plate



Time per analysis: Reading time; 3 hrs Control time: starting from 2hrs and 15 minutes Delvotest® Accelerator: 100-105 minutes





Special equipment: Delvoscan software available



Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: DSM

77

GENERAL DESCRIPTION Delvotest® SP-NT is a standard diffusion test for the detection of residues of antibacterial substances (such as antibiotics and Sulfonamides) in milk. It has been developed for a variety of applications ranging from a single test for testing the milk of an individual cow at the farmhouse to large-scale routine analysis in milk quality payment schemes and milk control laboratories. The test consists of multi-test plates containing a solid agar medium seeded a standardised number of spores of Geobacillus stearothermophilus var. calidolactis together with required nutrients for growth purposes and an antifolate trimethoprim. The medium is coloured purple by the pH indicator bromocresol purple. Milk samples which are free from antibacterial substances, or contain them below specified levels will, when added to the test at the level of 0.1 ml and incubated at 64°C allow germination and growth of the bacteria. This will lead to a change in colour of the indicator from purple to yellow. When the milk sample contains antibacterial substances at or above the test sensitivity, growth is inhibited and as a result the colour remains purple. GENERAL TEST FORMAT/PRESENTATION Ampoules: Polypropylene ampoules in 1 block of 25 ampoules or 4 blocks of 25 ampoules (total of 100 ampoules). The ampoules can be separated into single test ampoules dependent on the number of tests to be executed. A dosage syringe and disposable pipettes for taking samples are included. Plates: Polypropylene multi-test plates consisting of 6 blocks of 16 wells each (5 multi test plates per pack). The blocks may be separated dependent on the number of tests to be executed. Adhesive covers for sealing of the plates during incubation are included.

78

DELVOTEST SP-NT LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotics Reading at the control

time/Accelerator Reading at

3 hours ß-lactams 1-2 2-3 Penicillin 4 6-7 Ampicillin 2-3 3-5 Amoxicillin 4-6 6-8 Cefapirin 25-50 50-100 Ceftiofur 20 20-30 Cloxacillin 10 10-20 Dicloxacillin 10 10 Oxacillin Sulfonamides 25-50 100-150 Sulfadiazine 25-100 100-250 Sulfamethazine Tetracyclines 250-500 800 Tetracycline 250-500 800 Oxytetracycline Macrolides 40-80 200 Erythromycin 30 50 Tylosin 400-600 800-1000 Spiramycin Aminoglycosides 50 200 Gentamicin 100-200 300-600 Neomycin Others 50-100 200-300 Trimethoprim 0.5-1 1-2.5 Dapsone

The data indicated in the table are based upon readings judged as being yellow/purple to purple and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found. Data in the table may be used for validation purposes. Definitions Drug : Active ingredient of cattle medication such as antibiotics and Sulfonamides. Spectrum : Total number of different active ingredients that can be detected by Delvotest® SP - NT at or below the required detection level. Detection level: The lowest concentration at which a drug is still detected by the test, at control

time or at a fixed reading time of 3 hours. Control time : Time at which the test agar containing the negative control sample has just changed to yellow.

79

28 Delvotest SP

Name of the test: Delvotest® SP

Presentation: Delvotest SP: 100 ampoules Delvotest SP: 25 ampoules (mini) Delvotest SP: 5 pack multi plate



Time per analysis: Reading time; 3 hrs Control time: starting from 2hrs 15 min








Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: DSM

80

GENERAL DESCRIPTION Delvotest® SP is a standard diffusion test for the detection of residues of antibacterial substances (such as antibiotics and Sulfonamides) in milk. It has been developed for a variety of applications ranging from a single test for testing the milk of an individual cow at the farmhouse to large-scale routine analysis in milk quality payment schemes and milk control laboratories. The test consists of multi-test plates containing a solid agar medium seeded a standardised number of spores of Geobacillus stearothermophilus var. calidolactis together with required nutrients for growth purposes and an antifolate trimethoprim. The medium is coloured purple by the pH indicator bromocresol purple. Milk samples which are free from antibacterial substances, or contain them below specified levels will, when added to the test at the level of 0.1 ml and incubated at 64°C allow germination and growth of the bacteria. This will lead to a change in colour of the indicator from purple to yellow. When the milk sample contains antibacterial substances at or above the test sensitivity, growth is inhibited and as a result the colour remains purple. GENERAL TEST FORMAT/PRESENTATION Ampoules: Polypropylene ampoules in 1 block of 25 ampoules or 4 blocks of 25 ampoules (total of 100 ampoules). The ampoules can be separated into single test ampoules dependent on the number of tests to be executed. One bottle with nutrient tablets, a pair of tweezers and a dosage syringe with disposable pipettes for taking samples are included. Plates: Polypropylene multi-test plates consisting of 6 blocks of 16 wells each (5 multi test plates per pack). The blocks may be separated dependent on the number of tests to be executed. One bottle with nutrient tablets and a sufficient number of adhesive covers for sealing of the plates during incubation are included.

81

DELVOTEST SP LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotics Reading at control

time

Reading between 2h45 and 3 hours

ß-lactams Penicillin 2 2.5 Ampicillin 2-3 3-5 Amoxicillin 2 3-5 Cephacetrile 20 20-40 Cephalexin 40-60 60-100 Cefalonium 5-10 10-25 Cefoperazonee 40 60-100 Cefapirin 5 5-10 Ceftiofur <50 50-70 Cloxacillin 15 15-25 Dicloxacillin 10 10-15 Oxacillin 5 10 Nafcillin 5 5-10 Sulfonamides Sulfadiazine 50 50-100 Sulfadimethoxine 50 50-100 Sulfamethazine 25 50-200 Sulfathiazole 50 50-150 Tetracyclines Tetracycline 100 200-600 Oxytetracycline 100 200-500 Chlortetracycline 100-150 200-600 Macrolides Erythromycin 50 100-250 Tylosin 10-20 30-100 Spiramycin 200 350-ns Aminoglycosides Gentamicin 100-300 200-500 Neomycin 100-200 300-2000 Dihydrostreptomycin 300-500 1500-10000 Kanamycin 2500 7500-ns Lincosamides Lincomycin 100 200-400 Others Chloramphenicol 2500 7500-10000 Trimethoprim 50 100-500 Dapsone 1 1-8

The data indicated in the table are based upon readings judged as being yellow/purple to purple and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found. Data in the table may be used for validation purposes.

82

Definitions Drugs:

Active ingredient of cattle medication such as antibiotics and sulfonamides.

Spectrum:

An indication of the number of different active ingredients that can be detected by a given method at or below the required detection level.

Detection level:

The lowest concentration at which a drug is still detected by the test, especially at control time and reading time.

Control time:

Time at which the test agar containing the negative control sample has just changed to yellow. At this time the best sensitivity for the test system is obtained.

Fixed Reading time:

For use without the requirement of running a negative control sample a fixed reading time between 2 hours 45 minutes and 3 hours can be used. This may decrease the sensitivity for bacteriostatic drugs.

83

29 Delvotest P

Name of the test: Delvotest® P

Presentation: Delvotest P: 100 ampoules Delvotest P: 25 ampoules (mini) Delvotest P: 5 pack multi plate

Group of drugs detected: Wide range of antibiotics


Time per analysis: Reading time; 2hrs 30 minutes Control time: starting from 2.15








Yes


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: DSM

84

GENERAL DESCRIPTION Delvotest® P is a standard diffusion test for the detection of residues of antibacterial substances in milk. It has been developed for a variety of applications ranging from a single test for testing the milk of an individual cow at the farmhouse to large-scale routine analysis in milk quality payment schemes and milk control laboratories. The test consists of multi-test plates containing a solid agar medium seeded a standardised number of spores of Geoacillus stearothermophilus var. calidolactis together with required nutrients for growth purposes and an antifolate trimethoprim. The medium is coloured purple by the pH indicator bromocresol purple. Milk samples which are free from antibacterial substances, or contain them below specified levels will, when added to the test at the level of 0.1 ml and incubated at 64°C allow germination and growth of the bacteria. This will lead to a change in colour of the indicator from purple to yellow. When the milk sample contains antibacterial substances at or above the test sensitivity, growth is inhibited and as a result the colour remains purple. GENERAL TEST FORMAT/PRESENTATION Ampoules: Polypropylene ampoules in 1 block of 25 ampoules or 4 blocks of 25 ampoules (total of 100 ampoules). The ampoules can be separated into single test ampoules dependent on the number of tests to be executed. One bottle with nutrient tablets, a pair of tweezers and a dosage syringe with disposable pipettes for taking samples are included. Plates: Polypropylene multi-test plates consisting of 6 blocks of 16 wells each (5 multi test plates per pack). The blocks may be separated dependent on the number of tests to be executed. One bottle with nutrient tablets and a sufficient number+ of adhesive covers for sealing of the plates during incubation are included.

85

DELVOTEST P


Antibiotics Reading at control

time Reading at 2h30 ß-lactams Penicillin 2 3 Ampicillin 2-3 3-5 Amoxicillin 2 3-5 Cephacetrile 20 20-40 Cephalexin 40-60 60-100 Cefalonium 5-10 10-25 Cefoperazonee 40 60-100 Cefapirin 8 8-10 Ceftiofur <50 50-70 Cloxacillin 15 25 Dicloxacillin 10-15 20 Oxacillin 5 10-15 Nafcillin 5 5-10 Tetracyclines Tetracycline 100 200-300 Oxytetracycline 100 200-300 Chlortetracycline 100-150 200-300 Macrolides Erythromycin 500 500-1500 Tylosin 50-100 100-300 Spiramycin 200 350-ns Aminoglycosides Gentamicin >500 >1000 Neomycin >500 >1000 Dihydrostreptomycin 300-500 1500-10000 Kanamycin 2500 7500-ns Lincosamides Lincomycin 400 400-1000 Others Chloramphenicol 2500 7500-10000 Trimethoprim 100 500-1500

The data indicated in the table are based upon readings judged as being yellow/purple to purple and are to be regarded as an indication. It is recommended to always confirm positive test results. Variations in incubation times and temperatures, operators and storage conditions may have an influence on the reading result found. Data in the table may be used for validation purposes. Definitions Drugs: Active ingredient of cattle medication such as antibiotics and sulfonamides. Spectrum:

An indication of the number of different active ingredients that can be detected by a given method at or below the required detection level.

Detection level:

The lowest concentration at which a drug is still detected by the test, especially at control time and reading time.

Control time:

Time at which the test agar containing the negative control sample has just changed to yellow. At this time the best sensitivity for the test system is obtained.

Fixed Reading time:

For use without the requirement of running a negative control sample a fixed reading time of 2 hours 30 minutes can be used. This may decrease the sensitivity for bacteriostatic drugs.

86

30 ECLIPSE FARM

Name of the test: ECLIPSE FARM

Presentation: ECLIPSE FARM 24: Pack of 24 single tubes ECLIPSE FARM 96: pack of 96 single tubes

Group of drugs detected: Wide range of antibiotics and Sulfonamides

Type of reaction: Bacterial inhibition growth

Time per analysis: 2.15-2.30 hours

Number of samples at a time: No limit

Detection limits: See table attached

Equipment: Incubator/Water bath

Lorry incubator availability: Yes (lorry adaptor can be used, but no provided )

Special equipment: None



Milk containing penicillin G with 5ppb

Applicable at: Lorry: yes

Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer ZEU-INMUNOTEC Maria de Luna 11, Building 19 50018 Zaragoza, SPAIN Tel:+34 976731533


[email protected]

Internet site: www.zeu-inmunotec.com

Reference: Technical note from ZEU-INMUNOTEC

87

GENERAL DESCRIPTION ECLIPSE FARM is a qualitative test for detection of inhibitors substances in raw, heated and powder milk. It is a simple method to check weather milk contains antibiotics at concentrations exceeding the Maximum Limits of Residues (MRL). The test is used as screening method for cow, sheep and goat milk allowing the detection of a broad-range of antibiotics in a large number of samples. Eclipse follows the method adopted by the European Union in the decision of the Commission 91/180/CEE on the 14th of February 1991. It is based on the growth inhibition of Geobacillus stearothermophilus var. calidolactis. The kit is supplied in single tubes, where each one contains agar medium spread with Geobacillus stearothermophilus spores containing a pH indicator. Heating at 65ºC will produce the spores’ germination and microorganism growth producing acid and changing the agar pH. The blue (purple) colour of the agar will turn to yellow with the G. sterarothermophilus growth as the agar pH decreases. Samples containing antibiotics at concentrations above the detection limit will inhibit the microorganisms growth and the colour of the agar will remain the same. GENERAL TEST FORMAT/PRESENTATION ECLIPSE FARM test kit is supplied in 1 block of 24 or 96 single tubes. The table below shows the limits of detection of ECLISE test for cow´s milk.

ECLIPSE FARM LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limitAmoxicillin 5 Ampicillin 5 Cephalexin 75 Cefapirin 8 Cloxacillin 40 Oxacillin 25 Penicillin 4 Oxytetracycline 150 Tetracycline 150 Erythromycin 400 Tylosin 100 Gentamicin 200 Neomycin 1500 Sulfamethazine 200 Sulfanilamide 600 Sulfathiazole 100

88

31 ECLIPSE 50

Name of the test: ECLIPSE 50

Presentation: ECLIPSE 50: 96-well microtiter plate divisible into single wells. Packs of 1, 3 plates or in bulk.




Number of samples at a time: 96 samples



Lorry incubator availability : Milk containing Penicillin with 5ppb




No


Farm: Yes

Milk reception: Yes

Laboratory: Yes



Elena Dominguez [email protected]


Reference: Technical report from ZEU-INMUNOTEC

89

GENERAL DESCRIPTION ECLIPSE is a qualitative test for detection of inhibitors substances in raw, heated and powder milk. It is a simple method to check weather milk contains antibiotics at concentrations exceeding the Maximum Limits of Residues (MRL). The test is used as screening method for cow, sheep and goat milk allowing the detection of a broad-range of antibiotics in a large number of samples. Eclipse follows the method adopted by the European Union in the decision of the Commission 91/180/CEE on the 14th of February 1991. It is based on the growth inhibition of Geobacillus stearothermophilus var. calidolactis. The kit is supplied in microtiter plate format, where each well contains agar medium spread with Geobacillus stearothermophilus spores containing a pH indicator. Heating at 65ºC will produce the spores’ germination and microorganism growth producing acid and changing the agar pH. The blue (purple) colour of the agar will turn to yellow with the G. sterarothermophilus growth as the agar pH decreases. Samples containing antibiotics at concentrations above the detection limit will inhibit the microorganisms growth and the colour of the agar will remain the same. GENERAL TEST FORMAT/PRESENTATION ECLIPSE test kit is supplied in 96-well microtiter plates. Plates can be divided into single wells allowing the end user to run just the exact number of samples required. Kits of 1 or 3 plates are available. For large number of test plates in bulk can be also provided. The table below shows the limits of detection of ECLISE test for cow´s milk.

90

ECLIPSE 50


Antibiotic Detection limitAmoxicillin 5 Ampicillin 5 Cephalexin 75 Cefapirin 8 Cefalonium 20 Ceftiofur 100 Cefazolin 50 Cloxacillin 40 Oxacillin 25 Penicillin 4 Doxycycline 100 Oxytetracycline 150 Tetracycline 150 Erythromycin 400 Tylosin 100 Spiramycin >400 Streptomycin 2000 Gentamicin 300 Neomycin 1500 Kanamycin >2000 Sulfadiazine 100 Sulfamethazine 200 Sulfamethoxypyridazine 100 Sulfanilamide 600 Sulfamethoxazole 50 Sulfathiazole 100 Lincomycin 400 Chloramphenicol 5000

91

32 ECLIPSE 100

Name of the test: ECLIPSE 100

Presentation: ECLIPSE 100: 96-well microtiter plate divisible into single wells. Packs of 1, 3 plates or in bulk.




Number of samples at a time: No limit. 96 samples per plate









Farm: Yes

Milk reception: Yes

Laboratory: Yes



[email protected]



92

GENERAL DESCRIPTION ECLIPSE is a qualitative test for detection of inhibitors substances in raw, heated and powder milk. It is a simple method to check weather milk contains antibiotics at concentrations exceeding the Maximum Limits of Residues (MRL). The test is used as screening method for cow, sheep and goat milk allowing the detection of a broad-range of antibiotics in a large number of samples. Eclipse follows the method adopted by the European Union in the decision of the Commission 91/180/CEE on the 14th of February 1991. It is based on the growth inhibition of Geobacillus stearothermophilus var. calidolactis. The kit is supplied in microtiter plate format, where each well contains agar medium spread with Geobacillus stearothermophilus spores containing a pH indicator. Heating at 65ºC will produce the spores’ germination and microorganism growth producing acid and changing the agar pH. The blue (purple) colour of the agar will turn to yellow with the G. sterarothermophilus growth as the agar pH decreases. Samples containing antibiotics at concentrations above the detection limit will inhibit the microorganisms growth and the colour of the agar will remain the same. GENERAL TEST FORMAT/PRESENTATION ECLIPSE test kit is supplied in 96-well microtiter plates. Plates can be divided into single wells allowing the end user to run just the exact number of samples required. Kits of 1 or 3 plates are available. For large number of test plates in bulk can be also provided. The table below shows the limits of detection of ECLISE test in cow´s milk.

