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Efficacy of Intramuscular Oxytetracycline as a Dry Cow Treatment for Staphylococcus aureus Mastitis
Efficacy of Intramuscular Oxytetracycline as a Dry Cow Treatment for Staphylococcus aureus Mastitis R. J. ERSKINE, P. C. BARTLETT, P. C. CRAWSHAW, and D. M. GOMBAS Department of Large Animal Clinical Sciences Michigan State University
East Lansing 48824 ABSTRACT
To determine the efficacy of intramuscular oxytetracycline as a supplemental dry cow treatment for Staphylococcus aureus mastitis, 37 Holstein cows were randomly assigned to two treatment groups: intracisternal infusion with a commercial preparation of cephapirin benzathine at drying off (20 cows) and infusion with cephapirin benzathine at drying off and intramuscular oxytetracycline at 11 mgkg once daily on d 7, 8, 9, and 10 after drying off (17 cows). Milk samples collected 7, 14, 30, and 60 d after calving were plated for bacterial isolation within 24 h after collection and after 24 to 72 h of storage at -2022. Quarters were defined as infected if S. aureus was isolated from the fresh and frozen cultures from any one sample collected before drying off. An infected quarter was defined as cured if S. aureus was not isolated from the fresh or frozen culture from milk samples obtained following calving. The rate of cure by 30 d after calving for systemic oxytetracycline (in combination with cephapirin treatment) was 29.4% for infected quarters and 29.4% for infected cows, compared with 27.5 and 25.0%.respectively, for the cephapirin treatment only. Results including the culture at 60 d after calving were 21.2 and 22.5%, respectively, for combination therapy and cephapirin therapy only. Systemic oxytetracycline, in combination with intramammary dry cow treatment, did not improve the rate of cure for S. aureus mastitis.
Received February 22. 1994. Accepted May 31, 1994. 1994 J Dairy Sci 77:3347-3353
(Key words: Staphylococcus aureus, mastitis, dry cow treatment, oxytetracycline) Abbreviation key: CEPH = cephapirin benzathine, DCT = dry cow treatment, MH = Mantel-Haenszel, OXY = oxytetracycline. INTRODUCTION
Dry cow treatment (DCT) has been accepted as an integral part of mastitis control for over 25 yr (11). Benefits of DCT include the elimination of existing IMI and prevention of new IMI during the dry period. Therapeutic cure rates for Sraphylococcus aureus IMI during the dry period were typically reported in initial studies to be about 75% (3, 4, 8, 18, 19, 23, 26, 29). However, this degree of therapeutic success may vary among herds, ranging from 25 to 75%, and averaging 50% (13, 25). Soback et al. (24) determined that cure rates for quarters infected by S. aureus and treated with intramammary cephapirin (30.8%) did not differ from those for cows receiving no DCT (33.3%).Additionally, Cummins et al. (4)determined that DCT with cloxacillin at drying off or a second treatment 2 wk after drying off did not increase the cure rate for coagulasepositive staphylococcal IMI, including IMI caused by S. aureus, compared with that for cows receiving no DCT. Systemic antimicrobials have been administered to enhance the therapeutic success of intramammary infusions as a treatment for S. aureus mastitis. For lactating cows, intramuscular procaine penicillin G and oxytetracycline (OXY)administered in conjunction with commercial intramammary antibiotics improved the cure rate of S.aureus IMI (17, 25). Subcutaneous norfloxacin nicotinate, administered at drying off, increased cure rates for S. aureus IMI compared with that for untreated cows or for cows ad-
3347
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ERSKlNE ET AL.
ministered intramammary preparations of cephapirin benzathine (CEPH) (24). A single dose of intramuscular OXY did not increase cure rate but reduced the new IMI rate during the dry period compared with those for untreated or CEPH-treated cows (24). The purpose of this study was to determine the efficacy of systemic OXY as an adjunct to DCT for cows with S. aureus mastitis. The OXY was selected as a supplemental DCT because of favorable pharmacokinetics in the mammary gland and the relatively inexpensive cost of treatment. MATERIALS AND METHODS
A separate milk sample from each quarter was collected from all cows that were approaching the dry period in two Holstein dairies with known high prevalence of S. aureus IMI. Samples were aseptically collected 3 wk before, 1 wk before, and at the last milking before drying off. Samples were then transported to the laboratory on ice, refrigerated at 4"C,and cultured for bacterial isolation within 24 h after collection. Following the initial culture, the samples were stored at -2O'C for 24 to 72 h, thawed at 23°C and cultured again. Each quarter milk sample (10 pl) was inoculated on one-fourth of a plate of trypticase soy agar with 5% sheep blood. Quarters were considered to be infected if S. aureus was isolated from the fresh and frozen cultures of any one milk sample taken before drying off. Cows that were determined to have at least 1 quarter infected with S. aureus were assigned to OXY or control DCT according to computer-randomized assignment lists: 1) first lactation, 2) second and greater lactations, 3) instances in which only 1 quarter was infected, and 4) instances in which more than 1 quarter was infected. One group of 20 cows (40 infected quarters) was infused with a commercial preparation of 300 mg of CEPH (Tomorrow@; Franklin Laboratories, Inc., Fort Dodge, IA) in all 4 quarters at drying off; the second group of 17 cows (34 infected quarters) was also infused with CEPH but also administered intramuscular OXY (Liquamycin LA-200@; Pfizer, Inc., New York. NY) at 11 mg/kg once daily on d 7, 8,9, and 10 after drying off. Prior to any treatments, S. aureus isolates were collected from quarter samples of 3 cows from Journal of Dairy Science Vol. 77, No. 11, 1994
