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Efficacy Evaluations on Five Chlorhexidine Teat Dip Formulations
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Efficacy Evaluations on Five Chlorhexidlne Teat Dip Formulations P. A. DRECHSLER, J. K. O'NEIL, P. A. MURDOUGH, A. R. LAFAYETTE, E. E. WILDMAN, and J. W. PANKEY Department of Animal and Food Sciences University of Vermont BUrlington 05405 ABSTRACT
Experimental and natural exposure protocols with negative controls are recommended by the National Mastitis Council (5) to evaluate efficacy of teat dip formulations. Chlorhexidine formulations have been evaluated and used in mastitis control programs since 1955 (2, 4, 8, 10). The objectives of this study were to evaluate the efficacy of three chlorhexidine formulations under experimental exposure and two additional formulations under natural exposure conditions.
Three developmental postmilking teat dip formulations containing chlorhexidine digluconate were evaluated against Staphylococcus aureus and Streptococcus agalactiae in sequential experimental exposure trials. Two additional commercial chlorhexidine digluconate teat dip products were evaluated in natural exposure trials. Under conditions of experimental challenge, the developmental formulations were efficacious against Staph. aureus but did not significantly reduce incidence of new 1MI by Strep. agalactiae. None of the three formulations of a conventional germicide used as teat sanitizers effectively reduced incidence of new Strep. agalactiae IMI under experimental challenge conditions. In the natural exposure trials with negative controls, a .35% chlorhexidine teat sanitizer had efficacy of 88.7% against Staph. aureus and 51.4% against Strep. agalactiae. The .5% chlorhexidine product reduced Staph. aureus and Strep. agalactiae 1MI by 86 and 56%, respectively. (Key words: mastitis, teat dip, efficacy, chlorhexidine dips)
MATERIALS AND METHODS
Three developmental chlorhexidine formulations were evaluated in sequential experimental exposure trials (5). Two formulations contained .5% chlorhexidine digluconate with .5% glycerin; the third formulation contained 1% chlorhexidine digluconate and 1% glycerin (Table 1). Two commercial teat dips, a .35% chlorhexidine digluconate with 2.6% glycerin and a .5% chlorhexidine digluconate with 5% emollients, were evaluated by the natural exposure protocol (5). experimental Exposure Trials
INTRODUCTION
Prevention of mastitis by postmilking teat sanitation has been reviewed widely (1, 2, 4, 6, 7, 8, 9, 10). Conventional germicidal postmilking teat sanitizers are designed to kill bacteria that contaminate teats during milking (8). Postmilking teat sanitization with an effective teat dip formulation significantly reduced the rate of new IMI by contagious pathogens (6).
Received January 11, 1993. Accepted March 18. 1993. 1993 J Dairy Sci 76:2783-2788
The Wheelock Quality Milk Research Herd (University of Vermont, Burlington) was used in all three experimental exposure trials and contained between 40 and 46 lactating Jersey cows housed in a tie-stall barn with a high line milking system. Two challenge suspensions of Staphylococcus aureus (ATCC 29740) and Streptococcus agalactiae (ATCC 27956) were prepared daily and mixed to provide one challenge suspension at a concentration of approximately 5 x 107 cfulml of each pathogen (9). Bacterial concentrations were determined for Strep. agalactiae by adjustment of the optical density of cultures to between 70 and 75% transmittance at 620 nrn on a Spectronic-20 (Bausch & Lomb,
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DRECHSLER ET AL.
TABLE I. Summary of experimenlal challenge teat dip efficacy data.
Trial
Treatment group
Staphylococcus aureus IMI Eligible quarters
Quarters
Reduction (%)
- - (00.)--
Streptococcus agalactiae IMI Eligible quarters
Quarters
Reduction (%)
-(00.)--
]I
Dipped Control
61 56
8 27
13.1 48.2
72.8"
68 69
18 22
26.5 31.9
16.9b
22
Dipped Control
82 SO
8 15
9.8 18.8
47.9b
88 86
4 7
4.5 8.1
44.4 b
33
Dipped Control
80 74
7 13
8.8 17.6
50.Db
88 86
5 8
5.7 9.3
38.7b
"P < .01. Between-trial comparisons are invalid. bp> .05. 1.5% chlorhexidine digluconate with .5% glycerin. 2.5% chlorhexidine digluconate with .5% glycerin. A refonnulation of the trial 1 product. 31.0% chlorhexidine digluconate with 1% glycerin. A refonnulation of the trial 2 product.
