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 di-
number of new Strep. agalactiae IMI by 65.1%. chloroisocyanurate ( NaDCC) . The lower concentra-
Product 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% effec-
studies. 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 formula-
TSA = trypticase soy agar, TSB = trypticase soy tion reduced new Staph. aureus IMI by 75.9% and
broth. new Strep. agalactiae IMI by 63.2%; a 1.7% concen-
tration of SDT yielded a 48.1% efficacy against Strep.
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 liber-
disinfection 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 conjunc-
tion with a 3-yrprogram of dry cow therapy decreased MATERIALS AND METHODS
the prevalence of mastitis from 28 to 7% of quarters
( 10). Subsequently, a commercial bleach diluted to
Products 1 and 2 were evaluated in two separate
4% sodium hypochlorite was tested in three separate trials. Product 1 was evaluated from September
experimeintal challenge studies ( 1 1). The hypoch- through October 1994, and product 2 was evaluated
lorite dip exhibited efficacies of 96.3,83.3,and 50% from March through May 1995.
against Staphylococcus aureus and 60, 70, and 50%
The bacteriological status of mammary quarters
Received. January 22, 1996. was determined at the initiation of each trial by
Accepted March 22, 1996. collection and culture of duplicate milk samples. A
'Approved for publication by the director of the Louisiana
Agricultural Experiment Station as Manuscript Number 95-80- third sample was collected from specific quarters and
9219. cultured when results from the first two samples
1996 J Dairy Sci 79:1-1688 1683
1684 BODDIE AND NICKERSON
differed. Milk samples were collected and analyzed (Agrisept'" Tabs; Mick Doyle Marketing Int. Ltd.,
bacteriologically during each week of the trial. Naas, Ireland). Two tablets were added to 1 L of tap
Whenever Staph. aureus or Strep. agalactiae was iso- water and allowed to dissolve thoroughly. This solu-
lated for the first time in a previously uninfected tion provided 2800 ppm of available chlorine as
quarter, a second sample was collected and cultured hypochlorous acid CpH 5.5 to 6.0). The teat dip was
within 48 h after first isolation to confirm new IMI. stored in the dairy parlor in a light-resistant con-
All quarters were eligible for new Staph. aureus and tainer for up to 7 d.
Strep. agalactiae IMI during each trial except those Product 2 also was provided in tablet form, and
infected with organisms of the same species as each effervescent tablet contained 3.0 g of NaDCC
challenge organisms and those with deformed or ab- (EfferceptVet; manufactured for Effercept Products, a
normal teats. division of Micrel Ltd., Inc., Laramie, WY by Olin
Chemical Co., Lavonia, MI). Six tablets were added
Collection of Milk Samples to 3.78 L of tap water and allowed to dissolve
thoroughly, and then 28 cc of a solution containing a
Prior to quarter milk sampling, the ventral sur- dye and skin conditioner (Aquablue Soft-Teat Dip
faces of udders and teats that were excessively dirty Additive; Effercept Products, Phoenix, AZ) were
were was:hed using a hand-held hose and paper added. This solution provided 3000 ppm of available
towels. After being washed, udders and teats were chlorine as hypochlorous acid ( p H 6.7). The teat dip
dried thoroughly with additional paper towels, and was stored i n the dairy parlor in a light-resistant
two or three streams of foremilk were discarded. Each container for up to 5 d.
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 Treatment Method
were sanitized first, and milk samples were collected The milking herd of the Hill Farm Research Sta-
in reverse order in sterile snap-cap plastic tubes and tion was used for the experimental challenge studies.
then refrigerated a t 5°C. For teats that were visibly In the 5-wk trial to test product 1, 139 cows were
clean prior to collection, washing was omitted, and used, and, in the 9-wk trial to test product 2, 75 cows
only cotton. pledgets moistened with 70% alcohol were were used. During the afternoon milking, Monday
used to sanitize teat ends. 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 af-
aliquot was streaked on trypticase soy agar ( T S A ter milking machines were removed. Immediately fol-
Becton 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 experimen-
48 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
confirmed by tube coagulase test. Colonies of Strep. Suspensions of Staph. aureus and Strep. agalactiae
agalactiae were identified to serogroup by the Phade- were prepared as described by Boddie et al. ( 3 ) .
bact Strept,ococcus Test (Boule Diagnostics AB, Hud- Stock suspensions of Staph. aureus were prepared
dinge, Sweden). A new IMI was confirmed when 1) weekly. The contents of one lyophilized vial of Staph.
