An evaluation of the Delvo X-press beta-L test for detecting beta-lactams in ex-farm raw milks


Scannella, D.; Neaves, P.; Keedy, K.; Bell, C.

International Dairy Journal 7(1): 93-96

1997


The detection of antimicrobial residues in ex-farm, bulk, raw, cows' milk by the Delvo X-Press beta-lactam test and by the Delvotest P was compared. Of 500 samples, 499 gave the same results by the two procedures. One sample classified as Delvotest P-negative gave a borderline positive result with the Delvo X-Press test but a negative result upon re-test. Fifty samples giving positive results by the Delvotest P procedure were also positive by the Delvo X-Press test; all contained penicillinase-sensitive beta-lactams. The results of this study indicate that the Delvo X-Press test could accurately detect beta-lactams in bulked, ex-farm raw milks.

Int.
Dairy
Journal
7
(1997)
93-96
(E)
1997
Elsevier
Science
Limited
Printed
in
Great
Britain.
All
rights
reserved
ELSEVIER
PII: S0958-6946(96)00036-2
0958-6946/97/$17.00
+
0.00
An
Evaluation
of
the
Delvo
X-Press
13L
Test
for
Detecting
13-Lactams
in
Ex-farm
Raw
Milks
D.
Scannella,
P.
Neaves,*
K.
Keedy
and
C.
Bell
Microbiology
Department,
Technical
Division
Laboratories,
Leatherhead
Food
Research
Association,
Randalls
Road,
Leatherhead,-
Surrey,
KT22
7RY,
UK
(Received
22
November
1995;
accepted
23
August
1996)
ABSTRACT
The
detection
of
antimicrobial
residues
in
ex-farm,
bulk,
raw,
cows'
milk
by
the
Delvo
X-Press
P-lactam
test
and
by
the
Delvotest
P
was
compared.
Of
500
samples,
499
gave
the
same
results
by
the
two
procedures.
One
sample
classified
as
Delvotest
P-negative
gave
a
borderline
positive
result
with
the
Delvo
X-Press
test
but
a
negative
result
upon
re-test.
Fifty
samples
giving
positive
results
by
the
Delvotest
P
procedure
were
also
positive
by
the
Delvo
X-Press
test;
all
contained
penicillinase-sensitive
13-lactams.
The
results
of
this
study
indicate
that
the
Delvo
X-Press
test
could
accurately
detect
0-lactams
in
bulked,
ex-farm
raw
milks.
©
1997
Elsevier
Science
Limited.
All
rights
reserved.
Keywords:
Penicillin;
immune-receptor
assay;
raw
milk;
P-lactam
antibiotics;
detection
INTRODUCTION
An
increasing
volume
of
legislation
from
the
European
Union
(EU)
is
designed
to
limit
the
presence
of
specified
veterinary
residues
in
foods.
Milk
is
subject
to
such
legislation;
the
Maximum
Residue
Limits
(MRLs)
of
antibiotic
residues
are
defined
by
Commission
Regulations
EEC
2377/90
(Anon.,
1990a),
EEC
675/92
(Anon.,
1992a)
and
EEC
3093/92
(Anon.,
1992b),
although
a
number
of
amendments
have
subsequently
been
made,
primarily
to
extend
the
list
of
compounds
for
which
MRI,s
have
been
set.
A
variety
of
antibiotics
is
used
for
both
the
prophylactic
treatment
of
animals
and
for
treating
clinical
illness;
13-
lactams
(namely
penicillin
and
penicillin-related
compounds)
have
been
the
most
widely
used
antibiotic
types
in
dairy
husbandry.
Relatively
simple
test
methods
for
detecting
P-lactams
have
been
commercially
available
for
many
years.
Most
available
methods
are
based
on
the
inhibition
of
microbial
growth
and
hence
require
several
hours
to
obtain
a
result.
Such
tests
may
also
be
non-specific
because
they
can
detect
the
presence
of
any
substance
that
inhibits
the
growth
of
the
test
organism.