Rochester, NY) spectrophotometer. An aliquot of resting cell suspension of Staph. aureus was added to the Strep. agalactiae culture to achieve the appropriate concentration. A standard plate count was conducted daily on the pathogen suspension. The bacterial suspensions were taken to the Wheelock Research Herd immediately after preparation 5 d1wk. One suspension was used immediately to challenge teats during the p.m. milking. The second challenge suspension was held overnight at 5°C and used to challenge teats at the a.m. milking. The lower third of all four teats of each cow was immersed in the challenge suspension immediately after removal from the milking machine. Diagonal teats were dipped full length in the chlorhexidine formulations immediately after bacterial challenge, and the opposite set of diagonal teats served as undipped controls. In trial 1, Staph. aureus challenge was for 4 wk; Strep. agalactiae was used for 8 wk to obtain a sufficient number of 1M] for analysis. Teats were challenged with both pathogens for 5 and 6 wk in trials 2 and 3, respectively. Natural Exposure with Negative Control Trials
Two commercial dairy herds were used for trials 4 and 5 and met the following criteria: 1) Staph. aureus and Strep. agalactiae were present in the herd; 2) all lactating cows had permanent, visible identification; 3) milking Journal of Dairy Science Vol. 76, No.9, 1993
equipment met industry standards; and 4) all quarters of all cows were treated at drying off with a commercial, intramammary infusion antibiotic product. Trials 4 and 5 lasted for 9 and 10 mo. The herd used for trial 4 consisted of approximately 65 lactating Holstein cows, and the herd for trial 5 contained approximately 54 lactating Holstein cows. Both herds were housed in tie-stall barns with high line milking systems. A split-herd design was used in each herd. Treatment groups were balanced for lactation number, stage of lactation, and bacteriological status of quarters as by Pankey et al. (11). Cows were identified by treatment group with different-colored leg bands. Cows entering each herd were assigned alternately to each group to maintain balance. Treatments were the following: group 1, teats were wiped dry and forestripped prior to milking, and teats were dipped in the chlorhexidine formulation after milking; group 2, teats were wiped dry and forestripped prior to milking, and no postmilking teat antisepsis was used. In trial 4, premilking udder preparation was changed during the 5 mo. For the remainder of the trial, teats were cleaned with a paper towel moistened with sanitizer and air dried, instead of only being wiped dry. Collection of Milk Samples
All quarter milk samples were collected aseptically (3). Bacteriological status of in-
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dividual quarters was determined at initiation of each trial with duplicate milk cultures. When culture results of the first two samples differed, a second set of duplicate quarter milk samples was collected and cultured. All quarters were considered to be eligible for new IMI except nonlactating quarters and quarters with injured teats. Quarters with teats injured during the study were ineligible for the remainder of that lactation. If the injury healed, the quarter was eligible for IMI in the subsequent lactation. Also, an eligible quarter infected with a major pathogen was eligible for IMI with a different major pathogen during that lactation (5).
During the experimental exposure studies, quarter milk samples were collected and analyzed weekly. When either Staph. aureus or Strep. agalactiae was first present in an eligible quarter, a second sample was collected within 3 d and cultured to confirm diagnosis. In the natural exposure trials, duplicate quarter milk samples were collected by laboratory staff approximately every 2 mo on each herd to monitor incidence of new 1MI. Cooperators collected duplicate quarter milk samples from all quarters of cows entering or leaving the herd, at calving, at drying off, or from cows that developed clinical mastitis prior to administration of antibiotics. Samples were collected by fanners, stored frozen, and picked up approximately every 2 wk for bacteriological testing. Analysis of Milk Samples
Bacteriological analysis was conducted on all milk samples using recommended procedures (3). Samples were plated on tryptose blood agar containing 5% washed bovine red blood cells with .1 % esculin. In addition, for each milk sample from a clinical quarter, a .1ml aliquot of milk was smear plated on onehalf of a MacConkey agar plate, and an additional .1 ml of milk was plated on one-half of a tryptose blood agar plate to increase sensitivity of diagnosis. Identification of all staphylococcal isolates were confirmed with the Rapid Mastitis Tesr®, a latex agglutination test for Staph. aureus (Immucell, Portland, ME), and the STAPHTrac Staphylococcus Identification System® (Analytab Products, Plainview, NY). Streptococci were identified with the Phadehact ABCG Streptococcus Test
2785
Kit® (Phannacia Diagnostics, Phannacia Inc., Piscataway, NJ). Diagnosis of IMI
For the experimental exposure trials, an IMI was confmned by one of the following criteria: 1) Staph. aureus or Strep. agalactiae was isolated from a clinical quarter, 2) two consecutive samples yielded ~500 cfulml of the same pathogen, or 3) three consecutive samples contained <500 cfulml of the same pathogen. In the natural exposure trials, an IMI was diagnosed by one of the following criteria: 1) isolation of ~ 100 cfulml of a pathogen from a clinical sample, 2) isolation of ~500 cfulml of a pathogen from two consecutive samples, or 3) isolation of <400 cfulml of a pathogen from three consecutive samples. In the natural exposure trials, bacterial isolates were grouped as major and minor pathogens. Major pathogens, which commonly cause SCC increases >5 x lOs/ml, included Staph. aureus, Strep. agalactiae, streptococci other than Strep. agalactiae, and coliforms. Minor pathogens, which commonly cause moderate SCC increases <3 x IOS/ml, included coagulase-negative staphylococci and Corynebacterium bovis. Data Analysis
The 1MI data from the experimental exposure studies and the natural exposure evaluations were analyzed based on percentage of eligible quarters that became infected. The following statistic was applied (8):
where t = approximates a Student's t statistic, P = percentage of quarters becoming infected, Q percentage of quarters not becoming infected, and N = number of eligible quarters. Subscripts refer to treatment groups.