each herd, and the isolates were determined to be susceptible in vitro to cephalothin and tetracycline (1). Quarter milk samples were again collected at 7, 14, 30, and 60 d after calving. An infected quarter was defined as cured at 30 d after calving if no S. aureus were isolated from fresh and frozen culture from a sample within 30 d following calving. Similarly, an infected quarter was defined as cured at 60 d after calving if no isolation of S. aureus occurred from a sample within 60 d after calving. New IMI during the dry period were determined to occur when a quarter was not infected before drying off but yielded S. aureus from fresh and frozen culture of a sample collected after calving. Statistical Analysis
Because both treatments, OXY or control, and the outcome variable, cure or not cured, were dichotomous, Mantel-Haenszel (MH) chisquare analysis was used to evaluate statistical significance and to estimate relative risk. Separate analyses were performed for microbiologic cure determined at 30 and 60 d following calving. Treatment was compared with cure by MH chi-square analysis; quarter was used as the experimental unit. Relative risk and Taylor series 95% confidence limits for relative risk and the significance of the MH ch-square value were reported. Because the number of infected quarters per cow was associated with cure rate, this potential confounder was included in an MH chisquare analysis that compared treatment group with cure in each of three strata: 1) infected quarters, when the other three quarters were not infected; 2) infected quarters, when two quarters on the same cow were infected; and 3) infected quarters, when three or more quarters of the same cow were infected. Because parity was also suspected as a confounding factor, the results were also analyzed with adjustment for two-parity strata (first lactation and older cows). The results of the stratified analyses were reported as MH weighted odds ratio with Cornfield 95% confidence limits and significance of MH summary chi-square values. The data were also analyzed by simple chisquare; the 37 cows, rather than the 74 quarters, were the experimental units, and the two
3349
OXYTETRACYCLINE AS A DRY COW TREATMENT
TABLE 1. Frequency of cure 30 d after calving for quarters infected with Sfaphylococcus oureus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and slratified by number of quarters infected at drying off. Quarters infected at drying off1 1
2 3 4 Total
Quarter cure rate 30 d postcalving CEPH (no.) 2 of 6 of 1 of 2 of 1 1 of
(%)
9 12 3 16 40
22.2 50.0 33.3 12.5 27.5
CEPH
+ OXY
(no.) 4 of 4 of 2 of 0 of 10 of
6 14 6 8 34
Total (%)
66.7 28.6 33.3 0 29.4
(no.) 6 of 10 of 3 of 2 of 21 of
(%)
15 26 9 24 74
40.0 38.5 33.3 8.3 28.4
~
lThe number of quarters infected with S aureus at drying off was determined for each cow in the trial before assignment of treatment group.
treatment groups were compared by the cure rate (all four quarters negative) at 30 and 60 d postcal ving .
the association between cure rate and the number of infected quarters per cow, the MH analysis was repeated with adjustment for possible confounding by this variable. The resultant MH weighted odds ratio was 1.03; confidence RESULTS limits were .31 and 3.36 (P = 33). An MH analysis was also performed with adjustment 304 Cure Rates for parity (first lactation or older cows) as the Systemic OXY did not improve the fre- confounding variable (Table 2). The MH quency of cure for quarters infected with S. weighted odds ratio was 1.07, confidence aureus during the dry period when cure rate limits were .34 and 3.34, and MH summary was assessed after 30 d postcalving (Table 1). chi-square was P = .90. In the OXY treatment group (34 quarters), 10 cures (29.4%) were achieved; in the control 60-d Cure Rates group (40 quarters), 11 cures (27.5%) were When the 60-d culture was also considered, achieved. The relative risk was 1.05; 95% confidence limits were .61 and 1.80 (P = 36). 21.2% (7 of 33) of the OXY quarters were Quarters from cows with 1 to 3 infected quar- considered to be cured, compared with 22.5% ters infected had a significantly higher cure (9 of 40)of the control quarters (relative risk = rate of 38%, 19 of 31, compared with 8.3%, 2 .96; confidence limits = SI and 1.79; P = .89; of 24, for quarters from cows for which all 4 Table 3). Adjustment for three strata regarding quarters were infected (P = .0085). Because of number of quarters infected per cow yielded an TABLE 2. Frequency of cure 30 d after calving for quarters infected with Staphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPIT) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by parity.
.
Quarter cure rate 30 d postcalving
_.. .~
number'
CEPH
1 2 23 Total
(no.) 1 of 2 of 8 of 11 of
(45)
5 8 27 40
20.0 25.0 29.6 27.5
CEPH
+ OXY
(no.) 3 of 4 of 3 of 10 of
6 14 14 34
Total (46)
50.0 28.6 21.4 29.4
(no.) 4 of 6 of 11 of 21 of
(S) 11 22 41 74
36.4 27.3 26.8 28.4
lThe number of quarters infected with S. aureus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
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ERSKINE ET AL.
TABLE 3. Frequency of cure 60 d after calving for quarters infected with Sruphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Quarters infected at drying off' 1
2 3 4 Total
Quarter cure rate 60 d postcalving CEPH (no.) I Of9 5 of 12 1 of 3 2 of 16 9 of 40
CEPH
+ OXY
(no.) 2 of 5 3 of 14 2 of 6 0 of 8 7 of 33
11.1 41.7 33.3 12.5 22.5
Total
(W 40.0 21.4 33.3 0 21.2
(no.) 3 of 8 of 3 of 2 of 16 of
(%)
14 26
9 24 73
21.4 30.8 33.3 8.3 21.9
~~
'The number of quarters infected with S. aureus at drylng off was determined for each cow in the trial before assignment of treatment group.
MH weighted odds ratio of .85 and confidence limits of .23 and 3.04 (P = .W). The MH analysis with adjustment for parity (first lactation or older cows) as the extraneous variable (Table 4) yielded an MH weighted odds ratio of .77 (confidence limits = .21 and 2.69; MH summary chi-square, P = .86). The incidence of new S. uureus IMI during the dry period was 2 of 40 quarters in the control group compared with 1 of 34 quarters in the OXY group. Although sample size was small, this finding suggests that CEPH and OXY were no more effective than CEPH alone in preventing quarters that were uninfected at drying off from becoming infected sometime during the dry period or subsequent early lactation.