93

ECLIPSE 100


Antibiotic Detection limit

Amoxicillin 5 Ampicillin 4 Cephalexin 75 Cefapirin 8 Cefalonium 20 Ceftiofur 100 Cefazolin 50 Cloxacillin 40 Oxacillin 25 Penicillin 4 Doxycycline 50 Oxytetracycline 150 Tetracycline 150 Erythromycin 500 Tylosin 80 Spiramycin >400 Streptomycin 3000 Gentamicin 300 Neomycin 1500 Kanamycin >2000 Sulfadiazine 150 Sulfamethazine 150 Sulfamethoxypyridazine 100 Sulfanilamide 500 Sulfamethoxazole 50 Sulfathiazole 100 Lincomycin 300 Chloramphenicol 5000

94

33 EIA Chloramphenicol

Name of the test: Chloramphenicol EIA 5091CAP1p

Presentation: EIA (12 x 8 break 4 wells)

Group of drugs detected: Chloramphenicol

Type of reaction: Competitive enzyme immunoassay ELISA

Time per analysis: 1.5 hour

Number of samples at a time: 40 samples in duplicate


Equipment: Micro titre plate reader,

Lorry incubator availability : -

Special equipment: Centrifuge

Special reagents: -


-

Applicable at: Lorry: -

Farm: -

Milk reception: -

Laboratory: X

Manufacturer EuroProxima


P.J.M van Wichen [email protected]

Internet site: www.europroxima.com

Reference:

TEST Chloramphenicol EIA 5091CAP1p LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Chloramphenicol direct 0.1 ng/ml Chloramphenicol after ethyl acetate extraction 0.01 ng/ml

95

34 EIA Enrofloxacin

Name of the test: Enrofloxacin EIA 5101ERFX1p


Group of drugs detected: Enrofloxacin





Equipment: Micro titre plate reader


Special equipment: Head over head rotor

Special reagents: Methanol


-


Farm: -

Milk reception: -

Laboratory: X





Reference:

TEST Enrofloxacin LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Enrofloxacin 6 ng/ml

96

35 Flumequine EIA

Name of the test: Flumequine EIA 5101FLUM1p

Presentation: EIA (12 x 4 wells)

Group of drugs detected: Flumequine







Special equipment: Head over head rotor, Centrifuge



-


Farm: -

Milk reception: -

Laboratory: X

Price: On request



P.J.M. van Wichen [email protected]


Reference:

TEST Flumequine EIA LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Flumequine 13 ng/ml

97

36 EIA Fluoroquinolones

Name of the test: Fluoroquinolones EIA 5101FLUQG1p


Group of drugs detected: Broad range of Fluoroquinolones







Special equipment: Head over head rotor, centrifuge



-


Farm: -

Milk reception: -

Laboratory: X





Reference:

98

TEST FLUOROQUINOLONES EIA LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Norfloxacin 3 Enrofloxacin 3 Ciprofloxacin 3 Nadifloxacin 3 Pefloxacin 4 Enoxacin 5 Piromidic acid 5 Lomefloxacin 8 Danofloxacin 5 Oxolinic acid 5

99

37 EIA Gentamicin

Name of the test: Gentamicin EIA 5111GEN1p


Group of drugs detected: Gentamicin








Special reagents: -


-


Farm: -

Milk reception: -

Laboratory: X





Reference:

TEST Gentamicin EIA 5111GEN1p LIMIT OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Gentamicin 2 ng/ml

100

38 EIA Neomycin

Name of the test: Neomycin EIA 5111NEO1p


Group of drugs detected: Neomycin








Special reagents: -


-


Farm: -

Milk reception: -

Laboratory: X





Reference:

TEST Neomycin EIA 5111NEO1p LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Neomycin 18

101

39 EIA Streptomycin

Name of the test: Streptomycin EIA 5111STREP1p


Group of drugs detected: Streptomycin








Special reagents: -


-


Farm: -

Milk reception: -

Laboratory: X





Reference:

TEST STREPTOMYCIN EIA LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Streptomycin 4 ng/ml

102

40 EIA Sulfamethazine

Name of the test: Sulfamethazine EIA 5101SUL1p


Group of drugs detected: Sulfamethazine








Special reagents: -


-


Farm: -

Milk reception: -

Laboratory: X





Reference:

TEST Sulfamethazine EIA LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Sulfamethazine 8 ng/ml

103

Name of the test: Sulfonamides EIA 5101SULM1p


Group of drugs detected: Sulfonamides








Special reagents: Ethyl acetate


-


Farm: -

Milk reception: -

Laboratory: X

Price: On request





Reference:

TEST Sulfonamides EIA LIMITS OF DETECTION IN MILK

Results in µg/kg

Detection limit Sulfonamides <2.5 ng/ml

104

Name of the test: Sulfaquinoxaline EIA 5101SQUI1p


Group of drugs detected: Sulfaquinoxaline








Special reagents: -


-


Farm: -

Milk reception: -

Laboratory: X

Price: On request





Reference:

TEST Sulfaquinoxaline EIA LIMITS OF DETECTION IN MILK

Results in µg/kg

Detection limit Sulfaquinoxaline 20 ng/ml

105

41 Euroclone Chloramphenicol ELISA

Name of the test: Chloramphenicol ELISA

Presentation: Chloramphenicol ELISA kit (96 tests) (cat n. A34811)


Type of reaction: Competitive type ELISA

Time per analysis: 2 h 30 min


Detection limits: 0,05 ppb ( raw and skimmed milk)

Equipment: Bench Centrifuge Micropipettes

Lorry incubator availability : None

Special equipment: ELISA plate or strip reader

Special reagents: no

Applicable at: Lorry: no

Farm: no

Milk reception: yes

Laboratory: yes

Manufacturer Euroclone Spa Via Figino 20/22 20016 Pero (Milano)- Italy tel +39 02 38195378 fax +39 02 38195248


Chiara Tarantino [email protected]

Internet site: www.euroclone.net

Reference: See Euroclone data sheet

106

GENERAL DESCRIPTION The Euroclone CHLORAMPHENICOL ELISA KIT is a direct competitive enzyme immunoassay for the quantitative analysis of Chloramphenicol in various matrices. The test is suitable for milk, liver, eggs and honey. The assay is performed in plastic microwells, which have been pre-coated with goat anti-rabbit IgG. Chloramphenicol standard or sample, enzyme-marked chloramphenicol and rabbit anti-chloramphenicol antibodies are added to microwells. During the incubation, anti-chloramphenicol antibodies are bound by the immobilized IgG; free and enzyme-marked chloramphenicol competes for the chloramphenicol antibodies binding sites. After allowing this reaction to proceed, the unbound material is removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of colorimetric substrate, which develops a blue colour in the presence of peroxidase. The colour development is inversely proportional to the original chloramphenicol concentration in the sample, which is determined by reading off a calibration curve derived from standards of known chloramphenicol concentration. GENERAL TEST FORMAT/PRESENTATION Chloramphenicol ELISA kit is suitable for 96 determinations. Kit contents ! 96 wells microtiter plate (twelve 8-wells strips) coated with antibodies to rabbit IgG. ! Chloramphenicol standard solutions, 5 vials containing 1 ml each: 0.05 ng/ml, 0.3 ng/ml,

1.0 ng/ml, 2.5 ng/ml, 10.0 ng/ml ! Anti-Chloramphenicol antibody, 1 vial containing 11 ml red cap ! Peroxidase conjugated Chloramphenicol (CAP- HRP) ! 40X solution, 1 amber vial containing 300 µl white cap ! Wash buffer 10x concentrate, 1 bottle containing 50 ml ! Citrate buffer, 1 vial containing 25 ml blue cap ! Chromogen, 1 vial containing 3 ml black cap ! Conjugate diluent buffer, 1 vial containing 10ml green cap ! Stop Solution, 1 vial containing 6 ml white cap ! Plastic envelope for plate temporary storage ! Product Manual

TEST Chloramphenicol ELISA LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limits Chloramphenicol 0,05

107

42 Euroclone KALIDOS TB

Name of the test: KALIDOS TB

Presentation: Kalidos TB (96 test) (cat n. A21611)

Group of drugs detected: Antibiotics and Sulfonamides


Time per analysis: 3 h



Equipment: Water bath (65°C) or dry heater block





Farm: yes

Milk reception: yes

Laboratory: yes






108

GENERAL DESCRIPTION Kalidos is supplied in a tubes filled with an agar medium. The agar is pre-seeded with spores of Bacillus stearothermophilus var. calidolactis and incorporates a fermentable sugar: glucose and a pH indicator: bromocresol purple. The test is ready to use with no need to activate the product with additional reagents. The operator simply pipets the cow milk sample directly onto the surface of the agar and then incubates the test at 65 °C in a water bath or a dry heater block for 3 hours. The sample quickly diffuses throughout the agar medium. If no antimicrobial substances are present in the milk or the concentration is lower then the limits of detection, the Bacillus spores germinate, grow and metabolize the sugar. The acid produced from the fermentation of glucose changes the color of the indicator bromocresol purple to a intense yellow color. Alternatively, if antimicrobial substances are present in the milk sample then germination and growth of the Bacillus spores are inhibited. In this case, there is no fermentation of glucose, no acid production and therefore the pH indicator in the medium remains purple. GENERAL TEST FORMAT/PRESENTATION The format is 96 tubes

TEST KALIDOS TB LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limits Penicillin 2-4 Ampicillin 4 Amoxicillin 4 Oxacillin 15-20 Cloxacillin 30-40 Dicloxacillin 15-20 Nafcillin 10-15 Streptomycin 400-600 Dihydrostreptomycin 200-400 Kanamycin 150 Neomycin 50 Erythromycin 200 Tylosin 40-50 Spiramicin 600-800 Rifampicin 100-150 Tetracycline 100-150 Oxytetracycline 100-150 Chlortetracycline 100-150 Chloramphenicol 2500-3500 Bacitracin 1500-3000 Sulfamethazine 100-150 Sulfadiazine 25-100 Sulfadimetoxina 25-100 Sulfamerazine 25-100 Sulfamethoxazole 25-100 Sulfaquinoxaline 25-100 Trimethoprim 200-400

109

43 Euroclone Total Antibiotics

Name of the test: Total Antibiotics

Presentation: Total Antibiotics (96 test)(cat n. A20111) Total Antibiotics (3X96 tests)(breakable microplates) (cat n. A20811)

Group of drugs detected: Antibiotics





Equipment: Water bath (65°C)





Farm: yes

Milk reception: yes

Laboratory: yes






110

GENERAL DESCRIPTION “Total Antibiotics” is supplied in a multi-well microplates (A20811) or tubes (A20111) filled with an agar medium. The agar is pre-seeded with spores of Bacillus stearothermophilus var. calidolactis and incorporates a fermentable sugar: glucose and a pH indicator: bromocresol purple. The test is ready to use with no need to activate the product with additional reagents. The operator simply pipets the milk sample directly onto the surface of the agar and then incubates the test at 65 °C in a water bath for 3 hours. The sample quickly diffuses throughout the agar medium. If no antimicrobial substances are present in the milk or the concentration is lower then the limits of detection, the Bacillus spores germinate, grow and metabolize the sugar. The acid produced from the fermentation of glucose changes the color of the indicator bromocresol purple to a intense yellow color. Alternatively, if antimicrobial substances are present in the milk sample then germination and growth of the Bacillus spores are inhibited. In this case, there is no fermentation of glucose, no acid production and therefore the pH indicator in the medium remains purple. GENERAL TEST FORMAT/PRESENTATION The format is 3 x 96 breakable microplates for A20811 and 96 tubes for A20111

TEST A20111/A20811 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 2.5

Ampicillin 4

Amoxicillin 4

Oxacillin 15

Cloxacillin 20

Dicloxacillin 15

Nafcillin 80

Streptomycin 350

Dihydrostreptomycin 500

Erythromycin 500

Tetracycline 100

Oxytetracycline 100

Chlortetracycline 100

Rifampicin 150

Rifamycin 25

Spiramycin 500

Bacitracin 0.300 (U/ml)

Kanamycin 10.00

Neomycin 2.500

Chloramphenicol 5.000

Tylosin 100

111

44 Euroclone KALIDOS TB

Name of the test: KALIDOS TB

Presentation: Kalidos TB (96 test) (cat n. A21611)






Equipment: Water bath (65°C) or dry heater block





Farm: yes

Milk reception: yes

Laboratory: yes






112

GENERAL DESCRIPTION Kalidos is supplied in a tubes filled with an agar medium. The agar is pre-seeded with spores of Bacillus stearothermophilus var. calidolactis and incorporates a fermentable sugar: glucose and a pH indicator: bromocresol purple. The test is ready to use with no need to activate the product with additional reagents. The operator simply pipets the cow milk sample directly onto the surface of the agar and then incubates the test at 65 °C in a water bath or a dry heater block for 3 hours. The sample quickly diffuses throughout the agar medium. If no antimicrobial substances are present in the milk or the concentration is lower then the limits of detection, the Bacillus spores germinate, grow and metabolize the sugar. The acid produced from the fermentation of glucose changes the color of the indicator bromocresol purple to a intense yellow color. Alternatively, if antimicrobial substances are present in the milk sample then germination and growth of the Bacillus spores are inhibited. In this case, there is no fermentation of glucose, no acid production and therefore the pH indicator in the medium remains purple. GENERAL TEST FORMAT/PRESENTATION The format is 96 tubes

TEST KALIDOS TB LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limits Penicillin 2-4 Ampicillin 4 Amoxicillin 4 Oxacillin 15-20 Cloxacillin 30-40 Dicloxacillin 15-20 Nafcillin 10-15 Streptomycin 400-600 Dihydrostreptomycin 200-400 Kanamycin 150 Neomycin 50 Erythromycin 200 Tylosin 40-50 Spiramicin 600-800 Rifampicin 100-150 Tetracycline 100-150 Oxytetracycline 100-150 Chlortetracycline 100-150 Chloramphenicol 2500-3500 Bacitracin 1500-3000 Sulfamethazine 100-150 Sulfadiazine 25-100 Sulfadimetoxina 25-100 Sulfamerazine 25-100 Sulfamethoxazole 25-100 Sulfaquinoxaline 25-100 Trimethoprim 200-400

113

45 Euroclone KALIDOS MP

Name of the test: KALIDOS MP

Presentation: Kalidos MP (3x96 test) (breakable microplates) (cat n. A21411)






Equipment: Water bath (65°C)





Farm: yes

Milk reception: yes

Laboratory: yes






114

GENERAL DESCRIPTION Kalidos is supplied in a multi-well microplates filled with an agar medium. The agar is pre-seeded with spores of Bacillus stearothermophilus var. calidolactis and incorporates a fermentable sugar: glucose and a pH indicator: bromocresol purple. The test is ready to use with no need to activate the product with additional reagents. The operator simply pipets the milk sample directly onto the surface of the agar and then incubates the test at 65 °C in a water bath for 3 hours. The sample quickly diffuses throughout the agar medium. If no antimicrobial substances are present in the milk or the concentration is lower then the limits of detection, the Bacillus spores germinate, grow and metabolize the sugar. The acid produced from the fermentation of glucose changes the color of the indicator bromocresol purple to a intense yellow color. Alternatively, if antimicrobial substances are present in the milk sample then germination and growth of the Bacillus spores are inhibited. In this case, there is no fermentation of glucose, no acid production and therefore the pH indicator in the medium remains purple. GENERAL TEST FORMAT/PRESENTATION The format is 3 x 96 breakable microplates

TEST KALIDOS MP LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 2-3 Ampicillin 4 Amoxicillin 4 Oxacillin 10 Cloxacillin 15 Dicloxacillin 10 Nafcillin 10 Streptomycin 800 Dihydrostreptomycin 800 Kanamycin 300 Neomycin 150 Erythromycin 200 Tylosin 40 Spiramycin 900 Rifampicin 100 Tetracycline 100-150 Oxytetracycline 100-150 Chlortetracycline 100-150 Chloramphenicol 2500-3000 Bacitracin 1500-2000 Sulfamethazine 150-200 Sulfadiazine 100 Sulfadimetoxina 100 Sulfamerazine 100 Sulfamethoxazole 100 Sulfaquinoxaline 100

115

46 Parallux

Name of the test: Parallux

Presentation: Parallux beta lactam kit :100 tests Parallux combination A: 100 tests Parallux combination C: 100 tests

Group of drugs detected: Beta lactams SDM, SMZ Tetracyclines Neomycin Streptomycin Quinolones

Type of reaction: Competitive EIA method

Time per analysis: 4 min

Number of samples at a time: 1-4 samples

Detection limits: See the table

Equipment: Parallux processor





Yes


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer MEDEXX Co., Ltd #301 sanga, BUndang Technopark 151, Yatab-Dong, Seoungnam-City, Gyeonggi-Do, Korea. Tel:81-31-707-0181

Contact person HyunYong KIM

E-Mail: [email protected]

Internet site: www.medexx.co.kr

Reference: Journal of AOAC international Vol.85,No2,2002

GENERAL DESCRIPTION

116

PARALLUX ASSAY is a solid phase immunoassay method. It is a competitive immunoassay in which analyte in a sample inhibits the binding of a fluorescently labelled antibody to a glass capillary solid phase. Antibiotics moieties are immobilized on the surfaces dye. Milk samples are mixed with the fluorescently labelled antibodies, and this mixture is drawn into the antibiotic coated capillaries. The amount of binding of labelled antibody to the glass capillary is inversely proportional to the concerntration of antibiotic in the milk sample. GENERAL TEST FORMAT/PRESENTATION The assay requires the use of Parallux processor instrument. This instrument serves to: 1. Mix samples and antibody;2. Pipette sample into capillaries: 3. Incubate samples, 4. wash the capillaries:5. Dry the capillaries; 6. Read the fluorescent signal of capillaries; 7. Print test result.

TEST PARALLUX SYSTEM LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Ceftiofur 33.7 Ampicillin 2.9 Amoxicillin 3.6 Penicillin 2.3 Cloxacilin 30 Cephapirin 16.3 Sulfamethazine 10 Sulfadimethoxine 10 Tetracycline 75 Neomycin 30 Streptomycin 50 Norfloxacin 20 Enrofloxacin 25 Danofloxacin 50

117

47 Penzym®100

Name of the test: Penzym®100 Test for Beta-Lactam Drugs in Cow Milk

Presentation: Penzym®100 – 1 vial lyophilized enzyme, 1 vial of 100 tablets, tweezers, color chart


Type of reaction: Enzymatic, colorimetric assay




Equipment: Dry bath 47°C Rotary vortex mixer 1 ml syringe Adjustable 0-50µl pipette with disposable tips





Available at 5 ppb Penicillin


Farm: Yes

Milk reception: Yes

Laboratory: Yes



Gary White; [email protected]

Internet site: www.neogen.com

Reference: AFNOR Validation AOAC-RI 930707

118

GENERAL DESCRIPTION Penzym®100 is an enzymatic test for detection of beta-lactam antibiotic residues in raw, commingled cow milk. The test is based o a DD-carboxypeptidase enzyme that has the two following properties:

a. It is specifically and quantitatively inhibited by beta-lactam antibiotics, and as a result, the more the sample is contaminated with antibiotics, the lower the residual activity will be.

b. It specifically hydrolyses substrates of the R-D-Ala-D-Ala type with liberation of D-alanine.

In order to measure the activity of the enzyme, the liberated D-alanine is transformed by a stereospecific oxidase into pyruvic acid with liberation of hydrogen peroxide. The produced peroxide oxidizes, under the action of peroxidase, an organic dye with resulting change in color. GENERAL TEST FORMAT/PRESENTATION 1. Addition of the milk sample to the DD-carboxypeptidase enzyme in the assay tube. 2. Mixing, and incubation of the assay tube at 47°C. During this step, any antibiotics which

may be present in the milk sample will bind to the enzyme and inactivate it. 3. Addition of a tablet containing the color reagents to the assay tube and further incubation at

47°C. During this step, there will be a color generation which will be greater when more active DD-carboxypeptidase remains in the assay tube.

4. Interpretation of the color in the assay tube by comparison with the color chart provided in

the test kit.

119

TEST PENZYM®100 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Penicillin 4-6 Ampicillin 4-7 Amoxicillin 4-6 Oxacillin 30-50 Cloxacillin 60-100 Ceftiofur 40-70 Cephapirin 5-7 Cephalonium 10-15 Cefuroxime 50-100 Cephalothin 4-6 Cephaloridine 6-8 Cephalexin 20-40

120

48 Penzym®100 S

Name of the test: Penzym®100 S Test for Beta-Lactam Drugs in Cow Milk

Presentation: Penzym®100 S – 1 vial lyophilized enzyme, 1 vial of 100 tablets, tweezers, color chart


Type of reaction: Enzymatic, colorimetric assay




Equipment: Dry bath 47°C Rotary vortex mixer 1 ml syringe Adjustable 0-50µl pipette with disposable tips





Available at 5 ppb Penicillin


Farm: Yes

Milk reception: Yes

Laboratory: Yes




Internet site: www.neogen.com

Reference: AFNOR AOAC-RI 930707

121

GENERAL DESCRIPTION Penzym®100 S is an enzymatic test for detection of beta-lactam antibiotic residues in raw, commingled cow milk. The test is based o a DD-carboxypeptidase enzyme that has the two following properties:

c. It is specifically and quantitatively inhibited by beta-lactam antibiotics, and as a result, the more the sample is contaminated with antibiotics, the lower the residual activity will be.

d. It specifically hydrolyses substrates of the R-D-Ala-D-Ala type with liberation of D-alanine.

