Efficacies of Teat Germicides Containing 0.
5% Chlorhexidine
and 1% Iodine During Experimental Challenge
with Staphylococcus aureus and Streptococcus agalactiae1
R. L. BODDIE,* S. C. NICKERSON,* and R. W. ADKINSON†
ABSTRACT
Two germicides, one containing 0.5% chlorhexidine
and one containing 1% iodine, were tested for efficacy
against new intramammary infections (IMI) caused
by Staphylococcus aureus and Streptococcus agalac-
tiae. The model for the experimental challenge in the
trials that were designed to test the efficacy of the
two postmilking teat dips was recommended by the
National Mastitis Council. The 0.5% chlorhexidine
product reduced the number of new IMI caused by
Staph. aureus by 73.2% and reduced the number of
new IMI caused by Strep. agalactiae by 53.9%. The
1% iodine product reduced the number of new IMI
caused by Staph. aureus by 75.6% and reduced the
number of new IMI caused by Strep. agalactiae by
53.5%. In both trials, the incidence of clinical mastitis
was also reduced in dipped quarters compared with
control quarters. Use of the two teat dips over the
trial periods had no effect on the condition of teat skin
or teat ends.
( Key words: chlorhexidine, iodine, Staphylococcus
aureus, Streptococcus agalactiae)
Abbreviation key: TSA = trypticase soy agar, TSB
= trypticase soy broth.
INTRODUCTION
Disinfection is one of the most important aspects of
disease prevention. Many disinfectants have been de-
veloped specifically for the dairy industry to prevent
the spread of infectious diseases. One of the most
expensive diseases for the dairy farmer to combat is
Received February 3, 1997.
Accepted June 16, 1997.
1Approved for publication by the director of the Louisiana
Agricultural Experiment Station as manuscript number 97-
15-0040.
1997 J Dairy Sci 80:2809–2814
*Mastitis Research Laboratory, Hill Farm Research Station,
Louisiana Agricultural Experiment Station,
Louisiana State University Agricultural Center, Homer 71040
†Department of Dairy Science,
Louisiana Agricultural Experiment Station,
Louisiana State University Agricultural Center, Baton Rouge 70803
mastitis. This infectious disease can be controlled to a
manageable extent by the use of safe and effective
premilking and postmilking teat germicides, antibi-
otic therapy of all quarters at drying off, the culling of
cows with chronic infections, treatment of clinical
mastitis during lactation, and proper use of ade-
quately functioning milking machines (15).
An effective teat germicide should have certain
characteristics to identify the benefits of the product
to the dairy farmer. The germicide should have
proven antimicrobial activity on teat skin and should
reduce the incidence of new IMI. In addition, germici-
dal activity should not be affected by the presence of
organic matter, such as milk, manure, or urine. The
germicide should be nonirritating to teat skin, non-
toxic, and should promote healing of teat lesions.
Provided that reasonable udder hygiene practices are
followed, the germicide should not leave residues in
the milk or be absorbed into tissue (16).
The objective of these studies was to determine the
efficacies of chlorhexidine and iodine teat dip
products against experimental challenge with
Staphylococcus aureus and Streptococcus agalactiae.
MATERIALS AND METHODS
Cows
From the dairy herd at the Hill Farm Research
Station (Homer, LA), 117 lactating Jersey cows were
used in a 4-wk study to evaluate the 0.5% chlorhexi-
dine product, and 127 lactating Jersey cows were
used in a 5-wk study to evaluate the 1% iodine germi-
cide. Cows were maintained on pasture, and a free-
stall barn was used as a loafing and feeding area.
Hardwood shavings were used as bedding material.
Cows were milked in a double-2, side-opening, low
line parlor. A split udder design was used in each
study: 2 teats of each cow were dipped with the teat
germicide, and 2 teats served as undipped controls.
2809
2810 BODDIE ET AL.
Sampling Schedule
The bacteriologic status of each quarter was deter-
mined by collecting and culturing duplicate milk sam-
ples 1 wk before the bacterial challenge was initiated.