postcalving compared with 29% (5 of 17) of the cows treated with OXY (P = .76; relative risk = 1.13; confidence limits = .53 and 2.38). When samples taken 60 d postcalving were also considered (Table 6), cure rate was 15% (3 of 20 cows) for controls and 12.5% (2 of 16 cows) for cows treated with OXY (P = .83; relative risk = .89; confidence limits = .28 and 2.77). DISCUSSION
Culling is perhaps the most effective method for eliminating existing S. aureus IMI in a dairy herd (6). However, the need to cull infected cows for mastitis control can be limited by the need to maintain ample milk production for farm cash flow. Culling must be judicious with respect to production, reproduccows Cured tive performance, genetic potential, and other Analysis with cows as the unit of observa- health problems. Therapy is an alternative tion (Table 5 ) showed that 25% (5 of 20) of the method of eliminating IMI; but results are control cows had a microbiologic cure by 30 d usually poorer for IMI caused by S. aureus TABLE 4. Frequency of cure 60 d after calving for quarters infected with Sruphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPH)or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by parity. Quarter cure rate 60 d postcalving
Lactation number1
CEPH
1 2 23 Total
(no.) 1 of 1 of 7 of 9 of
CEPH (%)
5 8 27 40
20.0 12.5 25.9 22.5
(no.) 3 of 2 of 2 of 7 of
+ OXY
Total
(53 6 14 13 33
50.0 14.3 15.4 21.2
(no.) 4 of 3 of 9 of 16 of
(%)
11 22 40 73
36.4 27.3 26.8 21.9
'The number of quarters infected with S. uureus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
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OXYTETRACYCLINE AS A DRY COW TREATMENT
TABLE 5. Frequency of cure 30 d after calving for cows infected with Sraphylococcus aurcus during the dry period following treatment with intramammary cephapirin (CEPH) or intnunammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Cow cure rate 30 d postcalving
Quarters infected at drying off1
CEPH
1 2 3 4 Total
(no.) 2 of 3 of 0 of 0 of 5 of
9 6 1 4
20
CEPH
+ OXY
Total
(%)
(no.)
(%)
22.2 50.0 0 0 25.0
4 of 6
66.7 21.4 0 0 29.4
1 Of7 0 of 2 0 of 2 5 of 17
(no.) 6 of 4 of 0 of 0 of 10 of
(94 15 13 3 6 37
40.0 30.8 0 0 27.0
'The number of quarters infected with S. aurcus at drying off was determined for each cow in the trial before assignment of treatment group.
than by other pathogens because of a variety of virulence factors, including intracellular survival in phagocytes, microabscess formation, P-lactamase, and L-form induction (2, 13, 15, 16, 20, 21). Our objective in this study was to test a therapy that had a high probability of cure efficacy and was inexpensive. The DCT was considered because drug treatment of nonlactating cows allows more sustained regimens than would be practical for lactating cows because of concerns about drug residues in milk. Additionally, numerous studies determined that DCT cure rates were 65 to 75% for S. aureus IMI (3, 8, 18, 19, 25, 26, 29), which was higher than that typically reported for lactating cow therapy (12, 17, 28). The OXY was tested as a supplemental DCT for four reasons: 1) a majority of S. aureus isolates are sensitive to tetracyclines in
vitro (9, 10); 2) the lipophilic nature and relatively long pharmacokinetic half-life of OXY allows effective concentrations in mammary tissue (24); 3) tetracyclines are not detrimental to in vitro neutrophil phagocytosis or viability (7, 14); and 4) OXY is commercially available for nonlactating cows, although not approved for mastitis. Additionally, Sol et a]. (25) determined that 11 mgkg of OXY administered in conjunction with a commercial intramammary antibiotic improved the cure rate of S. aureus M I in lactating cows. In the present study, systemic OXY administered subsequent to intramammary antimicrobials did not affect the rate of cure or the incidence of new Ih4I during the dry period. Our results are in agreement with more recent studies suggesting that the probability of DCT enhancing the cure of S. aureus IMI
TABLE 6. Frequency of cure 60 d after calving for cows infected with Staphylococcus aurcus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Quarters infected at dryiig off* 1
2 3 4
Total
Cow cure rate 60 d postcalving1 CEPH (no.) 1 Of9 2 of 6 0 of 1 0 of 4 3 of 20
CEPH (96) 11.1
33.3 0 0 15.0
+ OXY
(no.) 2 of 5 0 of I 0 of 2 0 of 2 2 of 16
(%)
40.0
0 0 0 12.5
Total (no.) 3 of 2 of 0 of 0 of 5 of
(W 14
13 3 6 36
21.4 15.4 0 0 13.9
lone cow was culled between 30 and 60 d postcalving. 2The number of quarters infected with S. aurcus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
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ERSKINE ET AL.
during the dry period is lower than that reported in earlier studies (4, 24). Our study did not find any benefit from systemic OXY in the treatment of S. aureus IMI, which is in disagreement with previous studies (17, 24, 25) that reported a beneficial effect of systemic antibiotics. However, two of the previous studies were performed on lactating cows, one of which (17) involved experimental IMI (17, 25). In the other previous study (24), OTC did not increase the cure rate but decreased the incidence of new IMI during the dry period. One explanation for the relatively poor therapeutic success of DCT in the present and in other more recent studies could be the development of bacterial resistance to antimicrobials. This possibility is difficult to assess because S. aureus isolates typical of present IMI cannot be directly compared with those of 25 yr ago. However, Mackie et al. (9) determined that the percentage of S. aureus mastitis isolates that are susceptible in vitro to commonly used antimicrobials did not decrease from 1984 to 1987, and >85% of S. aureus isolates were susceptible to antimicrobials that did not have a structure vulnerable to P-lactamase (9, 10). Intramammary DCT have been routinely used in our study herds for several years. Thus, three S. aureus isolates were selected from each herd prior to the start of the trial to ensure in vitro susceptibility to CEPH and OXY. Because of the contagious nature of S. aurew IMI, such a sampling of isolates was expected to allow a reasonable assessment of the antimicrobial susceptibility pattern of this pathogen in these herds. Nonetheless, in vitro susceptibilities may not necessarily reflect the ability of antimicrobials to effect a cure in vivo, particularly in the mammary gland of a cow. The herds in this study had persistent problems with S. aureus mastitis and a high proportion of chronic IMI. The pathologic changes caused by IMI are probably the primary determinants in therapeutic failure in treatment of S. aureus IMI. This concept was supported by Newbould (12), who determined that quarter cure rates decreased from 87.5 to 62.5% as the duration of IMI increased from 3 to 60 d for experimental IMI. Thus, DCT of S. aureus IMI may be efficacious in some herds, although success may be limited in herds with a high proportion of chronically infected cows. Journal of Dairy Science Vol. 77. No. 11. 1994
