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Detection of Penicillin, Cephapirin, and Cloxacillin in Commingled Raw Milk by the Spot Test1
Detection of Penicillin, Cephapirin, and Cloxacillin in Commingled Raw Milk by the Spot Test 1 J. J. R Y A N , 2 E. E. W I L O M A N , A. H. O U T H I E , and H. V. A T H E R T O N Dairy Foods Program Department of Animal Sciences Vermont Agricultural Experiment Station University of Vermont Burlington 05405 J. A. A L E O N G Department of Mathematics and Statistics University of Vermont Burlington 05405 ABSTRACT
tests was 87.2% and false positive and negative percentages were 12.6 and .2%, respectively. In a field trial consisting of 823 bulk samples, Spot Test and disc assay agreement was 100%. The Spot Test is a rapid and reliable method for detecting penicillin, cephapirin, and cloxacillin residues in raw milk at concentrations that will produce a 16-mm zone.
The objective of this study was to compare Spot Test results with the results of the Bacillus stearotbermopbilus disc assay. Commingled raw milk samples were subdivided and spiked with penicillin, cephapirin, or cloxacillin. All subsamples, including unspiked subsamples, were analyzed by the Spot Test (3 or 9 replicates) and disc assay (9 replicates). Mean zone diameter for every subsample was calculated; subsamples having a mean zone diameter >/ 16 mm were considered positive. At penicillin concentrations ~< 3.7 ng/ml, agreement between the Spot Test and disc assay was 83.4%, and false positive and negative percentages were 14.4 and 2.2%, respectively. Above 3.7 ng/ml, agreement was 100%. At cephapirin concentrations < 12 ng/ml, agreement between the two tests was 67.9%, and false positive and negative percentages were 28.8 and 3.3%, respectively. Above 15 ng/ml, agreement was 99.3% with .7% false negatives. A t cloxacillin concentrations < 50 mg/ml agreement between the two tests was 54.5 with 45.5% false positives. At cloxacillin concentrations /> 62.5 ng/ml, agreement between the two
INTRODUCTION
Received December 2, 1985. Contribution Number 591 of the Vermont Agricultural Experiment Station. Approved by the Director. 2Present address: Department of Dairy Science, Louisiana State University, Baton Rouge 70803. 1986 J Dairy Sci 69:1510-1517
Intramammary and intramuscular drug products containing ~-lactam antibiotics are commonly used in the treatment of bovine mastitis. Penicillin and its synthetic derivatives are the most popular antimicrobial agents in these drug products. In an effort to minimize antibiotic contamination of fluid milk supplies, the United States F o o d and Drug Administration (FDA) limits the quantity of antibiotic in drug products and requires label instructions explaining product usage and drug withdrawal times (4). However, despite producer-oriented educational programs and financial penalties for shipping milk adulterated with antibiotics, contaminated milk is still a problem facing the dairy industry (3). Problems associated with antibiotic residues in the milk supply are well documented and have been reviewed (7), e.g., cultured dairy product manufacturers may experience partial or complete inhibition of acid production by starter cultures (5, 9), whereas hypersensitive individuals may exhibit adverse reactions (14). Considerable testing by processors and regulatory agencies is done to detect and minimize acceptance of antibiotic-contaminated milk and to assure consumers that milk and other dairy
1510
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK foods contaminated with antibiotics are not marketed. The ideal test for antibiotic residues in milk is one that combines simplicity and speed with low cost and that may be conducted at the farm prior to emptying the bulk tank (3). An on-farm test has yet to gain wide industry acceptance, and therefore, laboratory or receiving station tests are commonplace. Such methodology has been reviewed (2, 10). The Spot Test ® 3 is one of the latest receiving station antibiotic screening tests; this simple, 6-min monoclonal antibody-based test is designed to detect penicillin, cephapirin, and cloxacillin residues in raw milk. The test shows promise of becoming a routine antibiotic screening method. Thus, the purpose of this study was to compare the degree of agreement between the Spot Test and the B. stearotbermopbilus disc assay. MATERIALS AND METHODS Milk Samples
Commingled samples of milk were collected from transport trucks at several processing plants in Vermont. In the first set of experiments, 48 milk samples were collected in 1.89-L plastic milk containers and transported (at or below 4.4°C) to the University of Verm o n t laboratory where they were stored at 4.4°C. Antibiotic-treated (spiked) subsamples of each milk sample were prepared within 72 h of milk sample collection. One vial of frozen antibiotic stock solution, either penicillin, cephapirin, or cloxacillin, was thawed and a 1-ml aliquot transferred to a volumetric flask and diluted to 25 ml with phosphate buffer. The standard was further diluted b y transferring .5, .75, 1.0, 1.25, 2.0, 4.0, and 8.0-ml aliquots (1.5 instead of .5 for cloxacillin) to volumetric flasks and diluted to 100 ml with 1% phosphate buffer (6). From each 100 ml volumetric flask, 1-ml aliquots were transferred to separate
3Angenics, Inc., Cambridge, MA 02139. 4US Pharmacopeial Convention, Rockville, MD 20812. SDifco, Detroit, MI 48232. Lot Number 727046. 6 Schleicher and Schuell #740E, 12.7 ram.
1511
volumetric flasks and diluted to 100 ml with a portion of a commingled milk sample. The seven spiked subsamples were transferred to Whirl Pak bags and held in ice water prior to antibiotic testing. The milk sample spiking procedure was replicated 16 times with each of the three antibiotics. In the second experiment, 823 milk samples from transport trucks were screened for antibiotics by the disc assay and Spot Test procedures. Antibiotic Standards
Appropriate amounts of penicillin G and the sodium salts o f cephapirin and cloxacillin 4 were weighed to five decimal places then diluted with 1% phosphate buffer (6) to concentrations of 1.25 mg penicillin/ml, 3.0 mg cephapirin/ml, or 12.5 mg cloxacillin/ml. Each stock solution was subdivided in 1.2-ml amounts and stored frozen at - 6 4 ° C .
Bacillus stearotbermopbilus Disc Assay Treated (spiked) and untreated (unspiked) milk samples were assayed for antibiotic residues by the B. stearotbermopbilus disc assay screening procedure (8) as modified (6) to incorporate the use of a 90-pl pipettor. The PM indicator agar was prepared from individual ingredients, subdivided in 200 ml lots and autoclaved. The assay medium was seeded with Thermospore suspension PM. 5 Nine replicate discs 6 were prepared for each spiked milk subsample, and zone diameters were measured to the nearest .1 mm following incubation of the disc assay plates at 64 + 2°C. Six replicate discs were prepared for each unspiked control milk subsample. To compare results of the Spot Test to those obtained b y the disc assay, mean zone diameter in millimeters for every milk subsample was used to determine if the milk was positive or negative for antibiotics. Although any zone on the disc assay would indicate contamination with an inhibitor, a mean zone size of 16 mm was used, which is consistent with current National Conference on Interstate Milk Shipments and FDA actionable levels. Thus, milk subsamples having a mean zone diameter /> 16 m m were considered positive, and those having a mean zone diameter < 16 mm were considered negative. Journal of Dairy Science Vol. 69, No. 6, 1986
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RYAN ET AL.
