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Persistence of Antibiotics in Milk from Cows Treated Late in the Dry Period1
OUR INDUSTRY TODAY Persistence o f A n t i b i o t i c s in M i l k f r o m C o w s T r e a t e d L a t e in t h e D r y Period 1 M. E. JOHNSON ~, J. H. MARTIN, R. J. BAKER, and J. G. PARSONS Dairy Science Department South Dakota State University Brookings 57006
ABSTRACT
Nineteen cows were treated via intramammary infusion at various times prior to calving with one of two dry cow antibiotic preparations (cloxacillin or penicillin). Milk samples from these cows after calving were analyzed for antibiotic residues by the Sarcina lutea cylinder plate method. Eight cows were treated with cloxacillin. Cloxacillin was detected in the milk of only two cows after calving and then for only one milking, so no problem should exist with cloxacillin if the manufacturer's recommendations are followed. Penicillin residues were not detected in milk samples taken from cows treated more than 9 days prior to calving with the dry cow penicillin preparation. However, seven of nine cows treated with penicillin up to 9 days prior to calving had detectable penicillin residues in the milk from treated quarters after calving. Only one cow had detectable penicillin residues longer than the manufacturer's recommended milk withholding time of 96 h, and this cow was treated only 3 days prior to parturition. Normal routine on a dairy farm and adherence to manufacturer's recommendations should result in no antibiotic residue problems in milk from cows treated during the dry period for mastitis control with either cloxacillin or penicillin. INTRODUCTION
More and more dairy cows are being treated during their dry period with intramammary Received April 20, 1977. *Published with the approval of the Director, South Dakota Agricultural Experiment Station, as Publication No. 1497 o f the Journal Series. 2Department of Food Science, North Carolina State University, Raleigh 27607. 1977 J Dairy Sci 60:1655-1661
infusion of antibiotic preparations to aid mastiffs prevention. The manufacturers of dry cow antibiotic preparations recommend that dry cow treatments not be used for a time prior to the expected calving date. Also, a specific time is recommended for withholding milk from the market supply after the cow calves. Supposedly both these precautions are suggested to insure that no antibiotics will appear in milk which is offered for human consumption. However, what happens if the dairy farmer or veterinarian miscalculates the calving date, or if the calf is born prematurely? Only a few reports have been published on the persistence of antibiotics in the dry udder. Pearson (4) infused procaine penicillin in an oil base into the dry udder. The concentration of antibiotic declined rapidly in the dry udder but persisted at low levels for up to 12 days. Uvarov (8) in a similar trial found low concentrations of penicillin for 21 days. Retention of penicillin in dry udder secretions showed considerable variation but appeared to be greatest in cows that had been dry the longest. Antibiotics were in the milk of a few cows after calving, but again retention was variable. Smith et al. (6) detected cloxacillin in dry udder secretions for 3 wk after treatment when the antibiotic was administered in an oil base at the beginning of the dry period. Penicillin in a quick release base persisted in infused quarters for only 4 days (7). Pugh et al. (5) infused all quarters of 150 cows with a single infusion of 1 g procaine penicillin G and .5 g dihydrostreptomycin in an oil base. Penicillin was detected in dry cow secretions for at least 3 wk, and dihydrostreptomycin for up to 5 wk. However, 4 days after calving, no antibiotics were detectable in the milk from treated cows. The objective of this study was to determine if antibiotics in dry cow treatments persisted longer than the manufacturers' recommended time for withholding milk from the market supply if the cows calved earlier than expected.