Partly because of cyclic shedding of bacteria from infected glands, isolation of S. aureus from milk samples by routine bacteriologic methods can be erratic (22). The sensitivity of a .l-ml inoculum of a single quarter milk sample to determine IMI status was 74.5% but increased to 94 and 98% when second and third samples were included, respectively (22). Duplicate quarter samples collected at the same milking, compared with single quarter samples, do not improve the ability to isolate this bacteria (5). Additionally, freezing of composite milk samples prior to culture increased the frequency of isolation of Streprococcus agalacriae 2.5 times and S. aureus 1.48 times (27). Thus, in an effort to enhance sensitivity of IMI detection and to decrease the probability of false-positive results, we selected consecutive sampling with fresh and frozen cultures. Several previous DCT studies (7, 18, 26, 29) did not utilize repeat sampling to identify infected quarters before or after the dry period. However, whether this factor contributed to the differences in cure efficacy compared with those in our study is questionable because >90% of all S. aureus IMI identified prior to drying off were positive at all three samplings, and 82% of IMI detected after calving were positive at all four samplings. The repeatability of bacterial isolation may have been a function of the chronicity of IMI in the trial herds. Newbould (12) reported that recurrences of isolation of bacteria following treatment of S. aureus IMI in lactating cows could occur up to 28 d and therefore recommended a minimum of 3 wk after treatment, and preferably 30 d, of no bacterial isolation as a criterion for cure. To account for this recommendation and for the cyclic shedding of bacteria in milk during S. uureus IMI, we chose to sample for up to 60 d to determine a cure, thus allowing a conservative assessment of cure rates, although only a small proportion of cured quarters at 30 d were positive for S. aureus at 60 d. New IMI could have occuned during the interval between samples; however, the net result for the herd was that the cow continued to have an S. aureus IMI, despite DCT. CONCLUSIONS
Intramuscular OXY administered subsequent to administration of intramammary
OXYTETRACYCLINE AS A DRY COW TREATMENT
CEPH did not affect the rate of cure or the incidence of new S. aureus l M I during the dry period of dairy cows. Rates of cure were unaffected by parity but were affected by number of quarters infected at drying off. REFERENCES 1 Bauer, A. W.. W.M.M. Kirby, and J. C. Sherris. 1966.
Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493. 2Bramley. J. A,, and F. H. Dodd. 1984. Mastitis control-progress and prospects. I. Dairy Res. 51:481. 3 Clegg, F. G.. G. J. Halliday, and H. Hardie. 1975. Dry cow therapy: a comparative field trial using benzathine cloxacillin and erythromycin. Br. Vet. J. 131: 639. 4 Cummins, K. A,, and T. A. McCaskey. 1987. Multiple infusions of cloxacillin for treatment of mastitis during the dry period. J. Dairy Sci. 70:2658. 5Erskine. R. J., and R. J. Ebehart. 1988. Comparison of duplicate and single quarter milk samples for the identification of intramammaty infections. J. Dairy Sci. 71:854. 6 Fox, L. K., and D. D. Hancock. 1989. Effect of segregation on prevention of intramammary infections by Staphylococcus aureus. J. Dairy Sci. 72540. 7 Glette, J., S. Sandberg, G. Hoppen. and C. 0. Solberg. 1984. Influence of tetracyclines on human PMN leukocyte function. Antimicrob. Agents Chemother. 25: 354. 8 Hill, G. N., and T. 1.Keefe. 1974. Clinical efficacy of benzathine cloxacillin in dry cow mastitis treatment. Mod. Vet. Pract. 55843. 9 Mackie, D. P., E. F. Logan, D. A. Pollock, and S. P. Rodgers. 1988. Antibiotic sensitivity of bovine staphylococcal and coliform mastitis isolates over four years. Vet. Rec. 123515. 10Matthews. K. R., S . P. Oliver, and B. M. Jayarao. 1992. Susceptibility of staphylococci and streptococci isolated from bovine milk to antibiotics. A&-Practice 13:18. 11 Neave, F. K., F. H. Dodd. and R. G. Kingwill. 1966. A method of controlling udder disease. Vet. Rec. 78: 521. 12Newbould. F.H.S. 1974. Antibiotic treatment of experimental Staphylococcus ourew infections of the bovine mammary gland. Can.J. Comp. Med. 38:411. 13Nickerson. S. C. 1990. Mastitis and its control in heifers and dry cows. Page 82 in Roc. Int. Symp. Bovine Mastitis, Indianapolis, IN. Natl Mastitis Counc., Arlington, VA. 14 Nickerson, S. C., M. J. Pappe, and A. M.Dulin. 1985. Effect of antibiotics and vehicles on bovine mammary polymorphonuclear leukocyte morphologic features,
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viability, and phagocytic activity in vitro. Am. J. Vet. Res. 46:2259. 15 Owens, W. E. 1987. Isolation of Sraphylococcus aureus L-forms from experimentally induced bovine mastitis. J. Clin. Microbiol. 26:2187. 16 Owens, W. E. 1988. Evaluation of various antibiotics for induction of L-forms from Staphylococcus aureus strains isolated from bovine mastitis. J. Clin. Microbiol. 27: 1356. 17Owens, W. E., J. L. Watts, and R. L. Boddie. 1988. Antibiotic treatment of mastitis: comparison of intramammary and intramammary plus intramuscular therapies. J. Dairy Sci. 71:3143. 18 Postle, D. S., and R. P. Natzke. 1974. Efficacy of antibiotic treatment in the bovine udder as determined from field studies. Vet. Med. Small Anim. Clin. 69: 1535. 19 Schultze, W. D., and H. D. Mercer. 1976. Nonlactating cow therapy with formulation of penicillin and novobiocin: therapeutic and prophylactic effects. Am. J. Vet. Res. 37:1275. 20 Sears, P. M. 1993. Staphylococcus aureus mastitis. Page 4 in Proc. 32nd AMU. Mtg. Natl. Mastitis Counc. Natl. Mastitis Counc., Arlington, VA. 21 Sears, P. M.. M. Fettinger, and J. Salin-Marsh. 1987. Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis. J. Am. Vet. Med. Assoc. 191:681. 22Sears, P. M., B. S. Smith, and P. B. English. 1990. Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J. Dairy Sci. 73:2785. 23 Smith, A., D. R. Westgarth, M. R. Jones, F. K. Neave, and F. H. Dodd. 1967. Methods of reducing the incidence of udder infection in dry cows. Vet. Rec. 81:504. 24 Soback, S., G. Ziv, M. Winkler, and A. Saran. 1990. Systemic dry cow therapy-a preliminary report. J.