In order to measure the activity of the enzyme, the liberated D-alanine is transformed by a stereospecific oxidase into pyruvic acid with liberation of hydrogen peroxide. The produced peroxide oxidizes, under the action of peroxidase, an organic dye with resulting change in color. GENERAL TEST FORMAT/PRESENTATION 1. Addition of the milk sample to the DD-carboxypeptidase enzyme in the assay tube. 2. Mixing, and incubation of the assay tube at 47°C. During this step, any antibiotics which

may be present in the milk sample will bind to the enzyme and inactivate it. 3. Addition of a tablet containing the color reagents to the assay tube and further incubation at

47°C. During this step, there will be a color generation which will be greater when more active DD-carboxypeptidase remains in the assay tube.

4. Interpretation of the color in the assay tube by comparison with the color chart provided in

the test kit.

122

TEST PENZYM®100 S


Antibiotic Detection limit Penicillin 2-4 Ampicillin 3-4 Amoxicillin 3-4 Oxacillin 30-30 Cloxacillin 30-70 Ceftiofur 20-40 Cephapirin 3-5 Cephalonium 4-8 Cefuroxime 30-60 Cephalothin 2-4 Cephaloridine 2-4 Cephalexin 15-25

123

49 ROSA Enrofloxacin Test

Name of the test: ROSA Enrofloxacin Test for Raw Commingled Cow Milk

Presentation: LF-QUIN-500: 500 ROSA strips including positive controls LF-QUIN-100: 100 ROSA strips including positive controls LF-QUIN-20: 20 ROSA strips including positive controls

Group of antibiotics detected: Enrofloxacin

Type of reaction: Rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology


Number of samples at a time: As many as 4 tests per incubator

Detection limits: Enrofloxacin 10 ppb

Equipment: 56º C ROSA Incubator 300ul Pipette, 300ul Pipette tips


Special equipment: ROSA Reader

Special reagents: 10ppb Enrofloxacin Standard supplied with kits


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc 659 Andover Street Lawrence MA 01843


Charm Sciences Technical Support Support@charm,,com


Reference: Tech. Info. from Charm Sciences, Inc -

124

GENERAL DESCRIPTION The Enroflox Test is a rapid lateral flow immunoassay test for enrofloxacin detection utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect Enrofloxacin at the EU maximum residue limits (MRLs) and US regulatory limits (SL). As milk flows through the device, a line forms in the T (test) position when the sample contains no enrofloxacin. A weaker intensity T line forms when enrofloxacin is present in the sample. The T line is compared to the C (control) line which is designed to discriminate enrofloxacin close to maximum residue limits and safe levels. If the T line is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive. The strip may be inserted into a reader for interpretation. This test is designed for milk producers, from the farms to the final processing laboratory. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA ENROFLOXACIN TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Enrofloxacin 8

125

50 ROSA MRL Test for Beta-Lactam

Name of the test: ROSA MRL Test for Beta-Lactam Drugs in Bovine Milk

Presentation: LF-MRLBL-100K: 100 test strips including positive control. LF-MRLBL-500K: 500 test strips including positive control.

Group of antibiotics detected: Beta-Lactam family

Type of reaction: Lateral Flow- Rapid receptor assay utilizing ROSA (Rapid One Step Assay) technology. 2-line reaction.


Number of samples at a time: 2 samples per dual incubator 4 samples per quad incubator

Detection limits: Beta-lactam MRLs - See table

Equipment: 56°C ROSA incubator with timer 300uL pipette

Lorry incubator availability : Yes- 24V incubator available

Special equipment: ROSA reader

Special reagents: PG-4 10mL 4ppb Penicillin Positive Control Standard Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc. 659 Andover Street Lawrence, MA 01843 Fax: 978-687-9216


Technical Support [email protected]


References: Tech. Inf. from Charm Sciences Inc. D.D. Muir - Milk Quality Forum, 19 Cornwall Terrace, London, NW1 4QP Kroll et. Al - Proceddings 40. Arbeitstagung DVG Lebensmittelhygeine Presented at Garmish-Partenkichen, Sept. 1999 B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005

126

GENERAL DESCRIPTION The Charm MRL Beta-Lactam Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect beta-lactam drugs at the EU/Codex maximum residue limits, (MRLs). As milk flows through the device, a line forms in the T (test) position when the sample contains no beta-lactam. A weaker intensity T line forms when beta-lactam is present in the sample. The T line is compared to the C (control) line which is designed to discriminate beta-lactam close to safe levels. If the T line is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive. The strip may be inserted into a reader for interpretation. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA MRLBL TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Amoxicillin 3.0 – 4.0 Ampicillin 3.0 – 4.0 Cephacetrile 8 – 18 Cephalexin 30 – 60 Cefalonium 3.0 – 5.0 Cefazolin 12 – 20 Cefoperazonee 5 – 9 Cefquinomee 15 – 20 Ceftiofur and Metabolite 30 – 60 Cefuroxime 3.0 – 5.0 Cephapirin 6 – 10 Cloxacillin 25 – 35 Dicloxacillin 20 – 30 Penicillin 2.0 – 3.0

127

51 ROSA MRL3 Test for Beta-Lactam

Name of the test: ROSA MRL3 Test for Beta-Lactam Drugs in Cow Milk

Presentation: LF-MRL3-100K: 100 test strips including positive control. LF-MRL3-500K: 500 test strips including positive control.









Special reagents: PG-5 x 4 ppb Penicillin G Positive Control Tablets Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes





References: Tech. Inf. from Charm Sciences Inc. Validated 2007 at T&V-ILVO according to Commission Decision 2002/657/EC

GENERAL DESCRIPTION

128

The Charm MRL3 Beta-Lactam Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect beta-lactam drugs at the EU Maximum Residue Limits (MRL). As milk flows through the device, a line forms at the X (cloxacillin) and T (test) position when the sample contains no beta-lactam. A weaker intensity T line forms when beta-lactam is present in the sample and a weaker intensity X line forms when cloxacillin type molecules are present in the sample. The T line and X line are compared to the C (control) line which is designed to discriminate beta-lactam close to MRL. If the T line and X line is darker than or equal to the C line, the sample is negative. If the T line or X line are lighter than the C line, or the T line or X line do not form, the sample is positive. The strip is inserted into a reader for interpretation. GENERAL TEST FORMAT/PRESENTATION Individually sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA MRL3 LIMITS OF DETECTION IN MILK

Results in µg/kg* Antibiotic Detection limit Amoxicillin 4 Ampicillin 4 Cefacetrile 9 Cefalexin 10 Cefalonium 3 Cefazolin 16 Cefoperazonee 4 Cefquinomee 14 Ceftiofur and Metabolite 6 Cefuroxime Not evaluated Cephapirin 3 Cloxacillin 12 Dicloxacillin 14 Penicillin G 3 Oxacillin 18

*Values determined at T&V-IVLO

129

52 ROSA SDSM TEST

Name of the test: ROSA SDSM Test

Presentation: LF-SDSM-100K: 100 test strips including positive control. LF-SDSM-20K: 20 test strips including positive control.

Group of antibiotics detected: Sulfamethazine, Sulfadimethoxine

Type of reaction: Lateral Flow. Rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. 3-line reaction.

Time per analysis: 8 min incubation

Number of samples at a time: 4 per incubator


Equipment: Charm 56°C 2 or 4 well incubator 300uL fixed pipette



Special reagents: Sulfamethazine and Sulfadimethoxine Positive Control Standard supplied with kit.


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc 659 Andover St Lawrence,MA 01843




Reference: Tech. info. from Charm Sciences Inc. B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005

130

GENERAL DESCRIPTION The Charm SDSM ROSA Test uses receptors with binding to Sulfadimethoxine/sulfamethazine. As milk flows through the test strip, a Sulfadimethoxine and sulfamethazine line forms in the D and M position when the sample contains no Sulfadimethoxine/sulfamethazine. A weaker intensity D or M line forms when Sulfadimethoxine or sulfamethazine is present in the sample. The D and M lines are compared to the C (control) line that is designed to discriminate Sulfadimethoxine/sulfamethazine close to the safe level. If both the D and M lines are darker than or equal to the C line, the sample is negative. If either the D or M line is lighter than the C line, or the D or M line does not form, the sample is positive. If the C line does not form the test is flawed and must be repeated. An optional Reader is available to interpret completed test strips electronically. GENERAL TEST FORMAT/PRESENTATION 100-strip kits are delivered in a clear canister sealed in a transparent resealable plastic bag with blue desiccant. 20-strip kits are delivered in a capitol vial sealed in a transparent resealable plastic bag with desiccant.

ROSA SDSM TEST LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Sulfamethazine 6.2 Sulfadimethoxine 6.7

131

53 ROSA SL3 Test for Beta-Lactam

Name of the test: ROSA SL3 Test for Beta-Lactam Drugs in Cow Milk

Presentation: LF-SL3-100K: 100 test strips including positive control. LF-SL3-500K: 500 test strips including positive control.









Special reagents: PG-5 x 5 ppb Penicillin Positive Control Tablets Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes





References: Tech. Inf. from Charm Sciences Inc. AOAC-RI Performance Tested Method 040701- April 23, 2007 DHHS:PHS:FDA:CFSAN:OFS:DDFPS: DEB:MST M-a-85 (Rev #12) Oct. 2007

132

GENERAL DESCRIPTION The Charm SL3 Beta-Lactam Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect beta-lactam drugs at the US Safe Levels (SL). As milk flows through the device, a line forms in the T (test) position when the sample contains no beta-lactam. A weaker intensity T line forms when beta-lactam is present in the sample. The T line is compared to the C (control) line which is designed to discriminate beta-lactam close to safe levels. If the T line is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive. The strip may be inserted into a reader for interpretation. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA SL3 Test for Beta-Lactam LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Amoxicillin 7.8* Ampicillin 8.7* Cephacetrile 15 – 30 Cephalexin 50 – 70 Cefalonium 6 – 8 Cefazolin Not determined Cefoperazonee 10 – 15 Cefquinomee 50 – 75 Ceftiofur and Metabolite 51* Cefuroxime 10 – 15 Cephapirin 16* Cloxacillin 75 – 100 Dicloxacillin 50 – 75 Penicillin 4.2*

*90% detection level with 95% confidence

133

54 ROSA SLBL Test for Beta-Lactam

Name of the test: ROSA SLBL Test for Beta-Lactam Drugs in raw commingled cows milk

Presentation: LF-SLBL-100K: 100 test strips including positive control. LF-SLBL-500K: 500 test strips including positive control.

Group of antibiotics detected: Beta Lactam Family




Detection limits: US Safe Levels, see Table


Lorry incubator availability : Yes (24V Incubator and Car/Truck adapter available)

Special equipment: Rosa Reader

Special reagents: 5 ppb Penicillin Positive Control Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences Inc. 659 Andover St. Lawrence, MA 01843 Fax: 978-687-9216

Contact person/Email [email protected]

Internet site: http://www.charm.com

References: Tech. Inf. from Charm Sciences Inc. Beta-Lactam Methods for use under Appendix N of Past. Milk Ordinance- http://www.cfsan.fda.gov/~ear/m-a-85.html Salter et al., J. AOAC, V.84 No.1 2001 FDA-CVM approval for raw goat milk http://www.cfsan.fda.gov/~ear/mi-03-3.html Salter et al. P087 IAFP Conf. 2002 http://www.foodprotection.org/meetingsE

134

ducation/IAFP%202002/IAFP%202002%20Posters%20Abstracts.pdf

GENERAL DESCRIPTION The Charm SLTM Beta-Lactam Test is a receptor assay utilizing ROSATM (Rapid One Step Assay) technology. The SL Beta-Lactam Test is designed for use by milk intake, laboratory, field, and regulatory personnel. This test has been validated for use in raw, commingled, cows and goats milk. As milk flows through the device, a line forms in the T (test) position when the sample contains no beta-lactam. A weaker intensity T line forms when beta-lactam is present in the sample. The T line is compared to the C (control) line which is designed to discriminate beta-lactam close to safe levels. If the T line is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive. The strip may be inserted into a reader for interpretation. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA SLBL TEST LIMITS OF DETECTION IN MILK

Results in µg/kg (ppb) Antibiotic Detection limit Amoxicillin 5.6 Ampicillin 8.5 Ceftiofur 77 Cephapirin 13.7 Penicillin 3.6

Other beta-lactams are cross reactive with SL test. Only US approved beta-lactams are

reported.

135

55 ROSA MRLBLTET

Name of the test: ROSA MRL Test for Beta-Lactam and Tetracycline Drugs in Bovine Milk

Presentation: LF-MRLBLTET-100K: 100 test strips including positive control. LF-MRLBLTET-500K: 500 test strips including positive control.

Group of antibiotics detected: Beta-Lactam and Tetracycline families

Type of reaction: Rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. 3- line development.



Detection limits: MRLs - See table




Special reagents: PGOT 10mL 4ppb Penicillin and 100ppb Oxytetracycline Combined Positive Control Standard Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes





Reference: Tech. Info. from Charm Sciences Inc. A. Beck Henzelin et al. – Vth Int. Symposium on Hormone and Veterinary Residue Analysis –Antwerp 2005 Romney- CRA-W Grembloux- Département Qualité des Productions agricoles – Chimie analytique et Technologie - 2007

136

GENERAL DESCRIPTION The Charm MRL Beta-Lactam and Tetracycline Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect beta-lactam and tetracycline drugs at the EU/Codex maximum residue limits, (MRLs). As milk flows through the test strip, a beta-lactam and tetracycline line forms in the BL and T position when the sample contains no beta-lactam/tetracycline. A weaker intensity BL or T line forms when beta-lactam or tetracycline is present in the sample. The BL and T lines are compared to the C (control) line that is designed to discriminate beta-lactam/tetracycline close to the safe level. If both the BL and T lines are darker than or equal to the C line, the sample is negative. If either the BL or T line is lighter than the C line, or the BL or T line does not form, the sample is positive. A ROSA reader is available to interpret completed test strips electronically.

GENERAL TEST FORMAT/PRESENTATION 100 strip kits are delivered in a clear canister sealed in a transparent resealable plastic bag with blue desiccant. 20 strip kits are delivered in a snap top container sealed in a transparent resealable plastic bag with blue desiccant.

ROSA MRLBLTET LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Amoxicillin 2.5 – 4.0 Ampicillin 2.5 – 4.0 Cephacetrile 6 – 12 Cephalexin 15 – 30 Cefalonium 3.0 – 5.0 Cefazolin 8 – 16 Cefoperazonee 3 – 6 Cefquinomee 15 – 20 Ceftiofur and Metabolite 20 – 50 Cefuroxime 2.0 – 5.0 Cephapirin 4 – 8 Cloxacillin 25 – 35 Dicloxacillin 20 – 30 Penicillin 2.0 – 3.0 Chlortetracycline 50 – 100 Oxytetracycline 50 – 100 Tetracycline 7 - 15

137

56 ROSA 3 Minute MRL Test for Beta-Lactam and Tetracycline Drugs

Name of the test: ROSA 3 minute MRL Test for Beta-Lactam and Tetracycline Drugs for Cow Milk

Presentation: LF-MRLBLTET3-100K: 100 test strips including positive control. LF-MRLBLTET3-500K: 500 test strips including positive control.

Group of antibiotics detected: Beta-Lactam and Tetracycline families




Detection limits: Beta-lactam MRLs and Tetracycline MRLs- See table




Special reagents: PG-4 10mL 4ppb Penicillin and 100ppb Oxytetracycline Positive Control Standard Supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes





References:

138

GENERAL DESCRIPTION The Charm MRL Beta-Lactam and Tetracycline Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect beta-lactam and tetracycline drugs at the EU/Codex maximum residue limits, (MRLs). As milk flows through the device, a line forms in the T (tetracycline) and BL (Beta-lactam) position when the sample contains no beta-lactam or tetracycline. A weaker intensity T line forms when tetracycline is present in the sample and a weaker BL line forms when beta-lactam is present in the sample. The T line and BL line is compared to the C (control) line. If the T line and BL is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive for tetracycline at MRLs. If the BL line is lighter than the C line, or the BL line does not form, the sample is positive for beta-lactam at MRLs. The strip may be inserted into a reader for interpretation. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

ROSA 3 minute MRL Test for Beta-Lactam and Tetracycline


Antibiotic Detection limit Amoxicillin 2.5 - 4.0

Ampicillin 3.0 - 4.0

Cephacetrile 8 - 14

Cephalexin 30 - 60

Cefalonium 3.0 - 5.0

Cefazolin 12 - 20

Cefoperazonee 5 - 9

Cefquinomee 15 - 20

Ceftiofur and Metabolite 30 - 60

Cefuroxime 3.0 - 5.0

Cephapirin 6 - 10

Cloxacillin 25 - 35

Dicloxacillin 20 - 30

Penicillin 2.0 - 3.0

Tetracycline 50-100 Oxytetracycline 50-100 Chlortetracycline 50-100

139

57 ROSA Tetracycline Test

Name of the test: ROSA-Tetracycline Test at MRL or U.S. Safe level in Raw Cow Milk

Presentation: LF-TET-100K: 100 Test strips including positive control LF-TET-500K: 500 Test strips including positive controls

Group of antibiotics detected: Tetracyclines

Type of reaction: Rapid Binding assay utilizing ROSA (Rapid One Step Assay) lateral flow technology – 2 line reaction




Equipment: 56°C ROSA incubator with timer 300ul pipette



Special reagents: OT10-006 10ml 100ppb Oxytetracycline Standard supplied with kits.


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Charm Sciences, Inc. 659 Andover Street Lawrence, MA 01843


Charm Sciences, Inc. Technical Support [email protected]


Reference: Technical Information from CSI B. Quandt and A. Strasser -VIIIth Int. Conf. on AgriFood in Chester (UK) 2005

140

GENERAL DESCRIPTION The Charm MRL Tetracycline Test is a rapid receptor/immunoassay utilizing ROSA (Rapid One Step Assay) technology. This test is designed to detect tetracycline drugs at the EU/Codex maximum residue limits or US Safe Levels, (MRLs or SLs). As milk flows through the device, a line forms in the T (test) position when the sample contains no tetracycline. A weaker intensity T line forms when tetracycline is present in the sample. The T line is compared to the C (control) line which is designed to discriminate tetracyclines close to MRLs. If the T line is darker than or equal to the C line, the sample is negative. If the T line is lighter than the C line, or the T line does not form, the sample is positive. The strip may be inserted into a reader for interpretation. Initial positive samples may be diluted 1 part with 2 parts negative control buffer to confirm positives at the higher US-SL levels. GENERAL TEST FORMAT/PRESENTATION Individualy sealed test strips available in 20, or 100 strip canisters. Strips may be tested 2, or 4 at a time, depending on the size of the incubator purchased. 300 ul milk is added to a strip while in incubator. Closing incubator lid starts timer. At end of incubation time, strips are read visually or inserted into reader for positive or negative interpretation.

CHARM ROSA-TET TEST LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic US-SL Detection Limit

MRL Detection limit

Chlortetracycline 150-300 50 – 100 Oxytetracycline 150-300 50 – 100 Tetracycline 30-90 10 – 30

141

58 SCREENING PLUS

Name of the test: SCREENING PLUS

Presentation: SCREENING PLUS: 96-well microtiter plate divisible into single wells. Packs of 1, 3 plates

Group of drugs detected: Wide range of antibiotics and Sulfonamides.



