doses of procaine penicillin in lactating dairy cows - Europe PMC

!

Penicillin concentrations in serum, milk, and urine following intramuscular and subcutaneous administration of increasing doses of procaine penicillin in lactating dairy cows Pascal Dubreuil, Josee Daigneault, Yvon Couture, Patrick Guay, D. Landry

Abstract Eight healthy, non-pregnant, crossbred Holstein dairy cows (557-682 kg) within their first 3 months of lactation (13-21.5 kg of milk/day) were used. Cows were kept in tie stalls for the whole experiment. The 8 cows were randomly assigned to 2 (IM and SC) 4 x 4 balanced Latin square design experiments. Doses of procaine penicillin G (PPG) (300 000 IU/mL) in each square were 7000, 14 000, 21 000 and 28 000 IU/kg and were injected IM or SC once daily for 5 consecutive days. Volumes of PPG per site of injection never exceeded 20 mL. Blood was collected to determine the Cmax, Tmax, and AUC; urine and milk were also taken to measure the persistence of PPG in these fluids. Results show that serum Cmaxand Tmax were only slightly affected by increasing the doses or the route of administration, whereas the AUC was linearly increased in relation to the dose injected in both modes of injection. In the urine, Cmax varied from 160 to 388 IU/mL and Tmax from 72-120 h during 5 consecutive days of PPG injection. A dose effect in Cmax was observed only for the IM route of administration and no variation (P > 0.05) was found between the IM and SC routes. Milk Cmax concentrations were only increased by the dose regimen in the IM group. At doses of 21 000 and 28 000 IU/kg, the IM group had a higher (P > 0.05) Cmax when compared with the SC groups. Milk PPG residues were not detectable over 96 h following the last IM injection, independently of the dose injected. However milk PPG residues were detected for up to 132 h following the last SC injection. These results show that when PPG is injected IM once daily in volumes not exceeding 20 mL/site at doses as high as 28 000 IU/kg, the withdrawal period should be at least 96 h. Therefore, in the present model, there was no advantage to inject PPG by SC route to improve PPG kinetic parameters as the AUC, Cmax' or Tmax'

Resume Huit vaches laitieres non gravides de race Holstein croise'e (557-682 kg) a' l'inte'rieur de la pe'riode de leurs trois premiers mois de lactation (13-21,5 kg de lait/jour) furent gardees attachees durant toute la duree de l'experimentation. Les 8 vaches furent reparties au hasard a' l'un des deux modes d'administration (IM ou SC) selon un protocole experimental en carre' latin 4 x 4. Les doses de pe'nicilline G procaine (PPG) (300 000 UI/mL) administre'es a' l'inte'rieur de chacun des carre's e'taient 7000, 14 000, 21 000 et 28 000 UIIkg et furent administre'es IM ou SC SID durant 5 jours consecutifs. Le volume de PPG injecte n'exc6da jamais 20 mL par site. Du sang fut preleve afin de determiner les valeurs de Cmax de Tmax et la surface sous la courbe (AUC); de l'urine et du lait furent egalement preleves afin de mesurer la persistance de la PPG dans ces liquides. Les resultats montrent que pour le serum les valeurs de Cmax et de Tmax n'6taient que legerement influencees par une augmentation de la dose et la voie d'administration, alors qu'une augmentation line'aire de l'AUC en fonction de la dose injecte'e et de la voie d'administration fut observe'e. Dans l'urine, le C.. varia entre 160 et 388 UI/mL et le Tmax entre 72 et 120 h durant les 5 jours consecutifs d'injection de PPG. Un effet de dose sur les valeurs de Cmaxfut note'seulement pour la voie d'administration IM et aucune variation (P > 0,05) ne fut observee entre les voies d'administration IM et SC. Les valeurs de Ca,,x dans le lait ne furent que legerement augmentees en fonction de la dose administree par voie IM. A des doses de 21 000 et 28 000 UIIkg, les animaux ayant requ l'antibiotique par voie IM avaient une valeur de Cmax plus e'leve'e (P > 0,05) que ceux ayant requ l'antibiotique par voie SC. Les re'sidus de PPG dans le lait e'taient non d6tectables apres 96 h suivant la derniere injection IM, inde'pendamment de la dose administre'e. Toutefois, des re'sidus de PPG dans le lait furent d6tectes jusqu'a 132 heures suivant la derniere injection SC. Ces resultats demontrent que lorsque la PPG est injectee par voie IM SID dans des volumes ne de'passant pas 20 mL par site a' des doses aussi 6leve'es que 28 000 UI/kg, la pe'riode de retrait devrait etre d'au moins 96 h. Ainsi, dans le modele pre'sente', il n'y avait aucun avantage a injecter de la PPG par voie SC pour ameliorer les parame'tres de la cine'tique de la PPG, tels que l'AUC et les valeurs de Cmax et Tmax' (Traduit par docteur Serge Messier)

Faculte de m6decine veterinaire, Universite de Montreal, C.P. 5000, St-Hyacinthe, Quebec J2S 7C6 (Dubreuil, Couture, Guay); Pfizer Canada, Dorval, Quebec (Daigneault); Bureau of Veterinary Drugs, Health Canada, Ottawa, Ontario (Landry). Address correpondence and reprint requests to Dr. Pascal Dubreuil, telephone: 450-778-8100, fax: 450-778-8102, e-mail: [email protected] Received October 5, 2000. Accepted June 8, 2001.

