oxytocin were tested for effectiveness against experi- mental infection by Streptococcus uberis with the fol- lowing results from 54 animals: a) no treatment led to.
J. Dairy Sci. 85:1009–1014 American Dairy Science Association, 2002.
Effective Treatment of Streptococcus uberis Clinical Mastitis to Minimize the Use of Antibiotics J. Eric Hillerton and Kirsty E. Kliem Institute for Animal Health, Compton, United Kingdom
ABSTRACT Antibiotic regimens (intramammary antibiotic, penicillin-based parenteral treatment) and intramuscular oxytocin were tested for effectiveness against experimental infection by Streptococcus uberis with the following results from 54 animals: a) no treatment led to deterioration of infected quarters, requiring intervention within 48 h for cow health; b) aggressive intramammary antibiotic at every milking achieved 70% clinical cure in 3 d and 100% cure within 6 d; overall bacteriological cure was 80%; c) parenteral treatment alone used about 14 times as much antibiotic with 18% clinical cure in 3 d and 91% within 6 d; overall bacteriological cure was 80%; d) combination of aggressive intramammary and parenteral treatments achieved 61% clinical cure in 3 d and 100% within 6 d; overall bacteriological cure was 72%; e) intramammary antibiotic at labeled rates (1× for 3 d) achieved 27% clinical cure in 3 d but 91% within 6 d of treatment; overall bacteriological cure was 64%; f) use of oxytocin alone for 3 d failed to achieve clinical improvement with an increase in the severity of mastitis; g) combining oxytocin with labeled use of intramammary antibiotic (1× for 3 d) was unsuccessful: 0% clinical cures in 3 d, 10% in 6 d; significantly poorer than intramammary antibiotic alone. Extended treatment periods with parenteral or intramammary antibiotics resulted in positive inhibitory tests for milk from individual quarters up to 8 d after treatment. Aggressive intramammary antibiotic was the most effective treatment for fastest cure clinically and bacteriologically using least antibiotic. (Key words: mastitis, therapy, reduction of antibiotics) INTRODUCTION There is a widespread view that antibiotics are used unnecessarily or excessively in farm animals, especially in those directly in the food chain, and that the amount
Received July 5, 2001. Accepted November 16, 2001. Corresponding author: J. E. Hillerton; e-mail: eric.hillerton@ bbsrc.ac.uk.
and frequency should be reduced. One instance of major use is the treatment of clinical mastitis in dairy cows. In the United Kingdom, the incidence of clinical mastitis is approximately 40 cases/100 cows per year or 1,000,000 cases annually (Booth, 1997). Sales of intramammary antibiotic syringes approximated 6,000,000 in 1999, with parenteral treatment also used on up to 20% cows affected. The recommendations for approved antibiotic are to use 1 to 3 syringes of intramammary antibiotic per case. The use rate is therefore more than twice the approved level. This practical rate of use is a measure of the ineffectiveness of the recommended protocols in achieving a clinical cure. Yet even when a clinical cure is achieved, bacteriological elimination is rarely better than 60% (Hillerton et al., 1995). Dairy farm and practicing veterinary opinion of many is that the actual use of antibiotics on farms is required to achieve satisfactory cure of disease and restoration of milk quality. It is necessary to determine whether this is true, whether label recommendations are adequate and whether there are alternative strategies that are more effective. Research at the Institute for Animal Health and elsewhere has shown that effective elimination of bacteria can be achieved by use of 6 to 12 syringes, one per milking as opposed to poor elimination with one syringe every second milking for 3 d (Saran, 1995; Milner et al., 1997). The greatly increased effectiveness of this aggressive treatment reduces the rate of recurrence of disease by achieving a greater rate of bacteriological cure. Perhaps as many as 40% of cases are a recurrence of previous disease from failed therapy, when therapy has not achieved a bacteriological cure even if a clinical cure has resulted (Biggs, 1998). Failure to achieve a clinical cure may require even more extreme measures, often culling of the cow. Failed therapy also increases the amount of antibiotic necessary for dry cow treatment because the main reason to use dry cow treatment is to eliminate existing infections. Current protocols may not deliver an appropriate dose of antibiotic to the focus of infection (Erskine, 1998). Intramammary antibiotics may be used ineffectively because treatment protocols are inappropriate. Most protocols were designed to return milk to sale quickly when quality standards of bacterial content and