Number of samples at a time: No limit. 96 samples per plate









Farm: Yes

Milk reception: Yes

Laboratory: Yes



[email protected]



142

GENERAL DESCRIPTION Screening plus is a qualitative test for detection of inhibitors substances in raw, heated and powder milk. The test is used together with Eclipse as screening method for cow, sheep and goat milk allowing the detection of a broad-range of antibiotics in a large number of samples. Screening plus is more sensitive to macrolides and aminoglicosides. So, testing samples with Eclipse and Screening plus will allow a broad detection of antibiotic residues close to the MRL. Screening Plus follows the method adopted by the European Union in the decision of the Commission 91/180/CEE on the 14th of February 1991. It is based on the growth inhibition of Geobacillus stearothermophilus var. calidolactis. The kit is supplied in microtiter plate format, where each well contains agar medium spread with Geobacillus stearothermophilus spores containing a pH indicator. Heating at 65ºC will produce the spores’ germination and microorganism growth producing acid and changing the agar pH. The blue (purple) colour of the agar will turn to yellow with the G. sterarothermophilus growth as the agar pH decreases. Samples containing antibiotics at concentrations above the detection limit will inhibit the microorganisms growth and the colour of the agar will remain the same. GENERAL TEST FORMAT/PRESENTATION Screening Plus test kit is supplied in 96-well microtiter plates. Plates can be divided into single wells allowing the end user to run just the exact number of samples required. Kits of 1 or 3 plates are available. The table below shows the limits of detection of ECLISE test in cow´s milk.

SCREENING PLUS LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limitAmoxicillin 5 Cefalexin 80 Cefapirin 5 Penicillin G 4 Oxytetracycline 100 Tetracycline 100 Erythromycin 150 Tylosin 60 Spiramicin 400 Gentamicin 300 Neomycin 300 Kanamycin 3000 Sulfamethoxazole 50 Sulfathiazole 5000 Lincomycin 200 Bacitracin 400

143

59 SNAP Test Kits - IDEXX

Name of the test: IDEXX SNAP® Test Kits

Presentation: SNAP® MLR Beta-Lactam 4ppb 20 Test Kit SNAP® MLR Beta-Lactam 4ppb 100 Test Kit SNAP® Tetracycline 20 Test Kit SNAP® Sulfamethazine 20 Test Kit SNAP® Gentamicin 20 Test Kit

Group of drugs detected: Beta lactams Tetracyclines Sulfamethazine Gentamicin (SNAP® tests are drug family specific, no one SNAP® test will detect all drug families listed above.)

Type of reaction: Enzyme-linked receptor-binding assay


Number of samples at a time: One sample per test device, several test devices can be run simultaneously by staggering the start of timing.

Detection limits: In raw commingled bovine milk. See table below.

Equipment: IDEXX Heater Block capable of maintaining an operating temperature of 45° C ± 5 C°

Lorry incubator availability : Yes 220 volt

Special equipment: SNAPshot® reader available, but not necessary



Pen G positive control available for SNAP® Beta-Lactam


Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer

IDEXX LABORATORIES ISO 9001:2000 One IDEXX Drive Westbrook, Maine 04092 USA Tel: 1-207-556-0300 or 1-800-321-0207 Fax 1-207-556-0630


Laurent Depeige [email protected]

Internet site: www.idexx.com/dairy

Reference: Technical information from Idexx

144

GENERAL DESCRIPTION The SNAP® Beta-Lactam test is AOAC Research Institute approved and maintains Performance Tested Statussm. It is manufactured in an ISO 9001:2000 certified facility, as are all SNAP® tests. The test incorporates all the steps of an enzyme linked immunoassay by using a bi-directional flow device. The test is a competitive assay in which the conjugate is mixed with a sample, allowed to react with beta-lactams and then applied to the device. Generally sample spot develops color that is inversely proportional to the amount of beta-lactam present in the milk. Comparison to a control spot allows one to determine whether or not the beta-lactam is present at the claimed levels. The SNAP® test can be interpreted visually or with a SNAPshot® Reader.

GENERAL TEST FORMAT/PRESENTATION The SNAP® kits are packaged in either 20 or 100 test formats. The kit includes a SNAP® device, a Conjugate tube, and a pipette.

145

IDEXX SNAP TEST


Antibiotic Detection limit * SNAP® MRL Beta–Lactam: Peniclilin G 2-4

Cephalonium 2-3

Cephalexin 14-29

Nafcillin 52-111

Dicloxacillin 20-30

Cefazolin 13-17

Cefquinome 7-36

Cephacetrile 6-13

Amoxicillin 3.5-10

Ampicillin 3.5-5

Ceftiofur 5-13

Cephapirin 9-12

Cloxacillin 22-41

Oxacillin 24-90

Cefoperazone 3-7

SNAP® Tetracycline: Tetracycline At or below 50

Chlortetracycline At or below 100

Oxytetracycline At or below 50

SNAP® Gentamicin: Gentamicin At or below 30

SNAP® Sulfamethazine: Sulfamethazine At or below 10

*Detection limits were determined with the SNAPshot® Reader

146

60 TECNA - I’screen Tylosin

Name of the test: I’screen Tylosin, AB620/621

Presentation: EIA (12/6 strips x 8 breakable wells)

Group of drugs detected: Tylosin A

Type of reaction: Competitive enzyme immunoassay

Time per analysis: 50 min (sample preparation not included)

Number of samples at a time: Up to 40 in duplicate

Detection limits: Milk: 5 ppb

Equipment: ELISA plate, strip or microwell reader with 450 nm filter Bench centrifuge, possibly refrigerated (force required: 3000g) Micropipette (20-200µl and multichannel 20-300µl) Suitable tips

Lorry incubator availability: -




None

Difficulty: Low


Farm: No

Milk reception: Yes

Laboratory: Yes

Manufacturer: Tecna S.r.l. c/o Area Science Park Loc. Padriciano, 99 34012 Trieste Italy

Contact person: E-Mail:

Ms. Federica Tamburlini [email protected]

Internet site: www.tecnalab.com

Reference: Technical info from Tecna S.r.l.

147

GENERAL DESCRIPTION I’screen Tylosin is an EIA kit for the quantitative detection of tylosin in tissue, milk, honey, urine. The assay is performed in plastic microwells, which have been coated with anti-tylosin antibodies. The standards or the sample solutions and the enzyme conjugate (peroxidase labelled tylosin) are added to the microwells. During the incubation, the free tylosin molecules and the enzyme conjugate compete for the anti-tylosin antibodies coated to the solid phase. Any unbound enzyme conjugate is then removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of a chromogen. The enzyme converts the colorless chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The absorbance is measured by a microplate reader equipped with a 450 nm filter. The color development is inversely proportional to the tylosin concentration in the sample. GENERAL TEST FORMAT/PRESENTATION The test kit is available in 96 and 48 determination format Kit contents: Microtiter plate: 96/48 wells coated with anti-tylosin antibodies. As the strips are breakable, the wells can be used individually. For this purpose, it is sufficient to get out the wells from the sheath and break the joint. Standard Tylosin: 1 vial of tylosin solution at the concentration of 8 mg/ml. Enzyme conjugate: 1 vial of enzyme conjugate, concentrate. Enzyme conjugate diluent: 1 bottle, ready to use. Washing-buffer 10X: 1 bottle, concentrate. Developing solution: 1 bottle, ready to use. Stop solution: 1 bottle, ready to use. Dilution buffer 10X: 1 bottle, concentrate.

TEST I’screen Tylosin LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Tylosin 5

148

61 TECNA - I’screen Gentamicin

Name of the test: I’screen Gentamicin, AB670/671


Group of drugs detected: Gentamicin


Time per analysis: 1 hour (sample preparation not included)



Equipment: ELISA plate, strip or microwell reader with 450 nm filter Bench centrifuge possibly refrigerated (requested force: 3000g) Micropipettes (20-200µl, 200-1000µl and multichannel 20-300µl) Suitable tips





None

Difficulty: Low


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Diana F., Paleologo M., Persic L. (2007): “Validation of two enzyme immunoassays for aminoglycoside residues according to European Decision 657/2002”. Food Addit. Contam, 24(12): 1345-1352.

149

GENERAL DESCRIPTION I’screen Gentamicin is an EIA kit for the quantitative detection or gentamicin in milk, urine, serum and tissue. The assay is performed in plastic microwells, which have been coated with anti-gentamicin antibodies. The standards or the sample solutions and the enzyme conjugate (peroxidase labelled gentamicin) are added to the microwells. During the incubation, the free gentamicin molecules and the enzyme conjugate compete for the anti-gentamicin antibodies coated to the solid phase. Any unbound enzyme conjugate is then removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of a chromogen. The enzyme converts the colorless chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The absorbance is measured by a microplate reader equipped with a 450 nm filter. The color development is inversely proportional to the gentamicin concentration in the sample. GENERAL TEST FORMAT/PRESENTATION The test kit is available in 2 formats: 96 and 48 determinations. Kit contents: Microtiter plate: 96/48 wells coated with anti-gentamicin antibodies. As the strips are breakable, the wells can be used individually. For this purpose, it is sufficient to get out the wells from the sheath and break the joint. Standard Gentamicin: 1 vial of gentamicin solution at the concentration of 11 mg/ml. Standard Gentamicin: 7 vials of gentamicin solutions at different concentrations (3-250 ng/ml), ready to use. Enzyme conjugate: 1 vials of enzyme conjugate, concentrate. Enzyme conjugate diluent: 1 bottle, ready to use. Milk standard diluent 10X: 1 bottle, concentrate. Washing-buffer 10X: 1 bottle, concentrate. Developing solution: 1 bottle, ready to use. Stop solution: 1 bottle, ready to use. Dilution buffer: 1 bottle, ready to use.

TEST I’screen Gentamicin LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Gentamicin 50

150

62 TECNA - I’screen Neomycin

Name of the test kit: I’screen Neomycin, AB672/673


Group of drugs detected: Neomycin





Equipment: ELISA plate, strip or microwell reader with 450 nm filter Bench centrifuge, possibly refrigerated (requested force: 3000g) Micropipettes (20-200µl, 200-1000µl and multichannel 20-300µl) Suitable tips





None

Difficulty: Low


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Diana F., Paleologo M., Persic L. (2007): “Validation of two enzyme immunoassays for aminoglycoside residues according to European Decision 657/2002”. Food Addit. Contam, 24(12): 1345-1352.

151

GENERAL DESCRIPTION I’screen Neomycin is an EIA kit for the quantitative detection or neomycin in milk, urine, serum and tissue. The assay is performed in plastic microwells, which have been coated with anti-neomycin antibodies. The standards or the sample solutions and the enzyme conjugate (peroxidase labelled neomycin) are added to the microwells. During the incubation, the free neomycin molecules and the enzyme conjugate compete for the anti-neomycin antibodies coated to the solid phase. Any unbound enzyme conjugate is then removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of a chromogen. The enzyme converts the colorless chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The absorbance is measured by a microplate reader equipped with a 450 nm filter. The color development is inversely proportional to the neomycin concentration in the sample. GENERAL TEST FORMAT/PRESENTATION The test kit is available in 2 formats: 96 and 48 determinations. Kit contents: Microtiter plate: 96/48 wells coated with anti-neomycin antibodies. As the strips are breakable, the wells can be used individually. For this purpose, it is sufficient to get out the wells from the sheath and break the joint. Standard Neomycin: 1 vial of neomycin solution at the concentration of 10 mg/ml. Enzyme conjugate: 1 vials of enzyme conjugate, concentrate. Enzyme conjugate diluent: 1 bottle, ready to use. Milk standard diluent 10X: 1 bottle, concentrate. Washing-buffer 10X: 1 bottle, concentrate. Chromogen: 1 bottle, ready to use. Citrate buffer: 1 bottle, ready to use. Stop solution: 1 bottle, ready to use. Dilution buffer: 1 bottle, ready to use.

TEST I’screen Neomycin LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Neomycin 500

152

63 TECNA - I’screen CAP

Name of the test kit: I’screen CAP, AB630

Presentation: EIA (12 strips x 8 breakable wells)

Group of drugs detected: Chloramphenicol, chloramphenicol glucuronide




Detection limits: Milk: 0,01 ppb

Equipment: ELISA plate, strip or microwell reader with 450 nm filter Bench centrifuge, possibly refrigerated (force required: 2000g) Vortex Evaporation apparatus Micropipette (20-200µl, 200-1000µl and multichannel 20-300µl) Suitable tips



Special reagents: None (if direct method is used) Ethylacetate (if extraction method is used)


None; spiking solution included in the kit

Difficulty: Low


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Technical info from Tecna S.r.l.

153

GENERAL DESCRIPTION I’screen CAP is an EIA kit for the quantitative detection of chloramphenicol in milk, urine, tissue, serum, plasma, water, feed, eggs, honey, seafood. The assay is performed in plastic microwells, which have been coated with anti-IgG antibodies. The standards or the sample solutions, the enzyme conjugate (peroxidase labelled CAP) and anti-chloramphenicol antibodies are added to the microwells. During the incubation, the free CAP molecules and the enzyme conjugate compete for the anti-CAP antibodies, that has linked to the anti-IgG coated to the solid phase. Any unbound enzyme conjugate is then removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of a chromogen. The enzyme converts the colorless chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The absorbance is measured by a microplate reader equipped with a 450 nm filter. The color development is inversely proportional to the CAP concentration in the sample. GENERAL TEST FORMAT/PRESENTATION Kit contents: Microtiter plate: 96 wells coated with anti-IgG antibodies. As the strips are breakable, the wells can be used individually. For this purpose, it is sufficient to get out the wells from the sheath and break the joint. Standard CAP: 6 vials of CAP solutions at different concentrations (0,1-2 mg/ml), ready to use. CAP spiking solution: 1 vial of CAP solution at the concentration of 100 ng/ml in water, ready to use. Enzyme conjugate: 1 vial of enzyme conjugate, concentrate. Enzyme conjugate diluent: 1 vial, ready to use. Anti-chloramphenicol antibody: 1 bottle, ready to use. Milk standard diluent: 1 bottle, ready to use. Washing-buffer 10X: 1 bottle, concentrate. Developing solution: 1 bottle, ready to use. Stop solution: 1 bottle, ready to use. Dilution buffer 5X: 1 bottle, concentrate.

TEST I’screen CAP LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Chloramphenicol 0,01

154

64 TECNA – SuperScreen TETRA

Name of the test: SuperScreen TETRA, AB700/701

Presentation: Receptor assay (12/6 strips x 8 breakable wells)

Group of drugs detected: Tetracycline, oxytetracycline, chlortetracycline, doxytetracycline

Type of reaction: Binding assay



Detection limits: Raw milk: 50 ppb

Equipment: Microtiter plate, strip or microwell reader with 450 nm filter Bench centrifuge, possibly refrigerated (force required: 3000g) Micropipette (20-200µl and multichannel 20-300µl) Suitable tips





None

Difficulty: Mid


Farm: No

Milk reception: Yes

Laboratory: Yes





Reference: Puppini B., Tamburlini F., Persic L. and Paleologo M. (2008): “SuperScreen TETRA: Enzyme-receptor assay for high throughput detection of tetracyclines in foodstuffs”. Poster, AOAC Annual Meeting, Dallas (USA).

155

GENERAL DESCRIPTION SuperScreen TETRA is a receptor assay kit for the quantitative detection of tetracyclines in tissue, raw milk, honey, urine, seafood and eggs. The assay is performed in plastic microwells, which have been coated with tetracyclines competitor. The standards or the sample solutions and the tetracyclines receptor are added to the microwells. During the incubation, the receptor molecules can bind either the competitor coated to the solid phase or the free tetracyclines molecules. Any unbound molecule is then removed in a washing step. During the second incubation, the receptor bound on the solid phase is detected by an anti-receptor antibody conjugated to HRP. After a second washing step, the bound antibody-HRP conjugate is determined by adding a fixed amount of a chromogen. The enzyme converts the colorless chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The absorbance is measured by a microplate reader equipped with a 450 nm filter. The color development is inversely proportional to the tetracyclines concentration in the sample. GENERAL TEST FORMAT/PRESENTATION The test kit is available in 2 formats: 96 and 48 determinations. Kit contents: Microtiter plate: 96/48 wells coated with tetracyclines competitor. As the strips are breakable, the wells can be used individually. For this purpose, it is sufficient to get out the wells from the sheath and break the joint. Standard Tetracycline: 3 vials containing 100 ng of tetracycline. Standard diluent: 1 bottle, ready to use. Receptor: 3 vials of liophylized receptor. Receptor diluent: 1 bottle, ready to use. Enzyme conjugate: 1 vial, concentrate. Enzyme conjugate diluent: 1 bottle, ready to use. Washing-buffer 20X: 1 bottle, concentrate. Developing solution: 1 bottle, ready to use. Stop solution: 1 bottle, ready to use. Dilution buffer 10X: 1 bottle, concentrate.

TEST SuperScreen TETRA LIMITS OF DETECTION IN MILK

Results in µg/kg

Antibiotic Detection limit Tetracycline equivalent 50

156

65 Tetrasensor

Name of the test: Tetrasensor

Presentation: TK00543 (96pc)

Group of drugs detected: Tetracyclines

Type of reaction: Receptor assays in dipstick format

Time per analysis: Total 10 minutes

Number of samples at a time: 8 recommended 96 places on microwell tray


Equipment: None, no heating required

Lorry incubator availability : No incubator needed

Special equipment: Readsensor for optical measurement (optional)

Special reagents: None (pipette and tips are included)


Upon request not included into the kits

Applicable at: Yes

Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer UNISENSOR (BE) Zoning Industriel du dossay, 3, rue du dossay, B-4020 Wandre – Belgium Tel: + 32 42 52 66 02


Benoit Lemmens [email protected]

Internet site: www.tetrasensor.be

Reference:

157

GENERAL DESCRIPTION Tetrasensor is a one step competitive receptor based assay in a dipstick format. Tetracycline molecules present in the liquid sample interact with the receptor to form a stable non-active complex. In this form, the receptor is no longer able to bind the first capture line printed on the dipstick. In contrary, when the receptor is remaining free of antibiotic, binding at the first line occurs. After a few minutes, red lines are rising from the white background on the dipsticks. When the sample is free of antibiotics, a colour development occurs at the specific capture lines, indicating the absence of tetracycline in the milk sample. On the contrary, the presence of tetracycline in the sample will not cause the coloured signal to appear at the specific capture lines. There is no need to heat the milk and reaction occurs at room temperature of 20°C ± 5 °C. GENERAL TEST FORMAT / PRESENTATION Multi-test plate consisting of 12 single-break strips of 8 micro-wells each and 96 dipsticks. The microwells can be separated into single test wells dependant on the number of tests to be executed. Pipette of 200 µl and tips are included in each box.