Such
non-specificity
is
an
advantage
for
screening
purposes
but
may
be
a
disadvantage
when
identification
of
a
residue
is
required.
Enzyme
immunoassay
methods
are
currently
available
for
the
detection
of
specific
compounds
in
foodstuffs,
including
milk,
and
applications
for
several
of
the
kits
available
have
been
reviewed
by
Roulston
and
McEnroe
(1992).
These
methods
are
generally
*Corresponding
author.
Current
address:
Williams
and
Neaves,
`Moleview',
28,
Randalls
Road,
Leatherhead,
Surrey,
KT22
7TQ,
UK.
more
specific
than
microbial
inhibition
tests
and
give
results
in
minutes
rather
than
hours.
This
paper
reports
an
evaluation
of
the
Delvo
X-Press
assay
(Gist-brocades
BSD-BV,
Delft,
The
Netherlands)
for
detecting
P-lactams
in
ex-farm
raw
milks.
MATERIALS
AND
METHODS
Principle
and
use
of
the
Delvo
X-Press
test
The
Delvo
X-Press
test
for
P-lactams
is
a
qualitative,
competitive,
receptor-enzyme
assay.
P-Lactams
present
in
a
test
sample
are
'captured'
by
a
'tracer'
consisting
of
an
enzyme-linked,
receptor
protein.
After
capture
of
any
antibiotic
present
in
the
sample,
free
tracer,
which
was
not
bound
to
P-lactam
in
the
sample,
binds
to
the
inside
of
a
0-lactam-coated
tube.
The
addition
of
an
enzyme-substrate
(`colour
developer')
results
in
enzyme
activity
and
a
colour
reaction.
Colour
development
is
inversely
proportional
to
the
p-lactam
concentration
and
is
measured
spectrophotometrically.
A
comparison
of
the
absorbance
of
a
test
sample
with
that
of
a
control
sample
containing
a
standard
concentration
of
P-lactam
antibiotic
permits
the
classification
of
samples
as
either
'positive'
or
'negative'.
The
test
was
undertaken
using
a
proprietary
"Incubator-Shaker-
Reader-Printer"
(ISRP)
and
employed
3
incubation
stages
with
a
total
assay
time
of
about
10
min.
The
test
manufacturer's
claimed
sensitivities
to
the
following
antibiotics
are:
penicillin
G,
3-4
lig
kg
- '
(ppb);
ampicillin
or
amoxicillin,
4-8
p.g
kg
-1
;
cephapirin
or
ceftiofur,
2-4
1..tg
kg
-1
;
cloxacillin,
30-40
j.ig
kg
-1
;
dicloxacillin
or
oxacillin,
25-50
ps
kg
-1
.
For
all
samples,
Delvo
X-press
tests
were
undertaken
according
to
the
manufacturer's
instructions.
93
94
D.
Scannella
et
al.
The
routine
milk
quality
testing
procedure
A
routine
milk
quality
testing
laboratory
examined
raw
milk
samples
collected
from
farm
bulk
tanks
in
England
and
Wales.
All
samples
were
screened
initially
using
an
in-house
microbial
inhibitor
assay
that
employed
a
similar
principle
to
the
Delvotest
P
(Gist-brocades
BSD-
BV,
Delft,
Netherlands)
but
was
slightly
more
sensitive
to
13-lactams.
All
samples
showing
a
positive
result
in
the
in-house
test
were
re-tested
using
the
Delvotest
P
method
(Anon.,
1994)
and
farmers
incurred
a
financial
penalty
when
a
milk
sample
had
failed
the
Delvotest
P
which
has
a
sensitivity
to
penicillin
G
of
0.006
iu
mL
-
'.
Samples
showing
a
negative
response
with
the
in-house
test
were
not
re-tested.
