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Efficacy of Teat Dips Containing a Hypochlorous Acid Germicide Against Experimental Challenge with Staphylococcus aureus and Streptococcus agalactiae1
Efficacy of Teat Dips Containing a Hypochlorous Acid Germicide Against Experimental Challenge with Sfaphylococcus aureus and Strepfococcus agalactia@ R. L. BODDIE and S. C. NICKERSON Hill Farm Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Route 1, Box 10, Homer 71040
ABSTRACT
against Streptococcus agalactiae IMI ( 1 1). Using the experimental challenge model, Pankey et al. ( 12) Two teat dip formulations containing sodium di- evaluated 0.6 and 0.9% sodium hypochlorite teat dips chloroisocyanurate, which released hypochlorous acid and obtained efficacies of 28.3 and 56.870,respec(2800 ppm) as the active ingredient, were tested for tively, against Staph. aureus. In another study ( 13 ), efficacy against new Staphylococcus aureus and Strep- excised teats were challenged with Staph. aureus and tococcus agalactiae IMI using a n experimental Strep. agalactiae, followed by dipping with 0.3% and challenge model. Product 1 reduced the number of 1% formulations of sodium dichloro-s-triazinetrione new Staph. aureus IMI by 73.6% and reduced the (SDT), which were homologous to sodium dinumber of new Strep. agalactiae IMI by 65.1%. chloroisocyanurate ( NaDCC) . The lower concentraProduct 2 reduced the number of new Staph. aureus tion of 0.3% SDT was effective, exhibiting 95.2 and IMI by 69.0% and reduced the number of new Strep. 81.3% reductions in numbers of Staph. aureus and agalactiae IMI by 63.5%. No adverse effects on teat Strep. agalactiae organisms on teat skin, respectively. skin condition were observed over the course of the Similarly, a 1% formulation was 91 and 85.6% effecstudies. tive in reducing Staph, aureus and Strep. agalactiae ( Key words: hypochlorous acid, Staphylococcus organisms on teat skin. A 0.6% concentration of SDT aureus, Streptococcus agalactiae, teat dip) was tested against Staph. aureus in a n experimental Abbreviation key: NaDCC = sodium di-chloro- challenge trial and exhibited an efficacy of 79.0% isocyanurate, SDT = sodium dichloro-s-triazinetrione, ( 14). In a subsequent trial ( 121, a 1% SDT formulaTSA = trypticase soy agar, TSB = trypticase soy tion reduced new Staph. aureus IMI by 75.9% and new Strep. agalactiae IMI by 63.2%; a 1.7% concenbroth. tration of SDT yielded a 48.1% efficacy against Strep. agalactiae. INTRODUCTION The objective of this study was to evaluate the Chlorine and compounds containing chlorine have efficacies of two teat dip products containing a been used for many years for disease prevention and hypochlorous acid germicide, which had been liberdisinfection in dairy parlors. For example, teat dips ated from the dissociation of NaDCC in water, containing 4% sodium hypochlorite were prepared by against experimental challenge with Staph. aureus diluting commercial laundry bleaches with water and Strep. agalactiae. ( 10). Dipping teats with this formulation in conjunction with a 3-yrprogram of dry cow therapy decreased MATERIALS AND METHODS the prevalence of mastitis from 28 to 7% of quarters Products 1 and 2 were evaluated in two separate ( 10). Subsequently, a commercial bleach diluted to trials. Product 1 was evaluated from September 4% sodium hypochlorite was tested in three separate through October 1994, and product 2 was evaluated experimeintal challenge studies ( 1 1). The hypochfrom March through May 1995. lorite dip exhibited efficacies of 96.3,83.3,and 50% against Staphylococcus aureus and 60, 70, and 50% Sampling Schedule Received. January 22, 1996. Accepted March 22, 1996. 'Approved for publication by the director of the Louisiana Agricultural Experiment Station as Manuscript Number 95-809219. 1996 J Dairy Sci 79:1-1688
The bacteriological status of mammary quarters was determined at the initiation of each trial by collection and culture of duplicate milk samples. A third sample was collected from specific quarters and cultured when results from the first two samples
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differed. Milk samples were collected and analyzed bacteriologically during each week of the trial. Whenever Staph. aureus or Strep. agalactiae was isolated for the first time in a previously uninfected quarter, a second sample was collected and cultured within 48 h after first isolation to confirm new IMI. All quarters were eligible for new Staph. aureus and Strep. agalactiae IMI during each trial except those infected with organisms of the same species as challenge organisms and those with deformed or abnormal teats. Collection of Milk Samples
Prior to quarter milk sampling, the ventral surfaces of udders and teats that were excessively dirty were was:hed using a hand-held hose and paper towels. After being washed, udders and teats were dried thoroughly with additional paper towels, and two or three streams of foremilk were discarded. Each teat apex was scrubbed for several seconds with a cotton pledget moistened with 70% alcohol. Teats on the side of the udder opposite from the technician were sanitized first, and milk samples were collected in reverse order in sterile snap-cap plastic tubes and then refrigerated a t 5°C. For teats that were visibly clean prior to collection, washing was omitted, and only cotton. pledgets moistened with 70% alcohol were used to sanitize teat ends.