TEST TETRASENSOR TK00543 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic

Detection limit

Chlortetracycline 13 - 16 Doxycycline 5 - 8 Methacycline 10 - 13 Oxytetracycline 15 - 20 Tetracycline 20 - 25

158

66 Transia Plate Chloramphenicol

Name of the test: Transia Plate Chloramphenicol Presentation: 96 wells microplate


Type of reaction: Competitive-type ELISA

Time per analysis: 1h 40

Number of samples at a time: 40 samples

Detection limits: 0.05 ng/mL with direct method 0.01 ng/mL with extraction method

Equipment: Microplate reader - 450 nm


Special equipment: Centrifuge 2000g, 4°C for sample preparation mild stream of nitrogen for extraction method

Special reagents: Ethyl acetate for extraction method NaOH


FAPAS test milk powder

Applicable at: Lorry: Yes (reader available)

Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer BioControl Systems Inc. 12822 SE 32nd St. Bellevue, WA 98005 USA Tel: +011 425 603 1123

Contact person BioControl Technical Support

E-Mail: [email protected]

Internet site: www.biocontrolsys.com

Reference: AB0299

159

GENERAL DESCRIPTION Transia Plate Chloramphenicol is an ELISA kit for in vitro screening of chloramphenicol. The assay reliably recovers and detects low levels of chloramphenicol in milk. Sample preparation for milk is included in the package insert. Milk samples can be analysed with or without extraction depending on required limit of detection. GENERAL TEST FORMAT/PRESENTATION Transia Plate Chloramphenicol is based on a competitive-type ELISA (Enzyme Link ImmunoSorbent Assay). The solid support of the reaction is a microtitre plate with divisible strips coated with antibodies. The standards are ready to use. The chloramphenicol-peroxydase conjugate and anti-chloramphenicol antibody are lyophilised; and reconstituted in ready to use Reconstitution Buffer. These reconstituted solutions can be stored at 2-8°C or at -20°C for few months. After the preparation of samples, the milk samples are ready to be tested. The ELISA assay procedure is one hour incubation step, where samples, conjugate and antibody solutions are added into the well. The results are obtained after the 30 minutes incubation of the substrate.

TEST TRANSIA PLATE CHLORAMPHENICOLLIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limit Chloramphenicol 0.1

160

67 Twinsensor

Name of the test: Twinsensor BT

Presentation: BT00640 (96pc) BT00641 (24pc) BT00660 (96 pc) BT00661 (24 pc)

Group of drugs detected: Beta-lactams & Tetracyclines

Type of reaction: Receptor assays in dipstick format

Time per analysis: Total 6 minutes

Number of samples at a time: 8 recommended 48 places on Heatsensor

incubator.


Equipment: Heatsensor (or water batch)

Lorry incubator availability : 12V and 24V available

Special equipment: Readsensor for optical measurement (optional)

Special reagents: None (pipette and tips are included)


Upon request not included into the kits

Applicable at: Yes

Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer UNISENSOR (BE) Zoning Industriel du Dossay, 3, rue du Dossay, B-4020 Wandre – Belgium Tel: + 32 42 52 66 02


Benoit Lemmens [email protected]

Internet site: www.twinsensor.be

Reference: Fifth International Symposium on hormone and Veterinary Drug Residues Analysis www.vdra.ugent.be

161

GENERAL DESCRIPTION TwinsensorBT is a receptor-based assay in a dipstick format for rapid detection of ß-lactam and tetracycline molecules present in a milk sample. TwinsensorBT is a competitive test involving two receptors in one single operation. The test requires using of two elements. The first element is a microwell containing a certain amount of both receptors and antibodies linked to gold particles and the second is a dipstick made up of a set of membranes where there are capture lines. The “control” line printed in red is visible all the time and the other two are the specific “test” lines placed on both sides of the control line. The unique line for penicillins and cephalosporins is located below the “control” while the line relating to tetracyclines is located above it. When the sample is free of antibiotics, a colour development occurs at the specific capture lines, indicating the absence of the concerned analytes in the milk sample. On the contrary, the presence of antibiotics in the sample will not cause the coloured signal to appear at the specific capture lines. TwinsensorBT detects most drugs at or below the MRL and the test can be used in-situ for daily checks or in laboratories for analysis of series of milk samples. GENERAL TEST FORMAT / PRESENTATION Multi-test plate consisting of 12 single-break strips of 8 micro-wells each and 96 dipsticks. The microwells can be separated into single test wells dependant on the number of tests to be executed. Pipette of 200 µl and tips are included in each box.

TEST TWINSENSORBT 640 & 641 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotics

Detection Limit

Amoxicillin 3 - 5 Ampicillin 3 - 5 Benzylpenicillin 2 - 3 Cephacetrile 30 - 40 Cefalonium 3 - 5 Cefazolin 20 - 25 Cefoperazonee 3 - 4 Cefquinomee 30 - 35 Ceftiofur 10 - 15 Cephapirin 6 - 8 Cloxacillin 6 - 8 Dicloxacillin 6 - 8 Nafcillin 30 - 40 Oxacillin 12 - 18 Chlortetracycline 25 - 30 Oxytetracycline 30 - 40 Tetracycline 40 - 50

162

TEST TWINSENSORBT 660 & 661 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotics

Detection Limit

Amoxicillin 3 - 5 Ampicillin 3 - 5 Benzylpenicillin 2 - 3 Cephacetrile 30 - 40 Cefalonium 3 - 5 Cefazolin 20 - 25 Cefoperazonee 3 - 4 Cefquinomee 30 - 35 Ceftiofur 10 - 15 Cephapirin 6 - 8 Cloxacillin 6 - 8 Dicloxacillin 6 - 8 Nafcillin 30 - 40 Oxacillin 12 - 18 Chlortetracycline 50 - 60 Oxytetracycline 60 - 80 Tetracycline 80 - 100

163

68 Valio T 101 test

Name of the test: T 101 test

Presentation: T101 test field test: 10 tests T101 test 100 ampoules T101 test: 320 ampoules T101 test: Microwell strip 16 testsx10 T101 test microwells 96 tests

Group of drugs detected: Wide range of antibiotics


Time per analysis: 4 hrs 30 mn

Number of samples at a time: depends on incubator.


Equipment: Incubators for 10 ampoules, or for 30 ampoules or incubation chamber

Lorry incubator availability : yes

Special equipment: No



Farm: Yes

Milk reception: Yes

Laboratory: Yes

Manufacturer Valio Ltd PO Box 30 FIN-00039 Valio Finland


Commercial: [email protected] Technical: : [email protected]

Internet site: -

Reference: Technical information from Valio Ltd

164

GENERAL DESCRIPTION T101-TEST FOR DETECTING ANTIMICROBIAL RESIDUES IN MILK BACKGROUND The T101-test is based on the use of the Streptococcus thermophilus T101-strain. The species is used as a starter bacterium in Swiss (emmenthal) cheese and yoghurt production. This unique specific ‘dairy strain’ detects sensitively a very broad spectrum of antibiotics used in mastitis treatment. Growing in milk S. thermophilus T101 produces lactic acid from milk sugar (lactose). The acid produced reacts with a pH indicator in the culture media. This reaction leads to a colour change of the pH indicator related to the bacterial growth. If the milk sample contains antibiotic residues above the detection limit of the test, the growth of the bacteria is inhibited. The colour of the milk sample then remains blue. When the milk sample is free of antibiotic residues, bacterial growth occurs and the colour of the milk sample turns yellow or greyish yellow. The T101-test can be used throughout the testing steps from the farm to official laboratories, for testing of milk samples of individual cows or large milk batches e.g. tank milk and silo milk. Natural inhibitory substances which may interfere testing, are inactivated by heating the milk sample before testing. MAIN ADVANTAGES OF THE T101-TEST -the test detects sensitively a wide spectrum of conventionally used antibiotics in mastitis treatment. -the test microbe is commonly used in the dairy industry and the test result confirms successful starter growth and activity. -heating of the sample prevents interference of natural inhibitors of milk with the test result. -the test is easy to use and the result is easy to read. -the T101-test is produced in two different product forms: vials and microplates. GENERAL TEST FORMAT/PRESENTATION Start up -kit for the farm -includes a small incubator for 9 vials, a heating rack for the pipettes, 10 vials containing T101-bacteria strain and a pH indicator in freeze-dried form, and 10 disposable plastic pipettes. (The pipettes cannot be used more than once). Supply kit for the farm -includes 10 vials and 10 disposable pipettes. Test vials -100 and 320 vial packages Microplates -16 wells strips (10 strips/box) or 96 wells plate The vials and microplates must be stored at refrigerator temperature. The test should not be kept at higher temperatures, since this may damage the strain and thus give a false positive reaction. The expiration date is 12 months after the date of the production. T101-TEST PROCEDURE

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A control milk sample free of antibiotic residues is tested simultaneously with the milk samples. Vials: 1) 2 ml of the milk sample is sucked into the disposable pipette, turned upside down and set into the heating rack. Heating takes 5 minutes in boiling water (+90 - +95°C). After heating the milk samples are cooled to room temperature (about +22°C). 2) The sample is added to the test vial and shaken gently to completely dissolve the dry material. 3) The test vials are incubated for 4 ½ hours in the +42°C heat block. The test result is read according to the colour chart after shaking the vial to even out the colour. Microplates 1) Milk sample is poured or pipetted into the heating glass tubes set into the tube holder. Heat the samples 5 minutes in boiling water (+90 - +95°C). After heating the milk samples are cooled to room temperature (about +22°C). 2) 200 µl of milk samples are pipetted into the wells of the microplate. Seal the wells with the adhesive plastic strips or sheets. Mix the samples by turning the blocks upside down a few times to dissolve the freeze-dried material. 3) Incubate the plates for 4 ½ hours at +42°C in the temperature cabinet or in the special incubator equipped with a timer and a cooling system (the result can then be read when suitable). The result is read according to the colour chart. If the test is performed with tank milk or silo milk in a laboratory, the heating step can be left out. However, if the result is positive, the test should be repeated with heated samples. READING OF THE RESULTS In the absence of antibiotics the colour of the milk samples becomes greyish yellow (negative result) during the incubation at +42°C for 4 ½ hours. If the milk sample contains an inhibitory substance, such as an antibiotic residue within the detection limits of the T101-test, the colour stays blue (positive result). A green/yellow-green colour is considered as a negative or positive result according to the colour chart. A control milk sample free of antibiotic residues is tested along with the milk samples and should give a negative result (greyish yellow colour). The result should be read immediately after the incubation of 4 ½ hours. The detection limits of the test for antibiotics are changed if the incubation exceeds 5 hours. A doubtful (positive/negative) result may thus become a false negative result. SENSITIVITY The sensitivity spectrum of the T101-test strain to different antibiotics is wide and is shown in the table.

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Valio T101 LIMITS OF DETECTION IN MILK

Results in µg/kg Antibiotic Detection limitβ-Lactames Penicillin 2 - 4 Ampicillin 10 - 30 Amoxicillin 30 - 50 Cloxacillin 100 -150 Dicloxacillin 100 -150 Oxacillin 150 -200 Cephalexin 50 -100 Cephapirin 50 -100 Ceftiofur 20 - 30 Aminoglycosides Streptomycin 1000 -1500 Dihydrostreptomycin 1000 -1500 Neomycin 300 - 500 Gentamicin 500 -1000 Macrolides and lincosaminides Erythromycin 50 -100 Spiramycin 150 - 300 Pirlimycin 30 - 50 Tetracyclines Tetracycline 200 - 300 Oxytetracycline 200 - 300 Quinolones Enrofloxacin 1000 -1500 Danofloxacin 1500 - 2000 Sulfonamides Sulfadoxine 500 -1000 Sulfadiazine 300 - 500 Sulfacetamide 500 -1000 Sulfadimidine (Sulfamethazine) 500 -1500 Sulfamethizole 300 - 500 Sulfadimethoxine 300 - 500 Sulfamerazine 500 -1000 Sulfathiazole 200 - 500 Others Novobiocin 1000 -1500 Chloramphenicol 500 -1000 Trimethoprim 2000 - 5000 Dapsone 5000

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69 Alphabetical list of antibiotics and corresponding kits


in µg/kg Name of the test Type of test Amoxicillin 2-4 BetaStar Lateral flow Amoxicillin 2-3 Betatar Combo Lateral flow Amoxicillin 2 – 3 BRT Inhibitor Test Microbial inhibition Amoxicillin 2 – 3 BRT MRL-Screening Test Microbial inhibition Amoxicillin 3 - 4 BR-Test® AS Brilliant Microbial inhibition Amoxicillin 2-3 BR-Test® AS Special Microbial inhibition Amoxicillin 5-6 Charm Blue-Yellow Test Microbial inhibition Amoxicillin 5-6 Charm Blue-Yellow Test Microbial inhibition Amoxicillin 7.5 Charm BSDA Microbial inhibition Amoxicillin 6 Charm Cowside Microbial inhibition

Amoxicillin 4-5 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Amoxicillin 8.1 Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Amoxicillin 3-4 Copan Milk Test (3 hours) Microbial inhibition Amoxicillin 2 Copan Milk Test (control time) Microbial inhibition Amoxicillin 3-5 Delvotest P (3 hours) Microbial inhibition Amoxicillin 2 Delvotest P (control time) Microbial inhibition Amoxicillin 3-5 Delvotest SP (3 hours) Microbial inhibition Amoxicillin 2 Delvotest SP (control time) Microbial inhibition Amoxicillin 3-5 Delvotest SP- NT (3 hours) Microbial inhibition

Amoxicillin 2-3 Delvotest SP- NT (control time) Microbial inhibition

Amoxicillin 4-8 Delvo-X-press Receptor assay Amoxicillin 5 Eclipse 100 Microbial inhibition Amoxicillin 5 Eclipse 50 Microbial inhibition Amoxicillin 5 Eclipse farm Microbial inhibition Amoxicillin 4 KALIDOS MP Microbial inhibition Amoxicillin 4 KALIDOS TB Microbial inhibition Amoxicillin 4 KALIDOS TB Microbial inhibition

Amoxicillin 3.6 Parallux Solid phase immunoassay

Amoxicillin 4-6 Penzym 100 Lateral flow Amoxicillin 3-4 Penzym 100 S Lateral flow

Amoxicillin 2.5 - 4.0 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Amoxicillin 3.0 – 4.0 ROSA MRL Beta-Lactam Lateral flow Amoxicillin 4 ROSA MRL3 Beta-Lactam Lateral flow

Amoxicillin 2.5 – 4.0 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Amoxicillin 7.8 ROSA SL3 Beta-Lactam Lateral flow Amoxicillin 5.6 ROSA SLBL Beta-Lactam Lateral flow Amoxicillin 5 SCREENING PLUS Microbial inhibition Amoxicillin 3.5-10 SNAP MRL Beta –Lactam: Lateral flow Amoxicillin 4 Total Antibiotics Microbial inhibition Amoxicillin 3 - 5 Twinsensor 640 & 641 Lateral flow Amoxicillin 3 - 5 Twinsensor 660 & 661 Lateral flow Amoxicillin 30 - 50 Valio T 101 test Microbial inhibition Ampicillin 2-5 BetaStar Lateral flow Ampicillin 2-3 Betatar Combo Lateral flow

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in µg/kg Name of the test Type of test Ampicillin 2 – 3 BRT Inhibitor Test Microbial inhibition Ampicillin 2 – 3 BRT MRL-Screening Test Microbial inhibition Ampicillin 3 - 4 BR-Test® AS Brilliant Microbial inhibition Ampicillin 2-3 BR-Test® AS Special Microbial inhibition Ampicillin 5 Charm Blue-Yellow Test Microbial inhibition Ampicillin 5 Charm Blue-Yellow Test Microbial inhibition Ampicillin 6.7 Charm BSDA Microbial inhibition Ampicillin 5 Charm Cowside Microbial inhibition

Ampicillin 3-4 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Ampicillin 6.6 Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Ampicillin 4 Copan Milk Test (3 hours) Microbial inhibition Ampicillin 2 Copan Milk Test (control time) Microbial inhibition Ampicillin 3-5 Delvotest P (3 hours) Microbial inhibition Ampicillin 2-3 Delvotest P (control time) Microbial inhibition Ampicillin 3-5 Delvotest SP (3 hours) Microbial inhibition Ampicillin 2-3 Delvotest SP (control time) Microbial inhibition Ampicillin 6-7 Delvotest SP- NT (3 hours) Microbial inhibition

Ampicillin 4 Delvotest SP- NT (control time) Microbial inhibition

Ampicillin 4-8 Delvo-X-press Receptor assay Ampicillin 4 Eclipse 100 Microbial inhibition Ampicillin 5 Eclipse 50 Microbial inhibition Ampicillin 5 Eclipse farm Microbial inhibition Ampicillin 4 KALIDOS MP Microbial inhibition Ampicillin 4 KALIDOS TB Microbial inhibition Ampicillin 4 KALIDOS TB Microbial inhibition

Ampicillin 2.9 Parallux Solid phase immunoassay

Ampicillin 4-7 Penzym 100 Lateral flow Ampicillin 3-4 Penzym 100 S Lateral flow

Ampicillin 3.0 - 4.0 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Ampicillin 3.0 – 4.0 ROSA MRL Beta-Lactam Lateral flow Ampicillin 4 ROSA MRL3 Beta-Lactam Lateral flow

Ampicillin 2.5 – 4.0 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Ampicillin 8.7 ROSA SL3 Beta-Lactam Lateral flow Ampicillin 8.5 ROSA SLBL Beta-Lactam Lateral flow Ampicillin 3.5-5 SNAP MRL Beta –Lactam: Lateral flow Ampicillin 4 Total Antibiotics Microbial inhibition Ampicillin 3 - 5 Twinsensor 640 & 641 Lateral flow Ampicillin 3 - 5 Twinsensor 660 & 661 Lateral flow Ampicillin 10 - 30 Valio T 101 test Microbial inhibition Bacitracin 1500-2000 KALIDOS MP Microbial inhibition Bacitracin 1500-3000 KALIDOS TB Microbial inhibition Bacitracin 1500-3000 KALIDOS TB Microbial inhibition Bacitracin 0.300 (U/ml) Total Antibiotics Microbial inhibition Baitracin 400 SCREENING PLUS Microbial inhibition Benzylpenicillin 2 – 3 BRT Inhibitor Test Microbial inhibition Benzylpenicillin 1.5 – 2 BRT MRL-Screening Test Microbial inhibition Benzylpenicillin 2 - 3 Twinsensor 640 & 641 Lateral flow

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in µg/kg Name of the test Type of test Benzylpenicillin 2 - 3 Twinsensor 660 & 661 Lateral flow Cefacetrile 9 ROSA MRL3 Beta-Lactam Lateral flow Cefadroxil 5-25 Delvo-X-press Receptor assay Cefalexin 10 ROSA MRL3 Beta-Lactam Lateral flow Cefalexin 80 SCREENING PLUS Microbial inhibition Cefalonium 7-15 BetaStar Lateral flow Cefalonium 7-15 Betatar Combo Lateral flow Cefalonium 10 – 20 BRT Inhibitor Test Microbial inhibition Cefalonium 10 – 15 BRT MRL-Screening Test Microbial inhibition Cefalonium 8-10 Copan Milk Test (3 hours) Microbial inhibition Cefalonium 8 Copan Milk Test (control time) Microbial inhibition Cefalonium 10-25 Delvotest P (3 hours) Microbial inhibition Cefalonium 5-10 Delvotest P (control time) Microbial inhibition Cefalonium 10-25 Delvotest SP (3 hours) Microbial inhibition Cefalonium 5-10 Delvotest SP (control time) Microbial inhibition Cefalonium 20 Eclipse 100 Microbial inhibition Cefalonium 20 Eclipse 50 Microbial inhibition

Cefalonium 3.0 - 5.0 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cefalonium 3.0 – 5.0 ROSA MRL Beta-Lactam Lateral flow Cefalonium 3 ROSA MRL3 Beta-Lactam Lateral flow