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cell count in milk did not exist. Today, because milk price varies with cell count, a complete cure is needed. This appears in direct contradiction with political pressure to reduce antibiotic use in farm animals. Attempts to increase effective dosing have been made by the use of parenteral application of antibiotic, alone or in combination with intramammary syringes. This is no more effective in achieving a cure (Erskine et al., 1995) and may even be detrimental by exposing systemic bacteria to low levels of antibiotic, resulting in an increase in antibiotic resistance in gut enterococci. Intramammary pathogens do not show an increase in their resistance spectrum probably because intramammary antibiotic diffuses poorly from the udder (Erskine et al., 1995), and they are not exposed to a low dose. Also, the antibiotic contacts a separate population of strains in the mammary gland that rarely possess an efficient means of gene transfer (Lacey, 1980). Treatment of clinical mastitis appears to use significant amounts of antibiotic ineffectively and contrary to approved means of use. It challenges food quality and consumer perception of safety, and, because it is ineffective, it probably gives poor economic value while failing to promote animal welfare. It is necessary to understand and develop more effective treatment protocols for clinical mastitis to: a) eliminate infections and prevent recurrence of disease; b) reduce the total of antibiotic used to treat mastitis and so reduce the impact on resistance; and c) identify effective methods of control with limited or no use of antibiotics. The additional benefit is likely in guidance for the approval of veterinary medicines. A series of trials was conducted to determine the most effective method of treating clinical mastitis caused by Streptococcus uberis to achieve a clinical and bacteriological cure. This pathogen is responsible for some 35% clinical cases in the UK and has a reputation of being difficult to resolve in up to one quarter of cases. The trials included an established experimental infection model, and cows were treated using various commercially common and experimental combinations of methods. MATERIALS AND METHODS Suitable cows, free from previous intramammary infection in two or more quarters, and never having been infected with S. uberis, were selected by screening of the institute herd and health databases. They were further screened by examination of milk samples by bacteriology and cell counting. All cows, pregnant (to avoid fluctuations in yield and response due to estrus), Journal of Dairy Science Vol. 85, No. 4, 2002
yielded at least 15 L of milk per day. Cows selected for the experiment were milked through an experimental milking parlor in three groups of 18 animals. Baseline data were collected for 4 d on milking and milk quality measures including milk yield, quarter milk electrical conductivity, quarter milk bacteriology, quarter milk cell count, and rectal temperature, and then from infusion of the bacteria until treatment commenced. A bacterial suspension, approximately 1000 cfu S. uberis 0140J in quarter strength mammalian Ringers solution, was infused into two quarters of each cow and the response of the cows measured against the baseline data. On detection of disease, cows were allocated to a treatment group at random. The treatments were a) no treatment, unless animal welfare was compromised (negative control); b) aggressive use of intramammary antibiotic (2× daily for 3 d; positive control); c) parenteral antibiotic (1× daily i.m.); d) aggressive use of intramammary antibiotic and parenteral antibiotic in combination; e) label recommended use of intramammary antibiotics (1× daily for 3d); f) use of intramuscular oxytocin (2× daily), unless welfare was compromised; and g) oxytocin and label use of intramammary