, and A. van Uum. 1990. Factors affecting the result of dry cow treatment. Page 1 I8 in Roc.Int. Symp. Bovine Mastitis, Indianapolis, IN. Natl. Mastitis Counc., Arlington, VA. 26Uvarov. O., J. M. Evans, M. Marshall, and D. Sizer. 1967. Dry cow therapy. Vet. Rec. 81:241. 27 Villanueva. M. R., J. W. Tyler, and M. C. Thurmond. 1991. Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk. J. Am. Vet. Med. Assoc. 198:1398. 28Wilson, C. D., N. Agger, G. A. Gilbert, C. A. Thomasson, and S. T. Trolling. 1986. Field trials with cefoperazone in the treatment of bovine clinical mastitis. Vet. Rec. 118:17. 29 Ziv, G., M. Storper, and A. Saran. 1981. Comparative efficacy of three antibiotic products for the treatment and prevention of subclinical mastitis during the dry period. Vet. Q. 3:74.
Journal of Dairy Science Vol. 77, No. 11, 1994
East Lansing 48824 ABSTRACT
To determine the efficacy of intramuscular oxytetracycline as a supplemental dry cow treatment for Staphylococcus aureus mastitis, 37 Holstein cows were randomly assigned to two treatment groups: intracisternal infusion with a commercial preparation of cephapirin benzathine at drying off (20 cows) and infusion with cephapirin benzathine at drying off and intramuscular oxytetracycline at 11 mgkg once daily on d 7, 8, 9, and 10 after drying off (17 cows). Milk samples collected 7, 14, 30, and 60 d after calving were plated for bacterial isolation within 24 h after collection and after 24 to 72 h of storage at -2022. Quarters were defined as infected if S. aureus was isolated from the fresh and frozen cultures from any one sample collected before drying off. An infected quarter was defined as cured if S. aureus was not isolated from the fresh or frozen culture from milk samples obtained following calving. The rate of cure by 30 d after calving for systemic oxytetracycline (in combination with cephapirin treatment) was 29.4% for infected quarters and 29.4% for infected cows, compared with 27.5 and 25.0%.respectively, for the cephapirin treatment only. Results including the culture at 60 d after calving were 21.2 and 22.5%, respectively, for combination therapy and cephapirin therapy only. Systemic oxytetracycline, in combination with intramammary dry cow treatment, did not improve the rate of cure for S. aureus mastitis.
Received February 22. 1994. Accepted May 31, 1994. 1994 J Dairy Sci 77:3347-3353
(Key words: Staphylococcus aureus, mastitis, dry cow treatment, oxytetracycline) Abbreviation key: CEPH = cephapirin benzathine, DCT = dry cow treatment, MH = Mantel-Haenszel, OXY = oxytetracycline. INTRODUCTION
Dry cow treatment (DCT) has been accepted as an integral part of mastitis control for over 25 yr (11). Benefits of DCT include the elimination of existing IMI and prevention of new IMI during the dry period. Therapeutic cure rates for Sraphylococcus aureus IMI during the dry period were typically reported in initial studies to be about 75% (3, 4, 8, 18, 19, 23, 26, 29). However, this degree of therapeutic success may vary among herds, ranging from 25 to 75%, and averaging 50% (13, 25). Soback et al. (24) determined that cure rates for quarters infected by S. aureus and treated with intramammary cephapirin (30.8%) did not differ from those for cows receiving no DCT (33.3%).Additionally, Cummins et al. (4)determined that DCT with cloxacillin at drying off or a second treatment 2 wk after drying off did not increase the cure rate for coagulasepositive staphylococcal IMI, including IMI caused by S. aureus, compared with that for cows receiving no DCT. Systemic antimicrobials have been administered to enhance the therapeutic success of intramammary infusions as a treatment for S. aureus mastitis. For lactating cows, intramuscular procaine penicillin G and oxytetracycline (OXY)administered in conjunction with commercial intramammary antibiotics improved the cure rate of S.aureus IMI (17, 25). Subcutaneous norfloxacin nicotinate, administered at drying off, increased cure rates for S. aureus IMI compared with that for untreated cows or for cows ad-
3347
3348
ERSKlNE ET AL.
ministered intramammary preparations of cephapirin benzathine (CEPH) (24). A single dose of intramuscular OXY did not increase cure rate but reduced the new IMI rate during the dry period compared with those for untreated or CEPH-treated cows (24). The purpose of this study was to determine the efficacy of systemic OXY as an adjunct to DCT for cows with S. aureus mastitis. The OXY was selected as a supplemental DCT because of favorable pharmacokinetics in the mammary gland and the relatively inexpensive cost of treatment. MATERIALS AND METHODS
A separate milk sample from each quarter was collected from all cows that were approaching the dry period in two Holstein dairies with known high prevalence of S. aureus IMI. Samples were aseptically collected 3 wk before, 1 wk before, and at the last milking before drying off. Samples were then transported to the laboratory on ice, refrigerated at 4"C,and cultured for bacterial isolation within 24 h after collection. Following the initial culture, the samples were stored at -2O'C for 24 to 72 h, thawed at 23°C and cultured again. Each quarter milk sample (10 pl) was inoculated on one-fourth of a plate of trypticase soy agar with 5% sheep blood. Quarters were considered to be infected if S. aureus was isolated from the fresh and frozen cultures of any one milk sample taken before drying off. Cows that were determined to have at least 1 quarter infected with S. aureus were assigned to OXY or control DCT according to computer-randomized assignment lists: 1) first lactation, 2) second and greater lactations, 3) instances in which only 1 quarter was infected, and 4) instances in which more than 1 quarter was infected. One group of 20 cows (40 infected quarters) was infused with a commercial preparation of 300 mg of CEPH (Tomorrow@; Franklin Laboratories, Inc., Fort Dodge, IA) in all 4 quarters at drying off; the second group of 17 cows (34 infected quarters) was also infused with CEPH but also administered intramuscular OXY (Liquamycin LA-200@; Pfizer, Inc., New York. NY) at 11 mg/kg once daily on d 7, 8,9, and 10 after drying off. Prior to any treatments, S. aureus isolates were collected from quarter samples of 3 cows from Journal of Dairy Science Vol. 77, No. 11, 1994