2001;65:173-180

The Canadian Journal of Veterinary Research

173

Table 1. Pharmacokinetics serum parameters following the first and fifth IM or SC injections of PPG at 4 different doses in 4 lactating cows per route of administration

Contrastb Dose (lu/kg BW)a 14 000 21 000 28 000 3.42 2.81 1.58 3.09 3.56 2.33 7.13 4.38 3.50 2.63 3.75 4.63 23.3 36.5 46.1 29.0 47.0 39.3 2.47 3.52 4.88 4.86 3.45 4.13 2.13 2.00 2.75 4.13 3.25 3.75 44.7 56.8 31.8 58.6 36.4 49.6 NS - not significant (P > 0.05)

7000 Route lM 0.98 SC 1.29 IM 3.25 (h) Tmax SC 5.63 12.0 AUC (IUh/mL) IM SC 13.8 IM 1.32 5 Cmax (IU/mL) SC 1.53 IM 2.00 Tmax (h) 2.88 SC 14.5 AUC (llUh/mL) IM 17.5 SC IM intramuscular; SC - subcutaneous; a Values are least squares means for 4 cows bP value for the contrast between doses Day 1

Parameter C,ax (IU/mL)

In 1928, Alexander Fleming made a serendipitous discovery while examining Staphylococcus variants. In fact, he discovered penicillin, the first antibiotic of human history. However, it took more than a decade before the first human studies concerning penicillin were conducted on Staphylococcus infections. After being used as a therapeutic agent for many years in human and veterinary medicine (1), penicillin has been added in animal feed as a growth promoter. Because of its low toxicity and cost, and the increasing resistance of microorganisms to penicillin over the years, users started to increase the dosage. Such use of penicillin has contributed to residues in food, which presents possible health hazards to consumers, especially in sensitized individuals, where it could cause allergic reactions (2). Until the end of 1997, in Canada, procaine penicillin G (PPG) was approved for use in cattle at a dosage of 6600 to 7500 IU/kg of body weight (BW) by intramuscular (IM) injection once daily. Since then, the Bureau of Veterinary Drugs of Health Canada, the Canadian regulatory agency for veterinary drugs, recommended and allowed to increase the dosage to 21 000 IU/kg intramuscular (IM) once a day. However, in practice, the dosages used are sometimes higher and/or are injected twice daily for conditions not necessarily approved by the regulatory agencies (ie after surgical procedures). In textbooks of veterinary medicine, the suggested dosages of penicillin may be as high as 44 000 IU/kg twice daily for urinary tract

infections (3,4). There is very little information on the pharmacokinetics (5) and milk residues of PPG when administered to dairy cattle by IM or subcutaneous (SC) routes at doses higher than 7500 IU/kg (6). In fact, among the few studies conducted on PPG, the majority were performed in feedlot steers using PPG alone (7,8), in combination with other antibiotics (dihydrostreptomycin) (9) or with other forms of penicillin (benzathine) (10). 174


SEM 0.27 1.14 2.7

0.15

1.46 2.2

7000 vs 14 000 NS NS NS NS 0.0006 0.02 0.0014 0.0001 NS NS 0.0012 0.001

14 000 vs 21 000 0.007 NS NS NS 0.0003 NS 0.0021 0.02 NS NS 0.005 0.005

21 000 vs 28 000 NS NS NS NS 0.0013 NS 0.0005 0.01 NS NS 0.007 0.03

The administration route is an important factor which may affect the pharmacokinetics of an antibiotic (11). In one report, 2 dairy cows that were injected SC with 35 and 33 mL of PPG (300 000 IU/mL), ql2h, in one site for 4 and 6 consecutive days, respectively, had positive milk sample tests for up to 21 and 10 d, respectively, following the last PPG injection (12). Considering the discrepancy between the approved dosage of PPG and the dosage actually used in clinical practice, the aims of the present study were to 1) determine whether increasing doses of PPG administered IM or SC would provide corresponding increases in efficacy as estimated by the length of time during which serum concentration of PPG would exceed the minimal inhibitory concentration (MIC); 2) compare the IM and SC routes of administration for the presence of urine and milk residues in normal lactating dairy cows.