A third sample was collected when the results from
the first two samples did not agree. Quarters that had
an IMI caused by either of the challenge microorgan-
isms ( Staph. aureus or Strep. agalactiae) before the
start of the study were not considered eligible for new
IMI caused by that microorganism during the study.
The herd was sampled weekly, and, after cultures
were read, specific quarters were resampled within 48
h to confirm new IMI caused by the challenge bac-
teria. Abnormal or injured teats were not included as
eligible quarters.
Treatment Method and
Diagnostic Procedures
To increase the incidence of IMI, teats were im-
mersed daily, Monday through Friday, with the
challenge microorganisms at the p.m. milking im-
mediately after removal of the milking machine. For
the trial that evaluated the 0.5% chlorhexidine
product, the right front and left rear teats of each cow
were immersed in germicide to a depth of approxi-
mately 25 mm, and the 2 remaining teats served as
undipped controls. For the trial that evaluated the 1%
iodine product, the left front and right rear teats were
immersed in germicide and the 2 remaining teats
served as undipped controls.
An IMI was confirmed when Staph. aureus or
Strep. agalactiae was cultured from a quarter that
exhibited clinical mastitis, when 2 consecutive sam-
ples yielded >500 cfu/ml of either challenge microor-
ganism, or when 3 consecutive samples contained 100
to 400 cfu/ml of either challenge microorganism (12).
After a new IMI caused by Staph. aureus or Strep.
agalactiae was confirmed, the infected quarter was
treated with a commercially available antibiotic
product for lactating cows and was considered to be
ineligible for a new IMI with either microorganism for
the remainder of the study.
Udder Preparation and
Sample Collection
Prior to the collection of milk samples, the foremilk
was discarded, and the teat apex was sanitized using
cotton pledgets moistened with 70% ethyl alcohol.
Approximately 10 ml of milk were collected into
sterile plastic tubes prior to plating 0.01 ml of milk on
trypticase soy agar ( TSA) containing 5% bovine calf
blood and 0.1% esculin. Plates were incubated at 37°C
Journal of Dairy Science Vol. 80, No. 11, 1997
for 48 h. Isolates of Staph. aureus were identified
presumptively by the hemolytic pattern and were con-
firmed by the tube coagulase test. Isolates of Strep.
agalactiae were identified by serogroup using the
Phadebact Streptococcus Test (Boule Diagnostics AB,
Huddinge, Sweden).
Preparation of the Bacterial Challenge
Pooled challenge cultures of Staph. aureus and
Strep. agalactiae were prepared daily. A frozen stock
of Staph. aureus ATCC 29740 that had been stored in
trypticase soy broth ( TSB) and glycerin was thawed,
plated on TSA, and incubated at 37°C for 24 h. This
culture was used to inoculate six 6-ml tubes of TSB
that were incubated at 37°C for 8 h. This 8-h culture
was used to inoculate 500 ml of TSB, which was
incubated on a gyratory shaker for 16 h. Bacterial
cells were then pelleted by centrifugation, washed
twice with 0.1% proteose-peptone, and resuspended to
the original volume in proteose-peptone. A viable bac-
teria count was conducted; serial dilutions were made
in proteose-peptone, and a 0.1-ml aliquot was plated
on TSA. The plate count was incubated for 24 h at
37°C, and colonies were counted to determine the
microbial concentration of the stock suspension. This
stock suspension was stored at 5°C and was used
daily for 1 wk.