each herd, and the isolates were determined to be susceptible in vitro to cephalothin and tetracycline (1). Quarter milk samples were again collected at 7, 14, 30, and 60 d after calving. An infected quarter was defined as cured at 30 d after calving if no S. aureus were isolated from fresh and frozen culture from a sample within 30 d following calving. Similarly, an infected quarter was defined as cured at 60 d after calving if no isolation of S. aureus occurred from a sample within 60 d after calving. New IMI during the dry period were determined to occur when a quarter was not infected before drying off but yielded S. aureus from fresh and frozen culture of a sample collected after calving. Statistical Analysis
Because both treatments, OXY or control, and the outcome variable, cure or not cured, were dichotomous, Mantel-Haenszel (MH) chisquare analysis was used to evaluate statistical significance and to estimate relative risk. Separate analyses were performed for microbiologic cure determined at 30 and 60 d following calving. Treatment was compared with cure by MH chi-square analysis; quarter was used as the experimental unit. Relative risk and Taylor series 95% confidence limits for relative risk and the significance of the MH ch-square value were reported. Because the number of infected quarters per cow was associated with cure rate, this potential confounder was included in an MH chisquare analysis that compared treatment group with cure in each of three strata: 1) infected quarters, when the other three quarters were not infected; 2) infected quarters, when two quarters on the same cow were infected; and 3) infected quarters, when three or more quarters of the same cow were infected. Because parity was also suspected as a confounding factor, the results were also analyzed with adjustment for two-parity strata (first lactation and older cows). The results of the stratified analyses were reported as MH weighted odds ratio with Cornfield 95% confidence limits and significance of MH summary chi-square values. The data were also analyzed by simple chisquare; the 37 cows, rather than the 74 quarters, were the experimental units, and the two
3349
OXYTETRACYCLINE AS A DRY COW TREATMENT
TABLE 1. Frequency of cure 30 d after calving for quarters infected with Sfaphylococcus oureus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and slratified by number of quarters infected at drying off. Quarters infected at drying off1 1
2 3 4 Total
Quarter cure rate 30 d postcalving CEPH (no.) 2 of 6 of 1 of 2 of 1 1 of
(%)
9 12 3 16 40
22.2 50.0 33.3 12.5 27.5
CEPH
+ OXY
(no.) 4 of 4 of 2 of 0 of 10 of
6 14 6 8 34
Total (%)
66.7 28.6 33.3 0 29.4
(no.) 6 of 10 of 3 of 2 of 21 of
(%)
15 26 9 24 74
40.0 38.5 33.3 8.3 28.4
~
lThe number of quarters infected with S aureus at drying off was determined for each cow in the trial before assignment of treatment group.
treatment groups were compared by the cure rate (all four quarters negative) at 30 and 60 d postcal ving .
the association between cure rate and the number of infected quarters per cow, the MH analysis was repeated with adjustment for possible confounding by this variable. The resultant MH weighted odds ratio was 1.03; confidence RESULTS limits were .31 and 3.36 (P = 33). An MH analysis was also performed with adjustment 304 Cure Rates for parity (first lactation or older cows) as the Systemic OXY did not improve the fre- confounding variable (Table 2). The MH quency of cure for quarters infected with S. weighted odds ratio was 1.07, confidence aureus during the dry period when cure rate limits were .34 and 3.34, and MH summary was assessed after 30 d postcalving (Table 1). chi-square was P = .90. In the OXY treatment group (34 quarters), 10 cures (29.4%) were achieved; in the control 60-d Cure Rates group (40 quarters), 11 cures (27.5%) were When the 60-d culture was also considered, achieved. The relative risk was 1.05; 95% confidence limits were .61 and 1.80 (P = 36). 21.2% (7 of 33) of the OXY quarters were Quarters from cows with 1 to 3 infected quar- considered to be cured, compared with 22.5% ters infected had a significantly higher cure (9 of 40)of the control quarters (relative risk = rate of 38%, 19 of 31, compared with 8.3%, 2 .96; confidence limits = SI and 1.79; P = .89; of 24, for quarters from cows for which all 4 Table 3). Adjustment for three strata regarding quarters were infected (P = .0085). Because of number of quarters infected per cow yielded an TABLE 2. Frequency of cure 30 d after calving for quarters infected with Staphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPIT) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by parity.
.
Quarter cure rate 30 d postcalving
_.. .~
number'
CEPH
1 2 23 Total
(no.) 1 of 2 of 8 of 11 of
(45)
5 8 27 40
20.0 25.0 29.6 27.5
CEPH
+ OXY
(no.) 3 of 4 of 3 of 10 of
6 14 14 34
Total (46)
50.0 28.6 21.4 29.4
(no.) 4 of 6 of 11 of 21 of
(S) 11 22 41 74
36.4 27.3 26.8 28.4
lThe number of quarters infected with S. aureus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
3350
ERSKINE ET AL.
TABLE 3. Frequency of cure 60 d after calving for quarters infected with Sruphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Quarters infected at drying off' 1
2 3 4 Total
Quarter cure rate 60 d postcalving CEPH (no.) I Of9 5 of 12 1 of 3 2 of 16 9 of 40
CEPH
+ OXY
(no.) 2 of 5 3 of 14 2 of 6 0 of 8 7 of 33
11.1 41.7 33.3 12.5 22.5
Total
(W 40.0 21.4 33.3 0 21.2
(no.) 3 of 8 of 3 of 2 of 16 of
(%)
14 26
9 24 73
21.4 30.8 33.3 8.3 21.9
~~
'The number of quarters infected with S. aureus at drylng off was determined for each cow in the trial before assignment of treatment group.