1655
1656
JOHNSON ET AL. MATERIALS AND METHODS
Dry Cow Treatment
Nineteen dry Holstein dairy cows from the South Dakota State University dairy herd were treated via intramammary infusion of two quarters with one of two commercial dry cow antibiotic preparations designated here as antibiotic preparations A and B. The cows were treated approximately 10 to 12 days prior to the expected parturition date. Each treated quarter was infused once with one 10-ml plastet of the antibiotic preparation. The two quarters not treated with an antibiotic preparation were each given a placebo of lO-ml distilled, sterile water. This was to determine if antibiotics "would cross over from treated to nontreated quarters. Each 10-ml disposable polyethylene plastet of antibiotic preparation A contained the equivalent of 500 mg of benzathine cloxacillin in 7.5 g of suitable base. Each lO-ml disposable plastet of antibiotic preparation B contained 1 × 106 units of procaine penicillin G and 1 g of dihydrostreptomycin sulfate in an extended action base consisting of 1% wt/vol hydrogenated peanut oil, 3% wt/vol aluminum monostearate, and peanut oil USP. Milk samples from all four quarters were collected in sterile bottles beginning with the first milking postcalving. Samples were collected from all successive milkings until antibiotic tests were negative for two consecutive milkings. All samples were tested within 48 h after being taken. They were kept in a refrigerator at 4 C until analyzed. Sample Testing
All milk samples from cows treated with a dry cow antibiotic preparation were tested for the presence of antibiotics by the Sarcina lutea cylinder plate method (3). The test organism used throughout the analysis was derived from Sarcina lutea (ATCC 9341) obtained from the Land O'Lakes Laboratory in Volga, South Dakota. The original culture was streaked on an agar slant of Antibiotic Medium No. 1 (Difco), and allowed to incubate for 24 h at 32 C. The growth was washed from the slant with sterile physiological saline (.85% NaC1) and transferred to a small Roux bottle containing 12 ml of solidified Antibiotic Medium No. 1. The Roux Journal of Dairy Science Vol. 60, No. 10
b o t d e was incubated for 24 h at 32 C. After incubation the growth was washed from the agar surface with 100 ml of sterile physiological saline. The entire suspension was poured into a sterile bottle and stored in a refrigerator for no longer than 2 wk. This stock culture suspension served as the inoculum for the next stock culture prepared, instead of the slant culture as described by Carter (3). New stock cultures were prepared every 10 days. The contents of test tubes containing 12 ml of sterilized Antibiotic Medium No. 1 were poured into 100 × 15 mm, glass, fiat-bottomed petri dishes. After the agar had solidified, the petri dishes (with covers off) were dried in a 39 C incubator for 50 min. The covers were replaced after drying. One hundred and fifty milliliters of sterilized Antibiotic Medium No. 4 were inoculated with enough stock culture to obtain a concentration in the agar of about 7.5 x l 0 s organisms per ml. Four milliliters of the inoculated agar then were added to each plate, making sure that the agar was distributed evenly over the base layer of Antibiotic Medium No. 1. After the agar had solidified, the petri dishes (with covers off) were dried in a 39 C incubator for 25 min. The covers were replaced after drying and the plates were used immediately. Five stainless steel cylinders were dropped onto the agar surface of each plate from a height of about 4 mm with tweezers. The cylinders were not flamed before dropping them into the plate. The OD of the cylinders was 8 mm; the ID was 6 mm; and the length was 10 mm. Only five cylinders were dropped on a plate to avoid overlapping zones. Pasteur pipettes were used to fill three cylinders with test sample. The other two cylinders on opposite sides of the plate were filled with a standard solution of penicillin containing .05 /a/ml. Plates were incubated with covers off in a 32 C incubator for 16 to 18 h. The atmosphere in the incubator was kept moist with a sponge set in a pan of water to keep the plates from drying excessively. After incubation the diameters of the zones of inhibition were measured to the nearest .5 mm with a divider. To determine if the zones of inhibition resulted from penicillin, penicillinase-impregnated discs were placed near the cylinder with the test sample (1). If the zone of inhibition around the test sample was decreased near the penicillinase-impregnated disc, penicillin was
T A B L E 1. Cloxacillin c o n c e n t r a t i o n s in m i l k f r o m c o w s g i v e n 5 0 0 m g o f b e n z a t h i n e cloxacillin at v a r i o u s t i m e s p r i o r t o calving. Interval Cow number
to c a l v i n g after treatment
Treated quarters a
H o u r s a f t e r c a l v i n gc 0
12
24
83 1.65
0 0
0 0
(days) 3238
8
3307
8
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0
.165
0 0
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3031
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3030
7
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3167
18
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3276
9
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40
60
0 0
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72
(gg/ml) b LF RR
3021
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36
0
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a N o d e t e c t a b l e a n t i b i o t i c residues w e r e p r e s e n t in s a m p l e s f r o m n o n t r e a t e d q u a r t e r s , L F = l e f t f r o n t , L R = left rear, R F = r i g h t f r o n t , R R = r i g h t r e a r . b z e r o i n d i c a t e s n o d e t e c t a b l e a n t i b i o t i c r e s i d u e , (. , .) = s a m p l e n o t c o l l e c t e d . C
M a n u f a c t u r e r s r e c o m m e n d e d w i t h h o l d i n g t i m e f o r m i l k is 72 h a f t e r calving.