Cefalonium 3.0 – 5.0 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cefalonium 6 – 8 ROSA SL3 Beta-Lactam Lateral flow Cefalonium 3 - 5 Twinsensor 640 & 641 Lateral flow Cefalonium 3 - 5 Twinsensor 660 & 661 Lateral flow Cefapirin 8-16 BetaStar Lateral flow Cefapirin 8-16 Betatar Combo Lateral flow Cefapirin 4 – 5 BRT Inhibitor Test Microbial inhibition Cefapirin 4 – 5 BRT MRL-Screening Test Microbial inhibition Cefapirin 10 Copan Milk Test (3 hours) Microbial inhibition Cefapirin 5-10 Copan Milk Test (control time) Microbial inhibition Cefapirin 8-10 Delvotest P (3 hours) Microbial inhibition Cefapirin 8 Delvotest P (control time) Microbial inhibition Cefapirin 5-10 Delvotest SP (3 hours) Microbial inhibition Cefapirin 5 Delvotest SP (control time) Microbial inhibition Cefapirin 6-8 Delvotest SP- NT (3 hours) Microbial inhibition

Cefapirin 4-6 Delvotest SP- NT (control time) Microbial inhibition

Cefapirin 8 Eclipse 100 Microbial inhibition Cefapirin 8 Eclipse 50 Microbial inhibition Cefapirin 8 Eclipse farm Microbial inhibition Cefapirin 5 SCREENING PLUS Microbial inhibition Cefazolin 40-60 BetaStar Lateral flow Cefazolin 40-60 Betatar Combo Lateral flow Cefazolin 10 – 25 BRT Inhibitor Test Microbial inhibition Cefazolin 10 – 25 BRT MRL-Screening Test Microbial inhibition

Cefazolin 10-15 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Cefazolin ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Cefazolin 20-25 Copan Milk Test (3 hours) Microbial inhibition Cefazolin 15 Copan Milk Test (control time) Microbial inhibition

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in µg/kg Name of the test Type of test Cefazolin 50 Eclipse 100 Microbial inhibition Cefazolin 50 Eclipse 50 Microbial inhibition

Cefazolin 12 - 20 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cefazolin 12 – 20 ROSA MRL Beta-Lactam Lateral flow Cefazolin 16 ROSA MRL3 Beta-Lactam Lateral flow

Cefazolin 8 – 16 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cefazolin 13-17 SNAP MRL Beta –Lactam: Lateral flow Cefazolin 20 - 25 Twinsensor 640 & 641 Lateral flow Cefazolin 20 - 25 Twinsensor 660 & 661 Lateral flow Cefoperazon 5-8 BetaStar Lateral flow Cefoperazon 25 – 50 BRT Inhibitor Test Microbial inhibition Cefoperazon 20 – 30 BRT MRL-Screening Test Microbial inhibition Cefoperazone 5-8 Betatar Combo Lateral flow Cefoperazone 60 Copan Milk Test (3 hours) Microbial inhibition Cefoperazone 40 Copan Milk Test (control time) Microbial inhibition Cefoperazone 60-100 Delvotest P (3 hours) Microbial inhibition Cefoperazone 40 Delvotest P (control time) Microbial inhibition Cefoperazone 60-100 Delvotest SP (3 hours) Microbial inhibition Cefoperazone 40 Delvotest SP (control time) Microbial inhibition Cefoperazone 5-20 Delvo-X-press Receptor assay

Cefoperazone 5 - 9 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cefoperazone 5 – 9 ROSA MRL Beta-Lactam Lateral flow Cefoperazone 4 ROSA MRL3 Beta-Lactam Lateral flow

Cefoperazone 3 – 6 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cefoperazone 10 – 15 ROSA SL3 Beta-Lactam Lateral flow Cefoperazone 3-7 SNAP MRL Beta –Lactam: Lateral flow Cefoperazone 3 - 4 Twinsensor 640 & 641 Lateral flow Cefoperazone 3 - 4 Twinsensor 660 & 661 Lateral flow Cefotaxime 4-5 Delvo-X-press Receptor assay Cefquinom <20 BetaStar Lateral flow Cefquinom 100 – 200 BRT Inhibitor Test Microbial inhibition Cefquinom 80 – 100 BRT MRL-Screening Test Microbial inhibition Cefquinom 100 BR-Test® AS Brilliant Microbial inhibition Cefquinom 90 BR-Test® AS Special Microbial inhibition Cefquinome <20 Betatar Combo Lateral flow

Cefquinome 15-20 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Cefquinome ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Cefquinome 100 Copan Milk Test (3 hours) Microbial inhibition Cefquinome 75 Copan Milk Test (control time) Microbial inhibition

Cefquinome 15 - 20 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cefquinome 15 – 20 ROSA MRL Beta-Lactam Lateral flow Cefquinome 14 ROSA MRL3 Beta-Lactam Lateral flow

Cefquinome 15 – 20 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cefquinome 50 – 75 ROSA SL3 Beta-Lactam Lateral flow

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in µg/kg Name of the test Type of test Cefquinome 7-36 SNAP MRL Beta –Lactam: Lateral flow Cefquinome 30 - 35 Twinsensor 640 & 641 Lateral flow Cefquinome 30 - 35 Twinsensor 660 & 661 Lateral flow Ceftiofur 75-150 BetaStar Lateral flow Ceftiofur 75-100 Betatar Combo Lateral flow Ceftiofur 50 – 100 BRT Inhibitor Test Microbial inhibition Ceftiofur 50 – 100 BRT MRL-Screening Test Microbial inhibition Ceftiofur 50-100 Charm Blue-Yellow Test Microbial inhibition Ceftiofur 50-100 Charm Blue-Yellow Test Microbial inhibition Ceftiofur 50-70 Delvotest P (3 hours) Microbial inhibition Ceftiofur <50 Delvotest P (control time) Microbial inhibition Ceftiofur 50-70 Delvotest SP (3 hours) Microbial inhibition Ceftiofur <50 Delvotest SP (control time) Microbial inhibition Ceftiofur 50-100 Delvotest SP- NT (3 hours) Microbial inhibition

Ceftiofur 25-50 Delvotest SP- NT (control time) Microbial inhibition

Ceftiofur 4-8 Delvo-X-press Receptor assay Ceftiofur 100 Eclipse 100 Microbial inhibition Ceftiofur 100 Eclipse 50 Microbial inhibition

Ceftiofur 33.7 Parallux Solid phase immunoassay

Ceftiofur 40-70 Penzym 100 Lateral flow Ceftiofur 20-40 Penzym 100 S Lateral flow Ceftiofur 77 ROSA SLBL Beta-Lactam Lateral flow Ceftiofur 5-13 SNAP MRL Beta –Lactam: Lateral flow Ceftiofur 10 - 15 Twinsensor 640 & 641 Lateral flow Ceftiofur 10 - 15 Twinsensor 660 & 661 Lateral flow Ceftiofur 20 - 30 Valio T 101 test Microbial inhibition Ceftiofur (parent) 150 Charm BSDA Microbial inhibition Ceftiofur (total Metabolite) 20-40

Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Ceftiofur (total Metabolite) 40

Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Ceftiofur and Metabolite 30 - 60 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Ceftiofur and Metabolite 30 – 60 ROSA MRL Beta-Lactam Lateral flow Ceftiofur and Metabolite 6 ROSA MRL3 Beta-Lactam Lateral flow

Ceftiofur and Metabolite 20 – 50 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Ceftiofur and Metabolite 51 ROSA SL3 Beta-Lactam Lateral flow Ceftiofur§ 50-100 Charm Cowside Microbial inhibition Ceftiofur2) 50-70 Copan Milk Test (3 hours) Microbial inhibition Ceftiofur2) 50 Copan Milk Test (control time) Microbial inhibition Cefuroxime 200 – 300 BRT Inhibitor Test Microbial inhibition Cefuroxime 100 – 200 BRT MRL-Screening Test Microbial inhibition Cefuroxime 4-20 Delvo-X-press Receptor assay Cefuroxime 50-100 Penzym 100 Lateral flow Cefuroxime 30-60 Penzym 100 S Lateral flow

Cefuroxime 3.0 - 5.0 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cefuroxime 3.0 – 5.0 ROSA MRL Beta-Lactam Lateral flow

Cefuroxime 2.0 – 5.0 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

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in µg/kg Name of the test Type of test Cefuroxime 10 – 15 ROSA SL3 Beta-Lactam Lateral flow Cephacetrile 25 Copan Milk Test (3 hours) Microbial inhibition Cephacetrile 20 Copan Milk Test (control time) Microbial inhibition Cephacetrile 20-40 Delvotest P (3 hours) Microbial inhibition Cephacetrile 20 Delvotest P (control time) Microbial inhibition Cephacetrile 20-40 Delvotest SP (3 hours) Microbial inhibition Cephacetrile 20 Delvotest SP (control time) Microbial inhibition

Cephacetrile 8 - 14 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cephacetrile 8 – 18 ROSA MRL Beta-Lactam Lateral flow

Cephacetrile 6 – 12 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cephacetrile 15 – 30 ROSA SL3 Beta-Lactam Lateral flow Cephacetrile 6-13 SNAP MRL Beta –Lactam: Lateral flow Cephacetrile 30 - 40 Twinsensor 640 & 641 Lateral flow Cephacetrile 30 - 40 Twinsensor 660 & 661 Lateral flow Cephalexin 200 – 300 BRT Inhibitor Test Microbial inhibition Cephalexin 100 – 200 BRT MRL-Screening Test Microbial inhibition Cephalexin 25 - 50 BR-Test® AS Brilliant Microbial inhibition Cephalexin 20-30 BR-Test® AS Special Microbial inhibition Cephalexin 60-70 Copan Milk Test (3 hours) Microbial inhibition Cephalexin 50 Copan Milk Test (control time) Microbial inhibition Cephalexin 60-100 Delvotest P (3 hours) Microbial inhibition Cephalexin 40-60 Delvotest P (control time) Microbial inhibition Cephalexin 60-100 Delvotest SP (3 hours) Microbial inhibition Cephalexin 40-60 Delvotest SP (control time) Microbial inhibition Cephalexin 25-50 Delvo-X-press Receptor assay Cephalexin 75 Eclipse 100 Microbial inhibition Cephalexin 75 Eclipse 50 Microbial inhibition Cephalexin 75 Eclipse farm Microbial inhibition Cephalexin 20-40 Penzym 100 Lateral flow Cephalexin 15-25 Penzym 100 S Lateral flow

Cephalexin 30 - 60 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cephalexin 30 – 60 ROSA MRL Beta-Lactam Lateral flow

Cephalexin 15 – 30 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cephalexin 50 – 70 ROSA SL3 Beta-Lactam Lateral flow Cephalexin 14-29 SNAP MRL Beta –Lactam: Lateral flow Cephalexin 50 -100 Valio T 101 test Microbial inhibition Cephalonium 3-4 Delvo-X-press Receptor assay Cephalonium 10-15 Penzym 100 Lateral flow Cephalonium 4-8 Penzym 100 S Lateral flow Cephalonium 2-3 SNAP MRL Beta –Lactam: Lateral flow Cephaloridine 6-8 Penzym 100 Lateral flow Cephaloridine 2-4 Penzym 100 S Lateral flow Cephalothin 4-6 Penzym 100 Lateral flow Cephalothin 2-4 Penzym 100 S Lateral flow Cephapirin 10 Charm Blue-Yellow Test Microbial inhibition Cephapirin 10 Charm Blue-Yellow Test Microbial inhibition Cephapirin 11.7 Charm BSDA Microbial inhibition Cephapirin 10 Charm Cowside Microbial inhibition

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in µg/kg Name of the test Type of test

Cephapirin 3-4 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Cephapirin 4.1 Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Cephapirin 4-8 Delvo-X-press Receptor assay Cephapirin 5-7 Penzym 100 Lateral flow Cephapirin 3-5 Penzym 100 S Lateral flow

Cephapirin 6 - 10 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cephapirin 6 – 10 ROSA MRL Beta-Lactam Lateral flow Cephapirin 3 ROSA MRL3 Beta-Lactam Lateral flow

Cephapirin 4 – 8 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cephapirin 16 ROSA SL3 Beta-Lactam Lateral flow Cephapirin 13.7 ROSA SLBL Beta-Lactam Lateral flow Cephapirin 9-12 SNAP MRL Beta –Lactam: Lateral flow Cephapirin 6 - 8 Twinsensor 640 & 641 Lateral flow Cephapirin 6 - 8 Twinsensor 660 & 661 Lateral flow Cephapirin 50 -100 Valio T 101 test Microbial inhibition

Cephapirin 16.3 Parallux Solid phase immunoassay

Cephoxazole 75-100 Delvo-X-press Receptor assay Cephradine 25-50 Delvo-X-press Receptor assay Chloramphenicol 5000 – 7500 BRT Inhibitor Test Microbial inhibition Chloramphenicol 2500 – 5000 BRT MRL-Screening Test Microbial inhibition Chloramphenicol 1 Charm II Amphenicol Radio-labelled Assay Chloramphenicol 0.1 Charm II Chloramphenicol Radio-labelled Assay Chloramphenicol 0,05 Chloramphenicol ELISA ELISA Chloramphenicol 3000-5000 Copan Milk Test (3 hours) Microbial inhibition Chloramphenicol 3000 Copan Milk Test (control time) Microbial inhibition Chloramphenicol 7500-10000 Delvotest P (3 hours) Microbial inhibition Chloramphenicol 2500 Delvotest P (control time) Microbial inhibition Chloramphenicol 7500-10000 Delvotest SP (3 hours) Microbial inhibition Chloramphenicol 2500 Delvotest SP (control time) Microbial inhibition Chloramphenicol 5000 Eclipse 100 Microbial inhibition Chloramphenicol 5000 Eclipse 50 Microbial inhibition Chloramphenicol 0,01 I’screen CAP ELISA Chloramphenicol 2500-3000 KALIDOS MP Microbial inhibition Chloramphenicol 2500-3500 KALIDOS TB Microbial inhibition Chloramphenicol 2500-3500 KALIDOS TB Microbial inhibition Chloramphenicol 5 Total Antibiotics Microbial inhibition

Chloramphenicol 0.1 Transia Plate Chloramphenicol ELISA

Chloramphenicol 500 -1000 Valio T 101 test Microbial inhibition Chloramphenicol after ethyl acetate extraction 0.01 ng/ml Chloramphenicol EIA ELISA Chloramphenicol direct 0.1 ng/ml Chloramphenicol EIA ELISA Chloramphenicol glucuronide 1 Charm II Amphenicol Radio-labelled Assay Chlortetracicline2) 350-400 Copan Milk Test (3 hours) Microbial inhibition Chlortetracicline2) 300 Copan Milk Test (control time) Microbial inhibition Chlortetracycline 25-50 Betatar Combo Lateral flow Chlortetracycline 100 - 300 BR-Test® AS Brilliant Microbial inhibition

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in µg/kg Name of the test Type of test Chlortetracycline 100 - 300 BR-Test® AS Special Microbial inhibition Chlortetracycline 28 Charm II Tetracycline Radio-labelled Assay Chlortetracycline 200-300 Delvotest P (3 hours) Microbial inhibition Chlortetracycline 100-150 Delvotest P (control time) Microbial inhibition Chlortetracycline 200-600 Delvotest SP (3 hours) Microbial inhibition Chlortetracycline 100-150 Delvotest SP (control time) Microbial inhibition Chlortetracycline 100-150 KALIDOS MP Microbial inhibition Chlortetracycline 100-150 KALIDOS TB Microbial inhibition Chlortetracycline 100-150 KALIDOS TB Microbial inhibition

Chlortetracycline 50-100 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Chlortetracycline 50 – 100 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Chlortetracycline 50 – 100 ROSA Tetracycline (MRL) Lateral flow Chlortetracycline 150-300 ROSA Tetracycline (US) Lateral flow Chlortetracycline At or below 100 SNAP Tetracycline: Lateral flow Chlortetracycline 13 - 16 Tetrasensor Lateral flow Chlortetracycline 100 Total Antibiotics Microbial inhibition Chlortetracycline 25 - 30 Twinsensor 640 & 641 Lateral flow Chlortetracycline 50 - 60 Twinsensor 660 & 661 Lateral flow Ciprofloxacin 3 Fluoroquinolones EIA ELISA

Cloxacilin 30 Parallux Solid phase immunoassay

Cloxacillin 5-10 BetaStar Lateral flow Cloxacillin 5-10 Betatar Combo Lateral flow Cloxacillin 20 – 30 BRT Inhibitor Test Microbial inhibition Cloxacillin 10 – 20 BRT MRL-Screening Test Microbial inhibition Cloxacillin 30-50 Charm Blue-Yellow Test Microbial inhibition Cloxacillin 30-50 Charm Blue-Yellow Test Microbial inhibition Cloxacillin 50 Charm BSDA Microbial inhibition Cloxacillin 30-50 Charm Cowside Microbial inhibition

Cloxacillin 25-35 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Cloxacillin ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Cloxacillin 25 Copan Milk Test (3 hours) Microbial inhibition Cloxacillin 15 Copan Milk Test (control time) Microbial inhibition Cloxacillin 25 Delvotest P (3 hours) Microbial inhibition Cloxacillin 15 Delvotest P (control time) Microbial inhibition Cloxacillin 15-25 Delvotest SP (3 hours) Microbial inhibition Cloxacillin 15 Delvotest SP (control time) Microbial inhibition Cloxacillin 20-30 Delvotest SP- NT (3 hours) Microbial inhibition

Cloxacillin 20 Delvotest SP- NT (control time) Microbial inhibition

Cloxacillin 30-60 Delvo-X-press Receptor assay Cloxacillin 40 Eclipse 100 Microbial inhibition Cloxacillin 40 Eclipse 50 Microbial inhibition Cloxacillin 40 Eclipse farm Microbial inhibition Cloxacillin 15 KALIDOS MP Microbial inhibition Cloxacillin 30-40 KALIDOS TB Microbial inhibition Cloxacillin 30-40 KALIDOS TB Microbial inhibition Cloxacillin 60-100 Penzym 100 Lateral flow Cloxacillin 30-70 Penzym 100 S Lateral flow

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in µg/kg Name of the test Type of test

Cloxacillin 25 - 35 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Cloxacillin 25 – 35 ROSA MRL Beta-Lactam Lateral flow Cloxacillin 12 ROSA MRL3 Beta-Lactam Lateral flow

Cloxacillin 25 – 35 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Cloxacillin 75 – 100 ROSA SL3 Beta-Lactam Lateral flow Cloxacillin 22-41 SNAP MRL Beta –Lactam: Lateral flow Cloxacillin 20 Total Antibiotics Microbial inhibition Cloxacillin 6 - 8 Twinsensor 640 & 641 Lateral flow Cloxacillin 6 - 8 Twinsensor 660 & 661 Lateral flow Cloxacillin 100 -150 Valio T 101 test Microbial inhibition Danofloxacin 5 Fluoroquinolones EIA ELISA Danofloxacin 1500 - 2000 Valio T 101 test Microbial inhibition