Experimental
protocol
The
trial
was
divided
into
three
parts:
(a)
the
ability
of
the
Delvo
X-Press
test
to
detect
(3-lactams
was
compared
with
that
of
the
routine
milk
quality
testing
procedure,
as
described
above,
by
examining
500
randomly
selected,
routine
samples;
(b)
the
Delvo
X-
Press
test
was
used
to
assess
50
samples
that
had
given
positive
results
in
the
milk
quality
testing
laboratory
procedure;
and
(c)
the
sensitivity
of
the
Delvo
X-Press
test
to
ampicillin
and
cloxacillin,
which
are
widely
used
in
veterinary
formulations,
was
assessed
by
examining
artificially-contaminated
(spiked)
milk
samples.
Preparation
of
antibiotic
standards
A
stock
solution
of
sodium
benzyl-penicillin
G
(commercially-prepared
as
`Crystapen';
Glaxo,
UK)
containing
1000
iu
mL
-1
was
prepared
in
0.2
m
potassium
phosphate
buffer,
pH
6.0,
using
Grade
A
volumetric
glassware.
This
solution
was
diluted
volumetrically
in
deionised
water
to
provide
a
working
solution
containing
1
iu
mL
-1
which
was
dispensed
into
5-10
mL
aliquots
and
frozen
at
-20°C.
On
each
day
when
standards
were
required,
an
aliquot
of
the
working
solution
was
thawed
in
a
waterbath
at
45°C.
Bulked
raw
milk
standards,
containing
0.003,
0.004,
0.005,
0.006,
0.007
or
0.008
iu
mL
- '
penicillin
G,
were
prepared
by
appropriate
dilution
of
the
thawed
working
solution.
All
individual
milks
used
to
make
up
the
bulk
sample
had
passed
the
routine
milk
quality
testing
laboratory
in-house
screening
test.
Examination
of
standards
On
each
day
of
testing
the
randomly
selected
milks,
the
performance
of
the
Delvo
X-Press
test
was
monitored
using
the
raw
milk
penicillin
standards,
together
with
a
'zero'
milk
sample.
To
minimise
operator
variation,
the
examination
of
test
samples
and
standards
was
carried
out
by
two
technical
staff
according
to
an
internationally-accepted
accreditation
standard
(Anon.,
1990b).
Examination
of
samples
Randomly
selected,
routine,
raw
milk
samples
(500),
collected
and
examined
by
the
routine
milk
quality
testing
laboratory,
were
delivered
to
Technical
Division
Laboratories
(TDL)
overnight
at
<
4°C
in
insulated
boxes.
All
samples
found
positive,
either
by
the
Delvo
X-
Press
method
or
by
the
routine
milk
quality
testing
laboratory
(i.e.
failing
the
Delvotest
P),
were
subjected
to
confirmatory
testing
at
TDL
(see
below).
Positive
samples
(50),
that
had
failed
the
Delvotest
P
as
undertaken
by
the
routine
milk
quality
testing
laboratory,
were
examined
at
TDL
by
the
Delvo
X-
Press
and
by
the
additional
confirmatory
tests.
Confirmatory
tests
All
samples
that
had
shown
a
positive
reaction,
either
in
the
Delvo
X-Press
test
or
by
the
Delvotest
P
as
employed
in
the
routine
milk
quality
testing
laboratory
procedure,
were
re-examined
at
TDL
using
the
Delvotest
P
and
the
Lactek
13-lactam
test
(Idetek
Laboratories,
Sunnyvale,
CA,
USA).
Those
samples
showing
a
positive
result
when
examined
by
the
Delvotest
P
at
TDL
(TDL
Delvotest
P)
were
further
tested
using
Delvotest
P
with
the
addition
of
penicillinase
(penase;
Consolidated
Chemicals,
Wrexham,
UK),
prepared
by
re-
constituting
the
entire
contents
of
one
vial
containing
500,000
iu
of
penase
with
5
mL
distilled
water.
An
aliquot
(0.1
mL)
was
added
to
the
test
sample
(2.5
mL)
and
incubated
at
room
temperature
for
15
min.
The
penase-treated
sample
was
then
examined
using
Delvotest
P
according
to
manufacturer's
instructions.