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JOHNSON ET AL.
present. If no change in the zone of inhibition around the test sample was observed, the inhibitory substance was not penicillin.
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Preparation of Standard Curves
Sodium penicillin G was used as the standard for penicillin. Sodium cloxacillin was used as the standard for benzathine cloxacillin. A small quantity of the antibiotic was weighed and dissolved in 100 ml of 1% phosphate buffer, pH 6.0 + .1 (8.0 g monobasic potassium phosphate, 2.0 g dibasic potassium phosphate diluted to 1 liter with distilled water and adjusted to pH with 18 N phosphoric acid or 10 N potassium hydroxide) (2). A portion of the penicillin stock solution was diluted with 1% phosphate buffer to give a concentration of 10 gi/ml of penicillin. Further dilutions were made in milk containing 2% milkfat. A portion of the cloxacillin stock solution was diluted with heat treated milk containing 2% milk fat to give a concentration of 10 gig per ml. The 2% milk for this dilution and for all other dilutions was steamed for 2 h to caramelize the lactose and give the standard solutions a darker color than the samples that were tested. The darker color of the standard solutions made it easier to identify the cylinders with the reference concentration in the cylinder plate test. The standard concentrations of penicillin to establish the standard response line were .2, .1, .05, .025, and .0125 gi/ml (3). The standard concentrations of cloxacillin to establish the standard response line were 8.0, 4.0, 2.0, 1.0, .5, .25, and .125 gig/ml. A regression equation was calculated for each standard curve. These equations were used to determine the concentration of antibiotic in the unknown samples.
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RESULTS A N D DISCUSSION
Data from the dry cow experiments with cloxacillin as the antibiotic are in Table 1. Cloxacillin rarely was detected in milk from the eight cows treated by intramammary infusion of 500 mg of benzathine cloxacillin during the dry period. After the cows had calved, cloxacilfin was detected in the milk of only two cows and then for only one milking. Detectable penicillin residues were in the
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a N o d e t e c t a b l e a n t i b i o t i c residues w e r e p r e s e n t in s a m p l e s f r o m n o n t r e a t e d q u a r t e r s e x c e p t w h e r e i n d i c a t e d , L F = left f r o n t , L R = left rear, R F = r i g h t f r o n t , R R = right rear. bQuarters were not treated. CRight rear q u a r t e r t r e a t e d for an i n f e c t i o n a f t e r 24 h. d z e r o indicates no detectable antibiotic residue. ( . . . ) = sample n o t collected.
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1660
JOHNSON ET AL.
milk from 7 of 11 lactating cows that were treated by intramammary infusion of 1 x 106 units of procaine penicillin G during the dry period (Table 2). Penicillin residues were not detected in milk samples from cows treated more than 9 days prior to calving. With two exceptions, cows that were treated with penicillin less than 9 days prior to calving had detectable penicillin residues in the milk from treated quarters after calving. Penicillin crossed over from treated to nontreated quarters only in one cow (cow 3315); however, this cow calved 24 h after treatment. Due to the extremely short interval between treatment and calving the data from cow 3315 probably should be similar to results after treatment of a lactating cow. However, it does emphasize the importance of avoiding treatment of dry cows just prior to parturition to insure that no antibiotic is carried over into the milk. Ziv et al. (10) stated that in the dry udder the rate of disappearance of seven different antibiotic preparations was exponentially linear. Our results show that in the lactating cow, antibiotic residues were generally not excreted exponentially but were excreted slowly until complete disappearance. The minimum concentration of cloxacillin detectable by the Sarcina lutea cylinder plate test in this study was .25/~g of cloxacillin per ml of milk. Smith et al. (6) and Ziv et al. (11) detected cloxacillin residues in dry udder secretions of cows for 3 wk following the infusion of 500 mg of benzathine cloxacillin. Both groups (6, 11) used the Sarcina lutea cylinder plate method, which was used also in this study. In the current research, cloxacillin was detected in the milk of only two cows after calving, and then for only one milking, even though the time from treatment to calving was less than 3 wk. These findings tend to contradict the findings of Smith et al. (6) and Ziv et al. (11). However, they assayed dry udder secretions whereas in this work only secretions from lactating udders were assayed. The volume of milk secreted from the lactating cow is extremely large when compared to the 6 ml secretion taken from each quarter of the dry udder as in their work. Ziv et al. (11) observed that when penicillin G and cloxacillin were infused into the udders of cows producing 2 to 3 times more milk at drying-off than other cows, antibiotics in the secretions of the high Journal of Dairy Science Vol. 60, No. 10