Cultures of Strep. agalactiae ATCC 27956 were
prepared: a frozen stock vial of the microorganism
was thawed in TSB, and 0.01 ml of bacteria was
plated on each of five TSA plates. Plates were in-
cubated at 37°C for 24 h and stored at 5°C to serve as
stock cultures for a 1-wk period. Daily challenge sus-
pensions of Strep. agalactiae were prepared by in-
oculating five 6-ml tubes of TSB with six colonies
from a TSA stock plate. This culture was incubated
for about 15 h at 37°C and was used to inoculate 500
ml of TSB. The 500-ml culture was incubated for 7 h
at 37°C on a gyratory shaker. Aliquots based on the
optical density of the culture were added to pasteu-
rized milk to adjust the concentration of Strep.
agalactiae to approximately 5 × 107 cfu/ml. An aliquot
of the Staph. aureus stock suspension was added to
the Strep. agalactiae suspension to obtain a concen-
tration of approximately 5 × 107 cfu/ml of Staph.
aureus. This bacterial suspension was taken immedi-
ately to the milking parlor to challenge teats after the
p.m. milking. A plate count was conducted daily on
challenge suspensions.
Description of Teat Germicides
The teat germicides were provided ready to use.
The chlorhexidine germicide contained 0.5% chlor-
hexidine gluconate with 4% glycerin as a humectant
 EFFICACIES OF CHLORHEXIDINE AND IODINE TEAT DIPS
and skin conditioner (Blue Ribbon; IBA, Inc., Mill-
bury, MA). The iodine product contained a 5% nonyl-
phenoxypolyethoxyethanol-iodine complex, which
provided a minimum of 1% titratable iodine, 12 ppm
of free iodine, and 10% glycerin as a humectant and
skin conditioner (FS-103 X; IBA, Inc.).
Statistical Analysis of New IMI Data
The number of quarter milkings per new IMI was
determined using the following equation (14): Q = N
× M × D/I where Q = number of quarter milkings per
new IMI, N = number of quarters eligible for new IMI,
M = number of milkings per day, D = number of days,
and I = number of new IMI.
Differences between the percentages of quarters
that were infected in the treatment groups were
tested using an approximated t statistic defined by
Hogan et al. ( 1 2 ) as follows:
( x1/n1) – ( x2/n2) 0.5
t = ( x1 + x2) / ( n1/n2) 
where x1 = number new IMI in control quarters, x2 =
number new IMI in treated quarters, n1 = number of
control quarters × time unit, and n2 = number of
treated quarters × time unit. The denominators n1
and n2 were expressed as the sum of quarter days.
After developing a new IMI, a quarter was not consi-
dered to be at risk for another IMI for the remainder
of the study. The percentage of reduction in the rate
of new IMI for dipped quarters compared with that
for undipped control quarters was expressed as
100(x1/n1) – (x2/n2)/(x1/n1). Teat germicides gener-
ally are considered to be efficacious for preventing
new IMI when the mean percentage reduction in new
IMI is ≥40% and the lower confidence limit of the
mean is >25% (12).
Condition Scoring of Teat Skin
and Teat Ends
Characteristics of teat skin surfaces and teat ends
in both trials were scored immediately before and at
the conclusion of the trials to determine any effects of
the two germicides on teat condition. Condition scores
of teat skin and teat ends were characterized accord-
ing to the parameters established by Goldberg et al.
(11). Condition scores for teat skin and teat ends
were analyzed using least squares techniques and
linear methods of the general linear models procedure
of SAS (17). The design was a split plot in time
adapted from Gill and Hafs (10). The statistical
2811
model was
Yijkl = m + ai + bj( ai) + dk + adik + eijkl
where
Yijkl = observation of the dependent variable,
m = effect common to all observations,
ai = effect caused by treatment i,
bj( ai) = effect caused by cow j in treatment i,
dk = effect caused by time k,
adik = interaction of the effects between treat-
ment i and time k, and
eijkl = error term, assumed to be normally and
2
(
independently distributed 0, se . )
Cow within treatment was considered a random
effect, and all other effects, except error, were consi-
dered fixed effects. In the model statement, treatment
refers to control or dipped teats, and time refers to
before or after the trial. Effects were tested against
appropriate error terms based on expected mean
squares.