Danofloxacin 50 Parallux Solid phase immunoassay

Dapsone 20 Charm II Sulfa Drug (MRL) Radio-labelled Assay Dapsone 2 Charm II Sulfa Drug (US) Radio-labelled Assay Dapsone 2-4 Copan Milk Test (3 hours) Microbial inhibition Dapsone 2 Copan Milk Test (control time) Microbial inhibition Dapsone 1-8 Delvotest SP (3 hours) Microbial inhibition Dapsone 1 Delvotest SP (control time) Microbial inhibition Dapsone 0.5-1 Delvotest SP- NT (3 hours) Microbial inhibition Dapsone 1-2.5 Delvotest SP- NT (3 hours) Microbial inhibition Dapsone 5000 Valio T 101 test Microbial inhibition DH/Streptomycin 1000 – 1500 BRT Inhibitor Test Microbial inhibition DH/Streptomycin 400 – 600 BRT MRL-Screening Test Microbial inhibition DH-Streptomycin 1000-2000 Copan Milk Test (3 hours) Microbial inhibition DH-Streptomycin 1000 Copan Milk Test (control time) Microbial inhibition Dicloxacillin 5-10 BetaStar Lateral flow Dicloxacillin 5-10 Betatar Combo Lateral flow Dicloxacillin 10 – 20 BRT Inhibitor Test Microbial inhibition Dicloxacillin 5 – 10 BRT MRL-Screening Test Microbial inhibition

Dicloxacillin 15-20 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Dicloxacillin ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Dicloxacillin 20 Copan Milk Test (3 hours) Microbial inhibition Dicloxacillin 10 Copan Milk Test (control time) Microbial inhibition Dicloxacillin 20 Delvotest P (3 hours) Microbial inhibition Dicloxacillin 10-15 Delvotest P (control time) Microbial inhibition Dicloxacillin 10-15 Delvotest SP (3 hours) Microbial inhibition Dicloxacillin 10 Delvotest SP (control time) Microbial inhibition Dicloxacillin 10-20 Delvotest SP- NT (3 hours) Microbial inhibition

Dicloxacillin 10 Delvotest SP- NT (control time) Microbial inhibition

Dicloxacillin 25-50 Delvo-X-press Receptor assay Dicloxacillin 10 KALIDOS MP Microbial inhibition Dicloxacillin 15-20 KALIDOS TB Microbial inhibition Dicloxacillin 15-20 KALIDOS TB Microbial inhibition

Dicloxacillin 20 - 30 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Dicloxacillin 20 – 30 ROSA MRL Beta-Lactam Lateral flow

176


in µg/kg Name of the test Type of test Dicloxacillin 14 ROSA MRL3 Beta-Lactam Lateral flow

Dicloxacillin 20 – 30 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Dicloxacillin 50 – 75 ROSA SL3 Beta-Lactam Lateral flow Dicloxacillin 20-30 SNAP MRL Beta –Lactam: Lateral flow Dicloxacillin 15 Total Antibiotics Microbial inhibition Dicloxacillin 6 - 8 Twinsensor 640 & 641 Lateral flow Dicloxacillin 6 - 8 Twinsensor 660 & 661 Lateral flow Dicloxacillin 100 -150 Valio T 101 test Microbial inhibition

Dihydrostreptomycin 150 Charm II (Streptomycin Type) Aminoglycoside (MRL) Radio-labelled Assay

Dihydrostreptomycin 75 Charm II (Streptomycin Type) Aminoglycoside (US) Radio-labelled Assay

Dihydrostreptomycin 1500-10000 Delvotest P (3 hours) Microbial inhibition Dihydrostreptomycin 300-500 Delvotest P (control time) Microbial inhibition Dihydrostreptomycin 1500-10000 Delvotest SP (3 hours) Microbial inhibition Dihydrostreptomycin 300-500 Delvotest SP (control time) Microbial inhibition Dihydrostreptomycin 800 KALIDOS MP Microbial inhibition Dihydrostreptomycin 200-400 KALIDOS TB Microbial inhibition Dihydrostreptomycin 200-400 KALIDOS TB Microbial inhibition Dihydrostreptomycin 500 Total Antibiotics Microbial inhibition Dihydrostreptomycin 1000 -1500 Valio T 101 test Microbial inhibition Doxycycline 50 Eclipse 100 Microbial inhibition Doxycycline 100 Eclipse 50 Microbial inhibition Doxycycline 05 - 08 Tetrasensor Lateral flow Enoxacin 5 Fluoroquinolones EIA ELISA Enrofloxacin 6 Enrofloxacin EIA ELISA

Enrofloxacin 25 Parallux Solid phase immunoassay

Enrofloxacin 8 ROSA Enrofloxacin Lateral flow Enrofloxacin 1000 -1500 Valio T 101 test Microbial inhibition Enrofloxacine 3 Fluoroquinolones EIA ELISA Erythromycin 40 – 60 BRT Inhibitor Test Microbial inhibition Erythromycin 40 – 60 BRT MRL-Screening Test Microbial inhibition Erythromycin 100 BR-Test® AS Brilliant Microbial inhibition Erythromycin 100 BR-Test® AS Special Microbial inhibition Erythromycin 40 Charm II Macrolide Radio-labelled Assay Erythromycin 250-350 Copan Milk Test (3 hours) Microbial inhibition Erythromycin 250 Copan Milk Test (control time) Microbial inhibition Erythromycin 500-1500 Delvotest P (3 hours) Microbial inhibition Erythromycin 500 Delvotest P (control time) Microbial inhibition Erythromycin 100-250 Delvotest SP (3 hours) Microbial inhibition Erythromycin 50 Delvotest SP (control time) Microbial inhibition Erythromycin 200 Delvotest SP- NT (3 hours) Microbial inhibition

Erythromycin 40-80 Delvotest SP- NT (control time) Microbial inhibition

Erythromycin 500 Eclipse 100 Microbial inhibition Erythromycin 400 Eclipse 50 Microbial inhibition Erythromycin 400 Eclipse farm Microbial inhibition Erythromycin 150 SCREENING PLUS Microbial inhibition Erythromycin 50 -100 Valio T 101 test Microbial inhibition Erytromicin 200 KALIDOS MP Microbial inhibition Erytromicin 200 KALIDOS TB Microbial inhibition

177


in µg/kg Name of the test Type of test Erytromicin 200 KALIDOS TB Microbial inhibition Erytromicin 500 Total Antibiotics Microbial inhibition Florfenicol 40 Charm II Amphenicol Radio-labelled Assay Flumequine 13 Flumequine EIA ELISA Flumequine >10000 Copan Milk Test (3 hours) Microbial inhibition Flumequine >10000 Copan Milk Test (control time) Microbial inhibition Gentamicin 200 – 300 BRT Inhibitor Test Microbial inhibition Gentamicin 100 – 200 BRT MRL-Screening Test Microbial inhibition Gentamicin 300-400 Charm Blue-Yellow Test Microbial inhibition Gentamicin 300-400 Charm Blue-Yellow Test Microbial inhibition Gentamicin 500 Charm BSDA Microbial inhibition Gentamicin 300-400 Charm Cowside Microbial inhibition

Gentamicin 96 Charm II (Neomycin Type) Aminoglycoside (MRL) Radio-labelled Assay

Gentamicin 24 Charm II (Neomycin Type) Aminoglycoside (T/SL) Radio-labelled Assay

Gentamicin 50 Charm II (Streptomycin Type) Aminoglycoside (MRL) Radio-labelled Assay

Gentamicin 25 Charm II (Streptomycin Type) Aminoglycoside (US) Radio-labelled Assay

Gentamicin 250-350 Copan Milk Test (3 hours) Microbial inhibition Gentamicin 250 Copan Milk Test (control time) Microbial inhibition Gentamicin 300 Eclipse 100 Microbial inhibition Gentamicin 300 Eclipse 50 Microbial inhibition Gentamicin 200 Eclipse farm Microbial inhibition Gentamicin 2 Gentamicin EIA ELISA Gentamicin 50 I’screen gentamicin ELISA Gentamicin At or below 30 SNAP Gentamicin Lateral flow Gentamicin 500 -1000 Valio T 101 test Microbial inhibition Gentamycin 200 - 400 BR-Test® AS Brilliant Microbial inhibition Gentamycin 200 - 400 BR-Test® AS Special Microbial inhibition Gentamycin >1000 Delvotest P (3 hours) Microbial inhibition Gentamycin >500 Delvotest P (control time) Microbial inhibition Gentamycin 200-500 Delvotest SP (3 hours) Microbial inhibition Gentamycin 100-300 Delvotest SP (control time) Microbial inhibition Gentamycin 200 Delvotest SP- NT (3 hours) Microbial inhibition

Gentamycin 50 Delvotest SP- NT (control time) Microbial inhibition

Gentamycin 300 SCREENING PLUS Microbial inhibition Hetacillin 6-10 Delvo-X-press Receptor assay Kanamycin 4000-5000 Copan Milk Test (3 hours) Microbial inhibition Kanamycin 4000 Copan Milk Test (control time) Microbial inhibition Kanamycin 7500-ns Delvotest P (3 hours) Microbial inhibition Kanamycin 2500 Delvotest P (control time) Microbial inhibition Kanamycin 7500-ns Delvotest SP (3 hours) Microbial inhibition Kanamycin 2500 Delvotest SP (control time) Microbial inhibition Kanamycin >2000 Eclipse 100 Microbial inhibition Kanamycin >2000 Eclipse 50 Microbial inhibition Kanamycin 300 KALIDOS MP Microbial inhibition Kanamycin 150 KALIDOS TB Microbial inhibition Kanamycin 150 KALIDOS TB Microbial inhibition

178


in µg/kg Name of the test Type of test Kanamycin 3000 SCREENING PLUS Microbial inhibition Kanamycin 10 Total Antibiotics Microbial inhibition Lincomycin 150 – 300 BRT Inhibitor Test Microbial inhibition Lincomycin 75 – 150 BRT MRL-Screening Test Microbial inhibition Lincomycin 100 Charm II Macrolide Radio-labelled Assay Lincomycin 400-1000 Delvotest P (3 hours) Microbial inhibition Lincomycin 400 Delvotest P (control time) Microbial inhibition Lincomycin 200-400 Delvotest SP (3 hours) Microbial inhibition Lincomycin 100 Delvotest SP (control time) Microbial inhibition Lincomycin 300 Eclipse 100 Microbial inhibition Lincomycin 400 Eclipse 50 Microbial inhibition Lincomycin 200 SCREENING PLUS Microbial inhibition Lincomycin 300-500 Copan Milk Test (3 hours) Microbial inhibition Lincomycin 300 Copan Milk Test (control time) Microbial inhibition Lomefloxacin 8 Fluoroquinolones EIA ELISA Macrolides BR-Test® AS Brilliant Microbial inhibition Methacycline 10 - 13 Tetrasensor Lateral flow Methicillin 10-20 Delvo-X-press Receptor assay Nadifloxacin 3 Fluoroquinolones EIA ELISA Nafcillin 8-20 BetaStar Lateral flow Nafcillin 8-20 Betatar Combo Lateral flow Nafcillin 10 – 15 BRT Inhibitor Test Microbial inhibition Nafcillin 5 – 10 BRT MRL-Screening Test Microbial inhibition

Nafcillin 20-30 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Nafcillin ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Nafcillin 5-10 Delvotest P (3 hours) Microbial inhibition Nafcillin 5 Delvotest P (control time) Microbial inhibition Nafcillin 5-10 Delvotest SP (3 hours) Microbial inhibition Nafcillin 5 Delvotest SP (control time) Microbial inhibition Nafcillin 10 KALIDOS MP Microbial inhibition Nafcillin 10-15 KALIDOS TB Microbial inhibition Nafcillin 10-15 KALIDOS TB Microbial inhibition Nafcillin 52-111 SNAP MRL Beta –Lactam: Lateral flow Nafcillin 80 Total Antibiotics Microbial inhibition Nafcillin 30 - 40 Twinsensor 640 & 641 Lateral flow Nafcillin 30 - 40 Twinsensor 660 & 661 Lateral flow Naficillin 10 Copan Milk Test (3 hours) Microbial inhibition Naficillin 5 Copan Milk Test (control time) Microbial inhibition Neomycin 500 – 750 BRT Inhibitor Test Microbial inhibition Neomycin 500 – 750 BRT MRL-Screening Test Microbial inhibition Neomycin 400 - 800 BR-Test® AS Brilliant Microbial inhibition Neomycin 400 - 800 BR-Test® AS Special Microbial inhibition

Neomycin 40 Charm II (Neomycin Type) Aminoglycoside (MRL) Radio-labelled Assay

Neomycin 10 Charm II (Neomycin Type) Aminoglycoside (T/SL) Radio-labelled Assay

179


in µg/kg Name of the test Type of test Neomycin 1000-2000 Copan Milk Test (3 hours) Microbial inhibition Neomycin 1000 Copan Milk Test (control time) Microbial inhibition Neomycin >1000 Delvotest P (3 hours) Microbial inhibition Neomycin >500 Delvotest P (control time) Microbial inhibition Neomycin 300-2000 Delvotest SP (3 hours) Microbial inhibition Neomycin 100-200 Delvotest SP (control time) Microbial inhibition Neomycin 300-600 Delvotest SP- NT (3 hours) Microbial inhibition

Neomycin 100-200 Delvotest SP- NT (control time) Microbial inhibition

Neomycin 1500 Eclipse 100 Microbial inhibition Neomycin 1500 Eclipse 50 Microbial inhibition Neomycin 1500 Eclipse farm Microbial inhibition Neomycin 500 I’screen neomycin ELISA Neomycin 150 KALIDOS MP Microbial inhibition Neomycin 50 KALIDOS TB Microbial inhibition Neomycin 50 KALIDOS TB Microbial inhibition Neomycin 18 Neomycin EIA ELISA Neomycin 300 SCREENING PLUS Microbial inhibition Neomycin 2.5 Total Antibiotics Microbial inhibition Neomycin 300 - 500 Valio T 101 test Microbial inhibition

Neomycin 30 Parallux Solid phase immunoassay

Norfloxacin 3 Fluoroquinolones EIA ELISA

Norfloxacin 20 Parallux Solid phase immunoassay

Novobiocin 100 Charm II Novobiocin Radio-labelled Assay Novobiocin 1000 -1500 Valio T 101 test Microbial inhibition Oxacillin 5-10 BetaStar Lateral flow Oxacillin 5-10 Betatar Combo Lateral flow Oxacillin 10 – 20 BRT Inhibitor Test Microbial inhibition Oxacillin 5 – 10 BRT MRL-Screening Test Microbial inhibition

Oxacillin 25-35 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Oxacillin ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Oxacillin 10 Copan Milk Test (3 hours) Microbial inhibition Oxacillin 5 Copan Milk Test (control time) Microbial inhibition Oxacillin 10-15 Delvotest P (3 hours) Microbial inhibition Oxacillin 5 Delvotest P (control time) Microbial inhibition Oxacillin 10 Delvotest SP (3 hours) Microbial inhibition Oxacillin 5 Delvotest SP (control time) Microbial inhibition Oxacillin 10 Delvotest SP- NT (3 hours) Microbial inhibition

Oxacillin 10 Delvotest SP- NT (control time) Microbial inhibition

Oxacillin 25-50 Delvo-X-press Receptor assay Oxacillin 25 Eclipse 100 Microbial inhibition Oxacillin 25 Eclipse 50 Microbial inhibition Oxacillin 25 Eclipse farm Microbial inhibition Oxacillin 10 KALIDOS MP Microbial inhibition Oxacillin 15-20 KALIDOS TB Microbial inhibition Oxacillin 15-20 KALIDOS TB Microbial inhibition Oxacillin 30-50 Penzym 100 Lateral flow Oxacillin 30-30 Penzym 100 S Lateral flow

180


in µg/kg Name of the test Type of test Oxacillin 18 ROSA MRL3 Beta-Lactam Lateral flow Oxacillin 24-90 SNAP MRL Beta –Lactam: Lateral flow Oxacillin 15 Total Antibiotics Microbial inhibition Oxacillin 12 - 18 Twinsensor 640 & 641 Lateral flow Oxacillin 12 - 18 Twinsensor 660 & 661 Lateral flow Oxacillin 150 -200 Valio T 101 test Microbial inhibition Oxolinic acid 5 Fluoroquinolones EIA ELISA Oxytetracycline 25-50 Betatar Combo Lateral flow Oxytetracycline 500 – 750 BRT Inhibitor Test Microbial inhibition Oxytetracycline 250 – 500 BRT MRL-Screening Test Microbial inhibition Oxytetracycline 100 - 400 BR-Test® AS Brilliant Microbial inhibition Oxytetracycline 100 - 300 BR-Test® AS Special Microbial inhibition Oxytetracycline 300 Charm Blue-Yellow Test Microbial inhibition Oxytetracycline 300 Charm Blue-Yellow Test Microbial inhibition Oxytetracycline 400 Charm BSDA Microbial inhibition Oxytetracycline 200-300 Charm Cowside Microbial inhibition Oxytetracycline 19 Charm II Tetracycline Radio-labelled Assay Oxytetracycline 300-350 Copan Milk Test (3 hours) Microbial inhibition Oxytetracycline 250 Copan Milk Test (control time) Microbial inhibition Oxytetracycline 200-300 Delvotest P (3 hours) Microbial inhibition Oxytetracycline 100 Delvotest P (control time) Microbial inhibition Oxytetracycline 200-500 Delvotest SP (3 hours) Microbial inhibition Oxytetracycline 100 Delvotest SP (control time) Microbial inhibition Oxytetracycline 800 Delvotest SP- NT (3 hours) Microbial inhibition

Oxytetracycline 250-500 Delvotest SP- NT (control time) Microbial inhibition

Oxytetracycline 150 Eclipse 100 Microbial inhibition Oxytetracycline 150 Eclipse 50 Microbial inhibition Oxytetracycline 150 Eclipse farm Microbial inhibition Oxytetracycline 100-150 KALIDOS MP Microbial inhibition Oxytetracycline 100-150 KALIDOS TB Microbial inhibition Oxytetracycline 100-150 KALIDOS TB Microbial inhibition

Oxytetracycline 50-100 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Oxytetracycline 50 – 100 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Oxytetracycline 50 – 100 ROSA Tetracycline (MRL) Lateral flow Oxytetracycline 150-300 ROSA Tetracycline (US) Lateral flow Oxytetracycline 100 SCREENING PLUS Microbial inhibition Oxytetracycline At or below 30 SNAP Tetracycline: Lateral flow Oxytetracycline 15 - 20 Tetrasensor Lateral flow Oxytetracycline 100 Total Antibiotics Microbial inhibition Oxytetracycline 30 - 40 Twinsensor 640 & 641 Lateral flow Oxytetracycline 60 - 80 Twinsensor 660 & 661 Lateral flow Oxytetracycline 200 - 300 Valio T 101 test Microbial inhibition PABA 30 Charm II Sulfa Drug (MRL) Radio-labelled Assay PABA 3 Charm II Sulfa Drug (US) Radio-labelled Assay Pefloxacin 4 Fluoroquinolones EIA ELISA

Penethamate 2-3 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Penethamate ND Charm II Beta-lactam (Sequential) US Radio-labelled Assay

181


in µg/kg Name of the test Type of test Penicillin 2-4 BetaStar Lateral flow Penicillin 2-4 Betatar Combo Lateral flow Penicillin 3 - 4 BR-Test® AS Brilliant Microbial inhibition Penicillin 1-2 BR-Test® AS Special Microbial inhibition Penicillin 3-4 Charm Blue-Yellow Test Microbial inhibition Penicillin 3-4 Charm Blue-Yellow Test Microbial inhibition Penicillin 3.8 Charm BSDA Microbial inhibition Penicillin 3-4 Charm Cowside Microbial inhibition

Penicillin 2-3 Charm II Beta-lactam (Sequential) MRL Radio-labelled Assay

Penicillin 3.4 Charm II Beta-lactam (Sequential) US Radio-labelled Assay

Penicillin 3 Delvotest P (3 hours) Microbial inhibition Penicillin 2 Delvotest P (control time) Microbial inhibition Penicillin 2.5 Delvotest SP (3 hours) Microbial inhibition Penicillin 2 Delvotest SP (control time) Microbial inhibition Penicillin 2-3 Delvotest SP- NT (3 hours) Microbial inhibition