Eight non-pregnant, crossbred Holstein dairy cows were used, weighing between 557 and 682 kg at the beginning of the experiment. These cows (which were either in their third or fourth lactation) were all within their first 3 mo of lactation and produced between 15.0 and 27.0 kg of milk per day at the beginning of the experiment. None of these cows had a history of difficult calving, mastitis, or metritis. Cows were fed grain, haylage, and hay in quantities to cover their milk production and had free access to water. They were kept in tie stalls during all the experiment. Experiment 1 was performed during the months of May through September, and experiment 2 was performed in October. The protocol was approved by the Institutional Animal Care Committee.

Experiment 1 Eight Holstein cows were randomly assigned to 2, 4 X 4 balanced Latin square design experiments, using 4 cows per square. The 4 cows in Latin square I received IM PPG injections and the 4 cows

Table II. Urine penicillin concentrations during IM or SC injections of 4 different doses of PPG for 5 consecutive days Dose (lu/kg BW)a Parameter Route 7000 14 000 21 000 28 000 Cmax (IU/mL) IM 160 329 293 351 SC 167 244 302 388 Tmax (h) IM 78 84 99 81 SC 90 84 120 72 AUC (lU-h/mL) lM 9402 17 944 21 087 24 515 SC 10 893 16 341 22 834 30 555 IM - intramuscular; SC - subcutaneous; NS - not significant (P > 0.05) a Values are least squares means for 4 cows b P value for the contrast between doses

SEM 27

16 1640

7000 vs 14 000 0.0002 NS NS NS 0.0107 NS

Contrastb 14 000 vs 21 000

21 000 vs 28 000 0.0334 NS NS NS NS 0.0154

NS NS NS NS 0.0305

Table Ill. Milk penicillin concentrations during IM or SC injection of 4 different doses of PPG for 5 consecutive days

Contrastb Dose (lu/kg BW)a Parameter Route 7000 14 000 21 000 28 000 IM Cmax (IU/mL) 0.128 0.204 0.385 0.551 SC 0.148 0.178 0.226 0.283 IM Tmax (h) 81 84 96 96 SC 99 75 102 96 AUC (IU'h/mL) IM 9.24 16.32 27.96 33.36 SC 7.26 12.26 18.00 24.64 IM - intramuscular; SC - subcutaneous; NS - not significant (P > 0.05) a Values are least squares means for 4 cows b P value for the contrast between doses

in square II received SC injections. Doses of PPG (rogar STB; Pfizer Canada, Pointe-Claire, Dorval, Quebec. Lot 00231006, 300 000 IU/mL) were 7000, 14 000,21 000 and 28 000 IU/kg BW once daily for 5 consecutive days. The total volume of PPG injected was divided in volumes of 20 mL/site and the remaining volume (to complete the dose) was injected in a supplemental site. This procedure was applied for both routes of administration. After 21 d, i.e. 16 d of withdrawal time, the second period of the Latin square was performed. This procedure was repeated until all 4 cows of each square received each of the 4 dosages of PPG, ie 4 periods of 21 d. The IM injections were performed in an alternative manner beginning in the right side in 1) the gluteus muscles followed by 2) the semimembranosus-tendinosus muscles, 3) anconeus and the cleoidian muscles, then the same muscles on the left side were

SEM 0.03

10 0.68

7000 vs 14 000 NS NS NS NS 0.0012 0.0064

14 000 vs 21 000 0.0203 NS NS NS 0.0001 0.0033

21 000 vs 28 000 0.0279 NS NS NS 0.0046 0.0016

Urine and milk samples (pooled) were obtained from each cow following each milking starting before the first injection and thus for 11 consecutive days. Urine samples were filtered (0.22 p,m) before being frozen at -70°C until assayed. Standards of known concentrations of PPG in sera, urine and milk were frozen in the same conditions to verify any possible degradation during the freezing period.

Experiment 2 Following a 30-day washout period, the same 8 cows were injected IM with 21 000 IU/kg of PPG once a day for 5 consecutive days in order to verify milk PPG concentrations and residues. Injections and milk samples were performed as previously described.

injected, in the same order. The SC injections were performed in the

Analytical method

cervical and thoracic areas on the left and right sides alternatively. The day prior to the beginning of each period, a complete evaluation of each cow was performed, including a somatic cell count (SCC) using the California mastitis test (CMT) and a milk culture (pooled sample). Cows were milked twice a day (12 h apart) and PPG injections were administered following each morning milking. Samples - Blood samples were taken by venipuncture at 0 (before the injection) and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, and 24 h following the first and fifth injections only. Blood samples were allowed to clot at room temperature for less than 1 h, then sera were harvested and immediately frozen at -70°C until assayed.

The concentrations of PPG in serum and urine were measured using a biological assay (13) using Bacillus subtilis as the test organism. The sensitivity of the test was 0.12 IU/mL. Standard concentrations from pooled serum were 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25, and 0.12 IU/mL.