Spot Test Assay
Statistical Analyses
Spot Test reagents and test kits were received bimonthly from the manufacturer. 3 The reagents included an enzyme preparation (reagent A), buffer solution (reagent B), penicillin, cephapirin, and cloxacillin antibody (reagents C-PEN, C-CEPH, and C-CLOX, respectively), penicillin, cephapirin and cloxacillin-coated latex (reagents D-PEN, D-CEPH, and D-CLOX, respectively), and Spot Test positive control antibiotic standard. Prior to antibiotic screening, all Spot Test reagents were warmed to 22.2 to 24.4°C and vortexed. Spot Test antibiotic standards, which served as positive controls for the assay, were rehydrated with 10 ml of antibiotic-free, commingled milk. All milk samples to be assayed were held in an ice water bath until the assay was started. A 1.5-ml aliquot milk sample was transferred to a 12 × 75-ram disposable glass culture tube containing 10/al of reagent A. The tube was stoppered with a polyethylene plug and the contents were vigorously shaken by hand for 5 s. Following shaking, 200/~1 of reagent B were added to the tube. The tube was restoppered, inverted three times, then rolled in a horizontal position for 15 s. During the rolling period, an acid coagulum was formed that was subsequently separated using a disposable filter unit attached to a 3-ml syringe. The first 4 to 5 drops of clear filtrate were collected in a clean culture tube. Twenty microliters of filtrate were transferred to one of the circular areas of a glass agglutination slide. Depending on which antibiotic was being assayed, 10 ~tl of reagent C-PEN, C-CEPH, or C-CLOX were added to the slide and mixed with the filtrate. Ten microliters of the appropriate reagent D, either D-PEN, D-CEPH, or D-CLOX were added to the slide and mixed with the filtrate-antibody combination. At this point, the slide was placed on the agglutination test rocker and continuously rocked for the 4-min reaction time. When the reaction time had elapsed, the slide was checked for visible signs of agglutination. Degree of agglutination was subjectively compared with that resulting from the positive control antibiotic standard. Milk samples producing more agglutination than the positive control were considered negative and those equal to or less than the positive control antibiotic standard were considered positive.
Two different methods of statistical analysis were used in this study. For each antibiotic, the percent of positive results by each screening assay was calculated. These percentages were transformed to angles by the variance-stabilizing transformation to arc sine square root of percent positive. Screening method and concentration effects were determined by the analysis of variance program in SPSS-X (11, 12) and stepwise logistic regression from the BMDP package (13). The response variable in the analysis of variance was arc sine square root of percent positive. The effects were screening method, antibiotic concentration, day, screening method by concentration, and trial within day. Effects were assumed fixed. For the logistic regression, the dependent variable was the logit of percent positives, and the effects were screening method, concentration, trial, and screening method by concentration. Dummy variables were created for these effects, and stepwise logistic regression was used to determine the significant effects.
Journal of Dairy Science Vol. 69, No. 6, 1986
RESULTS AND DISCUSSION Penicillin
Precision of the disc assay procedure, defined as the pooled standard deviation (15), has been reported to range from .26 to .91 with an average precision among nine laboratories participating in a collaborative study of .45 (6). Precision in our laboratory for milk samples spiked with penicillin was .33. The mean zone diameter in millimeters of nine replicate disc assays was calculated for each of the 16 subsamples within each level of penicillin spiking. The range of mean zone diameters for unspiked control subsamples and subsamples spiked with penicillin are included in Table 1. No zones of inhibition were found with the unspiked control subsamples, which indicates that all milk samples were free of antibiotics or other inbibitors that could be detected by the disc assay procedure. As the penicillin concentration in the subsamples increased, mean zone diameters increased. Of the 16 subsamples containing 3.7 ng penicillin/ ml, means of the nine disc assay replicates per subsample ranged from 15.6 to 16.8 mm. Mean zone diameters of subsamples containing 5 ng
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
] 513
TABLE 1. N u m b e r of false positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific m i l k subsamples spiked w i t h penicillin. Range of mean (n=9) Penicillin
zone diameters x
(nglml)
(mm)
0 2.5 3.7 5.0 6.3 10.0 20.1 40.2
0 13.815.6 17.0 17.8 19.8 -20.1 -24.3 --
Disc assay
15.0 16.8 18.0 18.4 20.5 23.0 25.3
Spot Test
Agree
False +
False -
Agree
False +
False -
96 141 116 140 142 142 141 139
0 0 22 0 0 0 0 0
0 0 203 0 0 0 0 0
89 31 111 134 136 47 47 48
1 26 13 0 0 0 0 0
0 0 6 0 0 0 0 0
a Range of means from 16 different subsamples. ~When the mean o f nine replicate disc assays per subsample was < 16 mm, a false positive was recorded if one or mo re of th e replicates was > / 1 6 ram. 3When t h e mean o f nine replicate disc assays per subsample w a s / > 16 mm, a false negative was recorded if one or more o f the replicates was < 16 mm.
penicillin/ml or .008 IU ranged from 17.0 to 20.0 and fell within the recommended range of 17 to 20 mm as outlined in the 14th edition of
The Association of Official Analytical Chemists Official Methods of Analysis (1). The mean zone diameters were plotted against log10 penicillin concentration (Figure 1). The regression equation for the line was zone diameter
30A
E 206o O
N
10-
O9
0
I
0
.4
I
I
I
I
,8 1.2 1.6 2.0 Log Antibiotic (ng/ml)
I
2.4
]
2.8
Figure 1. Effect of varying c o n c e n t r a t i o n s of penicillin (A), cephapirin (B), and cloxacillin (C) on Bacillus stearotbermopbilus disc assay zone size. Means of nine replicate disc assays per spiked m i l k subsample (n=16) are shown.
= 11.18 + 8.67 (logl0 penicillin concentration). Table 1 contains a summary of the number of Spot Tests that agreed with the disc assay or yielded false positive or false negative results. A false positive Spot Test was defined as one in which the Spot Test w.as positive while the mean zone size on the disc assay was < 16 mm. A false negative test was one in which the Spot Test was negative while the disc assay was/> 16 mm. At 3.7 ng penicillin/ml, where the disc assay zone sizes ranged from 15.6 to 16.8, 111 of the 130 Spot Tests agreed with the disc assay. Thirteen false positive and six false negative results were also observed. At 3.7 ng penicillin/ml or less, agreement between the Spot Test and the disc assay was 83.4%. False positive and false negative percentages in this range were 14.4 and 2.2%, respectively. Above 3.7 ng penicillin/ml, agreement between the two tests was 100%. Due to inherent variability in zone size, false positive and false negative results were also observed with the disc assay, the standard against which the Spot Test was compared. It is for this reason that the mean of nine disc assays replicates was used to determine if the milk sample was above or below the 16-mm actionable level.