producers were at times 10 to 100 fold lower than in the secretions of cows with lower milk yield. In this study, dilution of the cloxacillin and penicillin in the lactating cow may have been the reason for no detectable antibiotic residues in the milk samples. The antibiotic concentrations listed in Tables 1 and 2 are approximate and must not be taken as absolute values. Colostrum is viscous compared to the milk containing 2% fat which was used as a diluent for the standard solutions. The colostrum samples did not diffuse into the agar from the cylinders as completely as the 2% milk in the standards. The cylinder plate method of assay depends on the diffusion of antibiotics into the agar. Thus, the cylinder plate test is not suited well for viscous products such as colostrum. Ziv and Rasmussen (9) indicated that the repeatability o f results in the assay of cream was small, and in recovery studies low and erratic values were common. Therefore, the antibiotic concentration observed may be lower than the actual concentration in the colostrum samples. With the exception of cow 3196, all milk samples from cows that were infused with a dry cow antibiotic preparation during the dry period showed no detectable antibiotic residue beyond the manufacturer's recommended period for withholding milk from the market supply. The manufacturer of the penicillin preparation recommends that the product not be used during the 6 wk prior to the expected date of parturition to ensure that the antibiotic would not be excreted longer than a recommended withholding time of 96 h after calving. The manufacturer of the cloxacillin preparation recommends that the product not be used during the 4 wk prior to the expected date of calving to ensure that cloxaciilin would not be excreted longer than the recommended withholding time of 72 h. CONCLUSIONS
It should be highly recommended to dairymen who use dry cow treatments that they dump the milk of cows treated during the dry period for the specified number of milkings to be sure of complete removal of antibiotics from the milk. It also should be recommended that cows that calve within 10 days after infusion of a dry cow antibiotic preparation have their milk
OUR INDUSTRY TODAY c h e c k e d for a n t i b i o t i c residues b e f o r e it e n t e r s t h e m a r k e t supply. ACKNOWLEDGMENT
The a u t h o r s express a p p r e c i a t i o n to F e n t o n L u d e n s , S o u t h D a k o t a State University Dairy R e s e a r c h U n i t Manager, f o r a d m i n i s t e r i n g t h e a n t i b i o t i c p r e p a r a t i o n s and for assistance in collecting t h e milk samples. REFERENCES
1 American Public Health Association. 1972. Standard methods for the examination of dairy products. 13th ed. Amer. Public Health Ass., Washington, DC. 2 Arret, B., D. Johnson, and A. Kirshbaum. 1971. Outlines of details for microbiological assays of antibiotics: second revision. J. Pharma. Sci. 60:1689/ 3 Carter, G. G. 1974. Detection of penicillin in dry powdered milk by the Sarcina lutea cylinder plate method. National Center for Antibiotic and Insulin Analysis, Food and Drug Administration, Washington, DC. 4 Pearson, J. K. J. 1951. Further experiments in the use of penicillin in the prevention of C. pyogenes infection of the non-lactating bovine udder. Vet.
1661
Rec. 68:215. 5 Pugh, K. E., A. M. Harris, M. J. Marshall, and J. M. Evans. 1973. Evaluation of a long-acting intramammary preparation containing high doses of penicillin and streptomycin for use in dry cows. Vet. Rec. 93:212. 6 Smith, A., F. K. Neave, F. H. Dodd, A. Jones, and D. N. Gore. 1967. The persistence of cloxacillin in the mammary gland when infused immediately after the last milking of lactation. J. Dairy Res. 34:47. 7 Smith, A., F. K. Neave, and A. Jones. 1967. The persistence of penicillin G in the mammary gland when infused immediately after the last milking of lactation. J. Dairy Res. 34: 59. 8 Uvarov, O. 1960. The concentration of some antibiotics in the milk after intramammary infusion. Vet. Rec. 72:1228. 9 Ziv, G., and F. Rasmussen. 1975. Distribution of labeled antibiotics in different components of milk following intramammary and intramuscular administrations. J. Dairy Sci. 58:938. 10 Ziv, G., A. Saran-Rosenzuaig, and E. Gluckmann. 1973. Kinetic considerations of antibiotic persistence in the udders of dry cows. Zbl. Vet. Med. Bull. 20:425. 11 Ziv, G. A., Saran-Rosenzuaig, and R. Risenberg. 1973. Retention of antibiotics in dry-udder secretions after the infusion of several dry cow antibiotic products. Zhl. Vet. Meal. Bull. 20:415.