=
RESULTS AND DISCUSSION Experimental Exposure Studies
Trial 1. A total of 35 Staph. aureus IMI were diagnosed during the study (Table 1): 8 in Journal of Dairy Science Vol. 76, No.9, 1993
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DRECHSLER ET AL.
quarters dipped with the developmental .5% chlorhexidine formulation and 27 in undipped quarters. The 73% reduction of Staph. aureus IMI was significant (P < .(01). A total of 40 Strep. agalactiae IMI were diagnosed during the study: 18 in dipped quarters and 22 in undipped quarters. The product was not efficacious against Strep. agalactiae under experimental exposure conditions. Trial 2. The .5% chlorhexidine product used in trial 1 was reformulated and evaluated in a second experimental exposure study. Twenty-three Staph. aureus IMI were diagnosed. Eight IMI occurred in dipped quarters and 15 in undipped quarters, which was a 48% reduction (P > .05). Eleven Strep. agalactiae IMI were detected during the 5 wk study: 4 in dipped quarters and 7 in undipped quarters. The trial was terminated when results suggested that the second formulation would not
effectively reduce IMI caused by Strep. agalactiae. Trial 3. A second reformulation was tested with the experimental exposure procedure and contained 1% chlorhexidine digluconate and I % glycerin. A total of 20 Staph. aureus IMI developed during the 6-wk study: 7 in dipped quarters and 13 in undipped quarters (P > .05). Thirteen Strep. agalactiae IMI were diagnosed: 5 in dipped and 8 in undipped quarters (P > .05). This trial was terminated because results suggested that the 1% chlorhexidine formulation also was ineffective against Strep. agalactiae under experimental exposure conditions. Efficacy, the ability of a postrnilking teat sanitizer to reduce the incidence of IMI, was demonstrated by numerous formulations with the experimental exposure protocol (7, 8, 9, 10); a few formulations were not efficacious
TABLE 2. Summary of teat dip efficacy data under natural exposure conditions.
Organism
Trial 52
Trial 4'
Treatment group
New IMI
Reduction (%)
(no.)
New IMI (no.)
Reduction (%) - -
Staphylococcus aureus
Dipped Control
2 18
1.7 15.0
88.78
2 14
1.9 13.5
86.oa
Streptococcus agalactiae
Dipped Control
10 21
8.5 17.5
51.48
8 18
7.7 17.5
56.oa
Contagious pathogens
Dipped Control
12 39
10.3 32.5
68.48
10 32
9.6 30.0
6&.&8
Dipped Control
7 8
6.0 6.8
11.8b
8 8
7.7 7.8
l.3 b
Coliforms
Dipped Control
2 4
1.7 3.3
48.5 b
2 3
1.9 2.9
33.3b
Environmental pathogens
Dipped Control
9 12
7.7 10.0
23.l b
10
9.6 10.7
1O.3b
11
Total major pathogens
Dipped Control
21 51
18.0 42.5
57.&-
20 43
19.2 41.7
53.8-
Staphylococcus spp.
Dipped Control
5 14
4.3 11.7
63.3-
4 12
3.& 11.6
67.oa
Corynebacterium bovis
Dipped Control
18.0 38.3
53.3-
27 35
26.0 34.0
23.5 b
Total minor pathogens
Dipped Control
21 46 26 60
22.2 50.0
55.68
31 48
29.8 46.6
35.8b
Streptococci other than Strep. agalactiae
8p < .05. Between-trial comparisons are invalid. bp
> .05.
'.35% chlorhexidine digluconate with 2.6% glycerin; number of eligible quarters: dipped = 117. control = 120. 2.5% chlorhexidine digluconate with 5% emollients; nurnber of eligible quarters: dipped = 104. control = 103. Journal of Dairy Science Vol. 76. No.9. 1993
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(12). The first chlorhexidine formulation used in these experimental exposure trials significantly reduced incidence of IMI caused by Staph. aureus (trial 1) but was ineffective against Strep. agalactiae. Trials 2 and 3 were conducted to evaluate reformulations but were terminated before conclusive data were generated because the distribution of new Strep. agalactiae 1MI among dipped and undipped quarters suggested that the reduction would not be statistically significant. These results suggest that some formulations of commonly used germicides are not effective in the reduction of new 1MI by specific pathogens under experimental challenge conditions. Additional studies are necessary to determine factors that affect efficacy. These experimental formulations are not marketed. Natural Exposure Studies
Trial 4. New 1MI with Staph. aureus and Strep. agalactiae were reduced 88.7 and 51.4% (P < .05) and in trial 5, Staph. aureus and Strep. agalactiae 1MI were reduced 86 and 56%, respectively (P < .05) (Table 2). Rate of new 1MI with environmental pathogens, streptococci other than Strep. agalactiae and coliforms, was not significantly reduced in either natural exposure trial. Postdipping with the .35% chlorhexidine product in trial 4 reduced IMI with major mastitis pathogens 57.8% (P < .05) compared with the undipped control group. Major mastitis pathogens were reduced 53.8% (P < .05) by the .5% chlorhexidine product in trialS. The number of IMI by coagulase-negative staphylococci was reduced 63.3 and 67.0%, respectively, in trials 4 and 5 (P < .05). Corynebacterium bovis WI were reduced 53.3% (P < .05) in trial 4 and 23.5% (P> .05) in trial 5. A premilking udder preparation procedure in which teats were wiped dry but without teat sanitization was used throughout trial 5 and may have affected efficacy against C. bovis. Udder infections by minor pathogens were reduced 55.6% (P < .05) in trial 4 and 35.8% (P > .05) in trial 5. In trial 4, rate of IMI for major mastitis pathogens was considered to be abnormally high for the initial 4 mo. During this period, rate of 1MI was approximately 1.5-fold higher in undipped than in dipped quarters. Manage-