Penicillin 1-2 Delvotest SP- NT (control time) Microbial inhibition

Penicillin 2-4 Delvo-X-press Receptor assay Penicillin 4 Eclipse 100 Microbial inhibition Penicillin 4 Eclipse 50 Microbial inhibition Penicillin 4 Eclipse farm Microbial inhibition Penicillin 2-3 KALIDOS MP Microbial inhibition Penicillin 2-4 KALIDOS TB Microbial inhibition Penicillin 2-4 KALIDOS TB Microbial inhibition

Penicillin 2.3 Parallux Solid phase immunoassay

Penicillin 4-6 Penzym 100 Lateral flow Penicillin 2-4 Penzym 100 S Lateral flow

Penicillin 2.0 - 3.0 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Penicillin 2.0 – 3.0 ROSA MRL Beta-Lactam Lateral flow

Penicillin 2.0 – 3.0 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Penicillin 4.2 ROSA SL3 Beta-Lactam Lateral flow Penicillin 3.6 ROSA SLBL Beta-Lactam Lateral flow Penicillin 2.5 Total Antibiotics Microbial inhibition Penicillin 2 - 4 Valio T 101 test Microbial inhibition Penicillin G 3 ROSA MRL3 Beta-Lactam Lateral flow Penicillin G 4 SCREENING PLUS Microbial inhibition Penicillin-Na salt 2-3 Copan Milk Test (3 hours) Microbial inhibition Penicillin-Na salt 2 Copan Milk Test (control time) Microbial inhibition Penicillin-V 3-5 Delvo-X-press Receptor assay Peniclilin G 2-4 SNAP MRL Beta –Lactam: Lateral flow Piperacillin 10 – 15 BRT Inhibitor Test Microbial inhibition Piperacillin 5 – 10 BRT MRL-Screening Test Microbial inhibition Piperacillin 5-10 Delvo-X-press Receptor assay Pirlimycin 100-200 Charm Cowside Microbial inhibition Pirlimycin 80 Charm II Macrolide Radio-labelled Assay Pirlimycin 30 - 50 Valio T 101 test Microbial inhibition Piromidic acid 5 Fluoroquinolones EIA ELISA Procaine-penicillin 3-5 Delvo-X-press Receptor assay

182


in µg/kg Name of the test Type of test Rifampicin 100 KALIDOS MP Microbial inhibition Rifampicin 100-150 KALIDOS TB Microbial inhibition Rifampicin 100-150 KALIDOS TB Microbial inhibition Rifampicin 150 Total Antibiotics Microbial inhibition Rifamycin 25 Total Antibiotics Microbial inhibition Spectinomycin 3000-4000 Copan Milk Test (3 hours) Microbial inhibition Spectinomycin 3000 Copan Milk Test (control time) Microbial inhibition Spiramicin 600-800 KALIDOS TB Microbial inhibition Spiramicin 600-800 KALIDOS TB Microbial inhibition Spiramicin 400 SCREENING PLUS Microbial inhibition Spiramycin 400 – 600 BRT Inhibitor Test Microbial inhibition Spiramycin 200 – 400 BRT MRL-Screening Test Microbial inhibition Spiramycin 400 - 1000 BR-Test® AS Brilliant Microbial inhibition Spiramycin 400 - 800 BR-Test® AS Special Microbial inhibition Spiramycin 50 Charm II Macrolide Radio-labelled Assay Spiramycin 1000-2000 Copan Milk Test (3 hours) Microbial inhibition Spiramycin 1000 Copan Milk Test (control time) Microbial inhibition Spiramycin 350-ns Delvotest P (3 hours) Microbial inhibition Spiramycin 200 Delvotest P (control time) Microbial inhibition Spiramycin 350-ns Delvotest SP (3 hours) Microbial inhibition Spiramycin 200 Delvotest SP (control time) Microbial inhibition Spiramycin 800-1000 Delvotest SP- NT (3 hours) Microbial inhibition

Spiramycin 400-600 Delvotest SP- NT (control time) Microbial inhibition

Spiramycin >400 Eclipse 100 Microbial inhibition Spiramycin >400 Eclipse 50 Microbial inhibition Spiramycin 900 KALIDOS MP Microbial inhibition Spiramycin 500 Total Antibiotics Microbial inhibition Spiramycin 150 - 300 Valio T 101 test Microbial inhibition

Streptomycin 40 Charm II (Streptomycin Type) Aminoglycoside (MRL) Radio-labelled Assay

Streptomycin 20 Charm II (Streptomycin Type) Aminoglycoside (US) Radio-labelled Assay

Streptomycin 1000-2000 Copan Milk Test (3 hours) Microbial inhibition Streptomycin 1000 Copan Milk Test (control time) Microbial inhibition Streptomycin 3000 Eclipse 100 Microbial inhibition Streptomycin 2000 Eclipse 50 Microbial inhibition Streptomycin 800 KALIDOS MP Microbial inhibition Streptomycin 400-600 KALIDOS TB Microbial inhibition Streptomycin 400-600 KALIDOS TB Microbial inhibition

Streptomycin 50 Parallux Solid phase immunoassay

Streptomycin 4 Streptomycin EIA ELISA Streptomycin 350 Total Antibiotics Microbial inhibition Streptomycin 1000 -1500 Valio T 101 test Microbial inhibition Sulfacetamide 500 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfacetamide 50 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfacetamide 100-150 Copan Milk Test (3 hours) Microbial inhibition Sulfacetamide 100 Copan Milk Test (control time) Microbial inhibition Sulfacetamide 500 -1000 Valio T 101 test Microbial inhibition Sulfachlorpyridazine 50 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfachlorpyridazine 5 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfadiazine 500 – 750 BRT Inhibitor Test Microbial inhibition

183


in µg/kg Name of the test Type of test Sulfadiazine 100 BRT MRL-Screening Test Microbial inhibition Sulfadiazine 25 - 100 BR-Test® AS Brilliant Microbial inhibition Sulfadiazine 50-150 BR-Test® AS Special Microbial inhibition Sulfadiazine 49 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfadiazine 4.9 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfadiazine 50-100 Copan Milk Test (3 hours) Microbial inhibition Sulfadiazine 50 Copan Milk Test (control time) Microbial inhibition Sulfadiazine 50-100 Delvotest SP (3 hours) Microbial inhibition Sulfadiazine 50 Delvotest SP (control time) Microbial inhibition Sulfadiazine 100-150 Delvotest SP- NT (3 hours) Microbial inhibition

Sulfadiazine 25-50 Delvotest SP- NT (control time) Microbial inhibition

Sulfadiazine 150 Eclipse 100 Microbial inhibition Sulfadiazine 100 Eclipse 50 Microbial inhibition Sulfadiazine 100 KALIDOS MP Microbial inhibition Sulfadiazine 25-100 KALIDOS TB Microbial inhibition Sulfadiazine 25-100 KALIDOS TB Microbial inhibition Sulfadiazine 300 - 500 Valio T 101 test Microbial inhibition Sulfadimethoxin 500 – 750 BRT Inhibitor Test Microbial inhibition Sulfadimethoxin 100 BRT MRL-Screening Test Microbial inhibition Sulfadimethoxin 50 Charm Blue-Yellow Test Microbial inhibition Sulfadimethoxin 50 Charm Blue-Yellow Test Microbial inhibition Sulfadimethoxin 50 Charm Cowside Microbial inhibition Sulfadimethoxin 40 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfadimethoxin 4 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfadimethoxin 50-100 Copan Milk Test (3 hours) Microbial inhibition Sulfadimethoxin 50 Copan Milk Test (control time) Microbial inhibition Sulfadimethoxin 50-100 Delvotest SP (3 hours) Microbial inhibition Sulfadimethoxin 50 Delvotest SP (control time) Microbial inhibition

Sulfadimethoxin 10 Parallux Solid phase immunoassay

Sulfadimethoxin 6.7 ROSA SDSM Lateral flow Sulfadimethoxin 300 - 500 Valio T 101 test Microbial inhibition Sulfadimetoxina 100 KALIDOS MP Microbial inhibition Sulfadimetoxina 25-100 KALIDOS TB Microbial inhibition Sulfadimetoxina 25-100 KALIDOS TB Microbial inhibition Sulfadoxine 70 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfadoxine 7 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfadoxine 100-200 Copan Milk Test (3 hours) Microbial inhibition Sulfadoxine 100 Copan Milk Test (control time) Microbial inhibition Sulfadoxine 500 -1000 Valio T 101 test Microbial inhibition Sulfamerazine 50 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfamerazine 5 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfamerazine 100 Copan Milk Test (3 hours) Microbial inhibition Sulfamerazine 70 Copan Milk Test (control time) Microbial inhibition Sulfamerazine 100 KALIDOS MP Microbial inhibition Sulfamerazine 25-100 KALIDOS TB Microbial inhibition Sulfamerazine 25-100 KALIDOS TB Microbial inhibition Sulfamerazine 500 -1000 Valio T 101 test Microbial inhibition Sulfamethazine 500 – 750 BRT Inhibitor Test Microbial inhibition Sulfamethazine 100 – 300 BRT MRL-Screening Test Microbial inhibition Sulfamethazine 50 - 200 BR-Test® AS Brilliant Microbial inhibition Sulfamethazine 100-200 BR-Test® AS Special Microbial inhibition

184


in µg/kg Name of the test Type of test Sulfamethazine 100-200 Charm Cowside Microbial inhibition Sulfamethazine 94 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfamethazine 9.4 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfamethazine 50-200 Delvotest SP (3 hours) Microbial inhibition Sulfamethazine 25 Delvotest SP (control time) Microbial inhibition Sulfamethazine 100-250 Delvotest SP- NT (3 hours) Microbial inhibition

Sulfamethazine 25-100 Delvotest SP- NT (control time) Microbial inhibition

Sulfamethazine 150 Eclipse 100 Microbial inhibition Sulfamethazine 200 Eclipse 50 Microbial inhibition Sulfamethazine 200 Eclipse farm Microbial inhibition Sulfamethazine 150-200 KALIDOS MP Microbial inhibition

Sulfamethazine 10 Parallux Solid phase immunoassay

Sulfamethazine 6.2 ROSA SDSM Lateral flow Sulfamethazine At or below 10 SNAP Sulfamethazine Lateral flow Sulfamethazine 8 Sulfamethazine EIA ELISA Sulfamethazine 500 -1500 Valio T 101 test Microbial inhibition Sulfamethazine5) 100-150 Copan Milk Test (3 hours) Microbial inhibition Sulfamethazine5) 100 Copan Milk Test (control time) Microbial inhibition Sulfamethizole 150 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfamethizole 15 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfamethizole 300 - 500 Valio T 101 test Microbial inhibition Sulfamethoxazole 30 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfamethoxazole 3 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfamethoxazole 50-100 Copan Milk Test (3 hours) Microbial inhibition Sulfamethoxazole 50 Copan Milk Test (control time) Microbial inhibition Sulfamethoxazole 50 Eclipse 100 Microbial inhibition Sulfamethoxazole 50 Eclipse 50 Microbial inhibition Sulfamethoxazole 100 KALIDOS MP Microbial inhibition Sulfamethoxazole 25-100 KALIDOS TB Microbial inhibition Sulfamethoxazole 25-100 KALIDOS TB Microbial inhibition Sulfamethoxazole 50 SCREENING PLUS Microbial inhibition Sulfamethoxypyridazine 50 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfamethoxypyridazine 5 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfamethoxypyridazine 100 Eclipse 100 Microbial inhibition Sulfamethoxypyridazine 100 Eclipse 50 Microbial inhibition Sulfamonometossina 50-100 Copan Milk Test (3 hours) Microbial inhibition Sulfamonometossina 50 Copan Milk Test (control time) Microbial inhibition Sulfanilamide 500 Eclipse 100 Microbial inhibition Sulfanilamide 600 Eclipse 50 Microbial inhibition Sulfanilamide 600 Eclipse farm Microbial inhibition Sulfapyridine 100 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfapyridine 10 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfaquinoxalina 25-100 KALIDOS TB Microbial inhibition Sulfaquinoxalina 25-100 KALIDOS TB Microbial inhibition Sulfaquinoxaline 30 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfaquinoxaline 3 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfaquinoxaline 100 KALIDOS MP Microbial inhibition Sulfathiazole 200 – 400 BRT Inhibitor Test Microbial inhibition Sulfathiazole 100 BRT MRL-Screening Test Microbial inhibition Sulfathiazole 73 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfathiazole 7.3 Charm II Sulfa Drug (US) Radio-labelled Assay

185


in µg/kg Name of the test Type of test Sulfathiazole 50-100 Copan Milk Test (3 hours) Microbial inhibition Sulfathiazole 50 Copan Milk Test (control time) Microbial inhibition Sulfathiazole 50-150 Delvotest SP (3 hours) Microbial inhibition Sulfathiazole 50 Delvotest SP (control time) Microbial inhibition Sulfathiazole 100 Eclipse 100 Microbial inhibition Sulfathiazole 100 Eclipse 50 Microbial inhibition Sulfathiazole 100 Eclipse farm Microbial inhibition Sulfathiazole 5000 SCREENING PLUS Microbial inhibition Sulfathiazole 200 - 500 Valio T 101 test Microbial inhibition Sulfisoxazole 60 Charm II Sulfa Drug (MRL) Radio-labelled Assay Sulfisoxazole 6 Charm II Sulfa Drug (US) Radio-labelled Assay Sulfonamides Copan Milk Test (control time) Microbial inhibition Sulfonamides <2.5 Sulfonamides EIA ELISA Sulphametazine 100-150 KALIDOS TB Microbial inhibition Sulphametazine 100-150 KALIDOS TB Microbial inhibition Sulphaquinoxaline 20 Sulphaquinoxaline EIA ELISA Sulphonamides BR-Test® AS Brilliant Microbial inhibition Tetracycline 50 Betatar Combo Lateral flow Tetracycline 200 – 400 BRT Inhibitor Test Microbial inhibition Tetracycline 100 – 200 BRT MRL-Screening Test Microbial inhibition Tetracycline 5 Charm II Tetracycline Radio-labelled Assay Tetracycline 250-300 Copan Milk Test (3 hours) Microbial inhibition Tetracycline 200 Copan Milk Test (control time) Microbial inhibition Tetracycline 200-300 Delvotest P (3 hours) Microbial inhibition Tetracycline 100 Delvotest P (control time) Microbial inhibition Tetracycline 200-600 Delvotest SP (3 hours) Microbial inhibition Tetracycline 100 Delvotest SP (control time) Microbial inhibition Tetracycline 800 Delvotest SP- NT (3 hours) Microbial inhibition

Tetracycline 250-500 Delvotest SP- NT (control time) Microbial inhibition

Tetracycline 150 Eclipse 100 Microbial inhibition Tetracycline 150 Eclipse 50 Microbial inhibition Tetracycline 150 Eclipse farm Microbial inhibition Tetracycline 100-150 KALIDOS MP Microbial inhibition Tetracycline 100-150 KALIDOS TB Microbial inhibition Tetracycline 100-150 KALIDOS TB Microbial inhibition

Tetracycline 50-100 ROSA 3 minute MRL Beta-Lactam and Tetracycline Lateral flow

Tetracycline 7 - 15 ROSA MRLBLTET Beta-Lactam and Tetracycline Lateral flow

Tetracycline 10 – 30 ROSA Tetracycline (MRL) Lateral flow Tetracycline 30-90 ROSA Tetracycline (US) Lateral flow Tetracycline 100 SCREENING PLUS Microbial inhibition Tetracycline At or below 50 SNAP Tetracycline: Lateral flow Tetracycline 50 SuperScreen TETRA ELISA Tetracycline 20 - 25 Tetrasensor Lateral flow Tetracycline 100 Total Antibiotics Microbial inhibition Tetracycline 40 - 50 Twinsensor 640 & 641 Lateral flow Tetracycline 80 - 100 Twinsensor 660 & 661 Lateral flow Tetracycline 200 - 300 Valio T 101 test Microbial inhibition

Tetracycline 75 Parallux Solid phase immunoassay

Tetracyclines BR-Test® AS Brilliant Microbial inhibition

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in µg/kg Name of the test Type of test Thiamphenicol 50 Charm II Amphenicol Radio-labelled Assay Thiamphenicol >100 Copan Milk Test (3 hours) Microbial inhibition Thiamphenicol 100 Copan Milk Test (control time) Microbial inhibition Thrimethoprim 200-400 KALIDOS TB Microbial inhibition Thrimethoprim 200-400 KALIDOS TB Microbial inhibition Ticarcillin 30-100 Delvo-X-press Receptor assay Tilmicosin 20 Charm II Macrolide Radio-labelled Assay Trimethoprim 50-100 Copan Milk Test (3 hours) Microbial inhibition Trimethoprim 50 Copan Milk Test (control time) Microbial inhibition Trimethoprim 500-1500 Delvotest P (3 hours) Microbial inhibition Trimethoprim 100 Delvotest P (control time) Microbial inhibition Trimethoprim 100-500 Delvotest SP (3 hours) Microbial inhibition Trimethoprim 50 Delvotest SP (control time) Microbial inhibition Trimethoprim 200-300 Delvotest SP- NT (3 hours) Microbial inhibition

Trimethoprim 50-100 Delvotest SP- NT (control time) Microbial inhibition

Trimethoprim 2000 - 5000 Valio T 101 test Microbial inhibition Tylmicosin 75-100 Copan Milk Test (3 hours) Microbial inhibition Tylmicosin 75 Copan Milk Test (control time) Microbial inhibition Tylomycin 30 - 40 BR-Test® AS Brilliant Microbial inhibition Tylosin 25 – 50 BRT Inhibitor Test Microbial inhibition Tylosin 25 – 50 BRT MRL-Screening Test Microbial inhibition Tylosin 20 BR-Test® AS Special Microbial inhibition Tylosin 75-100 Charm Blue-Yellow Test Microbial inhibition Tylosin 75-100 Charm Blue-Yellow Test Microbial inhibition Tylosin 200 Charm BSDA Microbial inhibition Tylosin 75-100 Charm Cowside Microbial inhibition Tylosin 50 Charm II Macrolide Radio-labelled Assay Tylosin 50-100 Copan Milk Test (3 hours) Microbial inhibition Tylosin 50 Copan Milk Test (control time) Microbial inhibition Tylosin 100-300 Delvotest P (3 hours) Microbial inhibition Tylosin 50-100 Delvotest P (control time) Microbial inhibition Tylosin 30-100 Delvotest SP (3 hours) Microbial inhibition Tylosin 10-20 Delvotest SP (control time) Microbial inhibition Tylosin 50 Delvotest SP- NT (3 hours) Microbial inhibition

Tylosin 30 Delvotest SP- NT (control time) Microbial inhibition

Tylosin 80 Eclipse 100 Microbial inhibition Tylosin 100 Eclipse 50 Microbial inhibition Tylosin 100 Eclipse farm Microbial inhibition Tylosin 5 I’screen tylosin ELISA Tylosin 40 KALIDOS MP Microbial inhibition Tylosin 40-50 KALIDOS TB Microbial inhibition Tylosin 40-50 KALIDOS TB Microbial inhibition Tylosin 60 SCREENING PLUS Microbial inhibition Tylosin 100 Total Antibiotics Microbial inhibition

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