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Rapid Test Kits for the Detection of

Antibiotics and Sulphonamides in Milk

June 2002

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CONTENTS

Page

DISCLAIMER 3

1. INTRODUCTION 4

2. MICROBIAL INHIBITION ASSAYS FOR THE DETECTION OF 6


ANTIBIOTICS IN MILK

3. RAPID TEST KITS FOR THE DETECTION OF ANTIBIOTICS IN 12


MILK

4. CONCLUSIONS 18

5. REFERENCES 19

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DISCLAIMER

The Food Safety Authority of Ireland cannot accept any liability for inaccuracies in the
test specifications which were based on information provided by the manufacturers.
The costs outlines were accurate at the time of publication.

The work reported does not imply that the Food Safety Authority of Ireland endorse
or in any other way promote the products and manufacturers featured.

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1. INTRODUCTION
Antibiotic contamination in milk can seriously affect consumers’ health by causing
allergic reactions to residues or by the development of resistant strains of
microorganisms. Therefore, subsequently antibiotic contamination in milk can also
cause significant economic losses for producers and manufacturers of milk and milk
products.

Therapeutic use of antibiotics, which is the use of antibiotics in the treatment of


disease, involves high dosages for a short period given by injection or orally in water
or, less often, given in feed. Sometimes, antibiotics are fed at lower levels to prevent
disease at critical times in the animal' s life, such as weaning, transport, mixing, this
type of antibiotic use is defined as prophylactic use of antibiotics.

When an antimicrobial is used in an animal in veterinary medicine, the animal cannot


be marketed or milk sent for human consumption before the specified withdrawal (or
withholding) period has expired. Sources of antibiotic residues in milk as outlined in
Radostitis et al. (1994) include:

• Extended usage or excessive dosage


• Failure to observe withdrawal times
• Poor records of treatment
• Prolonged drug clearance
• Failure to identify treated animals
• Contaminated milking equipment
• Products not used according to label directions
• Lack of advice on withdrawal period
• Withholding milk from treated milk only
• Early calving or short dry periods
• Purchase of treated cows
• Use of dry cow therapy to lactating cows
• Milking dry cows

Presently, the standard test method used for the detection of antibiotic residues in
liquid milk at the pasteurising plants in Ireland is the Delvo® SP (Fallon et al.,
1995/1996). The Delvo® SP is a test dependent on rapid growth and acid production
of Bacillus stearothermophilus var. calidolactis. This test method requires a lengthy
incubation time of 2 hours and 30 minutes. Thus, to avoid delays at milk intake
points, rapid antibiotic screening tests are regularly used on raw milk at the milk
pasteurising plants prior to completion of the Delvo® SP test.

The Delvo® SP is a microbial inhibition assay. Microbial inhibition assays test a broad
spectrum of antibiotics, whereas rapid test kits generally only detect a specific family
of antibiotics.

The Veterinary Inspectorate of the Department of Agriculture, Food and Rural


Development carry out frequent inspections of the EU approved pasteurising plants
in accordance with the requirements of EU Council Directive 92/46 and implemented
by European Communities Regulations, 1996 (S.I. No. 9 of 1996). At these

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inspections representative samples of the day’s production are collected for analysis
in the Department of Agriculture, Food and Rural Development Dairy Science
laboratories (Fallon et al., 1995/1996). The standard sampling protocol is 1 litre of
product sampled every 20 minutes (personal communication, Regional Veterinary
Laboratory, Sligo). The Delvo® SP test method is used by the DAFRD laboratories
for the determination of antibiotic residues in milk.

The Delvo® SP microbial inhibition test is required by DAFRD to be carried out for the
detection of antibiotics and sulphonamides in milk. However, rapid test kits are often
used by industry prior to the Delvo® SP test method, often for the positive release of
milk tankers.

The objective of the report is to provide an overview of the rapid test kits available for
the detection of antibiotics and sulphonamides in milk, thus enabling the dairy
industry to choose the test kit which is most appropriate to their use.

The report reviews test parameters such as the test sensitivity concerning the
maximum residue limits which have been set in accordance with EU Council
Regulation No. 2377/90 and amendments.

Residues of veterinary medicinal products are defined as all pharmacologically active


substances, whether active principles, excipients or degradation products, and their
metabolites which remain in foodstuffs obtained from animals to which the veterinary
medicinal product in question has been administered. A maximum residue limit
(MRL) is defined as the maximum concentration of residue resulting from the use of a
veterinary medicinal product which may be legally permitted or recognised as
acceptable in or on a food. The MRL is expressed in micrograms of the chemical per
kilogram of the food.

The MRL is based on the type and amount of residue considered to be without any
toxicological hazard for human health as expressed by the acceptable daily intake
(ADI), or on the basis of a temporary ADI that utilises an additional safety factor. It
also takes into account other relevant public health risks as well as food technology
aspects.

When establishing MRLs for veterinary medicinal products, consideration is also


given to residues that occur in food of plant origin and/or come from the environment.
Furthermore, the MRL may be reduced to be consistent with good practices in the
use of veterinary drugs and to the extent that practical analytical methods are
available.