antibiotic. Detection of disease was based on standard criteria used by the commercial herdsperson of clots in milk, swelling and/or hardness of the udder and general awareness of the animal’s well being. Deterioration in the clinical condition of any animal was assessed from the degree of milk abnormality, milk yield depression, rectal temperature, and any inappetence and general demeanor, especially lethargy or lameness. On welfare grounds, animals suffering such deterioration were removed from their experimental group and the trial, and reassigned to more appropriate treatment or received aggressive treatment to manage the disease and eliminate the infection. The effect of treatment was determined on clinical cure rate, bacteriological cure rate up to 21 d posttreatment, quarter milk cell count, milk yield, and systemic consequences. The work was conducted in three phases because of the physical limitations of the experimental facilities. In each phase, two quarters of each of 18 cows were infused to obtain sufficient quarters allocated to each test group. The infection model worked sufficiently in all phases, with 95% quarters exposed becoming infected. In phases 1 and 2, four treatment groups (a through d) were used. Initially, quarters were assigned at random on detection of mastitis to either no treat-
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ment, aggressive intramammary treatment only (6 × 12-h infusion of Leo Yellow, Leo Animal Health, Princes Risborough, UK), recommended dose of parenteral treatment only (3 × 24-h intramuscular injection Pen and Strep (Norbook Laboratories (GB) Ltd, Carlisle, UK) or a combination of aggressive intramammary and parenteral treatment. These treatments were appropriate, as the strain of S. uberis used is completely sensitive to the penicillins used. Leo Yellow syringes each contain 150 mg of penethemate hydroiodide, 150 mg of dihydrostreptomycin sulfate, 50 mg of framycetin sulfate, and 5 mg of prednisolone in an ointment base. The recommended dose is to infuse one syringe every 24 h for 3 d. The withholding time for human consumption is 84 h for milk and 28 d for meat. The Pen & Strep contains 200 mg of procaine penicillin and 250 mg of dihydrostreptomycin sulfate per milliliter. The recommended dose is 1 ml per 25 kg of body weight. The withholding time for human consumption is 60 h for milk and 18 d for meat. Allocations were made to make the first three groups of approximately equal size. Those receiving no treatment, on detection of disease, were assessed closely. Allocations were not always at random because allocation for one quarter only to a group receiving parenteral treatment meant that a case of mastitis occurring later could not be allocated to a group not receiving systemic treatment. There were no reassignments of animals or quarters to another treatment group. Where treatment was deemed to have failed, then subsequent action was outside the trial. In phase three, any quarter with clinical mastitis was allocated to treatment with intramammary antibiotic, intramuscular oxytocin, or intramuscular oxytocin plus intramammary antibiotic. The intramammary antibiotic was Leo Yellow at label recommended use (3 × 24 h). Intramuscular oxytocin (Intervet Ltd, Cambridge, UK) comprised 8 ml (80 i.u.) at the first milking on cluster removal, and then the cluster was reattached after 2 min to remove all residual milk. At each of the subsequent five milkings only 2 ml (20 i.u.) oxytocin was used. This dose regimen is that recommended on the datasheet for mastitis. There is no milk or meat withholding time for oxytocin. Treatments involving oxytocin were effectively allocated to the cow as the treatment is systemic and so both quarters had to show signs of disease at allocation. This was usual as 95% quarters were infected. Clinical cure was first assessed after 3 d unless cows were removed from the trial on welfare grounds. Clinical cure was when milk was of normal appearance and all or most of any udder swelling or firmness had dissipated. If there was no clinical cure, then the treatment was continued until clinical cure resulted or up to 6 d