(Agrisept'" Tabs; Mick Doyle Marketing Int. Ltd., Naas, Ireland). Two tablets were added to 1 L of tap water and allowed to dissolve thoroughly. This solution provided 2800 ppm of available chlorine as hypochlorous acid CpH 5.5 to 6.0). The teat dip was stored in the dairy parlor in a light-resistant container for up to 7 d. Product 2 also was provided in tablet form, and each effervescent tablet contained 3.0 g of NaDCC (EfferceptVet; manufactured for Effercept Products, a division of Micrel Ltd., Inc., Laramie, WY by Olin Chemical Co., Lavonia, MI). Six tablets were added to 3.78 L of tap water and allowed to dissolve thoroughly, and then 28 cc of a solution containing a dye and skin conditioner (Aquablue Soft-Teat Dip Additive; Effercept Products, Phoenix, AZ) were added. This solution provided 3000 ppm of available chlorine as hypochlorous acid ( p H 6.7). The teat dip was stored i n the dairy parlor in a light-resistant container for up to 5 d. Treatment Method
The milking herd of the Hill Farm Research Station was used for the experimental challenge studies. In the 5-wk trial to test product 1, 139 cows were used, and, in the 9-wk trial to test product 2, 75 cows were used. During the afternoon milking, Monday through Friday, all teats of each cow were immersed to a depth of approximately 25 mm in a challenge Culture and Diagnostic Procedures suspension containing Staph. aureus (ATCC 29740) Samples were mixed by shaking, and a 0.01-ml and Strep. agalactiae (ATCC 27956) immediately afaliquot was streaked on trypticase soy agar ( T S A ter milking machines were removed. Immediately folBecton Dickinson, Cockeysville, MD) containing 5% lowing challenge, the distal 25 mm of the left front bovine calf' blood. Plates were incubated at 37°C for and right rear teats were dipped with the experimen48 h and examined to identify the microorganisms tal teat dip; the remaining two teats were undipped that were present. Contaminated quarters were controls. resampled to confirm the presence or absence of challenge organisms. Colonies of Staph. aureus were Preparation of the Challenge Suspension identified presumptively by hemolytic pattern and Suspensions of Staph. aureus and Strep. agalactiae confirmed by tube coagulase test. Colonies of Strep. were prepared as described by Boddie et al. ( 3 ) . agalactiae were identified to serogroup by the PhadeStock suspensions of Staph. aureus were prepared bact Strept,ococcus Test (Boule Diagnostics AB, Hudweekly. The contents of one lyophilized vial of Staph. dinge, Sweden). A new IMI was confirmed when 1) were reconstituted in 6 ml of tripticase soy aureus Staph. aureus or Strep. agalactiae was isolated from a broth (TSB; Becton Dickinson) and incubated a t clinical quarter, 2 ) two consecutive samples yielded 37°C for 5 to 7 h. This culture was used to inoculate a 2500 cfdml of the same pathogen, or 3 ) three con500 ml of TSB, which was incubated on a volume of secutive samples contained 100 to 400 cfdml of the gyratory shaker for 16 h. After incubation, bacterial same pathogen ( 9 ) . cells were pelleted by centrifugation, washed twice with 0.1% proteose-peptone (Difco Laboratories, Description of Experimental Teat Dips Detroit, MI), and resuspended to the original volume Product 1 was provided in tablet form, and each in proteose-peptone. Serial dilutions were prepared in effervescent tablet contained 2.5 g of NaDCC proteose-peptone, and 0.1 ml was plated on TSA with Journal of Dairy Science Vol. 79, No. 9, 1996
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O U R INDUSTRY TODAY TABLE 1. Efficacy data of a hypochlorous acid teat dip (product 1) against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. Organism and treatment
Quarters eligible for new IMI
New
IMI
Quarter days New IMI per 100 quarter a t risk for new IMI days a t risk
(no.) Staph. aureus Dip Control Strep. agalactiae Dip Control
Reduction (%)
257 253
11 39
8188 7654
0.134 0.510
73.6*
270 290
9 27
8676 9114
0.104 0.296
65.1*
*P I 0.001.
5% bovine calf blood. Plates were incubated for 24 h at 37"C, and colonies were counted to ascertain the microbial concentration of the stock suspension. This suspension was stored at 5°C and used each day for 5 d to prepare challenge suspensions of Staph. aureus. Cultures of Strep. agalactiae were prepared by resuspension of a lyophilized vial of Strep. agalactiae in 6 ml of TSB, and a 0.01-ml aliquot was streakplated on each of five TSA plates. Plates were incubated a t 37°C for 16 h and stored at 5°C to serve as stock cultures for 5 d. Daily challenge suspensions of Strep. agalactiae were prepared by inoculation of 6 ml of TSB with six colonies from a TSA stock plate. The 6-ml culture was incubated for approximately 15 h a t 37°C and used to inoculate 500 ml of TSB. The 500-ml culture was incubated for 7 h at 37°C on a gyratory shaker. Aliquots of the culture were added t o approximately 150 ml of nonsterile pasteurized milk to adjust the concentration of Strep. agalactiae to approximately 5 x 107 cfdml. An aliquot of the Staph. aureus stock suspension was added t o the Strep. agalactiae suspension t o obtain a concentration of approximately 5 x 107 cfdml of Staph. aureus. This bacterial suspension was taken immediately to the milking parlor to challenge teats during the afternoon milking. A plate count was conducted daily on challenge suspensions. Statistical Methods
Differences between the percentage of quarters becoming infected in treatment groups were tested as described by Hogan et al. ( 9 ) using an approximated t statistic defined as t = [(xl/nl) - (x2/n2)l/[xl + x2)/ (nlna)]0.5, where XI = number of new IMI in control quarters, x2 = number of new IMI in treated quarters, n l = (number of control quarters)(time unit), and n2 = (number of treated quarters)(time unit). The denominators n l and n2 were expressed as the sum of
quarter days. A quarter was eligible for only one IMI per organism during each trial. The percentage reduction in rate of new IMI in the treated groups compared with that in the control groups was expressed as 100 [(xl/nl) - (x2/n2)]/(xl/nl). Teat germicides generally are considered to be efficacious when the mean percentage reduction of new IMI is 240% and the lower confidence limit of the mean is 225% ( 9 ) . Scoring of Teat Skin and Teat End Condition
In the trial that evaluated product 1, only a subjective evaluation of teat condition was made prior to, during, and after the trial. However, characteristics of teat skin surfaces and teat ends in the trial that evaluated product 2 were scored immediately before and at the conclusion of the trial to determine the effects of this germicide on teat condition. Teat skin and teat end condition were characterized according to the parameters established by Goldberg et al. ( 8 1, which are outlined in Tables 3 and 4. RESULTS AND DISCUSSION
Infection data collected during the trial for the evaluation of product 1 are summarized in Table 1. For both organisms, the percentage of dipped quarters becoming infected was less than that of control quarters. The teat dip reduced the number of new Staph. aureus IMI by 73.6% ( P I 0.001) during the 5-wk study. Reduction in new Strep. agalactiae IMI was 65.1% ( P 5 0.001). Infection rates for Staph. aureus were 15.4 and 4.3% for control and dipped quarters, respectively. Streptococcus agalactiae infection rates were 9.3 and 3.3% for control and dipped quarters, respectively. A total of 5 quarters exhibited symptoms of clinical mastitis during the trial that evaluated product 1, Journal of Dairy Science Vol. 79, No. 9, 1996
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TABLE 2. Efficacy data of a hypochlorous acid teat dip (product 2 ) against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. Organism and treatment Staph. aureus Dip Control Strep. agalactiae Dip Control
Quarters eligible for new IMI
New IMI
Quarter days at risk for new IMI
New IMI per 100 quarter days at risk
Reduction
137 140
10 30
8276 7690
0.121 0.390
69.0*
147 148
8 21
9020 8650
0.089 0.243
63.5**
*P 5 0.001. **P 5 0.01.