Journal of Dairy Science Vol. 60, No. 10
ABSTRACT
Nineteen cows were treated via intramammary infusion at various times prior to calving with one of two dry cow antibiotic preparations (cloxacillin or penicillin). Milk samples from these cows after calving were analyzed for antibiotic residues by the Sarcina lutea cylinder plate method. Eight cows were treated with cloxacillin. Cloxacillin was detected in the milk of only two cows after calving and then for only one milking, so no problem should exist with cloxacillin if the manufacturer's recommendations are followed. Penicillin residues were not detected in milk samples taken from cows treated more than 9 days prior to calving with the dry cow penicillin preparation. However, seven of nine cows treated with penicillin up to 9 days prior to calving had detectable penicillin residues in the milk from treated quarters after calving. Only one cow had detectable penicillin residues longer than the manufacturer's recommended milk withholding time of 96 h, and this cow was treated only 3 days prior to parturition. Normal routine on a dairy farm and adherence to manufacturer's recommendations should result in no antibiotic residue problems in milk from cows treated during the dry period for mastitis control with either cloxacillin or penicillin. INTRODUCTION
More and more dairy cows are being treated during their dry period with intramammary Received April 20, 1977. *Published with the approval of the Director, South Dakota Agricultural Experiment Station, as Publication No. 1497 o f the Journal Series. 2Department of Food Science, North Carolina State University, Raleigh 27607. 1977 J Dairy Sci 60:1655-1661
infusion of antibiotic preparations to aid mastiffs prevention. The manufacturers of dry cow antibiotic preparations recommend that dry cow treatments not be used for a time prior to the expected calving date. Also, a specific time is recommended for withholding milk from the market supply after the cow calves. Supposedly both these precautions are suggested to insure that no antibiotics will appear in milk which is offered for human consumption. However, what happens if the dairy farmer or veterinarian miscalculates the calving date, or if the calf is born prematurely? Only a few reports have been published on the persistence of antibiotics in the dry udder. Pearson (4) infused procaine penicillin in an oil base into the dry udder. The concentration of antibiotic declined rapidly in the dry udder but persisted at low levels for up to 12 days. Uvarov (8) in a similar trial found low concentrations of penicillin for 21 days. Retention of penicillin in dry udder secretions showed considerable variation but appeared to be greatest in cows that had been dry the longest. Antibiotics were in the milk of a few cows after calving, but again retention was variable. Smith et al. (6) detected cloxacillin in dry udder secretions for 3 wk after treatment when the antibiotic was administered in an oil base at the beginning of the dry period. Penicillin in a quick release base persisted in infused quarters for only 4 days (7). Pugh et al. (5) infused all quarters of 150 cows with a single infusion of 1 g procaine penicillin G and .5 g dihydrostreptomycin in an oil base. Penicillin was detected in dry cow secretions for at least 3 wk, and dihydrostreptomycin for up to 5 wk. However, 4 days after calving, no antibiotics were detectable in the milk from treated cows. The objective of this study was to determine if antibiotics in dry cow treatments persisted longer than the manufacturers' recommended time for withholding milk from the market supply if the cows calved earlier than expected.