Staph. aureus or Strep. agalactiae was isolated from a aureus were reconstituted in 6 ml of tripticase soy
clinical quarter, 2 ) two consecutive samples yielded broth (TSB; Becton Dickinson) and incubated a t
2500 cfdml of the same pathogen, or 3 ) three con- 37°C for 5 to 7 h. This culture was used to inoculate a
secutive samples contained 100 to 400 cfdml of the volume of 500 ml of TSB, which was incubated on a
same pathogen ( 9 ) . gyratory shaker for 16 h. After incubation, bacterial
cells were pelleted by centrifugation, washed twice
Description of Experimental Teat Dips
with 0.1% proteose-peptone (Difco Laboratories,
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
O U R INDUSTRY TODAY 1685
TABLE 1. Efficacy data of a hypochlorous acid teat dip (product 1) against experimental challenge
with Staphylococcus aureus and Streptococcus agalactiae.
Organism Quarters Quarter days New IMI
and eligible for New a t risk for per 100 quarter
treatment new IMI IMI new IMI days a t risk Reduction
Dip 257 11 8188 0.134
Control 253 39 7654 0.510 73.6*
Dip 270 9 8676 0.104
Control 290 27 9114 0.296 65.1*
*P I 0.001.
5% bovine calf blood. Plates were incubated for 24 h quarter days. A quarter was eligible for only one IMI
at 37"C, and colonies were counted to ascertain the per organism during each trial. The percentage reduc-
microbial concentration of the stock suspension. This tion in rate of new IMI in the treated groups com-
suspension was stored at 5°C and used each day for 5 pared with that in the control groups was expressed
d to prepare challenge suspensions of Staph. aureus. as 100 [(xl/nl) - (x2/n2)]/(xl/nl). Teat germicides
Cultures of Strep. agalactiae were prepared by generally are considered to be efficacious when the
resuspension of a lyophilized vial of Strep. agalactiae mean percentage reduction of new IMI is 240% and
in 6 ml of TSB, and a 0.01-ml aliquot was streak- the lower confidence limit of the mean is 225% ( 9 ) .
plated on each of five TSA plates. Plates were in-
cubated a t 37°C for 16 h and stored at 5°C to serve as Scoring of Teat Skin
stock cultures for 5 d. Daily challenge suspensions of and Teat End Condition
Strep. agalactiae were prepared by inoculation of 6 ml
of TSB with six colonies from a TSA stock plate. The In the trial that evaluated product 1, only a subjec-
6-ml culture was incubated for approximately 15 h a t tive evaluation of teat condition was made prior to,
37°C and used to inoculate 500 ml of TSB. The during, and after the trial. However, characteristics of
500-ml culture was incubated for 7 h at 37°C on a teat skin surfaces and teat ends in the trial that
gyratory shaker. Aliquots of the culture were added t o evaluated product 2 were scored immediately before
approximately 150 ml of nonsterile pasteurized milk and at the conclusion of the trial to determine the
to adjust the concentration of Strep. agalactiae to effects of this germicide on teat condition. Teat skin
approximately 5 x 107 cfdml. and teat end condition were characterized according
An aliquot of the Staph. aureus stock suspension to the parameters established by Goldberg et al. ( 8 1,
was added t o the Strep. agalactiae suspension t o ob- which are outlined in Tables 3 and 4.
tain a concentration of approximately 5 x 107 cfdml
of Staph. aureus. This bacterial suspension was taken RESULTS AND DISCUSSION
immediately to the milking parlor to challenge teats
during the afternoon milking. A plate count was con- Infection data collected during the trial for the
ducted daily on challenge suspensions. evaluation of product 1 are summarized in Table 1.