ment practices were reviewed and premillcing udder preparation procedures were modified during mo 5. For the remainder of the trial, udder preparation included cleaning of teats with a paper towel moistened with sanitizer and air dried instead of only wiping teats dry. Following changes in udder preparation, efficacy of the product improved, and incidence of new 1MI among undipped quarters was 2.5-fold higher than in dipped quarters. These results suggested that the method of premilking udder preparation can influence the efficacy of postmilking teat sanitation. CONCLUSIONS
In the experimental exposure studies, a .5% chlorhexidine digluconate was efficacious against Staph. aureus and ineffective against Strep. agalactiae. Reformulation of the .5% chlorhexidine product did not improve efficacy. Another reformation contained 1% chlorhexidine digluconate and remained ineffective against Strep. agalactiae. These three chlorhexidine digluconate formulations did not significantly reduce incidence of IMI by Strep. agalactiae under experimental exposure conditions. Under natural exposure conditions, two chlorhexidine digluconate teat dips significantly reduced incidence of 1MI by contagious mastitis pathogens and all major mastitis pathogens. The percentage of quarters diagnosed with environmental pathogens was smaller in the dipped group, but the reduction was nonsignificant. In trial 4, efficacy of the .35% chlorhexidine product increased when premilking udder preparation procedures were changed. Minor mastitis pathogens, coagulase-negative staphylococcal species and C. bovis, were controlled by postdipping in trial 4 when teats were sanitized prior to attachment of milking machines. In trial 5, rate of IMI by coagulase-negative staphylococci was reduced significantly by the .5% chlorhexidine product, but C. bovis WI were not controlled. These studies suggested that premilking udder preparation could affect efficacy of postmilking teat sanitizers. ACKNOWLEDGMENTS
The authors express appreciation to the two cooperator herds, Senesac Dairy and Fontaine Journal of Dairy Science Vol. 76, No.9, 1993
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Brothers Fann. for their patience throughout the course of these natural exposure studies. The partial support of these studies by the manufacturers of the teat dip formulations is appreciated. REFERENCES 1 Eberhart, R. J., and J. M. Buckalew. 1972. Evaluation of a hygiene and dry period therapy program for mastitis control. 1. Dairy Sci. 55: 1683. 2 Gerring, E. L., R. Hall, and A. J. Sandoe. 1968. The evaluation of a teat-dipping formulation chlorhexidine. Vet. Rec. 83:112. 3 Harmon, R. J., R. J. Eberhart, D. E. Jasper, B. E. Langlois, and R. A. Wilson. 1990. Microbiological Procedures for the Diagnosis of Bovine Udder Infection. 3rd ed. Natl. Mastitis Counc., Arlington, VA. 4 Hicks, W. G., T. J. Kennedy, D. M. Keister, and M. L. Miller. 1981. Evaluation of a teat dip of chlorhexidine digluconate (.5%) with glycerin (6%). J. Dairy Sci. 64: 2266. 5 Hogan. J. S.• D. M. Galton, R. J. Harmon, S. C. Nickerson. S. P. Oliver, and J. W. Pankey. 1990. Protocols for evaluating efficacy of postmilking teal dips. J. Dairy Sci. 73:2580.
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6 Neave. F. K.• F. H. Dodd. R. G. Kingwill, and D. R. Westgarth. 1969. Control of mastitis in the dairy herd by hygiene and management. J. Dairy Sci. 52:696. 7 Pankey, J. W.• R. L. Boddie, and S. C. Nickerson. 1985. Efficacy evaluation of two new teat dip formulations under experimental challenge. J. Dairy Sci. 68: 462. 8 Pankey. J. W., R. 1. Eberhart. A. L. Cuming. R. D. Daggett, R. J. Farnsworth, and C. K. McDuff. 1984. Update on postmilking teat antisepsis. J. Dairy Sci. 67:1336. 9 Pankey, 1. W.• S. C. Nickerson. and R. L. Boddie. 1984. Evaluation of linear dodecyl benzene sulfonic acid teat dip under experimental challenge. J. Dairy Sci. 70:867. 10 Pankey, J. W .• W. N. Philpot, R. L. Boddie, and J. L. Watts. 1983. Evaluation of nine teat dip formulations under experimental challenge 10 Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 66: 161. 11 Pankey, J. W., E. E. Wildman. P. A. Drechsler, and J. S. Hogan. 1987. Field trial evaluation of premilking teal disinfection. J. Dairy Sci. 70:867. 12 Philpot, W. N.• and 1. W. Pankey, Jr. 1975. Hygiene in the prevention of udder infections: II. Evaluation of oil-based teat dips. J. Dairy Sci. 58:205.