and frequency was similar for both challenge organ- the chlorine being present as hypochlorite ions rather isms in dilpped and control quarters (1 vs. 4). The than as undissociated hypochlorous acid ( 1, 2, 4) frequency of clinical mastitis in quarters diagnosed In solution, NaDCC forms a n equilibrium mixture with new Staph. aureus IMI was 1.4% among dipped of di- and monochlorocyanurates, hypochlorous acid, quarters and 1.67% among undipped control quarters. and hypochlorite ions. This equilibrium mixture acts The frequency of clinical mastitis in quarters diag- as a reservoir that converts to hypochlorous acid and nosed with new Strep. agalactiae IMI was 1.4% hypochlorite ions in response to their removal from among dipped quarters and 0.4% among undipped the system by the disinfection process. The lethal control quarters. action on microorganisms by hypochlorous acid is due Infection data collected during the trial for the to chlorination and oxidation of the cell wall and evaluation of product 2 are summarized in Table 2. internal proteins, including enzymes ( 1). The equilibrium between the components of this For both organisms, the percentage of dipped quarters becoming infected was significantly less than mixture remains stable until bacteria or organic that of control quarters. The teat dip reduced the material requires chlorine. This chlorine demand utinumber of new Staph. aureus IMI by 69.0% ( P I lizes hypochlorous acid, which, in turn, is rapidly 0.001) during the 9-wk study. Reduction in new replaced to satisfy the demand (1).The release of Strep. aga1,uctiae IMI was 63.5% ( P 5 0.01).Infection hypochlorous acid provides the teat dip with greater rates for Staph. aureus were 21.4and 7.3% for control biocidal activity than a sodium hypochlorite solution and dipped quarters, respectively. Infection rates for and is inactivated to a lesser extent by organic matter Strep. agalactiae were 14.2and 5.4% for control and ( 2 , 4). The teat dips tested in this study were also evaludipped quarters, respectively. A total of 7 quarters exhibited symptoms of clinical ated in laboratory trials using a lower strength solumastitis during the trial that evaluated product 2. tion against organisms known t o cause bovine mastiThe frequency of clinical mastitis in quarters diag- tis. A concentration of 1400 ppm of NaDCC was nosed with new Staph. aureus IMI was 0% among tested against Staph. aureus, Streptococcus uberis, dipped quarters and 2.9% among undipped control Escherichia coli, and Pseudomonas aeruginosa that quarters. The frequency of clinical mastitis in quar- had been isolated from mastitic cows. For all of the ters diagnosed with new Strep. agalactiae IMI was organisms tested, a log 3 reduction in colony-forming 0.7% among dipped quarters and 1.4% among un- units was achieved in the presence of 5% milk for 1 dipped control quarters. min of contact time ( 7 1. In another study, NaDCC at Solutions of NaDCC are slightly acidic, and, a t the 125 ppm of available chlorine was tested against concentrations used for surface disinfection, the pH is Staph. aureus, E . coli, P. aeruginosa, and Enterobac5.5 to 6.0. At this pH, about 95% of the free available ter aerogenes, and, for all species examined, disinfecchlorine is present as hypochlorous acid, the molecule tion was >io9 organismdm1 ( 1). During the trial that evaluated product 1, no that is primarily responsible for the biocidal activity of chlorine disinfectants. Sodium hypochlorite solu- detrimental effects were noted on teat skin from the tions (household bleach) have a lower biocidal ac- use of this teat dip, based on subjective evaluation of tivity because the pH is of the order of 12,resulting in teat skin by milkers and technicians. During the trial Journal of Dairy Science Vol. 79, No. 9, 1996
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that evaluated product 2, a n objective analysis of teat skin condition scores demonstrated that mean score before and after the trial for dipped and control quarters was approximately 1, which was characterized as normal, smooth skin that was free from scabs, cracks, or chapping. Similar analyses of teat end condition scores also showed that mean score across all variables was approximately 1, which was characterized as a normal, smooth teat end with no evidence of irritation. For product 2, a t least 94.7%of teats were characterized as having normal (score = 1 ) teat skin condition before and after the trial for dipped and control quarters (Table 3 ) . The percentages of teats with physical injuries (score = 0 ) were slightly lower after the trial than before the trial for dipped and control quarters. The percentage of teats showing some evidence of scaling (score = 2 ) decreased slightly for both dipped and control quarters from 0.7% before the trial to 0% after the trial. The percentage of teats showing chapping (score = 3 ) among dipped quarters decreased slightly from 2% before the trial to 0.7% after the trial; among control teats, there was no difference ( 0 . 7% 1. No significant differences were observed in teat skin condition between dipped and control teats before or after the trial. For product 2, a t least 94.4%of teats were characterized as having normal (score = 1 ) teat end condition before and after the trial for dipped and control quarters (Table 4 ) . The percentage of teats with normal teat end condition decreased slightly from before t o after the trial in dipped and control quarters. The percentage of teat ends with a raised ring TABLE 3. Frequency of teat skin condition scores1 before and after a trial that evaluated the efficacy of a hypochlorous acid teat dip (product 2 ) Before Score
Dipped
Control
0
2 3 4
27 94 7 07 20 0
5
0
40 34 7 07 07 0 0
After Dipped
Control
(9) 1
14 97 9 0 07 0 0
28 96 5 0 07 0 0
‘Teat skin condition scoring: 0 = teat skin has been subjected to a physical injury (e.g., stepped on or frostbitten) unrelated to the treatment, o r the quarter is nonlactating; 1 = teat skin is smooth (free from scales, cracks, or chapping); 2 = teat skin shows some evidence of scaling; 3 = teat skin is chapped and some small warts may be present; 4 = teat skin is chapped and cracked, redness (indicating inflammation) is present, and numerous warts may be present; and 5 = teat skin is severely damaged and ulcerative with scabs or open lesions, and large or numerous warts may be present that interfere with teat end function ( 8 ) .