For both organisms, the percentage of dipped quar-
Statistical Methods ters becoming infected was less than that of control
quarters. The teat dip reduced the number of new
Differences between the percentage of quarters be- Staph. aureus IMI by 73.6% ( P I 0.001) during the
coming infected in treatment groups were tested as 5-wk study. Reduction in new Strep. agalactiae IMI
described by Hogan et al. ( 9 ) using an approximated was 65.1% ( P 5 0.001). Infection rates for Staph.
t statistic defined as t = [(xl/nl) - (x2/n2)l/[xl + x2)/ aureus were 15.4 and 4.3% for control and dipped
(nlna)]0.5, where XI = number of new IMI in control quarters, respectively. Streptococcus agalactiae infec-
quarters, x2 = number of new IMI in treated quarters, tion rates were 9.3 and 3.3% for control and dipped
n l = (number of control quarters)(time unit), and n2 quarters, respectively.
= (number of treated quarters)(time unit). The A total of 5 quarters exhibited symptoms of clinical
denominators n l and n2 were expressed as the sum of mastitis during the trial that evaluated product 1,
Journal of Dairy Science Vol. 79, No. 9, 1996
1686 BODDIE AND NICKERSON
TABLE 2. Efficacy data of a hypochlorous acid teat dip (product 2 ) against experimental challenge
with Staphylococcus aureus and Streptococcus agalactiae.
Organism Quarters Quarter days New IMI
and eligible for New at risk for per 100 quarter
treatment new IMI IMI new IMI days at risk Reduction
Dip 137 10 8276 0.121
Control 140 30 7690 0.390 69.0*
Dip 147 8 9020 0.089
Control 148 21 8650 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).
For both organisms, the percentage of dipped quar- The equilibrium between the components of this
ters 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 uti-
number 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).
dipped quarters, respectively. The teat dips tested in this study were also evalu-
A total of 7 quarters exhibited symptoms of clinical ated in laboratory trials using a lower strength solu-
mastitis during the trial that evaluated product 2. tion against organisms known t o cause bovine masti-
The 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 Enterobac-
5.5 to 6.0. At this pH, about 95% of the free available ter aerogenes, and, for all species examined, disinfec-
chlorine is present as hypochlorous acid, the molecule tion was >io9 organismdm1 ( 1).
that is primarily responsible for the biocidal activity During the trial that evaluated product 1, no
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
OUR INDUSTRY TODAY 1687
that evaluated product 2, a n objective analysis of teat TABLE 4. Frequency of teat end condition scores1 before and after
a trial that evaluated the efficacy of a hypochlorus acid teat dip
skin condition scores demonstrated that mean score (product 2 ) .
before and after the trial for dipped and control quar-
ters was approximately 1, which was characterized as
normal, smooth skin that was free from scabs, cracks, Score
Dipped Control Dipped Control
or chapping. Similar analyses of teat end condition ~ (YC’
scores also showed that mean score across all varia- 0 13 0 14 0
bles was approximately 1, which was characterized as 1 96 0 97 3 94 4 95 1
2 13 13 21 35
a normal, smooth teat end with no evidence of irrita- 3 13 13 21 14
tion. 4 0 0 0 0
For product 2, a t least 94.7%of teats were charac- 5 0 0 0 0
terized as having normal (score = 1 ) teat skin condi- ‘Teat end condition scoring 0 = teat end has been subjected to a
tion before and after the trial for dipped and control physical injury le g , stepped on or frostbitten) unrelated to the
quarters (Table 3 ) . The percentages of teats with treatment or the quarter is nonlactating, 1 = teat end sphincter is
smooth with no evidence of irritation, 2 = teat end has a raised ring,
physical injuries (score = 0 ) were slightly lower after 3 = teat end sphincter is roughened with slight cracks, but no
the trial than before the trial for dipped and control redness IS present 4 = teat end sphincter is inverted with many
quarters. The percentage of teats showing some evi- cracks, giving a “flowered” appearance, and teat end may have old
but healing scabs, and 5 = teat end is severely damaged and
dence of scaling (score = 2 ) decreased slightly for ulcerative with scabs or open lesions, and large or numerous warts
both dipped and control quarters from 0.7% before the may be present that interfere with teat end function ( 8 )
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 (score = 2 ) increased slightly from 1.39~ before to
difference ( 0 . 7% 1. No significant differences were ob- 2.1%after the trial in dipped quarters, and, in control
served in teat skin condition between dipped and quarters, the increase was from 1.3 t o 3.5%. The
control teats before or after the trial. percentage of teat ends exhibiting a roughened
For product 2, a t least 94.4%of teats were charac- sphincter with slight cracks (score = 3 changed little
terized as having normal (score = 1 ) teat end condi- before and after the trial in dipped and control quar-
tion before and after the trial for dipped and control ters. No significant differences were observed in teat
quarters (Table 4 ) . The percentage of teats with end condition between dipped and control teats before
normal teat end condition decreased slightly from or after the trial.