Penicillin milk concentrations were measured by a disk assay methodology using Bacillus stearothermophilus as the test organism (14). Standard milk concentrations ranged from 6.4 to 0.008 IU/mL. When concentrations were expected to be greater than the standard, milk samples were diluted 1:3 or 1:10. All milk samples taken from day 1 (12 h sample) to day 5 were diluted. The Canadian Journal of Veterinary Research

175

0.600 -J

E

D

0.400

.2 ._

IL

.

0.200

0.000 0

1

2

3

2

3

4

10

9

8

7

6

5

Days

11

500 0.300

i`400 -J

E

I

c

300-

:a 0.200

I2

0

.

0.0 200 0

00

XL 0.100 Y:

_E

D 100 0

o.ooo 0

1

2

3

4

5

6

Days

7

8

9

10

11

12

Figure 1. Penicillin concentrations ± SD measured in the urine of 4 lactating injected IM (A) or SC (B) for 5 consecutive days with PPG at doses of 7000, 14 000, 21 000, and 28 000 IU/kg.

4

5

Days

6

7

8

9

10

11

Figure 2. Penicillin concentrations ± SD measured in the milk of 4 lactating injected IM (A) or SC (B) for 5 consecutive days with PPG at doses of 7000, 14 000, 21 000, and 28 000 IU/kg.

cows

cows

Statistical analysis

positive bacteriologic cultures of milk were obtained before and during both experiments, indicating an excellent udder health

The areas under the concentration-time curves (AUCs) were calculated using a trapezoidal summation method (15). The maximal PPG concentration (Cmax), the time of peak obtention (Tmax), the AUCs and the length of time during which sera PPG concentrations were maintained above the minimal inhibitory concentrations (MICs) of 0.4, 1.6, and 3.3 IU/mL were compared for the 4 different doses and for the 2 routes of administration. Data were analyzed by least squares analysis of variance, using the general linear model (GLM) of the statistical analysis software (SAS; SAS Institute, Cary, North Carolina, USA) (16), in a split-plot model in time using doses, periods, route of administration as main factors. Because of the significant interaction route X doses, dose contrasts for each route of administration were performed.

During both experiments, no cow exhibited any health problem as suggested by the very slight variation in body weights (taken 42 d apart) observed in Experiment 1 (618 ± 45 vs 625 ± 38 kg), and at the beginning of Experiment II, the average body weight was 647 ± 29 kg. The somatic cell count (SCC) evaluated by the CMT, and measured for each quarter before the beginning of every period, indicated that the cell count remained lower than 500 000 cells/mL of milk. No 176


status.

When PPG was injected IM at increasing doses, the maximal serum PPG concentration (Cmax) increased with the doses injected as well as AUCs of the first and fifth day of injection (Table I). However, increases in Cmax were not significantly different (P > 0.05) on the first day of injection between the 7000 to 14 000 IU/kg, and 21 000 to 28 000 IU/kg, doses. The time for Cmax obtention (Tmax) was unaffected by the dose injected. Subcutaneous injections of increasing doses of PPG produced nonsignificant increases (P > 0.05) in C Tmax and the AUC, with the exception of the AUC obtained between 7000 and 14 000 IU/kg doses (Table I) on the first day of injection. However, after 5 d of SC injections, significant dose-dependent increases (P < 0.05) in the Cmax and AUC were observed. Again, time of C obtention was unaffected by the dose and remained between 2 and 6 h following the SC max'

injection.

When Cmax and AUCs of the first and fifth days of injection were compared, a slight but non-significant (P > 0.08) cumulative effect was observed for both routes of administration. When parameters of the first and fifth days of IM injections were compared with those of SC injections, the C Tmax and AUCs observed were not significantly different (P > 0.05), indicating that IM or SC routes of administration might produce similar PPG profiles. max'

Table IV. Period of time (h) during which serum penicillin concentrations were over the minimal inhibitory concentration of 0.4, 1.6 and 3.3 IU/mL following the first and fifth penicillin injections Route First injection IM

SC

Dose (lu/kg)

7000 14000 21 000 28 000 7000 14000 21 000 28 000

Duration above 0.4 lU/mL meana + SD (range)b

12.5 ± 1.0 21 ±4 24 24 13± 2 21 ±4 22 ± 2 24

(12-14) (16-24) c

(10-14) (16-24) (20-24) -

Fifth injection IM

7000 14.5 ± 1.9 (12-16) 14 000 22 ± 4 (16-24) 21000 23± 2 (20-24) 28 000 23 ± 2 (20-24) SC 7000 16 14 000 22 ± 4 (16-24) 21 000 24 24 28 000 a Values are the mean ± standard deviation (SD) of 4 cows bRange value that includes all animals c No variation observed in the 4 time values

