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Risks of antibiotic residues in milk following intramammary and intramuscular treatments in dairy sheep
A. Pengov a, , A. Kirbis b
a b

Institute for Microbiology, Veterinary Faculty of Ljubljana, Gerbi eva 60, 1000 Ljubljana, Slovenia c Institute of Food Hygiene and Bromatology, Veterinary Faculty of Ljubljana, Gerbi eva 60, 1000 Ljubljana, Slovenia c

a r t i c l e
Article history:

i n f o

a b s t r a c t
Very few drugs on the market are approved for use in lactating ewes. Veterinarians in the European Union are allowed to prescribe drugs in an off-label manner but are then obligated to assure that residues do not enter the food chain. In case of mastitis treatment in dairy ewes antibiotic preparations designed and authorized for the bovine mammary gland are usually used. Due to inter-species differences, available bovine data cannot be accurately extrapolated for use in the dairy ewe. The objective of the study was therefore to determine appropriate withdrawal periods for ewes milk following mastitis treatment with two commercial lactating cow products. For the detection of all components standard agar plate diffusion techniques were used. Regardless of the therapy regime and the product used, residues of antibiotics in milk were detected up to 192 h after the last infusion. These results indicate that the required withholding periods for ewes milk are considerably longer than recommended on the label for bovine milk. 2008 Elsevier B.V. All rights reserved.

Received 11 June 2008 Received in revised form 2 September 2008 Accepted 8 September 2008 Published on line 16 September 2008 Keywords: Mastitis Treatment Ewes Withdrawal periods

1.

Introduction

Mastitis remains one of the most important causes of disease and economic loss in the dairy industry, despite the introduction of mastitis control programs over the last 30 years [8]. In ocks where ewes are milked commercially for the production of cheese, any factor that adversely affects the quantity and quality of ovine milk is of great nancial interest [2]. Where ewes are used primarily for meat production mastitis outbreaks, mostly associated with some predisposing factors (teat lesions), have been reported occasionally. The prevalence of clinical and subclinical mastitis is usually higher when ewes are milked commercially. As is the case in dairy cows, the increase in milk production by dairy ewes has resulted in a similar increase in the prevalence and severity of mastitis and other diseases of the udder [9].

Due to a shortage of appropriate and authorized preparations, antibiotic drugs generally formulated for the bovine mammary gland are often used to treat mammary gland infections in ewes. However, the withdrawal periods adapted to cows, are not necessarily valid for ewes [3]. The goal of the present study was to describe the excretion rate in lactating ewes following infusion of two antibiotic products registered for use in lactating cattle.

2.
2.1.

Materials and methods

Animals

Twenty lactating ewes (domestic dairy breed) with an elevated number of leucocytes in milk and without perceptible signs of inammation of the mammary gland (subclinical

Corresponding author. Tel.: +386 147 79164; fax: +386 128 32308. E-mail address: andrej.pengov@vf.uni-lj.si (A. Pengov). 0003-2670/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.09.021

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mastitis) were selected from a commercial milking ock. The rst group consisted of 10 animals with subclinical Streptococcus uberis mastitis. In the second group coagulase negative staphylococci (CNS) were determined as the causative agent of subclinical mastitis.

the medium were thoroughly mixed. A volume of 5 mL prepared medium was poured into Petri dishes 90 mm (Golias, Slovenia). Three assay plates were used for each sample.

2.7.

Sample preparation

2.2.

Treatment protocol

Animals in the rst group were treated with a cattle-labelled solution for intramuscular application containing benzylpenicillin (250,000 IU) and a cattle-labelled intramammary injector containing benzylpenicillin (1,000,000 IU). In the second group a solution for intramuscular application containing 35 mg of clavulanic acid in the form of potassium clavulanate and 140 mg of amoxycillin in the form of amoxycillintrihydrate per mL was used. Both drugs were also applied intramammary, the injector for intramammary application contained 50 mg of clavulanic acid in the form of potassium clavulanate, 200 mg of amoxycillin in the form of amoxycillintrihydrate and 10 mg of prednisolone. One syringe per udder-half was infused at ve consecutive milking. All animals received also two intramuscular infusions at 24 h intervals.

2.3.

Sampling intervals

In order to establish the excretion rate of the tested antibiotic products milk samples from the treated udder halves were collected at regular intervals. In each animal the rst sample was taken immediately after the nal antibiotic infusion and the subsequent samples were taken at 24-h intervals for 8 days (192 h).

The milk sample was mixed thoroughly, poured into the test tube (10 mL) and heated at 80 C for 5 min in water bath (Kambic, Slovenia) to inactivate non-specic inhibitory substances and the microora, which could have an inhibitory effect on the test culture. Afterwards the sample was cooled at room temperature. Paper discs 6 mm (Schleicher&Schuell, Germany) were inserted in the milk sample ve times and than slightly pressed onto the surface of the culture medium. Plates were incubated in a thermostat (Ehret, Germany) at the temperature of 65 1 C for 3 h 5 min. The growth of microorganisms and consequently their reaction to the applied sample differs between each series of test plates. Therefore, a correction factor for every series of plates must be determined. A reference concentration (16 g kg1 ) of penicillin and amoxycillin was applied. After incubation the diameter of an inhibition zone was measured with digital calliper (Stainless hardened, UK) and the concentrations were calculated from the calibration curve. The calibration curve for penicillin and amoxycillin was prepared with working standard solutions of the following concentrations ( g kg1 ): 4, 8, 16, 32, and 64. For amoxycillin and penicillin the limit of quantication (LOQ) was 4 g kg1 and the limit of detection (LOD) was 3 g kg1 . The decision limit (CC ) was 5.3 g kg1 and the detection capability (CC ) of method was 6.3 g kg1 .

2.4.