Efficacy Evaluations on Five Chlorhexidlne Teat Dip Formulations P. A. DRECHSLER, J. K. O'NEIL, P. A. MURDOUGH, A. R. LAFAYETTE, E. E. WILDMAN, and J. W. PANKEY Department of Animal and Food Sciences University of Vermont BUrlington 05405 ABSTRACT
Experimental and natural exposure protocols with negative controls are recommended by the National Mastitis Council (5) to evaluate efficacy of teat dip formulations. Chlorhexidine formulations have been evaluated and used in mastitis control programs since 1955 (2, 4, 8, 10). The objectives of this study were to evaluate the efficacy of three chlorhexidine formulations under experimental exposure and two additional formulations under natural exposure conditions.
Three developmental postmilking teat dip formulations containing chlorhexidine digluconate were evaluated against Staphylococcus aureus and Streptococcus agalactiae in sequential experimental exposure trials. Two additional commercial chlorhexidine digluconate teat dip products were evaluated in natural exposure trials. Under conditions of experimental challenge, the developmental formulations were efficacious against Staph. aureus but did not significantly reduce incidence of new 1MI by Strep. agalactiae. None of the three formulations of a conventional germicide used as teat sanitizers effectively reduced incidence of new Strep. agalactiae IMI under experimental challenge conditions. In the natural exposure trials with negative controls, a .35% chlorhexidine teat sanitizer had efficacy of 88.7% against Staph. aureus and 51.4% against Strep. agalactiae. The .5% chlorhexidine product reduced Staph. aureus and Strep. agalactiae 1MI by 86 and 56%, respectively. (Key words: mastitis, teat dip, efficacy, chlorhexidine dips)
MATERIALS AND METHODS
Three developmental chlorhexidine formulations were evaluated in sequential experimental exposure trials (5). Two formulations contained .5% chlorhexidine digluconate with .5% glycerin; the third formulation contained 1% chlorhexidine digluconate and 1% glycerin (Table 1). Two commercial teat dips, a .35% chlorhexidine digluconate with 2.6% glycerin and a .5% chlorhexidine digluconate with 5% emollients, were evaluated by the natural exposure protocol (5). experimental Exposure Trials
INTRODUCTION
Prevention of mastitis by postmilking teat sanitation has been reviewed widely (1, 2, 4, 6, 7, 8, 9, 10). Conventional germicidal postmilking teat sanitizers are designed to kill bacteria that contaminate teats during milking (8). Postmilking teat sanitization with an effective teat dip formulation significantly reduced the rate of new IMI by contagious pathogens (6).
Received January 11, 1993. Accepted March 18. 1993. 1993 J Dairy Sci 76:2783-2788
The Wheelock Quality Milk Research Herd (University of Vermont, Burlington) was used in all three experimental exposure trials and contained between 40 and 46 lactating Jersey cows housed in a tie-stall barn with a high line milking system. Two challenge suspensions of Staphylococcus aureus (ATCC 29740) and Streptococcus agalactiae (ATCC 27956) were prepared daily and mixed to provide one challenge suspension at a concentration of approximately 5 x 107 cfulml of each pathogen (9). Bacterial concentrations were determined for Strep. agalactiae by adjustment of the optical density of cultures to between 70 and 75% transmittance at 620 nrn on a Spectronic-20 (Bausch & Lomb,
2783
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DRECHSLER ET AL.
TABLE I. Summary of experimenlal challenge teat dip efficacy data.
Trial
Treatment group
Staphylococcus aureus IMI Eligible quarters
Quarters
Reduction (%)
- - (00.)--
Streptococcus agalactiae IMI Eligible quarters
Quarters
Reduction (%)
-(00.)--
]I
Dipped Control
61 56
8 27
13.1 48.2
72.8"
68 69
18 22
26.5 31.9
16.9b
22
Dipped Control
82 SO
8 15
9.8 18.8
47.9b
88 86
4 7
4.5 8.1
44.4 b
33
Dipped Control
80 74
7 13
8.8 17.6
50.Db
88 86
5 8
5.7 9.3
38.7b
"P < .01. Between-trial comparisons are invalid. bp> .05. 1.5% chlorhexidine digluconate with .5% glycerin. 2.5% chlorhexidine digluconate with .5% glycerin. A refonnulation of the trial 1 product. 31.0% chlorhexidine digluconate with 1% glycerin. A refonnulation of the trial 2 product.