TABLE 4. Frequency of teat end condition scores1 before and after a trial that evaluated the efficacy of a hypochlorus acid teat dip (product 2 ) . Before Score
Dipped
Control
13 96 0 13 13 0 0
0 97 3 13 13 0 0
After Dipped
Control
~
(YC’
~
0 1
2 3 4
5
14 94 4 21 21 0
0
0
95 1 35 14 0 0
‘Teat end condition scoring 0 = teat end has been subjected to a physical injury le g , stepped on or frostbitten) unrelated to the treatment or the quarter is nonlactating, 1 = teat end sphincter is smooth with no evidence of irritation, 2 = teat end has a raised ring, 3 = teat end sphincter is roughened with slight cracks, but no redness IS present 4 = teat end sphincter is inverted with many cracks, giving a “flowered” appearance, and teat end may have old but healing scabs, and 5 = teat end is severely damaged and ulcerative with scabs or open lesions, and large or numerous warts may be present that interfere with teat end function ( 8 )
(score = 2 ) increased slightly from 1.39~before to 2.1%after the trial in dipped quarters, and, in control quarters, the increase was from 1.3 t o 3.5%. The percentage of teat ends exhibiting a roughened sphincter with slight cracks (score = 3 changed little before and after the trial in dipped and control quarters. No significant differences were observed in teat end condition between dipped and control teats before or after the trial. The trial lengths of 5 and 9 wk were thought to be of sufficient duration to evaluate any effect of products 1 and 2 on teat skin condition. This contention is based on results of studies (5, 6 ) on the skin healing properties of various postmilking teat dips and emollients after the teat skin surface was experimentally chapped with VV sodium hydroxide. For example, in one study, Fox et al. ( 6 ) monitored the condition of teat skin daily over a 30-d period and found that some teat dips were superior to others in the ability to heal chapped teat skin; however, regardless of treatment, no chapping was observed after d 17 of the trial, and, by d 10, no differences were observed among treatments. In a similar, subsequent trial, Fox ( 5 ) monitored the condition of teat skin daily over 19 milkings ( 10 d ) in response to several postmilking teat disinfectants and found that this duration of exposure was sufficient for product evaluation. Thus, the germicidal effects of teat dip on teat skin condition became apparent in a matter of days, and the periods of 5 and 9 wk that were used in the present study were probably sufficient to evaluate the influence of the two NaDCC products. Journal of Dairy Science Vol. 79, No. 9, 1996
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CONCLUSIONS
The novel teat dip formulations containing NaDCC in tablet form reduced new IMI caused by Staph. aureus and Strep. agalactiae during the two efficacy trials conducted under experimental challenge conditions recommended by the National Mastitis Council (9). ACKNOWLEDGMENTS
We thank Nancy Boddie, Carol Pratt, Corinne Ray, and the Hill Farm Research Station dairy personnel for technical assistance and Frances Huff for secretarial support. Mick Doyle, Marketing Int. Ltd., and Monsanto are gratefully acknowledged for partial support of the teat dip efficacy trials. REFERENCES 1Bloomfield, S. F., and G. A. Miles. 1979. The antibacterial properties of sodium dichloroisocyanurate and sodium hypochlorite formulations. J. Appl. Bacteriol. 46:65. 2 Bloomfield, S. F., and E. E. Uso. 1985. The antibacterial properties of sodlium hypochlorite and sodium dichloroisocyanurate as hospital disinfectants. J. Hosp. Infect. 6:20. 3Boddie, R. L., S. C. Nickerson, and G. K. Kemp. 1994. Efficacy of two barrier teat dips containing chlorous acid germicides against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 77:3192.
Journal of Odry Science Vd. 79,No. 9,1996
4Coates, D. 1988. Comparison of sodium hypochlorite and sodium dichloroisocyanurate disinfectants: neutralization by serum. J. Hosp. Infect. 11:60. 5 Fox, L. K. 1992. Colonization by Staphylococcus aureus on chapped teat skm: effect of iodine and chlorhexidine postmilking disinfectants. J. Dairy Sci. 75:66. 6Fox, L. K., J. A. Nagy, J. K. Hillers, J. D. Cronrath, and D. A. Ratkowsky. 1991. Effects of postmilking teat treatment on the colonization of Staphylococcus aureus on chapped teat skin. Am. J. Vet. Res. 52:799. 7 Freedman, S. 1994. The bactericidal effect of troclosene sodium for dairy disinfection. A laboratory study. Israel J. Vet. Med. 49: 25. 8 Goldberg, J. J., P.A. Murdough, A. B. Howard, P. A. Drechsler, J. W. Pankey, G. A. Ledbetter, L. L. Day, and J. D.Day. 1994. Winter evaluation of a postmilking powdered teat dip. J. Dairy Sci. 77:748. 9Hogan, 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 teat dips. J. Dairy Sci. 73:2580. lONatzke, R. P., R. W. Everett, R. S.Guthrie, J. F. Keown, A. M. Meek, W. G. Merrill, S. J. Roberts. and G. H. Schmidt. 1972. Mastitis control program: effect on milk production. J. Dairy Sci. 55:1256. 11Pankey, J. W., and W. N. Philpot. 1975. Hygiene in the prevention of udder infections I. Comparative efficacy of four teat dips. J. Dairy Sci. 58:202 12 Pankey, J. W., W. N. Philpot. R. L. Boddie, and J. L. Watts. 1983. Evaluation of nine teat dip formulations under experimental challenge to Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 66:161. 13 Philpot, W. N., and J. W. Pankey. 1975. Hygiene in the prevention of udder infections 111. Effectiveness of 59 teat dips for reducing bacterial populations on teat skin. J. Dairy Sci. 58:209. 14Philpot, W. N., and J. W. Pankey. 1978. Hygiene in the prevention of udder infections. V. Efficacy of teat dips under experimental exposure to mastitis pathogens. J. Dairy SCI.61:956.