in total, whichever was shorter. Clinical failure was deemed for any quarter showing no improvement in clinical signs after 6 d treatment or on removal of the cow from the trial on welfare grounds. Bacteriological cure was assessed from examination of milk samples at 14 and 21 d after the last treatment and cell count recovery from samples taken 7, 14, and 21 d after the last treatment. Cell-count recovery rate was defined as the proportion of quarter milk cell counts returning to fewer than 400,000 cells/ml. Several samples were also examined 7, 8, and 9 d after the last of the extended treatments to determine if milk was free of β-lactam inhibition and hence, if a clinical cure had occurred, the milk was fit for sale. The commercial BetaStar kits (UCB-Bioproducts S.A., Braine l’Alleud, Belgium) were used. An aliquot of milk (0.2 ml) was incubated for 3 min at 47.5°C with β-lactam receptors then the reaction determined chromatographically on a dipstick after a further 2 min incubation. Bacterial recovery was determined by incubation of 50 µl of milk on esculin blood agar at 37°C for 48 h; cell count was measured by the Fossomatic method; both were standard International Dairy Federation methods as described fully by Milner et al. (1997). Data were analyzed by Fisher’s exact test using SAS (1999, version 8), with the advice and assistance of the Statistical Services Unit, University of Reading. All comparisons used quarter as the experimental unit as they are assumed to be independent both in response to bacteria and treatment. In each phase of the experiment, comparisons were made between treatments for clinical and bacteriological cure for 3 and 6 d of treatment. Comparisons were not made between phases or between times. RESULTS AND DISCUSSION In phases 1 and 2, all 11 quarters left untreated worsened significantly within 3 d, no clinical cure was achieved (Table 1), and the cows were removed from the trial to treat the mastitis. Thus, not treating S. uberis mastitis and awaiting a spontaneous cure cannot be recommended. In comparison, the aggressive, off-label intramammary treatment using one syringe per quarter at each milking, gave a 70% clinical cure in 3 d and 100% after up to 6 d of treatment, but antibiotic usage was up to four times the labeled recommendation. The average treatment period was 3.7 d (7.3 syringes) to achieve the 100% clinical cure. Overall, parenteral treatment achieved a significantly poorer clinical cure rate of 18% after 3 d (P < 0.005) but did cure 91% within 6 d of treatment. This was similar to the amount and speed of clinical cure Journal of Dairy Science Vol. 85, No. 4, 2002
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HILLERTON AND KLIEM Table 1. The clinical cure achieved for each of seven treatments of Streptococcus uberis mastitis. Clinical cure2 Treatment1
No. quarters
3 days (%)
6 days
Total
% cured in total
None
Mean no. treatments
Mean time (d)
No treatment (NT) Intramammary (A) Parenteral (P) Combined (A + P) Intramammary (L) Oxytocin (O) Combined (O + L)
11 10 11 18 11 10 10
0 (0)a 7 (70)b 2 (18)a 11 (61)b 3 (27)e 0 (0)f 0 (0)f
0 3 8 7 7 0 1
0a 10b 10b 18b 10e 0f 1f
0 100 91 100 91 0 10
11 0 1 0 1 10 9
... 7.3 4.2 6.8 (syringes) 4 ... ...
... 3.7 4.2 3 4 ... ...
1 Where L(abel) and P(arenteral) were treatments every 24 h and A(ggressive) and O(xytocin) were treatments every milking. The first four treatments were examined in phases 1 and 2 of the experiment and the last three treatments were examined in phase 3 of the experiment. 2 Statistically significant differences (P < 0.001) on the amount of clinical cure within each column and only within each section, i.e, phase of the experiment, of the table are shown by use of different superscripts (a vs. b and e vs. f). No comparisons across columns have been made.
with the labeled daily dosage of intramammary antibiotic treatment. In phases 1 and 2, five quarters treated with the combination of antibiotics were cured clinically after 3 d. Later, use of this combined treatment on a further group of animals increased the sample size to 18 quarters. For that combination strategy, 61% of quarters were clinically cured in 3 d and 100% within 6 d, not different from cure rates for the aggressive intramammary treatment. Inclusion of these extra data obtained outside of the trial, using the same model, seems realistic and appropriate to avoid bias from a small sample size. The best clinical cure rate in amount and speed was achieved by either