Rochester, NY) spectrophotometer. An aliquot of resting cell suspension of Staph. aureus was added to the Strep. agalactiae culture to achieve the appropriate concentration. A standard plate count was conducted daily on the pathogen suspension. The bacterial suspensions were taken to the Wheelock Research Herd immediately after preparation 5 d1wk. One suspension was used immediately to challenge teats during the p.m. milking. The second challenge suspension was held overnight at 5°C and used to challenge teats at the a.m. milking. The lower third of all four teats of each cow was immersed in the challenge suspension immediately after removal from the milking machine. Diagonal teats were dipped full length in the chlorhexidine formulations immediately after bacterial challenge, and the opposite set of diagonal teats served as undipped controls. In trial 1, Staph. aureus challenge was for 4 wk; Strep. agalactiae was used for 8 wk to obtain a sufficient number of 1M] for analysis. Teats were challenged with both pathogens for 5 and 6 wk in trials 2 and 3, respectively. Natural Exposure with Negative Control Trials
Two commercial dairy herds were used for trials 4 and 5 and met the following criteria: 1) Staph. aureus and Strep. agalactiae were present in the herd; 2) all lactating cows had permanent, visible identification; 3) milking Journal of Dairy Science Vol. 76, No.9, 1993
equipment met industry standards; and 4) all quarters of all cows were treated at drying off with a commercial, intramammary infusion antibiotic product. Trials 4 and 5 lasted for 9 and 10 mo. The herd used for trial 4 consisted of approximately 65 lactating Holstein cows, and the herd for trial 5 contained approximately 54 lactating Holstein cows. Both herds were housed in tie-stall barns with high line milking systems. A split-herd design was used in each herd. Treatment groups were balanced for lactation number, stage of lactation, and bacteriological status of quarters as by Pankey et al. (11). Cows were identified by treatment group with different-colored leg bands. Cows entering each herd were assigned alternately to each group to maintain balance. Treatments were the following: group 1, teats were wiped dry and forestripped prior to milking, and teats were dipped in the chlorhexidine formulation after milking; group 2, teats were wiped dry and forestripped prior to milking, and no postmilking teat antisepsis was used. In trial 4, premilking udder preparation was changed during the 5 mo. For the remainder of the trial, teats were cleaned with a paper towel moistened with sanitizer and air dried, instead of only being wiped dry. Collection of Milk Samples
All quarter milk samples were collected aseptically (3). Bacteriological status of in-
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dividual quarters was determined at initiation of each trial with duplicate milk cultures. When culture results of the first two samples differed, a second set of duplicate quarter milk samples was collected and cultured. All quarters were considered to be eligible for new IMI except nonlactating quarters and quarters with injured teats. Quarters with teats injured during the study were ineligible for the remainder of that lactation. If the injury healed, the quarter was eligible for IMI in the subsequent lactation. Also, an eligible quarter infected with a major pathogen was eligible for IMI with a different major pathogen during that lactation (5).
During the experimental exposure studies, quarter milk samples were collected and analyzed weekly. When either Staph. aureus or Strep. agalactiae was first present in an eligible quarter, a second sample was collected within 3 d and cultured to confirm diagnosis. In the natural exposure trials, duplicate quarter milk samples were collected by laboratory staff approximately every 2 mo on each herd to monitor incidence of new 1MI. Cooperators collected duplicate quarter milk samples from all quarters of cows entering or leaving the herd, at calving, at drying off, or from cows that developed clinical mastitis prior to administration of antibiotics. Samples were collected by fanners, stored frozen, and picked up approximately every 2 wk for bacteriological testing. Analysis of Milk Samples
Bacteriological analysis was conducted on all milk samples using recommended procedures (3). Samples were plated on tryptose blood agar containing 5% washed bovine red blood cells with .1 % esculin. In addition, for each milk sample from a clinical quarter, a .1ml aliquot of milk was smear plated on onehalf of a MacConkey agar plate, and an additional .1 ml of milk was plated on one-half of a tryptose blood agar plate to increase sensitivity of diagnosis. Identification of all staphylococcal isolates were confirmed with the Rapid Mastitis Tesr®, a latex agglutination test for Staph. aureus (Immucell, Portland, ME), and the STAPHTrac Staphylococcus Identification System® (Analytab Products, Plainview, NY). Streptococci were identified with the Phadehact ABCG Streptococcus Test
2785
Kit® (Phannacia Diagnostics, Phannacia Inc., Piscataway, NJ). Diagnosis of IMI
For the experimental exposure trials, an IMI was confmned by one of the following criteria: 1) Staph. aureus or Strep. agalactiae was isolated from a clinical quarter, 2) two consecutive samples yielded ~500 cfulml of the same pathogen, or 3) three consecutive samples contained <500 cfulml of the same pathogen. In the natural exposure trials, an IMI was diagnosed by one of the following criteria: 1) isolation of ~ 100 cfulml of a pathogen from a clinical sample, 2) isolation of ~500 cfulml of a pathogen from two consecutive samples, or 3) isolation of <400 cfulml of a pathogen from three consecutive samples. In the natural exposure trials, bacterial isolates were grouped as major and minor pathogens. Major pathogens, which commonly cause SCC increases >5 x lOs/ml, included Staph. aureus, Strep. agalactiae, streptococci other than Strep. agalactiae, and coliforms. Minor pathogens, which commonly cause moderate SCC increases <3 x IOS/ml, included coagulase-negative staphylococci and Corynebacterium bovis. Data Analysis
The 1MI data from the experimental exposure studies and the natural exposure evaluations were analyzed based on percentage of eligible quarters that became infected. The following statistic was applied (8):
where t = approximates a Student's t statistic, P = percentage of quarters becoming infected, Q percentage of quarters not becoming infected, and N = number of eligible quarters. Subscripts refer to treatment groups.