ABSTRACT
against Streptococcus agalactiae IMI ( 1 1). Using the experimental challenge model, Pankey et al. ( 12) Two teat dip formulations containing sodium di- evaluated 0.6 and 0.9% sodium hypochlorite teat dips chloroisocyanurate, which released hypochlorous acid and obtained efficacies of 28.3 and 56.870,respec(2800 ppm) as the active ingredient, were tested for tively, against Staph. aureus. In another study ( 13 ), efficacy against new Staphylococcus aureus and Strep- excised teats were challenged with Staph. aureus and tococcus agalactiae IMI using a n experimental Strep. agalactiae, followed by dipping with 0.3% and challenge model. Product 1 reduced the number of 1% formulations of sodium dichloro-s-triazinetrione new Staph. aureus IMI by 73.6% and reduced the (SDT), which were homologous to sodium dinumber of new Strep. agalactiae IMI by 65.1%. chloroisocyanurate ( NaDCC) . The lower concentraProduct 2 reduced the number of new Staph. aureus tion of 0.3% SDT was effective, exhibiting 95.2 and IMI by 69.0% and reduced the number of new Strep. 81.3% reductions in numbers of Staph. aureus and agalactiae IMI by 63.5%. No adverse effects on teat Strep. agalactiae organisms on teat skin, respectively. skin condition were observed over the course of the Similarly, a 1% formulation was 91 and 85.6% effecstudies. tive in reducing Staph, aureus and Strep. agalactiae ( Key words: hypochlorous acid, Staphylococcus organisms on teat skin. A 0.6% concentration of SDT aureus, Streptococcus agalactiae, teat dip) was tested against Staph. aureus in a n experimental Abbreviation key: NaDCC = sodium di-chloro- challenge trial and exhibited an efficacy of 79.0% isocyanurate, SDT = sodium dichloro-s-triazinetrione, ( 14). In a subsequent trial ( 121, a 1% SDT formulaTSA = trypticase soy agar, TSB = trypticase soy tion reduced new Staph. aureus IMI by 75.9% and new Strep. agalactiae IMI by 63.2%; a 1.7% concenbroth. tration of SDT yielded a 48.1% efficacy against Strep. agalactiae. INTRODUCTION The objective of this study was to evaluate the Chlorine and compounds containing chlorine have efficacies of two teat dip products containing a been used for many years for disease prevention and hypochlorous acid germicide, which had been liberdisinfection in dairy parlors. For example, teat dips ated from the dissociation of NaDCC in water, containing 4% sodium hypochlorite were prepared by against experimental challenge with Staph. aureus diluting commercial laundry bleaches with water and Strep. agalactiae. ( 10). Dipping teats with this formulation in conjunction with a 3-yrprogram of dry cow therapy decreased MATERIALS AND METHODS the prevalence of mastitis from 28 to 7% of quarters Products 1 and 2 were evaluated in two separate ( 10). Subsequently, a commercial bleach diluted to trials. Product 1 was evaluated from September 4% sodium hypochlorite was tested in three separate through October 1994, and product 2 was evaluated experimeintal challenge studies ( 1 1). The hypochfrom March through May 1995. lorite dip exhibited efficacies of 96.3,83.3,and 50% against Staphylococcus aureus and 60, 70, and 50% Sampling Schedule Received. January 22, 1996. Accepted March 22, 1996. 'Approved for publication by the director of the Louisiana Agricultural Experiment Station as Manuscript Number 95-809219. 1996 J Dairy Sci 79:1-1688
The bacteriological status of mammary quarters was determined at the initiation of each trial by collection and culture of duplicate milk samples. A third sample was collected from specific quarters and cultured when results from the first two samples
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differed. Milk samples were collected and analyzed bacteriologically during each week of the trial. Whenever Staph. aureus or Strep. agalactiae was isolated for the first time in a previously uninfected quarter, a second sample was collected and cultured within 48 h after first isolation to confirm new IMI. All quarters were eligible for new Staph. aureus and Strep. agalactiae IMI during each trial except those infected with organisms of the same species as challenge organisms and those with deformed or abnormal teats. Collection of Milk Samples
Prior to quarter milk sampling, the ventral surfaces of udders and teats that were excessively dirty were was:hed using a hand-held hose and paper towels. After being washed, udders and teats were dried thoroughly with additional paper towels, and two or three streams of foremilk were discarded. Each teat apex was scrubbed for several seconds with a cotton pledget moistened with 70% alcohol. Teats on the side of the udder opposite from the technician were sanitized first, and milk samples were collected in reverse order in sterile snap-cap plastic tubes and then refrigerated a t 5°C. For teats that were visibly clean prior to collection, washing was omitted, and only cotton. pledgets moistened with 70% alcohol were used to sanitize teat ends.