Cephapirin Spot Test and disc assay data from milk J ourna l of Dairy Science Vol. 69, No. 6, 1986
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RYAN ET AL.
samples spiked w i t h c e p h a p i r i n w e r e a n a l y z e d t h e same way as t h o s e f r o m penicillin-spiked samples. Disc assay precision for m i l k samples spiked w i t h c e p h a p i r i n was .34. T h e r a n g e o f m e a n z o n e d i a m e t e r s for u n s p i k e d c o n t r o l s u b s a m p l e s a n d s u b s a m p l e s spiked w i t h cephapirin are i n c l u d e d in T a b l e 2. No z o n e s of i n h i b i t i o n were f o u n d w i t h t h e u n s p i k e d c o n t r o l s u b s a m p l e s , a n d as t h e c e p h a p i r i n c o n c e n t r a t i o n in t h e s u b s a m p l e s increased, m e a n z o n e d i a m e t e r s increased. T h e m e a n z o n e d i a m e t e r s were p l o t t e d against log10 cephapirin c o n c e n t r a t i o n (Figure 1). T h e regression e q u a t i o n f o r t h e line was z o n e diam e t e r = 7.12 + 8.13 (log10 c e p h a p i r i n c o n c e n tration). Table 2 contains a summary of the number o f S p o t Tests t h a t agreed w i t h t h e disc assay or y i e l d e d false positive or false negative results. A t c e p h a p i r i n c o n c e n t r a t i o n s ~< 9 n g / m l , a g r e e m e n t b e t w e e n t h e S p o t Test a n d disc assay was 71%. In t h i s range, t h e S p o t Test h a d a 29% false positive rate. B e t w e e n 12 a n d 15 n g c e p h a p i r i n / m l , w h e r e disc assay z o n e sizes r a n g e d f r o m 15.7 to 17.0 m m , t h e S p o t Test agreed w i t h t h e disc assay 77% o f t h e t i m e , w h e r e a s false positive a n d false negative perc e n t a g e s were 15 a n d 8%, respectively. At
c e p h a p i r i n >/ 24 n g / m l , a g r e e m e n t b e t w e e n t h e t w o t e s t s was 99.3%. Cloxacillin
S p o t Test a n d disc assay d a t a f r o m m i l k samples s p i k e d w i t h cloxacillin were a n a l y z e d t h e same w a y as t h a t f r o m penicillin a n d c e p h a p i r i n - s p i k e d samples. Disc assay precision for m i l k samples spiked w i t h cloxacillin was .30. T h e r a n g e o f m e a n z o n e d i a m e t e r s for unspiked control subsamples and subsamples s p i k e d w i t h cloxacillin are i n c l u d e d in T a b l e 3. No z o n e s o f i n h i b i t i o n were f o u n d w i t h t h e u n s p i k e d c o n t r o l s u b s a m p l e s , a n d as t h e cloxacillin c o n c e n t r a t i o n in t h e s u b s a m p l e s increased, m e a n z o n e d i a m e t e r increased. T h e m e a n z o n e d i a m e t e r s were p l o t t e d against log10 cloxacillin concentration (Figure 1). T h e regression e q u a t i o n f o r t h e line was z o n e d i a m e t e r = .66 + 8.51 (log10 cloxacillin c o n c e n t r a t i o n ) . Table 3 contains a summary of the number o f S p o t T e s t s t h a t agreed w i t h t h e disc assay or y i e l d e d false positive or false negative results. A t cloxacillin c o n c e n t r a t i o n s < 50 n g / m l , a g r e e m e n t b e t w e e n t h e S p o t Test a n d disc assay was 54.5%. In this range, t h e S p o t Test h a d a 4 5 . 5 % false positive rate. A t 62.5 ng cloxacillin/ m l or greater, a g r e e m e n t b e t w e e n t h e t w o t e s t s
TABLE 2. Number of flase positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific milk subsamples spiked with cephapirin.
Cephapirin
Range of mean zone diameters 1 (n=9)
(ng/ml)
(mm)
0 6 9 12 15 24 48 96
0 0 14.6 15.7 16.4 18.2
----
14.5 15.5 16.7 17.0 19.0 2 0 . 5 -- 21.3 22.5 -- 23.6
Disc assay
Spot Test
Agree
False +
False --
Agree
False +
False --
90 141 131 94 140 142 136 133
0 0 12 23 0 0 0 0
0 0 1 193 1 0 0 0
88 38 76 88 134 47 47 48
3 10 68 42 0 0 0 0
0 0 0 14 9 1 0 0
J Range of means from 16 different subsamples. 2When the mean of n i n e replicate disc assays per subsample was < 16 mm, a flase positive was recorded if o n e or more of the replicates was ~> 16 mm. 3When the mean of nine replicate disc assays per subsample was >~ 16 ram, a false negative was recorded if one or more of the replicates was < 16 mm. Journal of Dairy Science Vol. 69, No. 6, 1986
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
1515
TABLE 3. Number of false positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific milk subsamples spiked with cloxacillin.
Cloxacillin
Range of mean zone diameters z (n=9)
(ng/ml)
(mm)
0 37.5 50.0 62.5 75 100 200 400
0 13.6 14.7 15.6 16.3 17.2 19.3 21.8
------
14.4 15.6 16.3 17.4 18.5 20.9 23.3
Agree
Disc assay False +
False --
Agree
Spot Test False +
False -
96 141 I39 113 143 140 144 143
0 0 32 5 0 0 0 0
0 0 0 223 1 0 0 0
96 44 17 89 144 48 47 40
0 4 127 53 0 0 0 0
0 0 0 1 0 0 0 0
, Range of means from 16 different subsamples. 2When the mean of nine replicate disc assays per subsamples was < 16 mm, a false positive was recorded if one or more of the replicates was >/ 16 mm. 3When the mean of nine replicate disc assays per subsample was/> 16 ram, a false negative was recorded if one or more of the replicates was < 16 mrn.
was 87.2%. False positive a n d false negative p e r c e n t a g e s in this r a n g e were 12.6 a n d .2%, respectively. Agreement Between the Disc Assay and Spot Test
An analysis o f variance on the percent positive results b y t h e S p o t Test a n d disc assay p r o c e d u r e s s h o w e d a significant ( P < . 0 0 1 ) method by antibiotic concentration interaction
I
60-
N
~0 l
Method I
0
2~S
3,7
5.0 6.3 10.0 Penicillin (nO/col)
Disc Test S~otT~s~
28.1 46.2
Figure 2. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of penicillin and unspiked control samples.