=
RESULTS AND DISCUSSION Experimental Exposure Studies
Trial 1. A total of 35 Staph. aureus IMI were diagnosed during the study (Table 1): 8 in Journal of Dairy Science Vol. 76, No.9, 1993
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quarters dipped with the developmental .5% chlorhexidine formulation and 27 in undipped quarters. The 73% reduction of Staph. aureus IMI was significant (P < .(01). A total of 40 Strep. agalactiae IMI were diagnosed during the study: 18 in dipped quarters and 22 in undipped quarters. The product was not efficacious against Strep. agalactiae under experimental exposure conditions. Trial 2. The .5% chlorhexidine product used in trial 1 was reformulated and evaluated in a second experimental exposure study. Twenty-three Staph. aureus IMI were diagnosed. Eight IMI occurred in dipped quarters and 15 in undipped quarters, which was a 48% reduction (P > .05). Eleven Strep. agalactiae IMI were detected during the 5 wk study: 4 in dipped quarters and 7 in undipped quarters. The trial was terminated when results suggested that the second formulation would not
effectively reduce IMI caused by Strep. agalactiae. Trial 3. A second reformulation was tested with the experimental exposure procedure and contained 1% chlorhexidine digluconate and I % glycerin. A total of 20 Staph. aureus IMI developed during the 6-wk study: 7 in dipped quarters and 13 in undipped quarters (P > .05). Thirteen Strep. agalactiae IMI were diagnosed: 5 in dipped and 8 in undipped quarters (P > .05). This trial was terminated because results suggested that the 1% chlorhexidine formulation also was ineffective against Strep. agalactiae under experimental exposure conditions. Efficacy, the ability of a postrnilking teat sanitizer to reduce the incidence of IMI, was demonstrated by numerous formulations with the experimental exposure protocol (7, 8, 9, 10); a few formulations were not efficacious
TABLE 2. Summary of teat dip efficacy data under natural exposure conditions.
Organism
Trial 52
Trial 4'
Treatment group
New IMI
Reduction (%)
(no.)
New IMI (no.)
Reduction (%) - -
Staphylococcus aureus
Dipped Control
2 18
1.7 15.0
88.78
2 14
1.9 13.5
86.oa
Streptococcus agalactiae
Dipped Control
10 21
8.5 17.5
51.48
8 18
7.7 17.5
56.oa
Contagious pathogens
Dipped Control
12 39
10.3 32.5
68.48
10 32
9.6 30.0
6&.&8
Dipped Control
7 8
6.0 6.8
11.8b
8 8
7.7 7.8
l.3 b
Coliforms
Dipped Control
2 4
1.7 3.3
48.5 b
2 3
1.9 2.9
33.3b
Environmental pathogens
Dipped Control
9 12
7.7 10.0
23.l b
10
9.6 10.7
1O.3b
11
Total major pathogens
Dipped Control
21 51
18.0 42.5
57.&-
20 43
19.2 41.7
53.8-
Staphylococcus spp.
Dipped Control
5 14
4.3 11.7
63.3-
4 12
3.& 11.6
67.oa
Corynebacterium bovis
Dipped Control
18.0 38.3
53.3-
27 35
26.0 34.0
23.5 b
Total minor pathogens
Dipped Control
21 46 26 60
22.2 50.0
55.68
31 48
29.8 46.6
35.8b
Streptococci other than Strep. agalactiae
8p < .05. Between-trial comparisons are invalid. bp
> .05.
'.35% chlorhexidine digluconate with 2.6% glycerin; number of eligible quarters: dipped = 117. control = 120. 2.5% chlorhexidine digluconate with 5% emollients; nurnber of eligible quarters: dipped = 104. control = 103. Journal of Dairy Science Vol. 76. No.9. 1993
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(12). The first chlorhexidine formulation used in these experimental exposure trials significantly reduced incidence of IMI caused by Staph. aureus (trial 1) but was ineffective against Strep. agalactiae. Trials 2 and 3 were conducted to evaluate reformulations but were terminated before conclusive data were generated because the distribution of new Strep. agalactiae 1MI among dipped and undipped quarters suggested that the reduction would not be statistically significant. These results suggest that some formulations of commonly used germicides are not effective in the reduction of new 1MI by specific pathogens under experimental challenge conditions. Additional studies are necessary to determine factors that affect efficacy. These experimental formulations are not marketed. Natural Exposure Studies
Trial 4. New 1MI with Staph. aureus and Strep. agalactiae were reduced 88.7 and 51.4% (P < .05) and in trial 5, Staph. aureus and Strep. agalactiae 1MI were reduced 86 and 56%, respectively (P < .05) (Table 2). Rate of new 1MI with environmental pathogens, streptococci other than Strep. agalactiae and coliforms, was not significantly reduced in either natural exposure trial. Postdipping with the .35% chlorhexidine product in trial 4 reduced IMI with major mastitis pathogens 57.8% (P < .05) compared with the undipped control group. Major mastitis pathogens were reduced 53.8% (P < .05) by the .5% chlorhexidine product in trialS. The number of IMI by coagulase-negative staphylococci was reduced 63.3 and 67.0%, respectively, in trials 4 and 5 (P < .05). Corynebacterium bovis WI were reduced 53.3% (P < .05) in trial 4 and 23.5% (P> .05) in trial 5. A premilking udder preparation procedure in which teats were wiped dry but without teat sanitization was used throughout trial 5 and may have affected efficacy against C. bovis. Udder infections by minor pathogens were reduced 55.6% (P < .05) in trial 4 and 35.8% (P > .05) in trial 5. In trial 4, rate of IMI for major mastitis pathogens was considered to be abnormally high for the initial 4 mo. During this period, rate of 1MI was approximately 1.5-fold higher in undipped than in dipped quarters. Manage-