Bacteriology

Before treatment, milk samples from udder halves were collected into autoclaved glass tubes. The samples were kept cool and streaked, using a sterile swab, within 24 h on quarter plates of washed ovine blood agar and were incubated at 37 C. Plates were examined at 24 and 48 h for aerobic bacterial growth. Gram-positive cocci were classied as Staphylococcus or Streptococcus species considering the results of the catalase test. In case of Staphylococcal isolates the coagulase test was performed to distinguish between CNS and Staphylococcus aureus. At the species level isolates were determined using the API-Identication System (Bio Merieux) [6].

3.

Results

Refer to Tables 13 and Figs. 13.

4.

Discussion

2.5.

Microbiological disc assay

The levels of penicillin and amoxycillin were estimated following the method of agar diffusion test using Bacillus stearotermophylus ATCC 7953 (Merck, Darmstadt, Germany) as test organism.

2.6.

Preparation of test plates

The basic medium, test agar for residue test according to Kundrat (Merck) was cooled down to 5060 C after autoclaving at 121 C for 15 min. One ampoule of the spore suspension of the test culture was added to 200 mL of the basic medium and

The increasing demand for ewes milk and milk products has been reected recently in fresh interest in breeding small ruminants [7]. Further intensication of the milk producing system will inevitably cause a higher incidence of ovine mastitis [1]. Since very few drugs are approved for use in lactating dairy sheep, in most cases preparations designed for the bovine mammary gland are used for therapy of ovine mastitis [5]. Within the European Union veterinary medical products may be placed on the market and used only when a marketing authorization has been issued by the competent authorities. Where no authorized medical product for a condition exists, member states may exceptionally and particularly in order to avoid causing unacceptable suffering to the animals concerned, permit administration of an unauthorized product by a veterinarian or a person under his/her direct responsibility. Under these circumstances a veterinary medical product authorized for use in another animal species or for another condition in the same species may be applied to an animal or a small group of animals on a particular holding. The

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Table 1 Mean concentrations of amoxycillin and penicillin in milk Time interval post-nal infusion (h) Mean concentrations of amoxycillin g kg1
64.0 19.1 10.2 9.1 7.0 5.9 5.0 6.0 4.5

Mean concentrations of penicillin g kg1

64.0 64.0 64.0 63.3 53.7 33.0 21.6 9.9 4.0

0 24 48 72 96 120 144 168 192

Table 2 Minimum and maximum concentrations of amoxycillin in milk Time interval post-nal infusion (h)
0 24 48 72 96 120 144 168 192

Maximum concentrations of amoxycillin ( g kg1 )

64.0 30.5 21.0 17.9 13.1 8.4 12.0 4.0 0

Minimum concentrations of amoxycillin ( g kg1 )

64.0 15.1 6.1 5.6 4.8 4.0 3.5 4.0 0

Table 3 Minimum and maximum concentrations of penicillin in milk Time interval post-nal infusion (h)
0 24 48 72 96 120 144 168 192

Maximum concentrations of penicillin ( g kg1 )

64.0 64.0 64.0 64.0 64.0 64.0 64.0 57.6 4.0

Minimum concentrations of penicillin ( g kg1 )

64.0 64.0 64.0 60.0 25.7 8.0 4.0 4.0 4.0

Fig. 1 Mean concentrations of amoxycillin and penicillin ( g kg1 ).

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Fig. 2 Maximum and minimum concentrations of amoxycillin ( g kg1 ).

Fig. 3 Maximum and minimum concentrations of penicillin ( g kg1 ).

prescribing veterinary surgeon is then responsible to dene an appropriate milk withholding period. However, the ofcial instructions concerning the length of withdrawal periods are very vague. Accordingly to the provisions in the directive of the European parliament 2001/82/EC in the described case the withdrawal period for milk shall not be less than 7 days. In the present study residues of antibiotic substances exceeding the MRL values were detected up to 192 h (8 days) after the last infusion regardless of the applied preparation (Table 1, Fig. 1). Differences regarding individual gland concentrations were also observed between ewes (Tables 2 and 3, Figs. 2 and 3). In a similar study [4], where a comparable preparation containing amoxycillin/clavulanic acid was used, ewes milk was not acceptable for human consumption or for the production of cheese and yoghurts until 136 h (about 6 days) following the nal infusion. However the MRL value of 10 g kg1 considered in this study was higher than the actual value of 4 g L1 . The results obtained in the present study are likely to be representative of other antibiotic products used in the ovine species following infusion with bovine antibiotic preparations. In order to prevent the possibility of antibiotic residues in ewes milk and milk products the prescribing veterinary surgeon should therefore extend the minimal 7 days withdrawal period prescribed by the European regulation after an out-oflabel intramammary treatment. It is also recommended to use

antimicrobial detection tests for milk whenever antimicrobial drugs are used to treat dairy ewes.

5.

Conclusions

The results of this study indicate that patterns of antibiotics excretion differ widely between the ovine and bovine mammary gland. The milk withholding time in ewes require a considerable longer period of retention than that recommended for dairy cows.

references

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[6] D.E. Jasper, F. Infante, J.D. Dellinger, Efcacy of the API Staph-Ident system for identication of staphylococcus species from milk, Am. J. Vet. Res. 46 (1985) 1263. [7] J.H. Kirk, J.S. Glenn, J.P. Maas, Mastitis in a ock of milking sheep, Small Rumin. Res. 22 (1996) 187191.

[8] J.P.T.M. Noordhuizen, G.H. Wentink, Developments in veterinary herd health programmes on dairy farms: a review, Vet. Quart. 23 (2001) 162169. [9] D.L. Watson, J.F. Buswell, Modern aspects of sheep mastitis, Br. Vet. J. 140 (1984) 529534.