for all t h r e e a n t i b i o t i c s tested. F u r t h e r investigation of these interactions showed that t h e disc assay a n d S p o t Test yield d i f f e r e n t results at c e r t a i n a n t i b i o t i c c o n c e n t r a t i o n s . T h e p e r c e n t positive results b y t h e S p o t Test was significantly ( P < . 0 0 1 ) higher t h a n t h e p e r c e n t positive b y t h e disc assay a t c o n c e n t r a t i o n s o f 2.5 a n d 3.7 ng p e n i c i l l i n / m l (Figure 2). A significantly ( P < . 0 0 1 ) h i g h e r p e r c e n t o f positive results b y t h e S p o t Test was at c e p h a p i r i n c o n c e n t r a t i o n s o f 6, 9, a n d 12 n g / m l (Figure 3). Similarly, t h e p e r c e n t positive results b y t h e S p o t Test were significantly h i g h e r t h a n p e r c e n t positive b y t h e disc assay at c o n c e n t r a t i o n s o f 37.5 ( P < . 0 0 5 ) , 50, a n d 62.5 ng c l o x a c i l l i n / m l (P<.OOI) (Figure 4). A n a l y s e s using t h e logistic regression also s h o w e d a significant ( P < . O 0 1 ) method by antibiotic concentration interaction for all t h r e e a n t i b i o t i c s t e s t e d . Disc assay results o n 823 c o m m i n g l e d m i l k samples f r o m t r a n s p o r t trailers i n d i c a t e d t h a t five m i l k s a m p l e s (.61%) c o n t a i n e d e n o u g h a n t i b i o t i c residue to p r o d u c e a 1 6 - m m z o n e or greater. S p o t Test results o n t h e s e five samples were all positive. Of t h e r e m a i n i n g samples t e s t e d , n o false positive r e s u l t s were r e c o r d e d w i t h t h e S p o t Test. T h u s , in a l i m i t e d field trial, a g r e e m e n t b e t w e e n t h e S p o t T e s t a n d B. stearotbermopbilus disc assay screening procedures was 100%. Journal of Dairy Science Vol. 69, No. 6, 1986
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RYAN ET AL.
CONCLUSIONS
F o r a d a i r y p r o c e s s o r to a c c e p t a n a n t i b i o t i c screening t e s t o t h e r t h a n t h e disc assay, t h e n e w test s h o u l d satisfy at least t h r e e general req u i r e m e n t s . T h e first t w o r e q u i r e m e n t s , n a m e l y cost per sample a n a l y z e d a n d simplicity o f t h e t e s t i n g p r o c e d u r e , m u s t be evaluated b y t h e specific processor. Variables such as l a b o r a t o r y budget, n u m b e r o f loads received per d a y o r p e r hour, and technician competence must be taken i n t o c o n s i d e r a t i o n . T h e t h i r d a n d m o s t imp o r t a n t r e q u i r e m e n t is h o w well t h e n e w test
1oo-
compares with the accepted and standardized disc assay s c r e e n i n g p r o c e d u r e . D a t a collected o n a n t i b i o t i c spiked milk samples i n d i c a t e t h a t t h e S p o t Test will d e t e c t m o s t m i l k samples c o n t a i n i n g penicillin, cephapirin, or cloxacillin at a m o u n t s sufficient to p r o d u c e a 1 6 - m m z o n e o n t h e disc assay. Based o n results o f t h e analysis o f variance a n d logistic regression, t h e test is m o r e sensitive t h a n t h e disc assay a n d f r e q u e n t l y d e t e c t s t h e s e antibiotics at amounts below those necessary to p r o d u c e a 1 6 - m m zone. C o n s i d e r i n g t h e p o t e n tial p u b l i c h e a l t h risks a n d f i n a n c i a l losses t h a t can o c c u r if o n e or m o r e silos of m i l k are c o n t a m i n a t e d w i t h a n t i b i o t i c - a d u l t e r a t e d milk, positive results b y t h e S p o t Test screening p r o c e d u r e will alert processors to suspect m i l k s h i p m e n t s , w h i c h m a y t h e n b e isolated a n d a n a l y z e d b y official testing p r o c e d u r e s .
~ 60] ~
REFERENCES
•
,oi
Method BIB Dise Test
Spot Test
0
6
9
12 15 24 CephoplHn (ng/rnl)
48
96
Figure 3. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of cephapirin and unspiked control samples.
*o0]
.oJ 60t
Method
°L
I Disc Test ~;~ Spot Test
6
37.5
50'.0
62'.5 75.0
100.0 200.0 400.0
Cloxocillln (ng/mt)
Figure 4. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of cloxacillin and unspiked control samples. Journal of Dairy Science Vol. 69, No. 6, 1986
1 Association of Official Analytical Chemists. 1984. Official methods of analysis. 14 ed. Assoc. Offic. Anal. Chem., Washington, DC. 2 Bishop, J. R., and C. H. White. 1984. Antibiotic residue detection in milk a review. J. Food. Prot. 47:647. 3 Claypool, L. L. 1984. Concerns and problems of cheese industry for milk quality and procurement today and future. J. Dairy Sci. 67:2091. 4 Code of Federal Regulations. 1984. Title 21 Food and drugs, Part 540. Office Fed. Reg., Washington, DC. 5 Everson, T. C. 1984. Concerns and problems of processing and manufacturing in super plants. J. Dairy Sci. 67:2095. 6 Ginn, R. E., R. A. Case, V. S. Packard, and S. R. Tatini. 1982. Quantitative estimates of beta-lactam residues in raw milk around a reference standard: collaborative study. J. Assoc. Offic. Anal. Chem. 65 : 1407. 7 Marth, E. H., and B. E. Ellickson. 1959. Problems created by the presence of antibiotics in milk and milk products. J. Milk Food Technol. 22:266. 8 Messer, J. W., J. E. Leslie, G. A. Houghtby, J. T. Peeler, and J. E. Barnett. 1982. Bacillus stearothermopbilus disc assay for detection of inhibitors in milk: collaborative study. J. Assoc. Offic. Anal. Chem. 65:1208. 9 Reinbold, G. W., and M. S. Reddy. 1974. Sensitivity or resistance dairy starter and associated microorganisms to selected antibiotics, J. Milk Food Technol. 37:517. 10 Ryan, J. J. 1983. Antibiotic tests screen raw milk. Dairy Field 166:48. 11 Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods. 6th ed. Iowa State Univ. Press, Ames. 12 Statistical Package for the Social Sciences, Inc.
-
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
1983. SPSS-X user's guide. McGraw Hill, New York, NY. 13 University of California-Los Angeles, Department of Biomathematics. 1979. BMDP Statistical software. Univ. California Press. 14 Van Arsdel, P. P., Jr. 1983. Adverse drug reactions.
15 17
Page 1389 in Allergy: principles and practice, Vol. 2. 2nd ed. E. Middleton, Jr., C. E. Reed, and E. F. Ellis, ed. C. V. Mosby, St. Louis, MO. 15 Youden, W. J., and E. H. Strominger. 1975. Statistical manual of the AOAC. Association of Official Analytical Chemists, Arlington, VA.