ment practices were reviewed and premillcing udder preparation procedures were modified during mo 5. For the remainder of the trial, udder preparation included cleaning of teats with a paper towel moistened with sanitizer and air dried instead of only wiping teats dry. Following changes in udder preparation, efficacy of the product improved, and incidence of new 1MI among undipped quarters was 2.5-fold higher than in dipped quarters. These results suggested that the method of premilking udder preparation can influence the efficacy of postmilking teat sanitation. CONCLUSIONS
In the experimental exposure studies, a .5% chlorhexidine digluconate was efficacious against Staph. aureus and ineffective against Strep. agalactiae. Reformulation of the .5% chlorhexidine product did not improve efficacy. Another reformation contained 1% chlorhexidine digluconate and remained ineffective against Strep. agalactiae. These three chlorhexidine digluconate formulations did not significantly reduce incidence of IMI by Strep. agalactiae under experimental exposure conditions. Under natural exposure conditions, two chlorhexidine digluconate teat dips significantly reduced incidence of 1MI by contagious mastitis pathogens and all major mastitis pathogens. The percentage of quarters diagnosed with environmental pathogens was smaller in the dipped group, but the reduction was nonsignificant. In trial 4, efficacy of the .35% chlorhexidine product increased when premilking udder preparation procedures were changed. Minor mastitis pathogens, coagulase-negative staphylococcal species and C. bovis, were controlled by postdipping in trial 4 when teats were sanitized prior to attachment of milking machines. In trial 5, rate of IMI by coagulase-negative staphylococci was reduced significantly by the .5% chlorhexidine product, but C. bovis WI were not controlled. These studies suggested that premilking udder preparation could affect efficacy of postmilking teat sanitizers. ACKNOWLEDGMENTS
The authors express appreciation to the two cooperator herds, Senesac Dairy and Fontaine Journal of Dairy Science Vol. 76, No.9, 1993
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Brothers Fann. for their patience throughout the course of these natural exposure studies. The partial support of these studies by the manufacturers of the teat dip formulations is appreciated. REFERENCES 1 Eberhart, R. J., and J. M. Buckalew. 1972. Evaluation of a hygiene and dry period therapy program for mastitis control. 1. Dairy Sci. 55: 1683. 2 Gerring, E. L., R. Hall, and A. J. Sandoe. 1968. The evaluation of a teat-dipping formulation chlorhexidine. Vet. Rec. 83:112. 3 Harmon, R. J., R. J. Eberhart, D. E. Jasper, B. E. Langlois, and R. A. Wilson. 1990. Microbiological Procedures for the Diagnosis of Bovine Udder Infection. 3rd ed. Natl. Mastitis Counc., Arlington, VA. 4 Hicks, W. G., T. J. Kennedy, D. M. Keister, and M. L. Miller. 1981. Evaluation of a teat dip of chlorhexidine digluconate (.5%) with glycerin (6%). J. Dairy Sci. 64: 2266. 5 Hogan. J. S.• D. M. Galton, R. J. Harmon, S. C. Nickerson. S. P. Oliver, and J. W. Pankey. 1990. Protocols for evaluating efficacy of postmilking teal dips. J. Dairy Sci. 73:2580.
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6 Neave. F. K.• F. H. Dodd. R. G. Kingwill, and D. R. Westgarth. 1969. Control of mastitis in the dairy herd by hygiene and management. J. Dairy Sci. 52:696. 7 Pankey, J. W.• R. L. Boddie, and S. C. Nickerson. 1985. Efficacy evaluation of two new teat dip formulations under experimental challenge. J. Dairy Sci. 68: 462. 8 Pankey. J. W., R. 1. Eberhart. A. L. Cuming. R. D. Daggett, R. J. Farnsworth, and C. K. McDuff. 1984. Update on postmilking teat antisepsis. J. Dairy Sci. 67:1336. 9 Pankey, 1. W.• S. C. Nickerson. and R. L. Boddie. 1984. Evaluation of linear dodecyl benzene sulfonic acid teat dip under experimental challenge. J. Dairy Sci. 70:867. 10 Pankey, J. W .• W. N. Philpot, R. L. Boddie, and J. L. Watts. 1983. Evaluation of nine teat dip formulations under experimental challenge 10 Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 66: 161. 11 Pankey, J. W., E. E. Wildman. P. A. Drechsler, and J. S. Hogan. 1987. Field trial evaluation of premilking teal disinfection. J. Dairy Sci. 70:867. 12 Philpot, W. N.• and 1. W. Pankey, Jr. 1975. Hygiene in the prevention of udder infections: II. Evaluation of oil-based teat dips. J. Dairy Sci. 58:205.