A
negative
reaction
in
this
test
confirmed
the
presence
of
a
13-lactam
that
had
been
inactivated
by
the
enzyme;
a
positive
reaction
suggested
the
presence
either
of
a
non
13-lactam
compound
or
a
penase-resistant
13-lactam.
Samples
were
stored
at
0-1°C
until
testing
was
complete,
then
stored
for
reference
at
-20°C.
Sensitivity
to
ampicillin
and
cloxacillin
Spiked
milk
standards
containing
ampicillin
concentrations
in
the
range
2-10
Lig
kg
- '
or
cloxacillin
concentrations
in
the
range
10-50
j.tg
kg
- '
were
prepared
as
described
above.
The
standards
were
examined
by
Delvo
X-Press,
in
triplicate,
and
single
determinations
were
made
using
Delvotest
P.
Spiked
samples
were
prepared
and
examined
on
two
test
occasions.
RESULTS
AND
DISCUSSION
Detection
of
penicillin
G
standards
by
the
Delvo
X-Press
test
Table
1
shows
the
day-to-day
variation
in
Delvo
X-
Press
results
and
the
overall
proportion
of
positives
at
each
concentration
tested.
As
expected,
the
proportion
of
positive
results
increased
with
antibiotic
concentration.
In
the
absence
of
penicillin
G,
negative
results
were
obtained
on
all
occasions
whilst
100%
of
samples
yielded
a
positive
result
at
0.006
iu
mL
-1
.
However,
at
intermediate
concentrations,
a
proportion
of
samples
tested
gave
positive
results,
indicating
that
the
end-point
of
the
test
was
not
as
precise
as
that
for
the
Delvotest
P,
i.e.
the
dose-response
curve
was
less
steep.
Immune-receptor
test
for
13-lactam
antibiotics
95
Table
1.
Detection
of
Penicillin
G
in
Raw
Milk
Standards
Using
the
Delvo
X-Press
13-lactam
Test
Concentration
No.
positive/testing
occasion
(1-13)
a
:-
Total
positives
(%)
(µg
kg
-I
)
(iu
m1L
-1
)
1
2
3
4
5
6
7
8
9
10
11
12
13
0.0
0.000
0
0
0
0 0
0
0 0
0 0
0
0
0
0/13
(0)
1.8
0.003
NT
NT
0
1
0 0 0
0
1
0
0
1
0
3/11
(27.3)
2.4
0.004
NT
NT
0
1
0
0
0
0
1
0
1
1
1
5/11
(45.5)
3.0
0.005
NT
NT
0
1
1
1
1
0
1
1
1
1
1
9/11
(81.8)
3.6
0.006
1 1
1
1
1
1
1
1
1
1
1
1
13/13
(100)
4.2
0.007
NT
NT
1
1
1
1
1
1
1
1
1
1
1
11/11
(100)
4.8
0.008
NT
NT
1 1
1 1
1 1
1
1
1
1
1
11/11
(100)
Total
1
3
6
4 4
4
3
6
4
5
6
5
52/81
NT
=
Not
tested.
1
iu
mL
-I
=600
pg
kg
-I
.
a
At
each
concentration,
1
sample
was
examined
on
each
test
occasion.
These
results
are
in
agreement
with
those
obtained
by
TDL
for
I3-lactam
test
kits
from
other
manufacturers
(unpublished
data).
This
is
a
well-
documented
phenomenon
associated
with
ELISA
or
immune-receptor
tests
and
indicates
a
potential
problem
that
applies
to
all
of
the
current
'fast
tests'
for
antibiotics.
Whilst
it
is
undesirable
for
milk
to
contain
any
antibiotic
residues,
the
EU
legislation
does
define
maximum
acceptable
concentrations
for
defined
antimicrobial
compounds
in
milk.
The
positive
test
results
observed
for
samples
containing
0.003-0.005
iu
mL
-1
penicillin
G
suggest
that
the
use
of
an
ELISA
or
immune-receptor
test
without
further
investigation
might
result
in
rejection
of
some
milk
supplies
that
would
otherwise
be
acceptable.