before t o after the trial in dipped and control quar- The trial lengths of 5 and 9 wk were thought to be
ters. The percentage of teat ends with a raised ring of sufficient duration to evaluate any effect of
products 1 and 2 on teat skin condition. This conten-
tion is based on results of studies (5, 6 ) on the skin
TABLE 3. Frequency of teat skin condition scores1 before and after healing properties of various postmilking teat dips
a trial that evaluated the efficacy of a hypochlorous acid teat dip
(product 2 )
and emollients after the teat skin surface was ex-
perimentally chapped with VV sodium hydroxide. For
Before After example, in one study, Fox et al. ( 6 ) monitored the
Score Dipped Control Dipped Control condition of teat skin daily over a 30-d period and
(9) found that some teat dips were superior to others in
0 27 40 14 28 the ability to heal chapped teat skin; however, regard-
1 94 7 34 7 97 9 96 5 less of treatment, no chapping was observed after d
2 07 07 0 0 17 of the trial, and, by d 10, no differences were
3 20 07 07 07
4 0 0 0 0 observed among treatments. In a similar, subsequent
5 0 0 0 0 trial, Fox ( 5 ) monitored the condition of teat skin
‘Teat skin condition scoring: 0 = teat skin has been subjected to daily over 19 milkings ( 10 d ) in response to several
a physical injury (e.g., stepped on or frostbitten) unrelated to the postmilking teat disinfectants and found that this
treatment, o r the quarter is nonlactating; 1 = teat skin is smooth duration of exposure was sufficient for product evalu-
(free from scales, cracks, or chapping); 2 = teat skin shows some
evidence of scaling; 3 = teat skin is chapped and some small warts ation. Thus, the germicidal effects of teat dip on teat
may be present; 4 = teat skin is chapped and cracked, redness skin condition became apparent in a matter of days,
(indicating inflammation) is present, and numerous warts may be and the periods of 5 and 9 wk that were used in the
present; and 5 = teat skin is severely damaged and ulcerative with
scabs or open lesions, and large or numerous warts may be present present study were probably sufficient to evaluate the
that interfere with teat end function ( 8 ) . influence of the two NaDCC products.
Journal of Dairy Science Vol. 79, No. 9, 1996
1688 BODDIE AND NICKERSON
CONCLUSIONS 4Coates, D. 1988. Comparison of sodium hypochlorite and so-
dium dichloroisocyanurate disinfectants: neutralization by se-
The novel teat dip formulations containing NaDCC rum. J. Hosp. Infect. 11:60.
5 Fox, L. K. 1992. Colonization by Staphylococcus aureus on
in tablet form reduced new IMI caused by Staph. chapped teat skm: effect of iodine and chlorhexidine postmilk-
aureus and Strep. agalactiae during the two efficacy ing disinfectants. J. Dairy Sci. 75:66.
trials conducted under experimental challenge condi- 6Fox, L. K., J. A. Nagy, J. K. Hillers, J. D. Cronrath, and D. A.
Ratkowsky. 1991. Effects of postmilking teat treatment on the
tions recommended by the National Mastitis Council colonization of Staphylococcus aureus on chapped teat skin.
(9). 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:
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.
We thank Nancy Boddie, Carol Pratt, Corinne Ray, Winter evaluation of a postmilking powdered teat dip. J. Dairy
and the Hill Farm Research Station dairy personnel Sci. 77:748.
for technical assistance and Frances Huff for secretar- 9Hogan, J. S., D. M. Galton, R. J. Harmon, S. C. Nickerson, S. P.
Oliver, and J. W. Pankey. 1990. Protocols for evaluating efficacy
ial support. Mick Doyle, Marketing Int. Ltd., and of postmilking teat dips. J. Dairy Sci. 73:2580.
Monsanto are gratefully acknowledged for partial 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.
support of the teat dip efficacy trials. Mastitis control program: effect on milk production. J. Dairy
11Pankey, J. W., and W. N. Philpot. 1975. Hygiene in the preven-
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Journal of Odry Science Vd. 79,No. 9,1996