the aggressive intramammary treatment (after every milking) or the combination of aggressive intramammary treatment and parenteral antibiotic (after every second milking). This suggests that the parenteral treatment makes no effective contribution to the amount of cure achieved by intramammary treatment. The similar clinical cure rate for parenteral treatment alone, within 6 d, suggests that the value of the intramammary route is the speed of effect in that the cure rate within 3 d favored the intramammary treatment. All quarters cured clinically by any of the treatments were assessed for bacteriological cure by examining milk samples after 14 and 21 d. Cure required that S. uberis was not isolated from either sample. The bacteriological cure achieved was calculated first for those quarters cured clinically and then for all quarters allocated to treatment (Table 2). The overall bacteriological cure rate was lowest for labeled intramammary treatment and highest by aggressive intramammary treatment. For only those quarters cured clinically, it varied between 70 and 80%. Quarters cured clinically earlier tended to have a higher bacteriological cure rate. The only contrary result was Journal of Dairy Science Vol. 85, No. 4, 2002
with parenteral treatment alone when clinical cure required extended treatment while bacteriological cure was at 80%. This supports the suggestion that the advantage of intramammary treatment is speed of effect. The cell-count recovery rate, defined as the return of the quarter milk cell count to fewer than 400,000 cells/ ml, was determined only for those quarters cured clinically and bacteriologically. All other quarters can be considered failures, and so the total failure rate was actually much higher. The number of quarters included was lower than the total eligible as data from some cows were unavailable because of an infection in another quarter, end of lactation, or culling. Cell-count recovery was slow and was always incomplete; 80% of quarters recovered following aggressive intramammary treatment, 73% of quarters recovered by the same time after parenteral treatment, while 94% of eligible quarters treated with the combination of antibiotics showed a cell-count recovery within 21 d. Failure of the cell count to recover is not due to persistent infection but is most likely a measure of residual damage to the mammary epithelial tissue. Use of oxytocin to ensure complete milk removal from the infected gland had no effect in resolution of clinical signs (Table 1); indeed, the clinical signs appeared to worsen. Treating quarters from which any residual milk had been removed, with intramammary antibiotic, only resulted in one of 10 quarters being cured. This was significantly worse than simply using label-recommended intramammary treatment. This confirms that use of oxytocin in the treatment of S. uberis mastitis has no value, as also shown by Hillerton and Semmens (1999). Differing levels of cure, clinical, bacteriological, and cell count, have been achieved at differing costs of time and/or amount of antibiotic used. The intramammary and parenteral treatments required very different
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OUR INDUSTRY TODAY Table 2. The amount of bacteriological cure (for those quarters cured clinically) achieved for each of seven treatments of Streptococcus uberis mastitis. Bacteriological cure2
Treatment1 No treatment (NT) Intramammary (A) Parenteral (P) Combined (A + P) Intramammary (L) Oxytocin (O) Combined (O + L)
No. quarters clinically cured 0 10 10 18 10 0 10
Effective cure after treatment for 3d a
0 6b 0a 11b 3 0 0
6d 0 2 8 2 4 0 1
Total cure (%) a
0 80b 80b 72b 70e 0f 10f
No cure
% Cured of all quarters infected
10 2 2 0 3 10 9
0 80 73 72 64 0 10
1 Where L(abel) and P(arenteral) were treatments every 24 h and A(ggressive) and O(xytocin) were treatments every milking. The first four treatments were examined in phases 1 and 2 of the experiment and the last three treatments were examined in phase 3 of the experiment. 2 Statistically significant differences (P < 0.005) on the amount of bacteriological cure within each column and only within each section, i.e. phase of the experiment, of the table are shown by use of different superscripts (a vs. b and e vs. f). No comparisons across columns have been made.