However,
the
results
of
the
study
of
randomly
selected,
ex-farm
raw
milks,
described
below,
did
not
confirm
this
theoretical
problem.
Out
of
499
commercial
milk
supplies
that
gave
a
negative
Delvotest
P
reaction,
only
1
was
positive
by
Delvo
X-Press
and
this
sample
was
negative
upon
re-test.
Overall,
the
results
indicate
that
the
Delvo
X-Press
13-
lactam
test
was
consistently
capable
of
detecting
penicillin
G,
artificially-inoculated
into
bulked
raw
milks
at
the
EU
MRL
(4
ug
kg
-1
or
0.0067
iu
mL
-1
)
and
above.
Comparison
of
the
Delvo
X-Press
test
with
Delvotest
P
for
the
detection
of
13-lactams
in
randomly
selected,
ex-farm
raw
milks
The
survey
results
for
500
randomly
selected,
ex-
farm
milks
are
shown
in
Table
2;
498
samples
(99.6%)
were
classified
as
negative
by
both
the
Delvo
X-Press
test
and
the
Delvotest
P,
while
1
sample
(0.2%)
was
identified
as
positive
by
both
tests.
One
sample
(0.2%)
gave
a
positive
Delvo
X-Press
test
result
but
a
negative
reaction
using
Delvotest
P.
Upon
re-test,
this
sample
yielded
a
negative
Delvo
X-
Press
result.
No
false
negative
results
were
observed
with
the
Delvo
X-Press
test
(i.e.
no
samples
were
classified
as
Delvotest
P
positive/Delvo
X-Press
test
negative).
The
randomly-selected
sample
that
yielded
positive
reactions
in
both
the
Delvo
X-Press
test
and
the
Delvotest
P
test
when
tested
by
the
routine
milk
Table
2.
Comparison
of
the
Delvo
X-Press
Test
with
the
Delvotest
P
°
for
the
Detection
of
13-lactams
in
500
Randomly
Selected,
Ex-farm
Raw
Milks
Number
(%)
of
samples
classified
as:-
Delvotest
P
Delvotest
P
positive
negative
Delvo
X-Press
positive
1
b
(0.2)
1'
(0.2)
Delvo
X-Press
negative
0
(0)
498
(996)
=
Delvotest
P
undertaken
by
the
routine
milk
quality
testing
laboratory.
b
=
Sample
classified
as
Delvo
X-Press
positive
in
duplicate
tests.
=
Sample
classified
as
Delvo
X-Press
positive
upon
initial
test,
but
negative
upon
re-test.
quality
laboratory
also
gave
positive
responses
in
a
repeat
Delvo
X-Press
test
and
in
a
Delvotest
P
test
undertaken
by
TDL;
the
Lactek
test
also
gave
a
positive
reaction.
In
the
Delvotest
P
+
penase
test,
a
negative
reaction
was
observed,
indicating
that
the
sample
contained
a
(3-lactam
antibiotic.
These
results
indicate
excellent
overall
agreement
between
the
Delvo
X-Press
P-lactam
test
and
the
Delvotest
P
for
bulked
raw
milk
samples
and
that
the
Delvo
X-Press
did
not
give
false-positive
reactions.
The
sample
classified
as
Delvo
X-Press
positive/
Delvotest
P
negative
in
the
routine
survey
gave
a
negative
Delvo
X-Press
result
upon
re-test.
A
negative
TDL
Delvotest
P
result
was
also
recorded.
We
therefore
conclude
that
this
sample
did
not
contain
a
13-lactam
antibiotic
at
a
concentration
at
or
above
the
EU
MRL.
Comparison
of
the
Delvo
X-press
test
with
Delvotest
P
for
'routine-positive'
samples
The
results
for
the
'routine-positive'
samples
are
shown
in
Table
3.
The
Delvo
X-Press
test
was
in
complete
agreement
with
the
'confirmatory'
Delvotest
P
and
close
agreement
with
the
Lactek
13-
lactam
test
results.