1655
1656
JOHNSON ET AL. MATERIALS AND METHODS
Dry Cow Treatment
Nineteen dry Holstein dairy cows from the South Dakota State University dairy herd were treated via intramammary infusion of two quarters with one of two commercial dry cow antibiotic preparations designated here as antibiotic preparations A and B. The cows were treated approximately 10 to 12 days prior to the expected parturition date. Each treated quarter was infused once with one 10-ml plastet of the antibiotic preparation. The two quarters not treated with an antibiotic preparation were each given a placebo of lO-ml distilled, sterile water. This was to determine if antibiotics "would cross over from treated to nontreated quarters. Each 10-ml disposable polyethylene plastet of antibiotic preparation A contained the equivalent of 500 mg of benzathine cloxacillin in 7.5 g of suitable base. Each lO-ml disposable plastet of antibiotic preparation B contained 1 × 106 units of procaine penicillin G and 1 g of dihydrostreptomycin sulfate in an extended action base consisting of 1% wt/vol hydrogenated peanut oil, 3% wt/vol aluminum monostearate, and peanut oil USP. Milk samples from all four quarters were collected in sterile bottles beginning with the first milking postcalving. Samples were collected from all successive milkings until antibiotic tests were negative for two consecutive milkings. All samples were tested within 48 h after being taken. They were kept in a refrigerator at 4 C until analyzed. Sample Testing
All milk samples from cows treated with a dry cow antibiotic preparation were tested for the presence of antibiotics by the Sarcina lutea cylinder plate method (3). The test organism used throughout the analysis was derived from Sarcina lutea (ATCC 9341) obtained from the Land O'Lakes Laboratory in Volga, South Dakota. The original culture was streaked on an agar slant of Antibiotic Medium No. 1 (Difco), and allowed to incubate for 24 h at 32 C. The growth was washed from the slant with sterile physiological saline (.85% NaC1) and transferred to a small Roux bottle containing 12 ml of solidified Antibiotic Medium No. 1. The Roux Journal of Dairy Science Vol. 60, No. 10
b o t d e was incubated for 24 h at 32 C. After incubation the growth was washed from the agar surface with 100 ml of sterile physiological saline. The entire suspension was poured into a sterile bottle and stored in a refrigerator for no longer than 2 wk. This stock culture suspension served as the inoculum for the next stock culture prepared, instead of the slant culture as described by Carter (3). New stock cultures were prepared every 10 days. The contents of test tubes containing 12 ml of sterilized Antibiotic Medium No. 1 were poured into 100 × 15 mm, glass, fiat-bottomed petri dishes. After the agar had solidified, the petri dishes (with covers off) were dried in a 39 C incubator for 50 min. The covers were replaced after drying. One hundred and fifty milliliters of sterilized Antibiotic Medium No. 4 were inoculated with enough stock culture to obtain a concentration in the agar of about 7.5 x l 0 s organisms per ml. Four milliliters of the inoculated agar then were added to each plate, making sure that the agar was distributed evenly over the base layer of Antibiotic Medium No. 1. After the agar had solidified, the petri dishes (with covers off) were dried in a 39 C incubator for 25 min. The covers were replaced after drying and the plates were used immediately. Five stainless steel cylinders were dropped onto the agar surface of each plate from a height of about 4 mm with tweezers. The cylinders were not flamed before dropping them into the plate. The OD of the cylinders was 8 mm; the ID was 6 mm; and the length was 10 mm. Only five cylinders were dropped on a plate to avoid overlapping zones. Pasteur pipettes were used to fill three cylinders with test sample. The other two cylinders on opposite sides of the plate were filled with a standard solution of penicillin containing .05 /a/ml. Plates were incubated with covers off in a 32 C incubator for 16 to 18 h. The atmosphere in the incubator was kept moist with a sponge set in a pan of water to keep the plates from drying excessively. After incubation the diameters of the zones of inhibition were measured to the nearest .5 mm with a divider. To determine if the zones of inhibition resulted from penicillin, penicillinase-impregnated discs were placed near the cylinder with the test sample (1). If the zone of inhibition around the test sample was decreased near the penicillinase-impregnated disc, penicillin was
T A B L E 1. Cloxacillin c o n c e n t r a t i o n s in m i l k f r o m c o w s g i v e n 5 0 0 m g o f b e n z a t h i n e cloxacillin at v a r i o u s t i m e s p r i o r t o calving. Interval Cow number
to c a l v i n g after treatment
Treated quarters a
H o u r s a f t e r c a l v i n gc 0
12
24
83 1.65
0 0
0 0
(days) 3238
8
3307
8
<
. .
. .
.
.
.
LF LR
0
.165
0 0
0 0
0 0
14
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... ...
0 0
0 0
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11
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3031
10
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.
.
.
.
.
.
.
.
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0
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3276
9
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... ...
0 0
0 0
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u~
40
60
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72
(gg/ml) b LF RR
3021
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36
0
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0 0
0 0
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a N o d e t e c t a b l e a n t i b i o t i c residues w e r e p r e s e n t in s a m p l e s f r o m n o n t r e a t e d q u a r t e r s , L F = l e f t f r o n t , L R = left rear, R F = r i g h t f r o n t , R R = r i g h t r e a r . b z e r o i n d i c a t e s n o d e t e c t a b l e a n t i b i o t i c r e s i d u e , (. , .) = s a m p l e n o t c o l l e c t e d . C
M a n u f a c t u r e r s r e c o m m e n d e d w i t h h o l d i n g t i m e f o r m i l k is 72 h a f t e r calving.
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1658
JOHNSON ET AL.
present. If no change in the zone of inhibition around the test sample was observed, the inhibitory substance was not penicillin.