amounts of antibiotic to be applied. Given that only 0.001% of penicillin applied systemically diffuses into the mammary gland but that procaine penicillin concentrates in the mammary gland over time (Debackere, 1995), it becomes clear that more antibiotic is available more quickly when applied by the intramammary route. Each quarter treated by intramammary infusion received on average 900 mg of penicillin equivalent, while 4 d of parenteral treatment comprised 20 g of penicillin. Estimating one recurrence in every quarter not cured bacteriologically suggests that daily intramammary treatment requires 1 g to cure each case, aggressive intramammary treatment 900 mg to cure each case, parenteral treatment requires 22 g to cure each case and the combined treatment 15.9 g to cure each case. Aggressive intramammary treatment was the cheapest in terms of least amount of antibiotic used. Overall, lost milk is the largest part of the cost of mastitis. The cost of antibiotic has been estimated as only 7% of the total cost of a case of mastitis (Kossaibati and Esslemont, 1997). Thus, the aggressive intramammary treatment appears the most cost effective because of the speed of effect. It was also the most effective in animal welfare, as there was minimal recurrence of disease. In the United Kingdom, any use of antibiotic to treat mastitis contrary to the label recommendation of the product requires milk to be withheld for 7 d after the last treatment. Quarter milks were tested for inhibitory substances from d 7. This showed that, in two of 10 quarters (two different cows) receiving intramammary treatment only and for 6 of 11 quarters (four different cows) receiving parenteral antibiotic, milk contained inhibitory substances for a maximum of 8 d after the last treatment. It is recommended that, following any extended antibiotic treatment for mastitis, milk be
withheld until achieving a negative inhibitory test for presence of inhibitory substances. CONCLUSIONS An aggressive use of intramammary antibiotic treatment at every milking appeared to be optimum, although not different overall in clinical or bacteriological cure from the other treatments. It resulted in a faster cure than daily treatment or parenteral treatment. It used significantly less antibiotic than parenteral treatment alone or the combined antibiotic treatment for a similar effect. On the basis of this study, it is important to use the correct amount of antibiotic applied by the correct route and for the correct time, if a complete cure is to be achieved. This study appears to justify the apparent use of aggressive or extended intramammary treatment by commercial dairy farmers. No obvious additional benefit was found from use of parenteral antibiotic alone or in combination. Therefore, reduction in the amount of antibiotic used to treat mastitis can best be obtained by avoiding use of parenteral treatment for S. uberis mastitis. Oxytocin appeared to be ineffective as an aid to cure S. uberis mastitis. ACKNOWLEDGMENTS The Ministry of Agriculture, Fisheries and Food (now the Department of the Environment, Food and Rural Affairs) funded this study. Zoe Penrose provided excellent technical help. REFERENCES Biggs, A. 1998. Mastitis therapy on farm–keeping up with the moving goalposts. Pages 15–21 in Proc. British Mastitis Conf., Axient/ Institute for Animal Health/Novartis/MDC. Journal of Dairy Science Vol. 85, No. 4, 2002
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Booth, J. M. 1997. The five-point control plan: Its impact and future. Pages 3–9 in Proc. British Mastitis Conf., Axient/Institute for Animal Health/Novartis/MDC. Debackere, M. 1995. Pharmacokinetics and pharmacodynamics of antimicrobials in relation to their residues in milk. Pages 41–53 in Residues of antimicrobial drugs and other inhibitors in milk. International Dairy Federation, Brussels, Belgium. Erskine, R. J. 1998. Making mastitis treatment decisions. Pages 9– 14 in Proc. British Mastitis Conf., Axient/Institute for Animal Health/Novartis/MDC. Erskine, R. J., R. C. Wilson, J. W. Tyler, K. A. McClure, R. S. Nelson, and H. J. Spears. 1995. Ceftiofur distribution in serum and milk from clinically normal cows with experimental Escherichia coliinduced mastitis. Am. J. Vet. Res. 56:481–485. Hillerton, J. E., and J. E. Semmens. 1999. Comparison of treatment of mastitis by oxytocin or antibiotics following detection by changes in milk electrical conductivity and prior to visible signs. J. Dairy Sci. 82:93–98.
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Hillerton, J. E., A. J. Bramley, R. T. Staker, and C. H. McKinnon. 1995. Patterns of infection over a 5-year period in a closely monitored herd applying mastitis control measures. J. Dairy Res. 62:39–50. Kossaibati, M. A., and R. J. Esslemont. 1997. The costs of production disease in dairy herds in England. Vet. J. 153:1–11. Lacey, R. W. 1980. Rarity of gene transfer between animal and human isolates of Staphylococcus aureus in vitro. J. Gen. Microbiol. 119:437–442. Milner, P., K. L. Page, and J. E. Hillerton. 1997. The effects of early antibiotic treatment following diagnosis of mastitis detected by a change in electrical conductivity of milk. J. Dairy Sci. 80:859–863. Saran, A. 1995. Intramammary and systemic antibiotic treatment in lactating and dry cows. Pages 85–96 in Residues of Antimicrobial Drugs and Other Inhibitors in Milk. International Dairy Federation, Brussels, Belgium. SAS. 1999. Version 8.0. SAS Institute Inc., Cary, NC.