The report also includes descriptions of the various test principles and the cost
(where supplied by the relevant company) of the rapid detection kits. Microbial
inhibition tests methods based on the EU reference method for the detection of
antibiotic residues in milk are described. The minimum reported sensitivities of the
individual rapid test kits for detection of antibiotic residues in milk are compared to
both the sensitivity of microbial inhibition tests (Delvo® SP) and to the EU MRL.

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2. MICROBIAL INHIBITION ASSAYS FOR THE DETECTION OF
ANTIBIOTICS IN MILK

2.1 REFERENCE METHOD.


The EU reference method for the determination of antibiotic residues in raw milk and
in heat-treated milk is the International Dairy Federation (IDF) microbial inhibition test
method using Bacillus stearothermophilus (IDF, 1970; IDF, 1991). The IDF microbial
inhibitor test method uses Bacillus stearothermophilus var. calidolactis, ATCC 10149
as the test organism due to its relatively high sensitivity to inhibitory substances (IDF,
1991) (Table 1). The IDF test procedure for detection of antibiotic residues in milk
has been chosen as representative of similar procedures which in principle use B.
stearothermophilus as the test organism. However, the IDF method is quite complex
and lengthy to carry out as it involves the continual growth of large quantities of B.
stearothermophilus spores. As the test involves a colour change, which is dependent
on the growth of B. stearothermophilus, if the organism fails to grow then a false
negative result may occur. Therefore, due to the aforementioned technical difficulties
in carrying out the reference method, microbial inhibitor test kit assays based on the
IDF method, using B. stearothermophilus are the routine methods used for the
determination of antibiotic residues in milk. These are described in Table 2 and
sections 2.2-2.5.

Table 1. Detectable concentrations of various antibiotics and sulphonamides using the


International Dairy Federation microbial inhibitor test method (IDF, 1991).

Antibiotic Test Sensitivity(1)


______________________________________________
All Negative All Positive

Benzylpenicillin 2 6
Ampicillin 2 5
Cloxacillin 15 35
Nafcillin 6 11
Tetracycline 100 400
Oxytetracycline 200 450
Chlortetracycline 150 500
Chloramphenicol 7000 15000
Dihydrostreptomycin 4000 13000
Neomycine 1000 22000
Kanamycin 9000 28000
Bacitracin 60 140
Erythromycin 1000 2250
Rifamycin 10 140
Diaphenylsulfone 10 100
Sulphamethazine (Sulphadimidine) 500 1000

(1)
Benzylpenicillin and bacitracin expressed as IU/kg, all others antibacterial substances as
µg/kg.

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Table 2. List of microbial inhibition assays for the detection of antibiotics in milk.

Name of Microbial Manufacturer Time per test Antibiotic tested Reaction type
Inhibition Assays
Delvotest SP Kit Gist-brocades/DSM 2. h 30 min Broad spectrum Colour change
Copan test (CH ATK) Copan Italia SpA 2 h 30 min -3 h Broad spectrum Colour change
Charm Farm Test Charm Sciences 2 h 30 min -3 hr Broad spectrum Colour change
Charm AIM-96 Charm Sciences 4 hrs Broad spectrum Colour change

2.2 DSM DELVOTEST® SP.


The Delvo® SP microbial inhibition assay for the detection of antibiotics and
sulphonamides in milk is based on the IDF reference method, however, it contains a
pre-defined number of B. stearothermophilus spores and agar wells which enables
the test to be carried out in 2 h 30 min (at the time the negative control has been
changed to yellow). Nutrients required for growth are in a tablet format for
maximizing shelf life performance. The Delvo® SP block method using the 96-well
plate supersedes the Delvo® P test which has been discontinued by the
manufacturing company. The Delvo® SP is a broad spectrum screening test for the
detection of antibiotic residues and sulphonamides in milk. The minimum sensitivities
for detection of beta-lactams, sulphonamides and other antibiotic residues, using the
Delvo® SP assay are shown in Table 3. The Delvo® SP is presently the standard test
method required by the DAFRD for the detection of antibiotics and sulphonamides in
milk, and is the current test method carried out in Ireland by dairy companies and the
DAFRD Dairy Science laboratories.

2.2.1 Test Outline

The growth of the B. stearothermophilus spores at 64°C initiates an acidification


process which causes the turning of a pH indicator from purple to yellow. The
presence of antibacterial substances will cause delay or inhibition of the spores,
depending on the concentration of the residues. In the presence of residues the
spores will not multiply and the pH indicator will remain purple.

1. Add 1 nutrient tablet to each of the agar wells in the strip.


2. Inoculate 100 µl of milk into the agar well plus nutrient tablet.
3. Seal the wells for incubation
4. Incubate the strip of wells in a waterbath at 64°C ± 0.5°C for 2 h 30 min* (at the
time the negative control has been changed to yellow)
5. Examine the strip for colour change from purple to yellow. A yellow reading
indicates that no inhibitory substances are present, a purple reading indicates that
antibiotic residues are present and a yellow/purple reading indicates a doubtful
result.

*For best sensitivity a control time reading is advised using a negative control
sample.