(Agrisept'" Tabs; Mick Doyle Marketing Int. Ltd., Naas, Ireland). Two tablets were added to 1 L of tap water and allowed to dissolve thoroughly. This solution provided 2800 ppm of available chlorine as hypochlorous acid CpH 5.5 to 6.0). The teat dip was stored in the dairy parlor in a light-resistant container for up to 7 d. Product 2 also was provided in tablet form, and each effervescent tablet contained 3.0 g of NaDCC (EfferceptVet; manufactured for Effercept Products, a division of Micrel Ltd., Inc., Laramie, WY by Olin Chemical Co., Lavonia, MI). Six tablets were added to 3.78 L of tap water and allowed to dissolve thoroughly, and then 28 cc of a solution containing a dye and skin conditioner (Aquablue Soft-Teat Dip Additive; Effercept Products, Phoenix, AZ) were added. This solution provided 3000 ppm of available chlorine as hypochlorous acid ( p H 6.7). The teat dip was stored i n the dairy parlor in a light-resistant container for up to 5 d. Treatment Method
The milking herd of the Hill Farm Research Station was used for the experimental challenge studies. In the 5-wk trial to test product 1, 139 cows were used, and, in the 9-wk trial to test product 2, 75 cows were used. During the afternoon milking, Monday through Friday, all teats of each cow were immersed to a depth of approximately 25 mm in a challenge Culture and Diagnostic Procedures suspension containing Staph. aureus (ATCC 29740) Samples were mixed by shaking, and a 0.01-ml and Strep. agalactiae (ATCC 27956) immediately afaliquot was streaked on trypticase soy agar ( T S A ter milking machines were removed. Immediately folBecton Dickinson, Cockeysville, MD) containing 5% lowing challenge, the distal 25 mm of the left front bovine calf' blood. Plates were incubated at 37°C for and right rear teats were dipped with the experimen48 h and examined to identify the microorganisms tal teat dip; the remaining two teats were undipped that were present. Contaminated quarters were controls. resampled to confirm the presence or absence of challenge organisms. Colonies of Staph. aureus were Preparation of the Challenge Suspension identified presumptively by hemolytic pattern and Suspensions of Staph. aureus and Strep. agalactiae confirmed by tube coagulase test. Colonies of Strep. were prepared as described by Boddie et al. ( 3 ) . agalactiae were identified to serogroup by the PhadeStock suspensions of Staph. aureus were prepared bact Strept,ococcus Test (Boule Diagnostics AB, Hudweekly. The contents of one lyophilized vial of Staph. dinge, Sweden). A new IMI was confirmed when 1) were reconstituted in 6 ml of tripticase soy aureus Staph. aureus or Strep. agalactiae was isolated from a broth (TSB; Becton Dickinson) and incubated a t clinical quarter, 2 ) two consecutive samples yielded 37°C for 5 to 7 h. This culture was used to inoculate a 2500 cfdml of the same pathogen, or 3 ) three con500 ml of TSB, which was incubated on a volume of secutive samples contained 100 to 400 cfdml of the gyratory shaker for 16 h. After incubation, bacterial same pathogen ( 9 ) . cells were pelleted by centrifugation, washed twice with 0.1% proteose-peptone (Difco Laboratories, Description of Experimental Teat Dips Detroit, MI), and resuspended to the original volume Product 1 was provided in tablet form, and each in proteose-peptone. Serial dilutions were prepared in effervescent tablet contained 2.5 g of NaDCC proteose-peptone, and 0.1 ml was plated on TSA with Journal of Dairy Science Vol. 79, No. 9, 1996
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O U R INDUSTRY TODAY TABLE 1. Efficacy data of a hypochlorous acid teat dip (product 1) against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. Organism and treatment
Quarters eligible for new IMI
New
IMI
Quarter days New IMI per 100 quarter a t risk for new IMI days a t risk
(no.) Staph. aureus Dip Control Strep. agalactiae Dip Control
Reduction (%)
257 253
11 39
8188 7654
0.134 0.510
73.6*
270 290
9 27
8676 9114
0.104 0.296
65.1*
*P I 0.001.
5% bovine calf blood. Plates were incubated for 24 h at 37"C, and colonies were counted to ascertain the microbial concentration of the stock suspension. This suspension was stored at 5°C and used each day for 5 d to prepare challenge suspensions of Staph. aureus. Cultures of Strep. agalactiae were prepared by resuspension of a lyophilized vial of Strep. agalactiae in 6 ml of TSB, and a 0.01-ml aliquot was streakplated on each of five TSA plates. Plates were incubated a t 37°C for 16 h and stored at 5°C to serve as stock cultures for 5 d. Daily challenge suspensions of Strep. agalactiae were prepared by inoculation of 6 ml of TSB with six colonies from a TSA stock plate. The 6-ml culture was incubated for approximately 15 h a t 37°C and used to inoculate 500 ml of TSB. The 500-ml culture was incubated for 7 h at 37°C on a gyratory shaker. Aliquots of the culture were added t o approximately 150 ml of nonsterile pasteurized milk to adjust the concentration of Strep. agalactiae to approximately 5 x 107 cfdml. An aliquot of the Staph. aureus stock suspension was added t o the Strep. agalactiae suspension t o obtain a concentration of approximately 5 x 107 cfdml of Staph. aureus. This bacterial suspension was taken immediately to the milking parlor to challenge teats during the afternoon milking. A plate count was conducted daily on challenge suspensions. Statistical Methods
Differences between the percentage of quarters becoming infected in treatment groups were tested as described by Hogan et al. ( 9 ) using an approximated t statistic defined as t = [(xl/nl) - (x2/n2)l/[xl + x2)/ (nlna)]0.5, where XI = number of new IMI in control quarters, x2 = number of new IMI in treated quarters, n l = (number of control quarters)(time unit), and n2 = (number of treated quarters)(time unit). The denominators n l and n2 were expressed as the sum of
quarter days. A quarter was eligible for only one IMI per organism during each trial. The percentage reduction in rate of new IMI in the treated groups compared with that in the control groups was expressed as 100 [(xl/nl) - (x2/n2)]/(xl/nl). Teat germicides generally are considered to be efficacious when the mean percentage reduction of new IMI is 240% and the lower confidence limit of the mean is 225% ( 9 ) . Scoring of Teat Skin and Teat End Condition
In the trial that evaluated product 1, only a subjective evaluation of teat condition was made prior to, during, and after the trial. However, characteristics of teat skin surfaces and teat ends in the trial that evaluated product 2 were scored immediately before and at the conclusion of the trial to determine the effects of this germicide on teat condition. Teat skin and teat end condition were characterized according to the parameters established by Goldberg et al. ( 8 1, which are outlined in Tables 3 and 4. RESULTS AND DISCUSSION
Infection data collected during the trial for the evaluation of product 1 are summarized in Table 1. For both organisms, the percentage of dipped quarters becoming infected was less than that of control quarters. The teat dip reduced the number of new Staph. aureus IMI by 73.6% ( P I 0.001) during the 5-wk study. Reduction in new Strep. agalactiae IMI was 65.1% ( P 5 0.001). Infection rates for Staph. aureus were 15.4 and 4.3% for control and dipped quarters, respectively. Streptococcus agalactiae infection rates were 9.3 and 3.3% for control and dipped quarters, respectively. A total of 5 quarters exhibited symptoms of clinical mastitis during the trial that evaluated product 1, Journal of Dairy Science Vol. 79, No. 9, 1996
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TABLE 2. Efficacy data of a hypochlorous acid teat dip (product 2 ) against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. Organism and treatment Staph. aureus Dip Control Strep. agalactiae Dip Control
Quarters eligible for new IMI
New IMI
Quarter days at risk for new IMI
New IMI per 100 quarter days at risk
Reduction
137 140
10 30
8276 7690
0.121 0.390
69.0*
147 148
8 21
9020 8650
0.089 0.243
63.5**
*P 5 0.001. **P 5 0.01.