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Preparation of Standard Curves
Sodium penicillin G was used as the standard for penicillin. Sodium cloxacillin was used as the standard for benzathine cloxacillin. A small quantity of the antibiotic was weighed and dissolved in 100 ml of 1% phosphate buffer, pH 6.0 + .1 (8.0 g monobasic potassium phosphate, 2.0 g dibasic potassium phosphate diluted to 1 liter with distilled water and adjusted to pH with 18 N phosphoric acid or 10 N potassium hydroxide) (2). A portion of the penicillin stock solution was diluted with 1% phosphate buffer to give a concentration of 10 gi/ml of penicillin. Further dilutions were made in milk containing 2% milkfat. A portion of the cloxacillin stock solution was diluted with heat treated milk containing 2% milk fat to give a concentration of 10 gig per ml. The 2% milk for this dilution and for all other dilutions was steamed for 2 h to caramelize the lactose and give the standard solutions a darker color than the samples that were tested. The darker color of the standard solutions made it easier to identify the cylinders with the reference concentration in the cylinder plate test. The standard concentrations of penicillin to establish the standard response line were .2, .1, .05, .025, and .0125 gi/ml (3). The standard concentrations of cloxacillin to establish the standard response line were 8.0, 4.0, 2.0, 1.0, .5, .25, and .125 gig/ml. A regression equation was calculated for each standard curve. These equations were used to determine the concentration of antibiotic in the unknown samples.
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Journal of Dairy Science Vol. 60, No. 10
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RESULTS A N D DISCUSSION
Data from the dry cow experiments with cloxacillin as the antibiotic are in Table 1. Cloxacillin rarely was detected in milk from the eight cows treated by intramammary infusion of 500 mg of benzathine cloxacillin during the dry period. After the cows had calved, cloxacilfin was detected in the milk of only two cows and then for only one milking. Detectable penicillin residues were in the
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a N o d e t e c t a b l e a n t i b i o t i c residues w e r e p r e s e n t in s a m p l e s f r o m n o n t r e a t e d q u a r t e r s e x c e p t w h e r e i n d i c a t e d , L F = left f r o n t , L R = left rear, R F = r i g h t f r o n t , R R = right rear. bQuarters were not treated. CRight rear q u a r t e r t r e a t e d for an i n f e c t i o n a f t e r 24 h. d z e r o indicates no detectable antibiotic residue. ( . . . ) = sample n o t collected.
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1660
JOHNSON ET AL.
milk from 7 of 11 lactating cows that were treated by intramammary infusion of 1 x 106 units of procaine penicillin G during the dry period (Table 2). Penicillin residues were not detected in milk samples from cows treated more than 9 days prior to calving. With two exceptions, cows that were treated with penicillin less than 9 days prior to calving had detectable penicillin residues in the milk from treated quarters after calving. Penicillin crossed over from treated to nontreated quarters only in one cow (cow 3315); however, this cow calved 24 h after treatment. Due to the extremely short interval between treatment and calving the data from cow 3315 probably should be similar to results after treatment of a lactating cow. However, it does emphasize the importance of avoiding treatment of dry cows just prior to parturition to insure that no antibiotic is carried over into the milk. Ziv et al. (10) stated that in the dry udder the rate of disappearance of seven different antibiotic preparations was exponentially linear. Our results show that in the lactating cow, antibiotic residues were generally not excreted exponentially but were excreted slowly until complete disappearance. The minimum concentration of cloxacillin detectable by the Sarcina lutea cylinder plate test in this study was .25/~g of cloxacillin per ml of milk. Smith et al. (6) and Ziv et al. (11) detected cloxacillin residues in dry udder secretions of cows for 3 wk following the infusion of 500 mg of benzathine cloxacillin. Both groups (6, 11) used the Sarcina lutea cylinder plate method, which was used also in this study. In the current research, cloxacillin was detected in the milk of only two cows after calving, and then for only one milking, even though the time from treatment to calving was less than 3 wk. These findings tend to contradict the findings of Smith et al. (6) and Ziv et al. (11). However, they assayed dry udder secretions whereas in this work only secretions from lactating udders were assayed. The volume of milk secreted from the lactating cow is extremely large when compared to the 6 ml secretion taken from each quarter of the dry udder as in their work. Ziv et al. (11) observed that when penicillin G and cloxacillin were infused into the udders of cows producing 2 to 3 times more milk at drying-off than other cows, antibiotics in the secretions of the high Journal of Dairy Science Vol. 60, No. 10