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The Public Health Unit of the DAFRD, Regional Veterinary Laboratory (RVL), Sligo
examine all biochemical and microbiological test methods carried out at the liquid
milk processing plants. The Public Health Unit of the DAFRD, RVL, Sligo make the
following recommendations regarding the Delvo® SP test methodology:

• The sample incubation period is crucial to the accuracy of the Delvo® SP test
method. This particularly applies to the detection of sulphonamides as the
sensitivity to sulphonamides is greatly reduced by an increased incubation time.
An incubation time of 2 hr 30 min (at the time the negative control has been
changed to yellow) is recommended with a 15 minute extension time of the test
in the case of a suspect sample. For reading times of 2 h 45 min or 3 hours the
sensitivity of the test will diminish. The use of antibiotic free skim milk powder as
a control is also advised.
• It is essential that the correct temperature is maintained in the waterbath (64°C ±
0.5°C)
• The use of a proper waterbath lid, a sloping lid is advised
• The temperature control in the waterbath should be a digital temperature readout
• Good circulation should be maintained in the waterbath

2.2.2 Cost per test


No instrumentation is required for this test method. However, an accurate, controlled
circulation waterbath with digital temperature readout is essential to ensure a
constant temperature of 64°C in the waterbath. A sloping lid for the waterbath is
advised.

The DSM, Delvo range of products is supplied by The Carbon Group, Ringaskiddy,
Co. Cork (Contact: Gerry O’Brien 021-4378988). The cost of the Delvo® SP test is: ~
€ 96.5 per 480 tests i.e., ~20 cent per test (standard flat price). It must be noted that
the above price quotation is for the materials only cost per test.

2.3 COPAN TEST.


The Copan test (CH ATK) P&S kit is a method for detection of antibiotic residues in
milk which has recently undergone a validation exercise in an independent Irish
laboratory. This method is also based on the IDF standard method for determination
of antibiotic residues in milk. The minimum sensitivities for detection of beta-lactams,
sulphonamides and other antibiotic residues, using the Copan antibiotic test kit are
shown in Table 3. This method is very similar to that of the Delvo® SP test method,
however, the nutrient tablet is already added to the agar, therefore, the procedure is
one step shorter than the Delvo® SP.

2.3.1 Cost per test

As for the Delvo® SP test, no instrumentation is required for this test method and an
accurate, controlled circulation waterbath is essential to ensure a constant
temperature of 64°C.

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The Copan Test is a product of Copan Italia SpA, via Perotti 10, 25125 Brescia, Italy
(phone: +39 030 2687201; +39 030 2687213; E-mail info@copanitalia.com). Davidson
and Hardy, 8 Pembroke Road, Dublin 4 (01-6600725) are the agents for the Copan
Test in Ireland. Pricing is subject to quantity discounts and volume usage.

2.4 CHARM FARM TEST


The Charm Farm test is a microbial inhibition test which uses a one step single
service vial. The Charm Farm test is a broad screening assay for five families of
veterinary drugs, including beta-lactams, sulphonamides, tetracyclines,
aminoglycosides and macrolides in raw, commingled, bovine milk. The results are
stable for 8 hours after assay completion and can be read by visual colour
comparison or optionally with a pH meter. The Charm Auto-Farm Equipment is
required to run this test. The test can be completed in approximately 3.5 hours. Up to
12 tests can be run simultaneously. The manufacturer’s declared sensitivities to
antimicrobials using the Charm Farm Test are outlined in Table 3.

2.5 CHARM AIM-96

The Charm AIM-96 Test is designed for high volume, broad spectrum screening of
raw, pasteurised, homogenised or skim milk. The results can be read by visual colour
comparison or optionally with a microplate reader. The Charm AIM-96 detects beta-
lactams, sulphonamides , tetracyclines, aminoglycosides and macrolides. 96 tests
can be completed simultaneously in approximately 4 hours. The manufacturer’s
declared sensitivity to antimicrobials using the Charm AIM-96 are outlined in Table 3.

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Table 3. Comparison of the EU maximum residue limits (EEC Regulation 2377/90 and
amendments) with the sensitivities of the microbial inhibition assays for the detection of
antibiotics in milk.

Antibiotic EU Maximum Sensitivity Sensitivity Charm Charm


Residue Limit(1) Delvo SP(2)* Copan(3)* AIM-96* Farm Test*
in Milk Test
µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)

β-lactams
Penicillin 4 2 1-2 4 3
Penethamat 4 (2) 4
Ampicillin 4 2-3 <2 6 4
Amoxicillin 4 2 2-4 4 4
Nafcillin 30 5 5-10
Cloxacillin 30 15 10-15 50
Dicloxacillin 30 10 10-15 30
Oxacillin 30 5 5-10 30
Cefecetril 125 20
Cefalexin 100 40-60 >45
Cefalonium 10 5-10
Ceftiofur(4) 100(5) 50 50-100 50 50
Cephapirin 10 5 2.5-5 10
Cephazolin 50 (10) 5-10
Cefquinom 20 (100) 30-100(8) 20
Cefaperazon 40 25-50

Tetracyclines
Chlortetracycline(4) 100(6) 150 250-500 150
Doxycycline(4) 0 (100) 150
Oxytetracycline(4) 100(6) 100 250-500 150 100-150
Tetracycline(4) 100(6) 100 250-500 100