Levels of PPG were measured in the urine 12 h apart for 11 consecutive days starting before the first PPG injection. Table II and Figure 1 show data obtained during 5 d of injection. The Cmax was significantly increased (P < 0.05) with the doses when PPG was injected IM, except between doses of 14 000 and 21 000 IU/kg. The SC administration produced no significant increases (P < 0.05) for the C max and T max However, increases (P < 0.05) in the AUC for both routes of administration were observed between different doses. Again, as observed with serum data, no difference in the IM and SC routes of administration was observed in the Cmax, Tmax, and AUCs of urine parameters. The IM-injected cows seemed to eliminate PPG more rapidly than the SC-injected cows. However, 170 h following the last injection, 6 out of 16 IM-injected cows had detectable urine PG residues, compared with 5 of 16 for the SC-injected cows. Milk Cmax were increased (P < 0.05) in a dose-related manner in the IM-injected cows but were not affected (P > 0.05) by the dose in the SC-injected cows (Table III). The Tmax ranged between 81 and 102 h after the PPG treatment was started and was not affected (P > 0.05) by the dose nor the route of administration. However, the PPG AUCs were increased (P < 0.05) in a dose-related manner in both IM and SC groups. Slight AUCs increases (P < 0.05) at the highest IM dose of PPG were observed when compared with the SC route. Figure 2 shows PPG milk concentrations for each dose injected and for both routes of injection. Milk residues for the 7000 and 14 000 IU/kg doses were similar for both routes of injection, and lasted for less than 60 h for the 7000 IU/kg dose and 84 h for the 14 000 IU/kg dose with concentrations less than 0.016 IU/mL.

Duration above 1.6 lU/mL mean ± SD (range)

0 0.75 ± 1.5 10.9 ± 3.7 13.5 ± 3.0 0 5.75 ± 3.2 10 ± 2 14.5 ± 4.0 0 9 ± 2.5 13± 2 14.5 ± 3.0

2.3±2.6 10.3 ± 2.8 14.5 ± 1.9 15.5 ± 1.0

(0-3) (7.5-14) (10-16) (0-9) (8-12) (12-20)

(7-12) (10-14) (10-16) (12-16) (14-16)

Duration above 3.3 lU/mL mean ± SD (range)

0 0 0 1.5 ± 1.7 0 0 0.9 ± 1.7 2.1 ± 2.0 0 0 1.4± 4.8 ± 0 0 3.0 ± 5.6 ±

(0-3.5)

(0-3.5) (0-4)

1.7 3.4

(0-3.5) (0.5-7.5)

1.1 1.1

(2-4.5) (4-6.5)

The 21 000 and 28 000 IU/kg doses, injected SC, produced PPG residues for a longer period when compared to the IM injections. At the dose of 28 000 IU/kg, 2 cows injected SC still had detectable level of PPG in their milk 132 h following the last injection. Table IV reports data in hours where penicillin concentrations were over MICs of 0.4, 1.6, and 3.3 IU/mL. These MICs correspond to bacteria very sensitive to resistant to 3-lactam, respectively. For MIC of 0.4 IU/mL, the dose of 7000 IU/kg produced serum concentration over that level for a period of at least 12 h. When the dose was increased to 14 000 IU/kg, the period increased to 16 h and for the 2 highest doses, sera concentrations over this MIC level were of 20 to 24 h. The 7000 IU/kg dose did not reach the MIC level of 1.6 IU/mL. Only 5 consecutive days of IM or SC injections at 14 000 IU/kg produced serum concentrations over this MIC for periods of 9-10 h. The 2 highest PPG doses did not produce more than 13 to 15 h duration over the 1.6 IU/mL level and this, by either route of injection, after the first and fifth day of injection. For resistant organisms that have MIC over 3.3 IU/mL, only the dose of 28 000 IU/kg produced sera levels over 3.3 IU/mL and for only 1.5 to 2 h following the first injection and for 5 h following 5 consecutive IM or SC

injections. Experiment 2 was conducted to verify the milk PPG concentrations of 8 cows injected with 21 000 IU/kg of PPG IM for 5 consecutive days. It was observed that only 1 cow still had detectable residue levels < 0.016 IU/mL 96 h after the PPG injections. Otherwise, no other cow had detectable penicillin residue levels past 84 h after the injections (Figure 3). The Canadian Journal of Veterinary Research

177

J11 111 J

E 0.300

I 21OOO0 I U/kgjI

f-f _

= 0.200

0)

a.

0X E~0.100

0.000

0

1

2

3

4

5

6

7

8

9

Days Figure 3. Penicillin concentrations ± SD measured in the milk of 8 lactating cows following 5 d of IM injections of 21 000 lU/kg of PPG.