MH weighted odds ratio of .85 and confidence limits of .23 and 3.04 (P = .W). The MH analysis with adjustment for parity (first lactation or older cows) as the extraneous variable (Table 4) yielded an MH weighted odds ratio of .77 (confidence limits = .21 and 2.69; MH summary chi-square, P = .86). The incidence of new S. uureus IMI during the dry period was 2 of 40 quarters in the control group compared with 1 of 34 quarters in the OXY group. Although sample size was small, this finding suggests that CEPH and OXY were no more effective than CEPH alone in preventing quarters that were uninfected at drying off from becoming infected sometime during the dry period or subsequent early lactation.
postcalving compared with 29% (5 of 17) of the cows treated with OXY (P = .76; relative risk = 1.13; confidence limits = .53 and 2.38). When samples taken 60 d postcalving were also considered (Table 6), cure rate was 15% (3 of 20 cows) for controls and 12.5% (2 of 16 cows) for cows treated with OXY (P = .83; relative risk = .89; confidence limits = .28 and 2.77). DISCUSSION
Culling is perhaps the most effective method for eliminating existing S. aureus IMI in a dairy herd (6). However, the need to cull infected cows for mastitis control can be limited by the need to maintain ample milk production for farm cash flow. Culling must be judicious with respect to production, reproduccows Cured tive performance, genetic potential, and other Analysis with cows as the unit of observa- health problems. Therapy is an alternative tion (Table 5 ) showed that 25% (5 of 20) of the method of eliminating IMI; but results are control cows had a microbiologic cure by 30 d usually poorer for IMI caused by S. aureus TABLE 4. Frequency of cure 60 d after calving for quarters infected with Sruphylococcus aureus during the dry period following treatment with intramammary cephapirin (CEPH)or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by parity. Quarter cure rate 60 d postcalving
Lactation number1
CEPH
1 2 23 Total
(no.) 1 of 1 of 7 of 9 of
CEPH (%)
5 8 27 40
20.0 12.5 25.9 22.5
(no.) 3 of 2 of 2 of 7 of
+ OXY
Total
(53 6 14 13 33
50.0 14.3 15.4 21.2
(no.) 4 of 3 of 9 of 16 of
(%)
11 22 40 73
36.4 27.3 26.8 21.9
'The number of quarters infected with S. uureus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
3351
OXYTETRACYCLINE AS A DRY COW TREATMENT
TABLE 5. Frequency of cure 30 d after calving for cows infected with Sraphylococcus aurcus during the dry period following treatment with intramammary cephapirin (CEPH) or intnunammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Cow cure rate 30 d postcalving
Quarters infected at drying off1
CEPH
1 2 3 4 Total
(no.) 2 of 3 of 0 of 0 of 5 of
9 6 1 4
20
CEPH
+ OXY
Total
(%)
(no.)
(%)
22.2 50.0 0 0 25.0
4 of 6
66.7 21.4 0 0 29.4
1 Of7 0 of 2 0 of 2 5 of 17
(no.) 6 of 4 of 0 of 0 of 10 of
(94 15 13 3 6 37
40.0 30.8 0 0 27.0
'The number of quarters infected with S. aurcus at drying off was determined for each cow in the trial before assignment of treatment group.
than by other pathogens because of a variety of virulence factors, including intracellular survival in phagocytes, microabscess formation, P-lactamase, and L-form induction (2, 13, 15, 16, 20, 21). Our objective in this study was to test a therapy that had a high probability of cure efficacy and was inexpensive. The DCT was considered because drug treatment of nonlactating cows allows more sustained regimens than would be practical for lactating cows because of concerns about drug residues in milk. Additionally, numerous studies determined that DCT cure rates were 65 to 75% for S. aureus IMI (3, 8, 18, 19, 25, 26, 29), which was higher than that typically reported for lactating cow therapy (12, 17, 28). The OXY was tested as a supplemental DCT for four reasons: 1) a majority of S. aureus isolates are sensitive to tetracyclines in
vitro (9, 10); 2) the lipophilic nature and relatively long pharmacokinetic half-life of OXY allows effective concentrations in mammary tissue (24); 3) tetracyclines are not detrimental to in vitro neutrophil phagocytosis or viability (7, 14); and 4) OXY is commercially available for nonlactating cows, although not approved for mastitis. Additionally, Sol et a]. (25) determined that 11 mgkg of OXY administered in conjunction with a commercial intramammary antibiotic improved the cure rate of S. aureus M I in lactating cows. In the present study, systemic OXY administered subsequent to intramammary antimicrobials did not affect the rate of cure or the incidence of new Ih4I during the dry period. Our results are in agreement with more recent studies suggesting that the probability of DCT enhancing the cure of S. aureus IMI
TABLE 6. Frequency of cure 60 d after calving for cows infected with Staphylococcus aurcus during the dry period following treatment with intramammary cephapirin (CEPH) or intramammary CEPH and intramuscular oxytetracycline (CEPH + OXY) and stratified by number of quarters infected at drying off. Quarters infected at dryiig off* 1
2 3 4
Total
Cow cure rate 60 d postcalving1 CEPH (no.) 1 Of9 2 of 6 0 of 1 0 of 4 3 of 20
CEPH (96) 11.1
33.3 0 0 15.0
+ OXY
(no.) 2 of 5 0 of I 0 of 2 0 of 2 2 of 16
(%)
40.0
0 0 0 12.5
Total (no.) 3 of 2 of 0 of 0 of 5 of
(W 14
13 3 6 36
21.4 15.4 0 0 13.9
lone cow was culled between 30 and 60 d postcalving. 2The number of quarters infected with S. aurcus at drying off was determined for each cow in the trial before assignment of treatment group. Journal of Dairy Science Vol. 77, No. 11, 1994
3352
ERSKINE ET AL.