Sulphonamides
Sulphadimidine 100(7) equal to sulfamethazine
Sulphadimethoxine 100(7) 50 50-100 15
Sulphamethazine 100(7) 25 100-200 10 50-100
Sulphathiazole 100(7) 50 50-100 5
Sulphadiazine 100(7) 50 50-100 5
Sulphadioxine (100) 100-200
Sulphamethoxazole (50) <50
Sulphamonometossina <50

Macrolides
Erythromycin 40 50 >200 100
Pirlimycin 150 (50-200)
Spiramycin 200 200 >2000 500
Tilmicosin 50 (100) 75-100 180
Tylosin 50 50 50-100 50 40-50

Aminoglycosides
Gentamicin 100 100-300 100-500 30 300-500
Neomycin 500 100-200 500-2000 100
Spectinomycin 200 (2500) >300
DH/Streptomycin 200 300-500 <1000 600/500
Kanamycin 2500

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Antibiotic EU Maximum Sensitivity Sensitivity Charm Charm
Residue Limit(1) Delvo SP(2)* Copan(3)* AIM-96* Farm Test*
in Milk Test
µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)

Quinolones
Enrofloxacin 100 (1000-1500)
Danofloxacin 30 (close to enrofloxacin)
Difloxacin 0 (close to enrofloxacin)
Flumequin 0 (no data available)
Marbofloxacin 75 (close to enrofloxacin)

Various
Bacitracin 150 (1000)
Baquiloprim 30 (60)
Chloramphenicol 0 2500 5000
Clavulanic acid 200 (not antibacterial)
Colistin 50 (no data available)
Dapsone 0 1 2-4 5
Lincomycin 150 100
Novobiocin 50 (1000)
Rifaximin 60 (50)
Thiamphenicol 50 (close to >100
chloramphenicol)
Trimethoprim 50 50 100-150 200

* It is important to note that the sensitivities of the individual test kits are based on
manufacturer’s declared levels.
(1)
Council Regulation (EEC) No. 2377/90 and amendments.
(2)
Values are for detection levels at control time. For reading times of 2 h 45 min or 3 hours
the sensitivity will diminish. Values in parenthesis are given as indication from the DSM
data-base.
(3)
Screening test, Bundesanstalt f r Milchforschung, September 2000.
(4)
Mother compound.
(5)
Mother compound and metabolites.
(6)
Mother compound and 4-epimer.
(7)
Sum of substances if this group
(8)
The lowest tested concentration (100 µg/kg) was evaluated as positive.

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3. RAPID TEST KITS FOR THE DETECTION OF ANTIBIOTICS IN
MILK
The following section of the report and Table 4 outline the test methods available for
the rapid detection of antibiotics in milk as distinct from section 2 above which
describes the microbial inhibition assays for the detection of antibiotics in milk.
Microbial inhibition assays are lengthy and test for a broad spectrum of antibiotics,
whereas rapid test kits generally only detect a specific family of antibiotics.

The standard test method used for the detection of antibiotic residues in liquid milk at
the pasteurising plants in Ireland is the Delvo® SP which is a microbial inhibition
assay. To avoid delays at milk intake points, rapid antibiotic screening tests are often
used on raw milk prior to completion of the Delvo® SP test.

Table 4. List of rapid tests for the detection of antibiotics in milk.

Name of Rapid Test Manufacturer Time per test Antibiotic tested Reaction type
Parallux β-Lactam Idexx Inc. 4 min β-lactams Fluorescence
Parallux Tetracyclines Idexx Inc. 4 min Tetracyclines Fluorescence
Parallux Sulphonamides Idexx Inc. 4 min Sulphonamides Fluorescence
SNAP β-Lactam Idexx Inc. < 10 min β-lactams Colour change
SNAP Tetracyclines Idexx Inc. < 10 min Tetracyclines Colour change
Charm MRL β-Lactam Charm Sciences < 9 min β-lactams Colour change
Charm MRL Charm Sciences < 9 min Tetracyclines Colour change
Tetracycline
Charm MRL Charm Sciences < 9 min Sulphonamides Colour change
Sulphonamides
Charm MRL Quinolone Charm Sciences < 9 min Quinolone Colour change
Beta s.t.a.r. UCB-Bioproducts 5 min β-lactams Colour change
Delvo-X-Press βL-II Gist-brocades/DSM < 7 min β-lactams Colour change
Fluorophos BetaScreen Advanced Instruments 10 min β-lactams Fluorescence
Test (EU) Inc.

 ANTIBIOTIC TEST KIT.


3.1 IDEXX PARALLUX

The Parallux Antibiotic Test Kit is a capillary based solid-phase fluorescent


immunoassay (Kumar et al., 1997) for the determination of beta-lactams,
sulphonamides and tetracyclines in bovine milk. The system consists of an assay
cartridge which contains four glass capillaries, a reagent tray with four wells of dried
reagents and a Parallux Processor which processes the assay, reads fluorescence
output, and reports test results. The Parallux antibiotic residue test takes <8
minutes to complete.

Parallux identifies an extensive range of antibiotics and sulphonamides, however, it


is important to note that the range of antibiotics and sulphonamides identified is
dependent on the specific assay cartridge used. Table 5 shows the Parallux assay
cartridges that are available. The IDEXX range of cartridges has been updated to
include the Beta/Tetra/Sulfa kit, which will test for all 3 drug families in one 4 minute

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test. The Parallux Test Kit identifies the specific residue detected. The minimum
sensitivities for detection of beta-lactams, sulphonamides and tetracyclines in milk
using the Parallux test kits are shown in Table 6.