The
penase
test
results
indicated
that
all
50
samples
contained
penase-sensitive
13-
lactams.
96
D.
Scannedla
et
al.
Table
3.
Confirmation
of
50
'Routine-positive'
Samples
No.
of
Delvo
Lactek
TDL TDL
samples
X-Press
13-lactam
Delvotest
Delvotest
P
(TDL)
test
P
+
penase°
44
Positive
Positive
Positive
Negative
2
Positive
Positive
Negative"
Negative
4
Positive
Negative
Positive
Negative
50
=
A
negative
result
confirms
the
presence
of
a
penicillinase-
sensitive
13-lactam
compound.
b
=
Borderline
Delvotest
P
results
indicated
the
presence
of
an
antimicrobial
compound
at
a
concentration
<0.006
iu
mL
-1
;
therefore
samples
were
classified
as
Delvotest
P-negative.
However,
the
Delvotest
P
+
penase
results
confirmed
the
presence
of
13-lactam
compounds.
Table
4.
Comparison
of
the
Delvo
X-Press
Test
Sensitivity
to
Ampicillin
and
Cloxacillin
with
that
of
the
Delvotest
P
Sample
Concentration
of
antimicrobial
Gig
kg
-1
)
No.
positive/no.
tested
on
occasion:-
Delvo
X-Press
Delvotest
P
1
2
1
2
Zero
milk
0
0/3
0/3
Y
Y
Ampicillin
2
2/3
0/3
Y
Y
4
3/3
1/3
YYP
YYP
6
3/3 3/3
YPP
P
8
3/3
3/3
NT
P
10
3/3
3/3
NT
P
Cloxacillin
10
2/3
0/3
Y
Y
20
3/3
2/3
YYP
YPP
30
3/3
3/3
YPP
P
40
3/3
3/3
NT
P
50
NT
3/3
NT
P
NT
=
Not
tested.
Separate
standards
were
prepared
on
the
two
occasions.
Y,
YYP,
YPP,
P
=
Range
of
colour
reactions:
Y
(entirely
yellow)
=
negative
result.
YYP
(mostly
yellow)
=
borderline,
negative
result.
YPP
(mostly
purple)
=
borderline,
negative
result.
P
(entirely
purple)
=
positive
result.
Sensitivity
of
the
Delvo
X-Press
test
to
ampicillin
and
cloxacillin
The
reaction
of
the
Delvo
X-Press
test
to
ampicillin
and
cloxacillin
is
compared
with
that
of
the
Delvotest
P
in
Table
4.
The
Delvo
X-Press
test
detected
4µg
kg
-1
ampicillin
(the
EU
MRL)
in
4/6
tests
and
6
kg
-1
in
6/6
tests.
Cloxacillin
at
20
lig
kg
-1
was
detected
in
5/6
tests
whilst
6/6
tests
were
positive
at
the
EU
MRL
(30
1.ig
kg
-1
).
These
data
concur
with
the
sensitivity
ranges
claimed
by
the
manufacturer.
However,
the
test
appeared
to
be
slightly
more
sensitive
to
ampicillin
and
cloxacillin
than
is
required
by
current
EU
legislation.
The
results
obtained
in
this
investigation
showed
excellent
agreement
between
the
Delvo
X-Press
13-
lactam
test
and
the
Delvotest
P
when
used
to
detect
antimicrobial
compounds
in
ex-farm
milks,
99.8%
agreement
being
achieved
for
routine
bulk
milk
samples.
The
Delvo
X-Press
test
showed
good
repeatability
and
the
confirmatory
test
results
demonstrated
the
reliability
of
the
Delvo
X-Press
test
reactions.
ACKNOWLEDGEMENTS
The
authors
are
grateful
to
the
staff
of
the
routine
milk
quality
testing
laboratory
for
their
co-operation
in
providing
raw
milk
samples.
The
assistance
of
Gist-
brocades
in
providing
kits
and
equipment
is
gratefully
acknowledged.
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