Journal of Dairy Science Vol. 69, No. 6, 1986
tests was 87.2% and false positive and negative percentages were 12.6 and .2%, respectively. In a field trial consisting of 823 bulk samples, Spot Test and disc assay agreement was 100%. The Spot Test is a rapid and reliable method for detecting penicillin, cephapirin, and cloxacillin residues in raw milk at concentrations that will produce a 16-mm zone.
The objective of this study was to compare Spot Test results with the results of the Bacillus stearotbermopbilus disc assay. Commingled raw milk samples were subdivided and spiked with penicillin, cephapirin, or cloxacillin. All subsamples, including unspiked subsamples, were analyzed by the Spot Test (3 or 9 replicates) and disc assay (9 replicates). Mean zone diameter for every subsample was calculated; subsamples having a mean zone diameter >/ 16 mm were considered positive. At penicillin concentrations ~< 3.7 ng/ml, agreement between the Spot Test and disc assay was 83.4%, and false positive and negative percentages were 14.4 and 2.2%, respectively. Above 3.7 ng/ml, agreement was 100%. At cephapirin concentrations < 12 ng/ml, agreement between the two tests was 67.9%, and false positive and negative percentages were 28.8 and 3.3%, respectively. Above 15 ng/ml, agreement was 99.3% with .7% false negatives. A t cloxacillin concentrations < 50 mg/ml agreement between the two tests was 54.5 with 45.5% false positives. At cloxacillin concentrations /> 62.5 ng/ml, agreement between the two
INTRODUCTION
Received December 2, 1985. Contribution Number 591 of the Vermont Agricultural Experiment Station. Approved by the Director. 2Present address: Department of Dairy Science, Louisiana State University, Baton Rouge 70803. 1986 J Dairy Sci 69:1510-1517
Intramammary and intramuscular drug products containing ~-lactam antibiotics are commonly used in the treatment of bovine mastitis. Penicillin and its synthetic derivatives are the most popular antimicrobial agents in these drug products. In an effort to minimize antibiotic contamination of fluid milk supplies, the United States F o o d and Drug Administration (FDA) limits the quantity of antibiotic in drug products and requires label instructions explaining product usage and drug withdrawal times (4). However, despite producer-oriented educational programs and financial penalties for shipping milk adulterated with antibiotics, contaminated milk is still a problem facing the dairy industry (3). Problems associated with antibiotic residues in the milk supply are well documented and have been reviewed (7), e.g., cultured dairy product manufacturers may experience partial or complete inhibition of acid production by starter cultures (5, 9), whereas hypersensitive individuals may exhibit adverse reactions (14). Considerable testing by processors and regulatory agencies is done to detect and minimize acceptance of antibiotic-contaminated milk and to assure consumers that milk and other dairy
1510
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK foods contaminated with antibiotics are not marketed. The ideal test for antibiotic residues in milk is one that combines simplicity and speed with low cost and that may be conducted at the farm prior to emptying the bulk tank (3). An on-farm test has yet to gain wide industry acceptance, and therefore, laboratory or receiving station tests are commonplace. Such methodology has been reviewed (2, 10). The Spot Test ® 3 is one of the latest receiving station antibiotic screening tests; this simple, 6-min monoclonal antibody-based test is designed to detect penicillin, cephapirin, and cloxacillin residues in raw milk. The test shows promise of becoming a routine antibiotic screening method. Thus, the purpose of this study was to compare the degree of agreement between the Spot Test and the B. stearotbermopbilus disc assay. MATERIALS AND METHODS Milk Samples
Commingled samples of milk were collected from transport trucks at several processing plants in Vermont. In the first set of experiments, 48 milk samples were collected in 1.89-L plastic milk containers and transported (at or below 4.4°C) to the University of Verm o n t laboratory where they were stored at 4.4°C. Antibiotic-treated (spiked) subsamples of each milk sample were prepared within 72 h of milk sample collection. One vial of frozen antibiotic stock solution, either penicillin, cephapirin, or cloxacillin, was thawed and a 1-ml aliquot transferred to a volumetric flask and diluted to 25 ml with phosphate buffer. The standard was further diluted b y transferring .5, .75, 1.0, 1.25, 2.0, 4.0, and 8.0-ml aliquots (1.5 instead of .5 for cloxacillin) to volumetric flasks and diluted to 100 ml with 1% phosphate buffer (6). From each 100 ml volumetric flask, 1-ml aliquots were transferred to separate
3Angenics, Inc., Cambridge, MA 02139. 4US Pharmacopeial Convention, Rockville, MD 20812. SDifco, Detroit, MI 48232. Lot Number 727046. 6 Schleicher and Schuell #740E, 12.7 ram.
1511
volumetric flasks and diluted to 100 ml with a portion of a commingled milk sample. The seven spiked subsamples were transferred to Whirl Pak bags and held in ice water prior to antibiotic testing. The milk sample spiking procedure was replicated 16 times with each of the three antibiotics. In the second experiment, 823 milk samples from transport trucks were screened for antibiotics by the disc assay and Spot Test procedures. Antibiotic Standards
Appropriate amounts of penicillin G and the sodium salts o f cephapirin and cloxacillin 4 were weighed to five decimal places then diluted with 1% phosphate buffer (6) to concentrations of 1.25 mg penicillin/ml, 3.0 mg cephapirin/ml, or 12.5 mg cloxacillin/ml. Each stock solution was subdivided in 1.2-ml amounts and stored frozen at - 6 4 ° C .
Bacillus stearotbermopbilus Disc Assay Treated (spiked) and untreated (unspiked) milk samples were assayed for antibiotic residues by the B. stearotbermopbilus disc assay screening procedure (8) as modified (6) to incorporate the use of a 90-pl pipettor. The PM indicator agar was prepared from individual ingredients, subdivided in 200 ml lots and autoclaved. The assay medium was seeded with Thermospore suspension PM. 5 Nine replicate discs 6 were prepared for each spiked milk subsample, and zone diameters were measured to the nearest .1 mm following incubation of the disc assay plates at 64 + 2°C. Six replicate discs were prepared for each unspiked control milk subsample. To compare results of the Spot Test to those obtained b y the disc assay, mean zone diameter in millimeters for every milk subsample was used to determine if the milk was positive or negative for antibiotics. Although any zone on the disc assay would indicate contamination with an inhibitor, a mean zone size of 16 mm was used, which is consistent with current National Conference on Interstate Milk Shipments and FDA actionable levels. Thus, milk subsamples having a mean zone diameter /> 16 m m were considered positive, and those having a mean zone diameter < 16 mm were considered negative. Journal of Dairy Science Vol. 69, No. 6, 1986
1512
RYAN ET AL.