Table 5. Parallux assay cartridges for the detection of antibiotic residues in milk*.

Assay Detects
BETA-LACTAM ASSAY β-Lactam drugs
BETA-LACTAM 2 X ASSAY Batch two samples for β-Lactam drugs
BL/SULFA/TETRA ASSAY B-Lactam, sulfonamides and tetracyclines
SULFA/TETRA ASSAY Sulfamethazine, sulfathiazole, sulfadimethoxine and
tetracyclines
BETA-LACTAM/TETRACYCLINE β-Lactam & tetracyclines
BETA-LACTAM/ SULFA β-Lactam & sulphonamides
CILLINS ASSAY Amoxicillin, ampicillin, cloxacillin & penicillin G
CEPHAPIRIN ASSAY Cephapirin
CEFTIOFUR ASSAY Ceftiofur
CLOXACILLIN ASSAY Cloxacillin
*Table 5 is based on manufacturer’s declared detection

The assay system was also demonstrated to be specific and sensitive for the
detection of incurred residues at FDA tolerance levels of antibiotic (penicillin-G, 5
ppb; ampicillin, 10 ppb; amoxicillin, 10 ppb; cloxacillin, 10 ppb; cephapirin, 20 ppb,
and ceftiofur, 50ppb). The Association of Official Analytical Chemists (AOAC) also
demonstrated that there was no interference in detection of minimum sensitivity
levels of antibiotic by the presence of somatic cells at approximately one million cells
per ml. Milk containing 300,000 cells/ml of bacteria commonly found in mastitic milk
was also tested, and no interference was found when testing milk containing
tolerance levels of antibiotic. There was no detectable interference of the method by
a wide variety of non-beta-lactam drugs (AOAC Performance Test 000402).

The Parallux test Kits are used as milk tanker positive release tests throughout the
UK and Northern Ireland. In the Republic of Ireland, a number of companies have
used Parallux as a screening test on milk at intake.

3.1.1 Technical Difficulties

IDEXX, the manufacturers of the Parallux test kit have stated that the Parallux
test is only for use in a laboratory situation as it is an instrument based test which
requires trained laboratory staff. The UK dairies that use the Parallux test as a
positive release for tankers carry out the test under laboratory conditions.

IDEXX also state that they work in consultation directly with the customer to ensure
that they have the correct test kit for their requirements. In a situation where
production staff may be carrying out the testing, IDEXX will always promote the
SNAP test procedure due to ease of use.

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3.1.2 Cost per test
The Parallux range of products are supplied in the Republic of Ireland by IDEXX
Laboratories, (Contact Sue Plant 0044 7970 245 068 / 0044 161 881 9184 or sue-
plant@idexx.com).

Equipment required:

(a) Parallux Processor for Beta Lactams, Sulfonamides and Tetracyclines. €5,680.00

(b) Pipette, 100ul fixed volume. €185.00

(c) Beta Lactam (100) Test Kits: €400.00 (€4.00 per test)
Beta Lactam 2x (200) Test Kits: €720.00 (€3.60 per test)
Beta Lactam / Sulfonamide (100) Test Kits: €440.00 (€4.40 per test)
Beta Lactam/Tetracycline/Sulfonamide (100) Test Kits: €570.00 (€5.70 per test)
for all 3 drug families

3.2 IDEXX SNAP® ANTIBIOTIC TEST KIT.


The SNAP® Beta-lactam Test is an enzyme-linked receptor-binding assay, which
detects a range of Beta-lactam antibiotics (see Table 6) in raw commingled bovine
milk. The minimum sensitivities for detection of beta-lactams using the SNAP® Test
are shown in Table 6. The SNAP® Antibiotic Test Kit is also available in a
Tetraclyline Test Kit which detects tetracycline, chlortetracycline and oxytetracycline
residues and an Aflatoxin M1 Test Kit. The SNAP® residue tests takes < 10 minutes
to complete.

The SNAP® test is a more user friendly test kit than the Parallux kit and is used by
some farmers to test milk at farm level.

3.2.1 Cost per test

The SNAP® range of products are supplied in the Republic of Ireland by IDEXX
Laboratories, (Contact Sue Plant 0044 7970 245 068 / 0044 161 881 9184 or sue-
plant@idexx.com

Equipment:

(a) SNAP heater block: €295.00

(b) Beta Lactam Test Kits (20): €72.00 (€3.60 per test),
€60.00 (€3.00 per test) for volumes > 15,000 tests p.a.
Tetracycline Test Kits (20): €72.00 (€3.60 per test)

(c) SNAPSHOT™ touch screen reader-printer: €4145.

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3.3 CHARM SCIENCES INC., CHARM MRL ANTIBIOTIC TEST KIT.
The Charm MRL antibiotic residue test is a rapid test kit for the determination of beta-
lactams, sulphonamides, tetracyclines and quinolones in bovine milk. There are
separate strips for testing each of the aforementioned antibiotic families. The
minimum sensitivities for detection of the beta-lactams are shown in Table 6. The
Charm MRL antibiotic residue test takes < 9 minutes to complete. The Charm MRL
test can be used for raw commingled bovine milk, individual cows milk and
homogenised whole milk (not for skim HTST milk or UHT milk).