To our knowledge, this study is the first to report serum, urine, and milk penicillin concentrations following increasing doses of PPG injected to lactating dairy cows by 2 different routes of administration for over 5 consecutive days. Many factors may interfere with the absorption of PPG (11) and the route of administration has been reported to be (17) or not to be (18,19) of importance. In the present study, no differences were observed after the first and fifth day of injection in the sera penicillin kinetics between the 2 routes of administration and this regardless of the doses injected, indicating that the IM and SC routes of administration (for PPG volumes of 20 mL/site) produced similar serum profiles, thus similar absorption. In experiments indicating variations between the SC and IM routes, the total volume per injection site of aqueous solution of 300 000 IU/mL of PPG varied from 33 to 37 mL in healthy crossbred yearling steers averaging 480 kg BW (17). These authors also reported that a single IM neck injection of 66 000 IU/kg produced a higher Cmax than 5 d of gluteal injections, which itself produced a higher Cmax than a SC injection of the same dose, indicating a difference in absorption among the IM injection sites. However, the limited number of animals per group (n = 3) was possibly responsible for the lack of significant difference in the experiment (17). This study did not report the type of housing nor the ambient temperature, 2 factors which might greatly interfere with the vascularization and drainage of PPG at the injection site (11). Luthman and Jacobsson (7) also reported that cattle injected in the neck muscles have a higher serum penicillin concentration than when 178


injected in any other site. In the present study, we followed an intermittent rotation of the IM site which enabled us to observe any difference among injection sites. In studies that reported differences between SC and IM routes, the volumes injected ranged from 33 to 37 mL (17) to 30 to 48 mL (20) which could play an important role in the absorption process (11). In companion animals (benzylpenicillin) (21) and calves (22), SC and IM injections of penicillin G produced similar blood profiles, but injected volumes were lower than those reported in steers. Korsrud et al (20) reported important differences in retention time when steers of different weight received the same dosage of PPG and they attributed this difference in part to the volume injected per site, which was 30 vs 42 mL. Their conclusions were that 30 mL/site of injection should not be exceeded, otherwise retention time might be increased. In normal calves, 20 000 IU/kg of PPG injected IM or SC did not produce different serum concentrations over 24 h of sampling (5). However, Papich et al (17) found that in steers injected IM or SC with similar volumes (33-37 mL/site), the neck muscle injection produced a 2.5-fold higher C max than the SC injection which was also 50% lower than the gluteal muscle injection site. Our results indicate no difference in the injection routes when volumes under 20 mL/site are used in dairy cows and, thus, independently of the site of

injection. The absence of a significant difference between the Cmax and

Tmax indicates the poor cumulative effect of PPG independently of the dose injected. This is in agreement with Papich et al (17), who compared PPG serum profiles of 2 groups of 3 steers, one injected IM daily for 5 consecutive days with 66 000 IU/kg of PPG and the other receiving a single IM injection of the same dose. No

difference in sera PPG profile was observed, indicating no cumulative effect. In the present study, increasing doses of PPG have been injected in lactating dairy cattle to document if in adult cows, these doses would produce higher PPG sera concentrations. In fact, an increase in serum PPG concentration was observed, generally related to the dose injected. In calves (5) and steers (17), this effect was also observed. English first reported that over a certain dose of penicillin, serum concentration plateaued and only the duration in serum was affected (21). In the present study, considering the doses injected, a plateau in the Cmax and AUC was not reached; however, an increase in the persistance of PPG in serum was observed. Plasma concentrations over MICs are important for adequate dosage and appropriate choice of the antibiotic. The purpose of this study was to observe the relationship between the dose and the effective level of penicillin by determining 3 different MIC levels corresponding to various types of bacteria ranging from very sensitive to resistant to penicillin. It is reported that the action of penicillin on bacteria is time-dependent and serum levels above the MIC should be maintained for the whole dosing interval (23). Consequently, to ensure that serum levels were maintained over the 3 predetermined MIC levels corresponding to the sensitivity of the invading microorganisms, increased frequency of injection must be used. However, it should be remembered that using doses higher than 21 000 IU/kg will not significantly prolong effective serum levels because of the short half-life (3.7 to 4.8 h) of penicillin in cattle (7,8). To our knowledge, PPG urine concentrations have been reported only once in normal cows following IM injection of PPG (5) at doses of 2000 and 60 000 IU/lb. The present study reports detectable PPG levels in urine for up to 170 h following the last injection independently of the dose injected. Cows receiving 2000 and 60 000 IU/lb of PPG had detectable urine levels for 4 and 14 d, respectively (5). These authors used the presence of penicillin in the urine to determine a tentative withdrawal period (TWP), calculated as follows: TWP = UP2 + (AWP-UP1), where UP2 is the number of positive days of penicillin in the urine, AWP is the approved withdrawal period and UPI is the number of positive days of penicillin in the urine of the approved drug. If we select the 7000 IU/kg as the approved dose, with a withdrawal time of 5 d, the calculated withdrawal times for meat of each PPG dose used in our study would be 10 d, > 12 d, > 12 d, for the IM route at doses of 14 000, 21 000, and 28 000 IU/kg, respectively. For these reasons, important considerations must be used when antibiotics are used for food producing animals and special attention must be given when extra-label use of antibiotics are prescribed and withdrawal times are not established. Hypersensitivity to penicillin is the most common side effect experienced by human patients with an incidence ranging from 0.7% to 10% in the population. Hypersensitivity reactions to penicillin are idiosyncratic, not dose-related and have no inheritance pattern (24). Another type of reaction would be the anaphylaxis which has been estimated to be 0.04 to 0.2% of all patients given penicillin with an incidence of death being 0.002% (1). Other side effects are the potential build-up of antibiotic resistant organisms in humans and, in the industry, the inhibition of starter cultures used to produce yogurt and cheese (1).