and frequency was similar for both challenge organ- the chlorine being present as hypochlorite ions rather isms in dilpped and control quarters (1 vs. 4). The than as undissociated hypochlorous acid ( 1, 2, 4) frequency of clinical mastitis in quarters diagnosed In solution, NaDCC forms a n equilibrium mixture with new Staph. aureus IMI was 1.4% among dipped of di- and monochlorocyanurates, hypochlorous acid, quarters and 1.67% among undipped control quarters. and hypochlorite ions. This equilibrium mixture acts The frequency of clinical mastitis in quarters diag- as a reservoir that converts to hypochlorous acid and nosed with new Strep. agalactiae IMI was 1.4% hypochlorite ions in response to their removal from among dipped quarters and 0.4% among undipped the system by the disinfection process. The lethal control quarters. action on microorganisms by hypochlorous acid is due Infection data collected during the trial for the to chlorination and oxidation of the cell wall and evaluation of product 2 are summarized in Table 2. internal proteins, including enzymes ( 1). The equilibrium between the components of this For both organisms, the percentage of dipped quarters becoming infected was significantly less than mixture remains stable until bacteria or organic that of control quarters. The teat dip reduced the material requires chlorine. This chlorine demand utinumber of new Staph. aureus IMI by 69.0% ( P I lizes hypochlorous acid, which, in turn, is rapidly 0.001) during the 9-wk study. Reduction in new replaced to satisfy the demand (1).The release of Strep. aga1,uctiae IMI was 63.5% ( P 5 0.01).Infection hypochlorous acid provides the teat dip with greater rates for Staph. aureus were 21.4and 7.3% for control biocidal activity than a sodium hypochlorite solution and dipped quarters, respectively. Infection rates for and is inactivated to a lesser extent by organic matter Strep. agalactiae were 14.2and 5.4% for control and ( 2 , 4). The teat dips tested in this study were also evaludipped quarters, respectively. A total of 7 quarters exhibited symptoms of clinical ated in laboratory trials using a lower strength solumastitis during the trial that evaluated product 2. tion against organisms known t o cause bovine mastiThe frequency of clinical mastitis in quarters diag- tis. A concentration of 1400 ppm of NaDCC was nosed with new Staph. aureus IMI was 0% among tested against Staph. aureus, Streptococcus uberis, dipped quarters and 2.9% among undipped control Escherichia coli, and Pseudomonas aeruginosa that quarters. The frequency of clinical mastitis in quar- had been isolated from mastitic cows. For all of the ters diagnosed with new Strep. agalactiae IMI was organisms tested, a log 3 reduction in colony-forming 0.7% among dipped quarters and 1.4% among un- units was achieved in the presence of 5% milk for 1 dipped control quarters. min of contact time ( 7 1. In another study, NaDCC at Solutions of NaDCC are slightly acidic, and, a t the 125 ppm of available chlorine was tested against concentrations used for surface disinfection, the pH is Staph. aureus, E . coli, P. aeruginosa, and Enterobac5.5 to 6.0. At this pH, about 95% of the free available ter aerogenes, and, for all species examined, disinfecchlorine is present as hypochlorous acid, the molecule tion was >io9 organismdm1 ( 1). During the trial that evaluated product 1, no that is primarily responsible for the biocidal activity of chlorine disinfectants. Sodium hypochlorite solu- detrimental effects were noted on teat skin from the tions (household bleach) have a lower biocidal ac- use of this teat dip, based on subjective evaluation of tivity because the pH is of the order of 12,resulting in teat skin by milkers and technicians. During the trial Journal of Dairy Science Vol. 79, No. 9, 1996
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that evaluated product 2, a n objective analysis of teat skin condition scores demonstrated that mean score before and after the trial for dipped and control quarters was approximately 1, which was characterized as normal, smooth skin that was free from scabs, cracks, or chapping. Similar analyses of teat end condition scores also showed that mean score across all variables was approximately 1, which was characterized as a normal, smooth teat end with no evidence of irritation. For product 2, a t least 94.7%of teats were characterized as having normal (score = 1 ) teat skin condition before and after the trial for dipped and control quarters (Table 3 ) . The percentages of teats with physical injuries (score = 0 ) were slightly lower after the trial than before the trial for dipped and control quarters. The percentage of teats showing some evidence of scaling (score = 2 ) decreased slightly for both dipped and control quarters from 0.7% before the trial to 0% after the trial. The percentage of teats showing chapping (score = 3 ) among dipped quarters decreased slightly from 2% before the trial to 0.7% after the trial; among control teats, there was no difference ( 0 . 7% 1. No significant differences were observed in teat skin condition between dipped and control teats before or after the trial. For product 2, a t least 94.4%of teats were characterized as having normal (score = 1 ) teat end condition before and after the trial for dipped and control quarters (Table 4 ) . The percentage of teats with normal teat end condition decreased slightly from before t o after the trial in dipped and control quarters. The percentage of teat ends with a raised ring TABLE 3. Frequency of teat skin condition scores1 before and after a trial that evaluated the efficacy of a hypochlorous acid teat dip (product 2 ) Before Score
Dipped
Control
0
2 3 4
27 94 7 07 20 0
5
0
40 34 7 07 07 0 0
After Dipped
Control
(9) 1
14 97 9 0 07 0 0
28 96 5 0 07 0 0
‘Teat skin condition scoring: 0 = teat skin has been subjected to a physical injury (e.g., stepped on or frostbitten) unrelated to the treatment, o r the quarter is nonlactating; 1 = teat skin is smooth (free from scales, cracks, or chapping); 2 = teat skin shows some evidence of scaling; 3 = teat skin is chapped and some small warts may be present; 4 = teat skin is chapped and cracked, redness (indicating inflammation) is present, and numerous warts may be present; and 5 = teat skin is severely damaged and ulcerative with scabs or open lesions, and large or numerous warts may be present that interfere with teat end function ( 8 ) .