during the dry period is lower than that reported in earlier studies (4, 24). Our study did not find any benefit from systemic OXY in the treatment of S. aureus IMI, which is in disagreement with previous studies (17, 24, 25) that reported a beneficial effect of systemic antibiotics. However, two of the previous studies were performed on lactating cows, one of which (17) involved experimental IMI (17, 25). In the other previous study (24), OTC did not increase the cure rate but decreased the incidence of new IMI during the dry period. One explanation for the relatively poor therapeutic success of DCT in the present and in other more recent studies could be the development of bacterial resistance to antimicrobials. This possibility is difficult to assess because S. aureus isolates typical of present IMI cannot be directly compared with those of 25 yr ago. However, Mackie et al. (9) determined that the percentage of S. aureus mastitis isolates that are susceptible in vitro to commonly used antimicrobials did not decrease from 1984 to 1987, and >85% of S. aureus isolates were susceptible to antimicrobials that did not have a structure vulnerable to P-lactamase (9, 10). Intramammary DCT have been routinely used in our study herds for several years. Thus, three S. aureus isolates were selected from each herd prior to the start of the trial to ensure in vitro susceptibility to CEPH and OXY. Because of the contagious nature of S. aurew IMI, such a sampling of isolates was expected to allow a reasonable assessment of the antimicrobial susceptibility pattern of this pathogen in these herds. Nonetheless, in vitro susceptibilities may not necessarily reflect the ability of antimicrobials to effect a cure in vivo, particularly in the mammary gland of a cow. The herds in this study had persistent problems with S. aureus mastitis and a high proportion of chronic IMI. The pathologic changes caused by IMI are probably the primary determinants in therapeutic failure in treatment of S. aureus IMI. This concept was supported by Newbould (12), who determined that quarter cure rates decreased from 87.5 to 62.5% as the duration of IMI increased from 3 to 60 d for experimental IMI. Thus, DCT of S. aureus IMI may be efficacious in some herds, although success may be limited in herds with a high proportion of chronically infected cows. Journal of Dairy Science Vol. 77. No. 11. 1994
Partly because of cyclic shedding of bacteria from infected glands, isolation of S. aureus from milk samples by routine bacteriologic methods can be erratic (22). The sensitivity of a .l-ml inoculum of a single quarter milk sample to determine IMI status was 74.5% but increased to 94 and 98% when second and third samples were included, respectively (22). Duplicate quarter samples collected at the same milking, compared with single quarter samples, do not improve the ability to isolate this bacteria (5). Additionally, freezing of composite milk samples prior to culture increased the frequency of isolation of Streprococcus agalacriae 2.5 times and S. aureus 1.48 times (27). Thus, in an effort to enhance sensitivity of IMI detection and to decrease the probability of false-positive results, we selected consecutive sampling with fresh and frozen cultures. Several previous DCT studies (7, 18, 26, 29) did not utilize repeat sampling to identify infected quarters before or after the dry period. However, whether this factor contributed to the differences in cure efficacy compared with those in our study is questionable because >90% of all S. aureus IMI identified prior to drying off were positive at all three samplings, and 82% of IMI detected after calving were positive at all four samplings. The repeatability of bacterial isolation may have been a function of the chronicity of IMI in the trial herds. Newbould (12) reported that recurrences of isolation of bacteria following treatment of S. aureus IMI in lactating cows could occur up to 28 d and therefore recommended a minimum of 3 wk after treatment, and preferably 30 d, of no bacterial isolation as a criterion for cure. To account for this recommendation and for the cyclic shedding of bacteria in milk during S. uureus IMI, we chose to sample for up to 60 d to determine a cure, thus allowing a conservative assessment of cure rates, although only a small proportion of cured quarters at 30 d were positive for S. aureus at 60 d. New IMI could have occuned during the interval between samples; however, the net result for the herd was that the cow continued to have an S. aureus IMI, despite DCT. CONCLUSIONS
Intramuscular OXY administered subsequent to administration of intramammary
OXYTETRACYCLINE AS A DRY COW TREATMENT
CEPH did not affect the rate of cure or the incidence of new S. aureus l M I during the dry period of dairy cows. Rates of cure were unaffected by parity but were affected by number of quarters infected at drying off. REFERENCES 1 Bauer, A. W.. W.M.M. Kirby, and J. C. Sherris. 1966.
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viability, and phagocytic activity in vitro. Am. J. Vet. Res. 46:2259. 15 Owens, W. E. 1987. Isolation of Sraphylococcus aureus L-forms from experimentally induced bovine mastitis. J. Clin. Microbiol. 26:2187. 16 Owens, W. E. 1988. Evaluation of various antibiotics for induction of L-forms from Staphylococcus aureus strains isolated from bovine mastitis. J. Clin. Microbiol. 27: 1356. 17Owens, W. E., J. L. Watts, and R. L. Boddie. 1988. Antibiotic treatment of mastitis: comparison of intramammary and intramammary plus intramuscular therapies. J. Dairy Sci. 71:3143. 18 Postle, D. S., and R. P. Natzke. 1974. Efficacy of antibiotic treatment in the bovine udder as determined from field studies. Vet. Med. Small Anim. Clin. 69: 1535. 19 Schultze, W. D., and H. D. Mercer. 1976. Nonlactating cow therapy with formulation of penicillin and novobiocin: therapeutic and prophylactic effects. Am. J. Vet. Res. 37:1275. 20 Sears, P. M. 1993. Staphylococcus aureus mastitis. Page 4 in Proc. 32nd AMU. Mtg. Natl. Mastitis Counc. Natl. Mastitis Counc., Arlington, VA. 21 Sears, P. M.. M. Fettinger, and J. Salin-Marsh. 1987. Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis. J. Am. Vet. Med. Assoc. 191:681. 22Sears, P. M., B. S. Smith, and P. B. English. 1990. Shedding pattern of Staphylococcus aureus from bovine intramammary infections. J. Dairy Sci. 73:2785. 23 Smith, A., D. R. Westgarth, M. R. Jones, F. K. Neave, and F. H. Dodd. 1967. Methods of reducing the incidence of udder infection in dry cows. Vet. Rec. 81:504. 24 Soback, S., G. Ziv, M. Winkler, and A. Saran. 1990. Systemic dry cow therapy-a preliminary report. J.
, and A. van Uum. 1990. Factors affecting the result of dry cow treatment. Page 1 I8 in Roc.Int. Symp. Bovine Mastitis, Indianapolis, IN. Natl. Mastitis Counc., Arlington, VA. 26Uvarov. O., J. M. Evans, M. Marshall, and D. Sizer. 1967. Dry cow therapy. Vet. Rec. 81:241. 27 Villanueva. M. R., J. W. Tyler, and M. C. Thurmond. 1991. Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk. J. Am. Vet. Med. Assoc. 198:1398. 28Wilson, C. D., N. Agger, G. A. Gilbert, C. A. Thomasson, and S. T. Trolling. 1986. Field trials with cefoperazone in the treatment of bovine clinical mastitis. Vet. Rec. 118:17. 29 Ziv, G., M. Storper, and A. Saran. 1981. Comparative efficacy of three antibiotic products for the treatment and prevention of subclinical mastitis during the dry period. Vet. Q. 3:74.
Journal of Dairy Science Vol. 77, No. 11, 1994