In 1997, the Bureau of Veterinary Drugs of Canada revised labeled recommendations for injectable PPG products. For dairy cows, the recommendation for dosage is 21 000 IU/kg IM once daily for a maximum of 5 d. Also a maximum of 15 mL/site should not be exceeded and the withdrawal periods are established to 96 h and 10 d for milk and meat, respectively. Penicillin residues in bulk tank milk may have many sources. In a study on drug residues in the dairy cattle industry, of the 614 positive samples, 30% were related to insufficient knowledge about withdrawal periods, 23% were due to errors from employees, 14% from poor identification or record, 12% to metritis treatment, 7% to dry cow treatment for mastitis, 4% for incorrect label reading, and 10% for undetermined cause (25). In this 10% of undetermined causes, we could include cows that individually have prolonged penicillin clearance times (26) and/or accident as contamination of the bedding by the urine of treated cows causing residues in the milk of neighboring untreated cows (27). Prolonged milk residues have been reported for up to 18 d in a cow injected SC ql2h with 35 mL per site (20 000 IU/kg) of PPG for 4 d and for up to 7 to 8 d in another cow injected SC with 33 mL PPG at one site over 6 consecutive days (12). Both cows developed firm subcutaneous masses at the injection site, which probably interfered with the absorption process. In 1961, Albright et al (18) reported different withdrawal times for milk residues in cows injected with 5000 to 10 000 IU/lb IM of different penicillin preparations. The withdrawal period varied between 48 to 96 h. However, the methods of detection were probably less sensitive than those now available. Seymour et al (6) reported that in 15 cows receiving PPG at a dose of 13 200 IU/kg IM, one had positive milk residues for up to 9 d following the last injection. The present study shows that following IM injection of increasing doses of PPG, milk penicillin was detectable for up to 96 h in one out of 8 cows in the 14 000 IU/kg group, otherwise all cows had detectable penicillin milk residues for 84 h or less, independently of the dose injected. When injected SC, the persistence of penicillin residues in the milk was measured for up to 132 h for the dose of 28 000 IU/kg and 108 h for the dose of 21 000 IU/kg. These results preclude the use of the SC administration route in dairy cows, since Canadian regulations do not allow for a withdrawal time of more than 96 h. The accepted penicillin residue levels tolerated in the milk is zero or the lowest non detectable presence of penicillin. The joint FAO/WHO Expert Committee on Food Additives (28) recommends a maximum daily intake of 30 pLg or IU of penicillin per day, which represents 1875 mL of milk containing 0.016 IU of PPG, to prevent harmful effects even in sensitized individuals. Anaphylaxis to penicillin in milk has been reported after the ingestion of as little as 300 IU (29). In a study on penicillin residues in meat, consumption of 450 to 900 IU in 9 volunteers allergic to penicillin produced itchy sensations in 2 subjects without other manifestations such as anaphylaxia, urticaria, angioedema and asthma, showing that the oral uptake of penicillin is less dangerous than the

parenteral route (30). In conclusion, this study demonstrates that PPG injected either IM or SC produces similar serum kinetics when injected at volumes not exceeding 20 mL/site. No cumulative effects of PPG were observed over 5 consecutive days of one daily IM or SC injection. Concerning The Canadian Journal of Veterinary Research

179

milk residues, if PPG is injected at doses equal or higher than 21 000 IU/kg by the SC route, it may present a human health hazard. If doses of PPG higher than 21 000 IU/kg need to be injected over a 5-day period or for more than once daily, it would be prudent to monitor the milk of the antibiotic- treated cows with an antibiotic residue test before adding the milk to the bulk tank or then again consider an alternative therapy with an approved product.