Spot Test Assay
Statistical Analyses
Spot Test reagents and test kits were received bimonthly from the manufacturer. 3 The reagents included an enzyme preparation (reagent A), buffer solution (reagent B), penicillin, cephapirin, and cloxacillin antibody (reagents C-PEN, C-CEPH, and C-CLOX, respectively), penicillin, cephapirin and cloxacillin-coated latex (reagents D-PEN, D-CEPH, and D-CLOX, respectively), and Spot Test positive control antibiotic standard. Prior to antibiotic screening, all Spot Test reagents were warmed to 22.2 to 24.4°C and vortexed. Spot Test antibiotic standards, which served as positive controls for the assay, were rehydrated with 10 ml of antibiotic-free, commingled milk. All milk samples to be assayed were held in an ice water bath until the assay was started. A 1.5-ml aliquot milk sample was transferred to a 12 × 75-ram disposable glass culture tube containing 10/al of reagent A. The tube was stoppered with a polyethylene plug and the contents were vigorously shaken by hand for 5 s. Following shaking, 200/~1 of reagent B were added to the tube. The tube was restoppered, inverted three times, then rolled in a horizontal position for 15 s. During the rolling period, an acid coagulum was formed that was subsequently separated using a disposable filter unit attached to a 3-ml syringe. The first 4 to 5 drops of clear filtrate were collected in a clean culture tube. Twenty microliters of filtrate were transferred to one of the circular areas of a glass agglutination slide. Depending on which antibiotic was being assayed, 10 ~tl of reagent C-PEN, C-CEPH, or C-CLOX were added to the slide and mixed with the filtrate. Ten microliters of the appropriate reagent D, either D-PEN, D-CEPH, or D-CLOX were added to the slide and mixed with the filtrate-antibody combination. At this point, the slide was placed on the agglutination test rocker and continuously rocked for the 4-min reaction time. When the reaction time had elapsed, the slide was checked for visible signs of agglutination. Degree of agglutination was subjectively compared with that resulting from the positive control antibiotic standard. Milk samples producing more agglutination than the positive control were considered negative and those equal to or less than the positive control antibiotic standard were considered positive.
Two different methods of statistical analysis were used in this study. For each antibiotic, the percent of positive results by each screening assay was calculated. These percentages were transformed to angles by the variance-stabilizing transformation to arc sine square root of percent positive. Screening method and concentration effects were determined by the analysis of variance program in SPSS-X (11, 12) and stepwise logistic regression from the BMDP package (13). The response variable in the analysis of variance was arc sine square root of percent positive. The effects were screening method, antibiotic concentration, day, screening method by concentration, and trial within day. Effects were assumed fixed. For the logistic regression, the dependent variable was the logit of percent positives, and the effects were screening method, concentration, trial, and screening method by concentration. Dummy variables were created for these effects, and stepwise logistic regression was used to determine the significant effects.
Journal of Dairy Science Vol. 69, No. 6, 1986
RESULTS AND DISCUSSION Penicillin
Precision of the disc assay procedure, defined as the pooled standard deviation (15), has been reported to range from .26 to .91 with an average precision among nine laboratories participating in a collaborative study of .45 (6). Precision in our laboratory for milk samples spiked with penicillin was .33. The mean zone diameter in millimeters of nine replicate disc assays was calculated for each of the 16 subsamples within each level of penicillin spiking. The range of mean zone diameters for unspiked control subsamples and subsamples spiked with penicillin are included in Table 1. No zones of inhibition were found with the unspiked control subsamples, which indicates that all milk samples were free of antibiotics or other inbibitors that could be detected by the disc assay procedure. As the penicillin concentration in the subsamples increased, mean zone diameters increased. Of the 16 subsamples containing 3.7 ng penicillin/ ml, means of the nine disc assay replicates per subsample ranged from 15.6 to 16.8 mm. Mean zone diameters of subsamples containing 5 ng
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
] 513
TABLE 1. N u m b e r of false positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific m i l k subsamples spiked w i t h penicillin. Range of mean (n=9) Penicillin
zone diameters x
(nglml)
(mm)
0 2.5 3.7 5.0 6.3 10.0 20.1 40.2
0 13.815.6 17.0 17.8 19.8 -20.1 -24.3 --
Disc assay
15.0 16.8 18.0 18.4 20.5 23.0 25.3
Spot Test
Agree
False +
False -
Agree
False +
False -
96 141 116 140 142 142 141 139
0 0 22 0 0 0 0 0
0 0 203 0 0 0 0 0
89 31 111 134 136 47 47 48
1 26 13 0 0 0 0 0
0 0 6 0 0 0 0 0
a Range of means from 16 different subsamples. ~When the mean o f nine replicate disc assays per subsample was < 16 mm, a false positive was recorded if one or mo re of th e replicates was > / 1 6 ram. 3When t h e mean o f nine replicate disc assays per subsample w a s / > 16 mm, a false negative was recorded if one or more o f the replicates was < 16 mm.
penicillin/ml or .008 IU ranged from 17.0 to 20.0 and fell within the recommended range of 17 to 20 mm as outlined in the 14th edition of
The Association of Official Analytical Chemists Official Methods of Analysis (1). The mean zone diameters were plotted against log10 penicillin concentration (Figure 1). The regression equation for the line was zone diameter
30A
E 206o O
N
10-
O9
0
I
0
.4
I
I
I
I
,8 1.2 1.6 2.0 Log Antibiotic (ng/ml)
I
2.4
]
2.8
Figure 1. Effect of varying c o n c e n t r a t i o n s of penicillin (A), cephapirin (B), and cloxacillin (C) on Bacillus stearotbermopbilus disc assay zone size. Means of nine replicate disc assays per spiked m i l k subsample (n=16) are shown.
= 11.18 + 8.67 (logl0 penicillin concentration). Table 1 contains a summary of the number of Spot Tests that agreed with the disc assay or yielded false positive or false negative results. A false positive Spot Test was defined as one in which the Spot Test w.as positive while the mean zone size on the disc assay was < 16 mm. A false negative test was one in which the Spot Test was negative while the disc assay was/> 16 mm. At 3.7 ng penicillin/ml, where the disc assay zone sizes ranged from 15.6 to 16.8, 111 of the 130 Spot Tests agreed with the disc assay. Thirteen false positive and six false negative results were also observed. At 3.7 ng penicillin/ml or less, agreement between the Spot Test and the disc assay was 83.4%. False positive and false negative percentages in this range were 14.4 and 2.2%, respectively. Above 3.7 ng penicillin/ml, agreement between the two tests was 100%. Due to inherent variability in zone size, false positive and false negative results were also observed with the disc assay, the standard against which the Spot Test was compared. It is for this reason that the mean of nine disc assays replicates was used to determine if the milk sample was above or below the 16-mm actionable level.