Penicillin G 4 ppb is used as the standard for verification of positives (3 % tolerance),


and is supplied as part of the test kit.

The Charm ROSA Reader can store up to 4,000 results and you can also download
the results from the reader onto a PC database (using Microsoft Excel) or to a printer.
The MRL test strips can be frozen for three months.

The Charm MRL Beta-lactam test is used in many milk plants as a positive release
for tankers. The Charm MRL Beta-lactam kit is the only one of the Charm antibiotic
residue rapid test kits in use presently in Ireland by dairy companies.

An advantage of the Charm MRL is that there are no moving parts in the kit.

3.3.1 Cost per test


Manufacturers of the Charm MRL test are Charm Sciences and the supplier of the
Charm MRL test in the Republic of Ireland is Mr. Darragh McGlynn, Sandyford, Co.
Dublin (01-2940233/087-2413077).

Equipment Required:
(a) The Charm ROSA Reader for beta-lactams, sulphonamides, tetracyclines,
quinolones and aflatoxin, each €6031.26.

(b) Dual place Incubator, each €612.00 /Quad Incubator, each €747.90.

(c) Pipette, 300ul fixed volume, each €235.00.

(d) Betalactam/100 Test Kit :€349.20 (€3.50 per test strip)


Tetracycline/100 Test Kit: €617.10 (€6.17 per test strip)
Quinolone/100 Test Kit: €772.00 (€7.72 per test strip)
Sulphonamides/100 Test Kit price: TBA This kit is at US Levels (10ppb) but the
sample can be diluted 1:10 to compensate.

The above kits quotes are for milk and the cost of the equipment and consumables
depends on the number of tests in a given workload. Charm MRL is also used for
testing tissue and urine samples, however, these kits are more expensive.

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3.4 UCB-BIOPRODUCTS BETA S.T.A.R. ANTIBIOTIC TEST KIT.
Beta s.t.a.r. is a receptor assay for rapid determination of beta-lactams antibiotics in
milk. The Beta s.t.a.r. antibiotic test kit supersedes the PENZYME® antibiotic test kit.
The minimum sensitivities for detection of the beta-lactams using the Beta s.t.a.r.
Test Kit are shown in Table 6.

The test involves a specific Beta-lactam receptor linked to gold particles. The
preliminary incubation of a specific amount of the receptor with milk containing
antibiotics results in the interaction of the antibiotics present with the receptor. In the
second stage the solution is transferred onto an immunochromatographic medium.
The first band of this medium will capture all the receptors which have not seen any
antibiotic during the first incubation. The second band on the
immunochromatographic medium serves as a reference band.

The Beta s.t.a.r. assay takes 5 minutes to complete and is used for rapid
determination of beta-lactams in milk.

3.4.1 Cost per test

UCB-Bioproducts Beta s.t.a.r. Kit for beta-lactams is supplied in Ireland by Davidson


and Hardy, 8 Pembroke Road, Dublin 4 (01-6600725). The kit usage by dairy
companies relates to seasonal factors. The cost per test is ~ €3.20.
Dry block heater: €190.50
Beta s.t.a.r. 25 Test Kit: €85.00
Beta s.t.a.r. 100 Test Kit: €197.00
The parent company (UCB-Bioproducts) examined the possibility of having a
combined kit for detection of beta-lactams and sulphonamides in milk, however, this
did not prove cost effective as the cost of the test would be increased to €19 per test.

3.5 DSM DELVO-X-PRESS βL-II ANTIBIOTIC TEST KIT.

The Delvo-X-Press is a rapid competitive receptor-enzyme assay that detects a


broad range of beta-lactams in milk in < 7 minutes. The minimum sensitivities for
detection of the beta-lactams using the Delvo-X-Press test kit are shown in Table 6.

The milk samples and reagents are mixed in tubes coated with a β-lactam receptor.
After washing the tube, a colour developer is added that detects residual binding
sites not occupied by antibiotic present in the milk sample. Colour development is
inversely proportional to the concentration of β-lactam residues in the sample. Six
samples and a standard can be evaluated at a time and all incubations are made
automatically in a compact Incubator-Shaker-Reader-Printer (Russell, 1997).

3.5.1 Cost per test


The DSM, Delvo range of products is supplied by The Carbon Group, Ringaskiddy,
Co. Cork (Contact: Gerry O’Brien 021-4378988). Few companies use this product in
Ireland, thus costs were unavailable.

16
3.6 ADVANCED INSTRUMENTS INC. FLUOROPHOS®
®
BETASCREEN TEST (EU).
The BetaScreen® is an ELISA-based test (using fluorescence and alkaline
phosphatase as a marker). The Fluorophos® BetaScreen® Test (EU) detects six
beta-lactams: penicillin, ampicillin, amoxicillin, oxacillin, cloxacillin, and dicloxacillin.
BetaScreen® test results are available in ten minutes. The test is instrument-based
with push-button operation, microprocessor-controlled prompts and diagnostic
menus, resulting in semi-quantitative results with no operator interpretation
necessary. The Fluorophos Test System offers a digital display plus a hard copy for
a permanent record. The computer interface allows connection to laboratory
computers for analysis and reporting of data.