TABLE 4. Frequency of teat end condition scores1 before and after a trial that evaluated the efficacy of a hypochlorus acid teat dip (product 2 ) . Before Score
Dipped
Control
13 96 0 13 13 0 0
0 97 3 13 13 0 0
After Dipped
Control
~
(YC’
~
0 1
2 3 4
5
14 94 4 21 21 0
0
0
95 1 35 14 0 0
‘Teat end condition scoring 0 = teat end has been subjected to a physical injury le g , stepped on or frostbitten) unrelated to the treatment or the quarter is nonlactating, 1 = teat end sphincter is smooth with no evidence of irritation, 2 = teat end has a raised ring, 3 = teat end sphincter is roughened with slight cracks, but no redness IS present 4 = teat end sphincter is inverted with many cracks, giving a “flowered” appearance, and teat end may have old but healing scabs, and 5 = teat end is severely damaged and ulcerative with scabs or open lesions, and large or numerous warts may be present that interfere with teat end function ( 8 )
(score = 2 ) increased slightly from 1.39~before to 2.1%after the trial in dipped quarters, and, in control quarters, the increase was from 1.3 t o 3.5%. The percentage of teat ends exhibiting a roughened sphincter with slight cracks (score = 3 changed little before and after the trial in dipped and control quarters. No significant differences were observed in teat end condition between dipped and control teats before or after the trial. The trial lengths of 5 and 9 wk were thought to be of sufficient duration to evaluate any effect of products 1 and 2 on teat skin condition. This contention is based on results of studies (5, 6 ) on the skin healing properties of various postmilking teat dips and emollients after the teat skin surface was experimentally chapped with VV sodium hydroxide. For example, in one study, Fox et al. ( 6 ) monitored the condition of teat skin daily over a 30-d period and found that some teat dips were superior to others in the ability to heal chapped teat skin; however, regardless of treatment, no chapping was observed after d 17 of the trial, and, by d 10, no differences were observed among treatments. In a similar, subsequent trial, Fox ( 5 ) monitored the condition of teat skin daily over 19 milkings ( 10 d ) in response to several postmilking teat disinfectants and found that this duration of exposure was sufficient for product evaluation. Thus, the germicidal effects of teat dip on teat skin condition became apparent in a matter of days, and the periods of 5 and 9 wk that were used in the present study were probably sufficient to evaluate the influence of the two NaDCC products. Journal of Dairy Science Vol. 79, No. 9, 1996
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BODDIE AND NICKERSON
CONCLUSIONS
The novel teat dip formulations containing NaDCC in tablet form reduced new IMI caused by Staph. aureus and Strep. agalactiae during the two efficacy trials conducted under experimental challenge conditions recommended by the National Mastitis Council (9). ACKNOWLEDGMENTS
We thank Nancy Boddie, Carol Pratt, Corinne Ray, and the Hill Farm Research Station dairy personnel for technical assistance and Frances Huff for secretarial support. Mick Doyle, Marketing Int. Ltd., and Monsanto are gratefully acknowledged for partial support of the teat dip efficacy trials. REFERENCES 1Bloomfield, S. F., and G. A. Miles. 1979. The antibacterial properties of sodium dichloroisocyanurate and sodium hypochlorite formulations. J. Appl. Bacteriol. 46:65. 2 Bloomfield, S. F., and E. E. Uso. 1985. The antibacterial properties of sodlium hypochlorite and sodium dichloroisocyanurate as hospital disinfectants. J. Hosp. Infect. 6:20. 3Boddie, R. L., S. C. Nickerson, and G. K. Kemp. 1994. Efficacy of two barrier teat dips containing chlorous acid germicides against experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 77:3192.
Journal of Odry Science Vd. 79,No. 9,1996
4Coates, D. 1988. Comparison of sodium hypochlorite and sodium dichloroisocyanurate disinfectants: neutralization by serum. J. Hosp. Infect. 11:60. 5 Fox, L. K. 1992. Colonization by Staphylococcus aureus on chapped teat skm: effect of iodine and chlorhexidine postmilking disinfectants. J. Dairy Sci. 75:66. 6Fox, L. K., J. A. Nagy, J. K. Hillers, J. D. Cronrath, and D. A. Ratkowsky. 1991. Effects of postmilking teat treatment on the colonization of Staphylococcus aureus on chapped teat skin. Am. J. Vet. Res. 52:799. 7 Freedman, S. 1994. The bactericidal effect of troclosene sodium for dairy disinfection. A laboratory study. Israel J. Vet. Med. 49: 25. 8 Goldberg, J. J., P.A. Murdough, A. B. Howard, P. A. Drechsler, J. W. Pankey, G. A. Ledbetter, L. L. Day, and J. D.Day. 1994. Winter evaluation of a postmilking powdered teat dip. J. Dairy Sci. 77:748. 9Hogan, 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 teat dips. J. Dairy Sci. 73:2580. lONatzke, R. P., R. W. Everett, R. S.Guthrie, J. F. Keown, A. M. Meek, W. G. Merrill, S. J. Roberts. and G. H. Schmidt. 1972. Mastitis control program: effect on milk production. J. Dairy Sci. 55:1256. 11Pankey, J. W., and W. N. Philpot. 1975. Hygiene in the prevention of udder infections I. Comparative efficacy of four teat dips. J. Dairy Sci. 58:202 12 Pankey, J. W., W. N. Philpot. R. L. Boddie, and J. L. Watts. 1983. Evaluation of nine teat dip formulations under experimental challenge to Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 66:161. 13 Philpot, W. N., and J. W. Pankey. 1975. Hygiene in the prevention of udder infections 111. Effectiveness of 59 teat dips for reducing bacterial populations on teat skin. J. Dairy Sci. 58:209. 14Philpot, W. N., and J. W. Pankey. 1978. Hygiene in the prevention of udder infections. V. Efficacy of teat dips under experimental exposure to mastitis pathogens. J. Dairy SCI.61:956.