Cephapirin Spot Test and disc assay data from milk J ourna l of Dairy Science Vol. 69, No. 6, 1986
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RYAN ET AL.
samples spiked w i t h c e p h a p i r i n w e r e a n a l y z e d t h e same way as t h o s e f r o m penicillin-spiked samples. Disc assay precision for m i l k samples spiked w i t h c e p h a p i r i n was .34. T h e r a n g e o f m e a n z o n e d i a m e t e r s for u n s p i k e d c o n t r o l s u b s a m p l e s a n d s u b s a m p l e s spiked w i t h cephapirin are i n c l u d e d in T a b l e 2. No z o n e s of i n h i b i t i o n were f o u n d w i t h t h e u n s p i k e d c o n t r o l s u b s a m p l e s , a n d as t h e c e p h a p i r i n c o n c e n t r a t i o n in t h e s u b s a m p l e s increased, m e a n z o n e d i a m e t e r s increased. T h e m e a n z o n e d i a m e t e r s were p l o t t e d against log10 cephapirin c o n c e n t r a t i o n (Figure 1). T h e regression e q u a t i o n f o r t h e line was z o n e diam e t e r = 7.12 + 8.13 (log10 c e p h a p i r i n c o n c e n tration). Table 2 contains a summary of the number o f S p o t Tests t h a t agreed w i t h t h e disc assay or y i e l d e d false positive or false negative results. A t c e p h a p i r i n c o n c e n t r a t i o n s ~< 9 n g / m l , a g r e e m e n t b e t w e e n t h e S p o t Test a n d disc assay was 71%. In t h i s range, t h e S p o t Test h a d a 29% false positive rate. B e t w e e n 12 a n d 15 n g c e p h a p i r i n / m l , w h e r e disc assay z o n e sizes r a n g e d f r o m 15.7 to 17.0 m m , t h e S p o t Test agreed w i t h t h e disc assay 77% o f t h e t i m e , w h e r e a s false positive a n d false negative perc e n t a g e s were 15 a n d 8%, respectively. At
c e p h a p i r i n >/ 24 n g / m l , a g r e e m e n t b e t w e e n t h e t w o t e s t s was 99.3%. Cloxacillin
S p o t Test a n d disc assay d a t a f r o m m i l k samples s p i k e d w i t h cloxacillin were a n a l y z e d t h e same w a y as t h a t f r o m penicillin a n d c e p h a p i r i n - s p i k e d samples. Disc assay precision for m i l k samples spiked w i t h cloxacillin was .30. T h e r a n g e o f m e a n z o n e d i a m e t e r s for unspiked control subsamples and subsamples s p i k e d w i t h cloxacillin are i n c l u d e d in T a b l e 3. No z o n e s o f i n h i b i t i o n were f o u n d w i t h t h e u n s p i k e d c o n t r o l s u b s a m p l e s , a n d as t h e cloxacillin c o n c e n t r a t i o n in t h e s u b s a m p l e s increased, m e a n z o n e d i a m e t e r increased. T h e m e a n z o n e d i a m e t e r s were p l o t t e d against log10 cloxacillin concentration (Figure 1). T h e regression e q u a t i o n f o r t h e line was z o n e d i a m e t e r = .66 + 8.51 (log10 cloxacillin c o n c e n t r a t i o n ) . Table 3 contains a summary of the number o f S p o t T e s t s t h a t agreed w i t h t h e disc assay or y i e l d e d false positive or false negative results. A t cloxacillin c o n c e n t r a t i o n s < 50 n g / m l , a g r e e m e n t b e t w e e n t h e S p o t Test a n d disc assay was 54.5%. In this range, t h e S p o t Test h a d a 4 5 . 5 % false positive rate. A t 62.5 ng cloxacillin/ m l or greater, a g r e e m e n t b e t w e e n t h e t w o t e s t s
TABLE 2. Number of flase positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific milk subsamples spiked with cephapirin.
Cephapirin
Range of mean zone diameters 1 (n=9)
(ng/ml)
(mm)
0 6 9 12 15 24 48 96
0 0 14.6 15.7 16.4 18.2
----
14.5 15.5 16.7 17.0 19.0 2 0 . 5 -- 21.3 22.5 -- 23.6
Disc assay
Spot Test
Agree
False +
False --
Agree
False +
False --
90 141 131 94 140 142 136 133
0 0 12 23 0 0 0 0
0 0 1 193 1 0 0 0
88 38 76 88 134 47 47 48
3 10 68 42 0 0 0 0
0 0 0 14 9 1 0 0
J Range of means from 16 different subsamples. 2When the mean of n i n e replicate disc assays per subsample was < 16 mm, a flase positive was recorded if o n e or more of the replicates was ~> 16 mm. 3When the mean of nine replicate disc assays per subsample was >~ 16 ram, a false negative was recorded if one or more of the replicates was < 16 mm. Journal of Dairy Science Vol. 69, No. 6, 1986
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
1515
TABLE 3. Number of false positive (+) and false negative ( - ) results for disc assay and Spot Test analyses when compared with the disc assay average of specific milk subsamples spiked with cloxacillin.
Cloxacillin
Range of mean zone diameters z (n=9)
(ng/ml)
(mm)
0 37.5 50.0 62.5 75 100 200 400
0 13.6 14.7 15.6 16.3 17.2 19.3 21.8
------
14.4 15.6 16.3 17.4 18.5 20.9 23.3
Agree
Disc assay False +
False --
Agree
Spot Test False +
False -
96 141 I39 113 143 140 144 143
0 0 32 5 0 0 0 0
0 0 0 223 1 0 0 0
96 44 17 89 144 48 47 40
0 4 127 53 0 0 0 0
0 0 0 1 0 0 0 0
, Range of means from 16 different subsamples. 2When the mean of nine replicate disc assays per subsamples was < 16 mm, a false positive was recorded if one or more of the replicates was >/ 16 mm. 3When the mean of nine replicate disc assays per subsample was/> 16 ram, a false negative was recorded if one or more of the replicates was < 16 mrn.
was 87.2%. False positive a n d false negative p e r c e n t a g e s in this r a n g e were 12.6 a n d .2%, respectively. Agreement Between the Disc Assay and Spot Test
An analysis o f variance on the percent positive results b y t h e S p o t Test a n d disc assay p r o c e d u r e s s h o w e d a significant ( P < . 0 0 1 ) method by antibiotic concentration interaction
I
60-
N
~0 l
Method I
0
2~S
3,7
5.0 6.3 10.0 Penicillin (nO/col)
Disc Test S~otT~s~
28.1 46.2
Figure 2. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of penicillin and unspiked control samples.