RESULTS AND DISCUSSION
Data collected during the two trials are summa-
rized in Table 1. Thirty-six new IMI caused by Staph.
aureus were confirmed in the 0.5% chlorhexidine
trial: 28 were in control quarters, and 8 were in
dipped quarters. Therefore, the number of new IMI
was reduced by 73.2% ( P < 0.001). Thirty-four new
IMI caused by Strep. agalactiae were confirmed: 23
were in control quarters, and 11 were in dipped
quarters. The reduction in the number of new IMI
was 53.9% ( P < 0.05). The rates of new IMI in
undipped control quarters for Staph. aureus and
Strep. agalactiae were 13.1 and 10.1%, respectively.
The 1% iodine germicide significantly reduced the
rate of new IMI caused by Staph. aureus and ap-
proached significance ( P < 0.1) for reducing the rate
of new IMI caused by Strep. agalactiae (Table 1).
Thirty new IMI caused by Staph. aureus were con-
firmed: 24 were in control quarters, and 6 were in
dipped quarters. The reduction in the number of new
IMI was 75.6% ( P < 0.001). Twenty-eight new IMI
caused by Strep. agalactiae were confirmed: 19 were
in control quarters, and 9 were in dipped quarters.
The reduction in the number of new IMI was 53.5%
( P < 0.1). Infection rates in undipped control quar-
ters were 10% for IMI caused by Staph. aureus and
7.5% for IMI caused by Strep. agalactiae.
Incidence of clinical mastitis caused by Staph.
aureus in the 0.5% chlorhexidine trial was 3.3% in
control quarters and 0.009% in dipped quarters. Inci-
Journal of Dairy Science Vol. 80, No. 11, 1997
2812 BODDIE ET AL.

dence of clinical mastitis caused by Strep. agalactiae
was 2.2% in control quarters and 0.009% in dipped
quarters.
Incidence of clinical mastitis caused by Staph.
aureus in the 1% iodine trial was 0.008% in control
quarters and 0% in dipped quarters. Incidence of
clinical mastitis caused by Strep. agalactiae was 1.2%
in control quarters and 0% in dipped quarters.
Chlorhexidine products previously tested at the
Hill Farm Research Station and at other sites under
experimental exposure conditions yielded the follow-
ing results. A 0.45% chlorhexidine germicide yielded
efficacies of 77.2 and 77.9% against Staph. aureus and
Strep. agalactiae, respectively, in a 4-wk study ( 4 ) .
Efficacies of 92.5 and 71.0% against Staph. aureus
and Strep. agalactiae, respectively, were generated by
a 0.55% chlorhexidine gluconate germicide (13). A
0.5% chlorhexidine gluconate dip yielded efficacies of
73.4 and 68.1% against Staph. aureus and Strep.
agalactiae, respectively ( 5 ) . Gerring et al. ( 9 ) also
found a 1% chlorhexidine formulation to be bacterici-
dal against Staph. aureus and Strep. agalactiae in the
presence of 50% milk in an in vitro study. Drechsler
et al. ( 8 ) tested two formulations of a 0.5% chlorhexi-
dine digluconate germicide with 0.5% glycerin under
conditions of experimental challenge; the highest ef-
ficacies were 72.8% against Staph. aureus and 44.4%
against Strep. agalactiae.
New technology allows teat dip products to be for-
mulated with higher free iodine concentrations ( 6 ) ,
and the ability of an iodine teat dip to kill freely
suspended vegetative microorganisms is highly de-
pendent on its free iodine concentration ( 7 ) ; the 1%
iodine product tested in the present investigation
provided 12 ppm of free iodine. Other iodophors that
released between 4 and 10 ppm of free iodine have
also been studied. For example, a 0.1% iodine product
( 5 to 6 ppm of free iodine) and a 0.175% iodine
product ( 4 to 6 ppm of free iodine) yielded efficacies
of 80.7 and 56.6% and 65.0 and 33.3% against Staph.
aureus and Strep. agalactiae, respectively ( 3 ) . A 0.5%
iodine product (6.8 ppm of free iodine) and a 1.0%
iodine product (6.0 ppm of free iodine) yielded effica-
cies of 79.3 and 49.7% and 74.6 and 72.5% against
Staph. aureus and Strep. agalactiae, respectively ( 4 ) .