3.6.1 Cost per test


Advanced Instruments Inc. Fluorophos® BetaScreen® Test (EU) for beta-lactams is
supplied in Ireland by AGB Scientific Ltd., Dublin Industrial Estate, Glasnevin, Dublin
11 (01-8309533).

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4. CONCLUSIONS
Antibiotic residue test methods based on the IDF reference method other than Delvo
SP® are appearing on the Irish market. For example, the Copan Test Kit and the
ELIPSE test kit which is manufactured by Z.E.U.-INMUNOTEC, Spain (www.zeu-
inmunotec.com) have both recently undergone validation exercises in Irish
laboratories. The ELIPSE test method is also a microbial inhibition assay for the
detection of antibiotics and sulphonamides in milk based on the IDF standard method
for determination of antibiotic residues and sulphonamides in milk.

The Parallux test kit is one of the most rapid test to carry out (< 8 minutes) and is
capable of identifying the specific antibiotic(s) detected. However, the range of
antibiotics identified is dependent on the specific assay cartridge used. Also this
method is relatively technical as it is an instrument-based test and thus, is not
suitable for use at milk intake where no laboratory facilities are available.

The Charm MRL kit has minimum sensitivities close to the EU MRL, with the
exception of Nafcillin and Cefquinom. However, the Charm MRL kit requires
separate strips for testing each of the antibiotic families as do many other rapid test
kits for the detection of residues in milk. It would be advisable to use the Charm
ROSA Reader for determination of the results as visual interpretation of test results
which involve colour change are often subjective in nature. The cost of the Charm
MRL kits escalates for sulphonamides, tetracyclines and quinolones.

The SNAP beta-lactam test, Delvo-X-Press and Beta s.t.a.r. tests only measure beta-
lactam residues in the milk. However, a separate SNAP test for tetracycline is also
available. The detection was less than or equal to the EU MRL for 8, 8, 6 and 8
compounds for the Charm MRL for beta-lactams, SNAP test for beta-lactams, Delvo-
X-Press and Beta s.t.a.r. respectively. Regarding the use of the rapid test kits as a
rapid screen for beta-lactams in raw milk, the Charm MRL for beta-lactam, SNAP test
beta-lactam assay and Beta s.t.a.r. appear to be suitable. It has been reported that
the Delvo-X-Press test is not recommended, mainly due to its insufficient test
sensitivity (Kroll, 1999). However, caution must be taken in the interpretation of
screening results on raw milk from rapid test kits which are designed to detect beta-
lactams alone, as the legal requirements for the detection of residues in milk require
the detection of a range of antibiotic residues and sulphonamides in milk, in
accordance with the EU MRL’s set out in Council Regulation No. 2377/90 and
amendments (as shown in Table 6).

It should be noted that the rapid test kits outlined are only useful for screening
purposes or positive release of tankers as inevitably the milk will have to pass a
broad spectrum microbial inhibition test (e.g. Delvo SP® test) prior to sale. Also, such
factors as high or low pH values, increased somatic cell counts, increased bacterial
counts, natural inhibitors in colostrum and compositional changes in the milk can
adversely affect the minimum sensitivities of the test methods described. However,
the examination of the influence of these factors was outside the scope of this study.

18
5. REFERENCES
AOAC Performance Test 000402. Paralluxtm Beta-lactam: A Capillary Based
Fluorescent Immunoassay For The Determination Of Penicillin-G, Ampicillin,
Amoxicillin, Cloxacillin, Cephapirin, And Ceftiofur In Bovine Milk.
[http://www.aoac.org/RI/000402.htm]

Council Directive 92/46/EEC of 16 June 1992. Laying down the health rules for the
production and placing on the market of raw milk, heat-treated milk and milk
products.

Council Regulation 2377/90/EEC of 26 June 1990 laying down a Community


procedure for the establishment of maximum residue limits of veterinary medicinal
products in foodstuffs of animal origin. Official Journal European Communities No.
L224, (18.8.90).

Fallon, M., O’Nuallain, N. & Hogan, G.P. (1995/1996) An outline of the current
position in relation to residues in milk and the control measures operating in this
industry in Ireland. Scientific Papers, The Food Safety Advisory Board, Ireland.

International Dairy Federation (1970) Detection of penicillin in milk by a disk assay


technique. International Dairy Federation Standard 57, International Dairy
Federation, Brussels, Belgium.

International Dairy Federation (1991) Detection and confirmation of inhibitors in milk


and milk products. International Dairy Federation Bulletin 258, International Dairy
Federation, Brussels, Belgium.

Kroll, S. (1999) Suitability of rapid test methods for the detection of residues of beta-
lactam antibiotics in milk. Dissertation, University of Munchen, Germany.

Kumar, A., Kharadia, S. & Piani, M. (1997) Immunoassays in capillaries. United


States Patent Application, 5,624,850.

Radostitis, O.M., Blood, D.C. & Gay, C.C. (1994) Principles of antimicrobial therapy.
In: Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats
and Horses. 8th edition, pp. 153-156, Bailliere Tindall Publishers.

Russell, P. (1997) Antibiotic detection reviewed. Milk Industry International, 99(4),


16-18, 20.