for all t h r e e a n t i b i o t i c s tested. F u r t h e r investigation of these interactions showed that t h e disc assay a n d S p o t Test yield d i f f e r e n t results at c e r t a i n a n t i b i o t i c c o n c e n t r a t i o n s . T h e p e r c e n t positive results b y t h e S p o t Test was significantly ( P < . 0 0 1 ) higher t h a n t h e p e r c e n t positive b y t h e disc assay a t c o n c e n t r a t i o n s o f 2.5 a n d 3.7 ng p e n i c i l l i n / m l (Figure 2). A significantly ( P < . 0 0 1 ) h i g h e r p e r c e n t o f positive results b y t h e S p o t Test was at c e p h a p i r i n c o n c e n t r a t i o n s o f 6, 9, a n d 12 n g / m l (Figure 3). Similarly, t h e p e r c e n t positive results b y t h e S p o t Test were significantly h i g h e r t h a n p e r c e n t positive b y t h e disc assay at c o n c e n t r a t i o n s o f 37.5 ( P < . 0 0 5 ) , 50, a n d 62.5 ng c l o x a c i l l i n / m l (P<.OOI) (Figure 4). A n a l y s e s using t h e logistic regression also s h o w e d a significant ( P < . O 0 1 ) method by antibiotic concentration interaction for all t h r e e a n t i b i o t i c s t e s t e d . Disc assay results o n 823 c o m m i n g l e d m i l k samples f r o m t r a n s p o r t trailers i n d i c a t e d t h a t five m i l k s a m p l e s (.61%) c o n t a i n e d e n o u g h a n t i b i o t i c residue to p r o d u c e a 1 6 - m m z o n e or greater. S p o t Test results o n t h e s e five samples were all positive. Of t h e r e m a i n i n g samples t e s t e d , n o false positive r e s u l t s were r e c o r d e d w i t h t h e S p o t Test. T h u s , in a l i m i t e d field trial, a g r e e m e n t b e t w e e n t h e S p o t T e s t a n d B. stearotbermopbilus disc assay screening procedures was 100%. Journal of Dairy Science Vol. 69, No. 6, 1986
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RYAN ET AL.
CONCLUSIONS
F o r a d a i r y p r o c e s s o r to a c c e p t a n a n t i b i o t i c screening t e s t o t h e r t h a n t h e disc assay, t h e n e w test s h o u l d satisfy at least t h r e e general req u i r e m e n t s . T h e first t w o r e q u i r e m e n t s , n a m e l y cost per sample a n a l y z e d a n d simplicity o f t h e t e s t i n g p r o c e d u r e , m u s t be evaluated b y t h e specific processor. Variables such as l a b o r a t o r y budget, n u m b e r o f loads received per d a y o r p e r hour, and technician competence must be taken i n t o c o n s i d e r a t i o n . T h e t h i r d a n d m o s t imp o r t a n t r e q u i r e m e n t is h o w well t h e n e w test
1oo-
compares with the accepted and standardized disc assay s c r e e n i n g p r o c e d u r e . D a t a collected o n a n t i b i o t i c spiked milk samples i n d i c a t e t h a t t h e S p o t Test will d e t e c t m o s t m i l k samples c o n t a i n i n g penicillin, cephapirin, or cloxacillin at a m o u n t s sufficient to p r o d u c e a 1 6 - m m z o n e o n t h e disc assay. Based o n results o f t h e analysis o f variance a n d logistic regression, t h e test is m o r e sensitive t h a n t h e disc assay a n d f r e q u e n t l y d e t e c t s t h e s e antibiotics at amounts below those necessary to p r o d u c e a 1 6 - m m zone. C o n s i d e r i n g t h e p o t e n tial p u b l i c h e a l t h risks a n d f i n a n c i a l losses t h a t can o c c u r if o n e or m o r e silos of m i l k are c o n t a m i n a t e d w i t h a n t i b i o t i c - a d u l t e r a t e d milk, positive results b y t h e S p o t Test screening p r o c e d u r e will alert processors to suspect m i l k s h i p m e n t s , w h i c h m a y t h e n b e isolated a n d a n a l y z e d b y official testing p r o c e d u r e s .
~ 60] ~
REFERENCES
•
,oi
Method BIB Dise Test
Spot Test
0
6
9
12 15 24 CephoplHn (ng/rnl)
48
96
Figure 3. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of cephapirin and unspiked control samples.
*o0]
.oJ 60t
Method
°L
I Disc Test ~;~ Spot Test
6
37.5
50'.0
62'.5 75.0
100.0 200.0 400.0
Cloxocillln (ng/mt)
Figure 4. Comparison of the percent positive results by the Spot Test and Bacillus stearotbermopbilus disc assay screening procedures on milk samples spiked with known concentrations of cloxacillin and unspiked control samples. Journal of Dairy Science Vol. 69, No. 6, 1986
1 Association of Official Analytical Chemists. 1984. Official methods of analysis. 14 ed. Assoc. Offic. Anal. Chem., Washington, DC. 2 Bishop, J. R., and C. H. White. 1984. Antibiotic residue detection in milk a review. J. Food. Prot. 47:647. 3 Claypool, L. L. 1984. Concerns and problems of cheese industry for milk quality and procurement today and future. J. Dairy Sci. 67:2091. 4 Code of Federal Regulations. 1984. Title 21 Food and drugs, Part 540. Office Fed. Reg., Washington, DC. 5 Everson, T. C. 1984. Concerns and problems of processing and manufacturing in super plants. J. Dairy Sci. 67:2095. 6 Ginn, R. E., R. A. Case, V. S. Packard, and S. R. Tatini. 1982. Quantitative estimates of beta-lactam residues in raw milk around a reference standard: collaborative study. J. Assoc. Offic. Anal. Chem. 65 : 1407. 7 Marth, E. H., and B. E. Ellickson. 1959. Problems created by the presence of antibiotics in milk and milk products. J. Milk Food Technol. 22:266. 8 Messer, J. W., J. E. Leslie, G. A. Houghtby, J. T. Peeler, and J. E. Barnett. 1982. Bacillus stearothermopbilus disc assay for detection of inhibitors in milk: collaborative study. J. Assoc. Offic. Anal. Chem. 65:1208. 9 Reinbold, G. W., and M. S. Reddy. 1974. Sensitivity or resistance dairy starter and associated microorganisms to selected antibiotics, J. Milk Food Technol. 37:517. 10 Ryan, J. J. 1983. Antibiotic tests screen raw milk. Dairy Field 166:48. 11 Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods. 6th ed. Iowa State Univ. Press, Ames. 12 Statistical Package for the Social Sciences, Inc.
-
DETECTION OF ANTIBIOTICS IN COMMINGLED MILK
1983. SPSS-X user's guide. McGraw Hill, New York, NY. 13 University of California-Los Angeles, Department of Biomathematics. 1979. BMDP Statistical software. Univ. California Press. 14 Van Arsdel, P. P., Jr. 1983. Adverse drug reactions.
15 17
Page 1389 in Allergy: principles and practice, Vol. 2. 2nd ed. E. Middleton, Jr., C. E. Reed, and E. F. Ellis, ed. C. V. Mosby, St. Louis, MO. 15 Youden, W. J., and E. H. Strominger. 1975. Statistical manual of the AOAC. Association of Official Analytical Chemists, Arlington, VA.
Journal of Dairy Science Vol. 69, No. 6, 1986