A 0.5% iodine product (10 ppm of free iodine) and a
1.0% iodine product (10 ppm of free iodine) yielded
efficacies of 78.2 and 73.2% and 43.5 and 46.4%
against Staph. aureus and Strep. agalactiae, respec-
tively ( 2 ) . Similarly, a 0.18% iodine product ( 8 ppm
of free iodine) yielded efficacies of 93.6 and 51.7%
against Staph. aureus and Strep. agalactiae, respec-
tively ( 1 ) .
Characteristics of lateral teat skin surfaces and
those of teat ends were scored in both trials immedi-
ately before the initiation of the teat dip trials and at
the conclusion of the trials to determine any effects of
these germicides on teat condition.
An analysis of condition scores for the lateral teat
skin (six-point scale where 0 = teat skin has been

TABLE 1. Summary of efficacy data of 0.5% chlorhexidine and 1% iodine teat germicides against
Staphylococcus aureus and Streptococcus agalactiae.
Quarters New IMI
days at per
Quarters risk 100 quarter
Challenge microorganism eligible for New for new days at
and germicide new IMI IMI IMI risk Reduction
(no.) (%)
Staph. aureus
0.5% Chlorhexidine 219 8 5284 0.1514 73.2***
Control 213 28 4963 0.5642
Strep. agalactiae
0.5% Chlorhexidine 227 11 5528 0.1990 53.9**
Control 227 23 5326 0.4318
Staph. aureus
1% Iodine 239 6 7897 0.0760 75.6***
Control 241 24 7721 0.3108
Strep. agalactiae
1% Iodine 250 9 8247 0.1091 53.5*
Control 252 19 8101 0.2345
*P < 0.1.
**P < 0.05.
***P < 0.001.

Journal of Dairy Science Vol. 80, No. 11, 1997

 EFFICACIES OF CHLORHEXIDINE AND IODINE TEAT DIPS 2813
TABLE 2. Mean condition score of teat skin and teat ends before CONCLUSIONS
and after the trials for teat germicides containing 0.5% chlorhexi-
dine and 1% iodine.1
A chlorhexidine teat germicide and an iodophor
Teat skin2 Teat end3 teat germicide reduced new IMI caused by Staph.
0.5% Chlorhexidine aureus and Strep. agalactiae under conditions of ex-
Before trial perimental exposure to these mastitis pathogens.
Dipped 1.012 1.048
Control 1.006 1.054 New IMI caused by Staph. aureus were reduced by
After trial the chlorhexidine and the iodophor products 73.2 and
Dipped 1.000 1.089 75.6%, respectively. The chlorhexidine and iodophor
Control 0.994 1.071
SE 0.00851– 0.01857– products reduced new IMI caused by Strep. agalactiae
0.00855 0.01866 53.9 and 53.5%, respectively. Characterization of the
1% Iodine condition of the teat skin and teat ends before and
Before trial
Dipped 1.014 1.152 after each trial showed that both products had no
Control 1.033 1.124 effects on the parameters measured.
After trial
Dipped 1.019 1.105
Control 1.005 1.100 ACKNOWLEDGMENTS
SE 0.01911 0.01966
1No significant differences ( P ≤ 0.05) were observed. Appreciation is expressed to IBA, Inc. for providing
2The condition of the teat skin was scored according to Goldberg partial financial support of this study; to Nancy Bod-
et al. ( 1 1 ) using a six-point scale: 0 = teat skin has been subjected die, Corinne Ray, and the dairy personnel at the Hill
to physical injury (e.g., stepped on or frostbitten) that is not
related to the treatment, or the quarter is nonlactating; 1 = teat Farm Research Station for technical support; and to
skin is smooth, free from scales, cracks, or chapping; 2 = teat skin Sondra Blackwell for secretarial assistance.
shows some evidence of scaling; 3 = teat skin is chapped, and some
small warts may be present; 4 = teat skin is chapped and cracked;
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