producers were at times 10 to 100 fold lower than in the secretions of cows with lower milk yield. In this study, dilution of the cloxacillin and penicillin in the lactating cow may have been the reason for no detectable antibiotic residues in the milk samples. The antibiotic concentrations listed in Tables 1 and 2 are approximate and must not be taken as absolute values. Colostrum is viscous compared to the milk containing 2% fat which was used as a diluent for the standard solutions. The colostrum samples did not diffuse into the agar from the cylinders as completely as the 2% milk in the standards. The cylinder plate method of assay depends on the diffusion of antibiotics into the agar. Thus, the cylinder plate test is not suited well for viscous products such as colostrum. Ziv and Rasmussen (9) indicated that the repeatability o f results in the assay of cream was small, and in recovery studies low and erratic values were common. Therefore, the antibiotic concentration observed may be lower than the actual concentration in the colostrum samples. With the exception of cow 3196, all milk samples from cows that were infused with a dry cow antibiotic preparation during the dry period showed no detectable antibiotic residue beyond the manufacturer's recommended period for withholding milk from the market supply. The manufacturer of the penicillin preparation recommends that the product not be used during the 6 wk prior to the expected date of parturition to ensure that the antibiotic would not be excreted longer than a recommended withholding time of 96 h after calving. The manufacturer of the cloxacillin preparation recommends that the product not be used during the 4 wk prior to the expected date of calving to ensure that cloxaciilin would not be excreted longer than the recommended withholding time of 72 h. CONCLUSIONS
It should be highly recommended to dairymen who use dry cow treatments that they dump the milk of cows treated during the dry period for the specified number of milkings to be sure of complete removal of antibiotics from the milk. It also should be recommended that cows that calve within 10 days after infusion of a dry cow antibiotic preparation have their milk
OUR INDUSTRY TODAY c h e c k e d for a n t i b i o t i c residues b e f o r e it e n t e r s t h e m a r k e t supply. ACKNOWLEDGMENT
The a u t h o r s express a p p r e c i a t i o n to F e n t o n L u d e n s , S o u t h D a k o t a State University Dairy R e s e a r c h U n i t Manager, f o r a d m i n i s t e r i n g t h e a n t i b i o t i c p r e p a r a t i o n s and for assistance in collecting t h e milk samples. REFERENCES
1 American Public Health Association. 1972. Standard methods for the examination of dairy products. 13th ed. Amer. Public Health Ass., Washington, DC. 2 Arret, B., D. Johnson, and A. Kirshbaum. 1971. Outlines of details for microbiological assays of antibiotics: second revision. J. Pharma. Sci. 60:1689/ 3 Carter, G. G. 1974. Detection of penicillin in dry powdered milk by the Sarcina lutea cylinder plate method. National Center for Antibiotic and Insulin Analysis, Food and Drug Administration, Washington, DC. 4 Pearson, J. K. J. 1951. Further experiments in the use of penicillin in the prevention of C. pyogenes infection of the non-lactating bovine udder. Vet.
1661
Rec. 68:215. 5 Pugh, K. E., A. M. Harris, M. J. Marshall, and J. M. Evans. 1973. Evaluation of a long-acting intramammary preparation containing high doses of penicillin and streptomycin for use in dry cows. Vet. Rec. 93:212. 6 Smith, A., F. K. Neave, F. H. Dodd, A. Jones, and D. N. Gore. 1967. The persistence of cloxacillin in the mammary gland when infused immediately after the last milking of lactation. J. Dairy Res. 34:47. 7 Smith, A., F. K. Neave, and A. Jones. 1967. The persistence of penicillin G in the mammary gland when infused immediately after the last milking of lactation. J. Dairy Res. 34: 59. 8 Uvarov, O. 1960. The concentration of some antibiotics in the milk after intramammary infusion. Vet. Rec. 72:1228. 9 Ziv, G., and F. Rasmussen. 1975. Distribution of labeled antibiotics in different components of milk following intramammary and intramuscular administrations. J. Dairy Sci. 58:938. 10 Ziv, G., A. Saran-Rosenzuaig, and E. Gluckmann. 1973. Kinetic considerations of antibiotic persistence in the udders of dry cows. Zbl. Vet. Med. Bull. 20:425. 11 Ziv, G. A., Saran-Rosenzuaig, and R. Risenberg. 1973. Retention of antibiotics in dry-udder secretions after the infusion of several dry cow antibiotic products. Zhl. Vet. Meal. Bull. 20:415.
Journal of Dairy Science Vol. 60, No. 10