1. Goldstein SL, Kaplan SL, Feigin RD. Penicillin update. Ped Rev 1995;16:83-90. 2. DeWeck AL. Allergology Proc 7th Cong Int Assoc Allergology, Florence, Italy. Frankland AW, Masala C, Jamar JM, eds. Excerpta Medica, Amsterdam, The Netherlands, 1971:208-215. 3. Smith BP. Large Animal Internal Medicine. 2nd ed. St. Louis: Mosby, 1996. 4. Blood DC, Radostits OM. Veterinary Medicine. 8th ed. Toronto: Balliere Tindall, 1994. 5. Hjerpe CA, Routen TA. Practical and theoretical considerations concerning treatment of bacterial pneumonia in feedlot cattle, with special reference to antimicrobic therapy. In: Proc 9th Annu Meet Am Assoc Bovine Pract 1976:97-140. 6. Seymour EH, Jones GM, McGillard ML. Persistence of residues in milk following antibiotic treatment of dairy cattle. J Dairy Sci 1988;1 :2292-2296. 7. Luthman J, Jacobsson SO. Distribution of penicillin G in serum and tissue cage fluid in cattle. Acta Vet Scand 1986;27:313-325. 8. Bengtsson B, Franklin A, Luthman J, Jacobsson SO. Concentrations of sulphadimidine, oxytetracycline and penicillin G in serum, synovial fluid and tissue cage fluid after parenteral administration to calves. J Vet Pharm Therap 1989; 12:37-45. 9. Teske RH, Rollins LD, Carter GG. Penicillin and dihydrostreptomycin serum concentrations after administration in single and repeated doses to feeder steers. J Am Vet Med Assoc 1972;160:873-878. 10. Huebner, R.A. Bovine penicillin blood levels obtained with parenteral benzethacil. Cornell Vet, 1952;42:457-461. 11. MacDiarmid SC. The absorption of drugs from subcutaneous and intramuscular injection sites. Vet Bull 1983;53:9-23. 12. Krainock RJ. Prolonged milk residue in two cows after subcutaneous injections of penicillin at an extra-label dose. J Am Vet Med Assoc 1991;198:862-863. 13. Bennett JV, Brodie JI, Benner EJ, Kirby WM. Simplified accurate method for an antibiotic assay of clinical specimens. Applied Microb 1966;14:170-177. 14. AOAC. Official methods of analysis. Beta-lactam antibiotics in milk: Quantitative Bacillus stearothermophilus disc method. 1984:295-296.

180


15. Abramovitz M, Slegun IA. Handbook of Mathematical Functions. New York: Dover Publications, 1972:885-887. 16. Statistical Analysis System Institute. SAS User's Guide: Statistics. Cary, North Carolina SAS Institute, 2000. 17. Papich MG, Korsrud GO, Boison JO, et al. A study of the disposition of procaine penicillin G in feedlot steers following intramuscular and subcutaneous injection. J Vet Pharm Therap 1993;16:317-327. 18. Albright JL, Tuckey SL, Woods GT. Antibiotics in milk - a review. J Dairy Sci 1961;44:779. 19. Cannon RY, Hawkins GE, Wiggons AM. Duration of secretion of bacteriostatic drugs in milk. 1. Penicillin, following oral and parenteral administration. J Dairy Sci 1962;45:769. 20. Korsrud GO, Boison JO, Papich MG, et al. Depletion of intramuscularly and subcutaneously injected procaine penicillin G from tissues and plasma of yearling beef steers. Can J Vet Res 1993;57:223-230. 21. English PB. The therapeutic use of penicillin. The relationship between dose rate and plasma concentration after parenteral administration of benzylpenicillin (Penicillin G). Vet Rec 1965; 77:810-814. 22. Mercer HD, Rollins LD, Garth MA, Carter GG. A residue study and comparison of penicillin and dihydrostreptomycin concentrations after intramuscular and subcutaneous administration in cattle. J Am Vet Med Assoc 1971;158:776-779. 23. Prescott JF, Baggot JD. r-Lactam antibiotics: Penam penicillins. In: Prescott JF, Baggott JD, eds. Antimicrobial Therapy in Veterinary Medicine. 2nd ed. Ames: Iowa State University Press, 1993:74-97. 24. Jones GM, Seymour EH. Cowside antibiotic residue testing. J Dairy Sci 1988;71:1691-1699. 25. Kaneene JB, Ahl A. Drug residues in dairy cattle industry: epidemiological evaluation of factors influencing their occurrence. J Dairy Sci 1987;70:2176-2180. 26. McQueen RD. Managing for antibiotic residue-free milk. In: Proc 26th Annu Meet Natl Mastitis Council, 1987:160-177. 27. Vanos JL. Verontreeniging van melk met penicilline-bevahende urine. Tijdschr Diergeneesk 1973;98:547-548. 28. FAO/WHO Expert Committee on Food Additives. Technical Report Series: Evaluation of certain veterinary drug residues in food. Geneva: World Health Organization, 1990;799:37-41. 29. Wicker K. Allergic reaction to penicillin present in milk. J Am Vet Med Assoc 1969;268:143-148. 30. Lindemayr H, Knobler R, Kraft D, Baumgartner W. Challenge of penicillin-allergic volunteers with penicillin-contaminated meat. Allergy 1981;36:471-478.