Statutory Instrument No. 9 of 1996. European Communities (Hygienic production


and placing on the market of raw milk, heat-treated milk and milk-based products)
Regulations, 1996.

19
Table 6. Comparison of the EU maximum residue limits (MRL) with the sensitivities of different antibiotic test kits.

Set Limits Microbial Rapid Test Kits


Inhibition Assay
Antibiotic EU MRL FDA Delvo Parallux Charm SNAP Delvo Beta
MRL(1) Codex(2) USA(3) SP* Test(4)* MRL(5) * Test* X-Press(6) * star*
µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)
(µ µg/kg)

β-lactams
Penicillin 4 4 5 2 3.2 4 2-4 2-4 2-4
Penethamat 4 (2)
Ampicillin 4 10 2-3 2.9 4 4-5 4-8 2-5
Amoxicillin 4 10 2 3.6 4 6 4-8 2-4
Nafcillin 30 5 60 75-85 8-20
Cloxacillin 30 10 15 7.4 30 30 30-60 5-10
Dicloxacillin 30 10 20 25 30 25-50 5-10
Oxacillin 30 5 10 30 35-40 25-50 5-10
Cefacetril 125 20 62.5
Cefalexin 100 40-60 tbc(7) 25-50
Cefalonium 10 5-10 tbc 3-4 7.5-15
Ceftiofur(8) 100(9) 100 50 50 33.7 100 50 4-8 75-150
Cephapirin 10 20 5 16.3 10 5 4-8 8-16
Cephazolin 50 (10) 40-50 25-27.5
Cefquinom 20 (100) 20 22.5-27.5 5-20
Cefaperazon 40 tbc 5-8

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Antibiotic EU MRL FDA Delvo Parallux Charm SNAP Delvo Beta
MRL Codex USA SP* Test* MRL* Test* X-Press* star*

µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg)

Sulphonamides
Sulphadimidine 100(10) 25 10 equal to sulfamethazine
Sulphadimethoxine 100(10) 10 50 10
Sulphamethazine 100(10) 10 25 10
Sulphathiazole 100(10) 10 50 10
Sulphadiazine 100(10) 10 50

Tetracyclines
Chlortetracycline(8) 100(11) 100 300 150 125 30
Doxycycline 0 (100) tbc
Oxytetracycline(8) 100(11) 100 300 100 100 30
Tetracycline(8) 100(11) 100 300 100 100 30

Macrolides
Erythromycin 40 50 50
Pirlimycin 150 (50-200)
Spiramycin 200 100 200
Tilmicosin 50 50 (100)
Tylosin 50 50 50

Aminoglycosides
Gentamicin 100 200 30 100-300
Neomycin 500 500 150 100-200
Spectinomycin 200 200 (2500)
DH/Streptomycin 200 200 125 300-500
Kanamycin 2500

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Antibiotic EU MRL FDA Delvo Parallux Charm SNAP Delvo Beta
MRL Codex USA SP* Test* MRL* Test* X-Press* star*

µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg) (µ
µg/kg)

Quinolones
Enrofloxacin 100 (1000-1500)
Danofloxacin 30 (close to enrofloxacin)
Difloxacin 0 (close to enrofloxacin)
Flumequin 0 (no data available)
Marbofloxacin 75 (close to enrofloxacin)

Various
Bacitracin 150 (1000)
Baquiloprim 30 (60)
Chloramphenicol 0 0 2500
Clavulanic acid 200 (not antibacterial)
Colistin 50 (no data available)
Dapsone 0 1
Lincomycin 150 100
Novobiocin 50 100 (1000)
Rifaximin 60 (50)
Thiamphenicol 50 (close to chloramphenicol)
Trimethoprim 50 50
Ticarcillin N/a(12) 50
Cefadroxyl N/a 50

* It is important to note that the sensitivities of the individual test kits are based on manufacturer’s declared levels. In the case
of the Delvo SP test the values are for detection levels at control time. For reading times of 2 h 45 min or 3 hours the
sensitivity will diminish. Values for Delvo SP in parenthesis are given as indication from the DSM data-base.

22
Footnotes to Table 6.
(1)
Council Regulation (EEC) No. 2377/90 and amendments..
(2)
Codex Committee on Residues of Veterinary Drugs in Foods
(3)
Code of Federal Regulations (21 CFR 556) and Centre for Veterinary Medicine.
(4)
Different individual test kits for β-lactams, tetracyclines and sulphonamides
(5)
Different individual test kits for β-lactams, tetracyclines, sulphonamides and Quinolones, however, MRL’s unavailable for some groups to date.
(6)
Delvo-X-Press rapid test kit also detects a number of other cephalosporins not mentioned above.
(7)
tbc, denotes to be confirmed
(8)
Mother compound.
(9)
Mother compound and metabolites.
(10)
Sum of substances if this group.
(11)
Mother compound and 4-epimer.
(12)
N/a, denotes not applicable.

FURTHER INFORMATION
Further information can be obtained from:

Food Safety Information Centre,


Food Safety Authority of Ireland,
Abbey Court, Lower Abbey Street, Dublin 1
Tel: 1890 336677 Fax: (01) 817 1301
E-mail: info@fsai.ie. Website: www.fsai.ie

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