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Chlorous Acid Study in Treating Veterinary Mastitis

22nd October 2007 by Arrow Durfee Posted in Uncategorized

This article demonstrates the efficacy of a chloride dioxide product in veterinary care.

Efficacy of Two Barrier Teat Dips Containing Chlorous Acid
Germicides Against Experimental Challenge
with Staphylococcus aureus and Streptococcus agalactiael
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
G. K. KEMP
Alcide Corp.
One Willard Road
Norwalk, CT 06851
ABSTRACT
Two postmilking teat dips were rested
for efficacy against Staphylococcus
aureus and Streptococcus agalactiae us-
ing experimental challenge procedures
recommended by the National Mastitis
Council. Both dips contained chlorous
acid as the primary germicidal agent and
lactic acid or mandelic acid as the chlo-
rous acid activator. The dip activated
with mandelic acid significantly reduced
new IMI by Staph. aureus and Strep.
agalactiae. The IMI rate was reduced
68.7% for Staph. aureus and 56.4% for
Strep. agalactiae. The dip activated with
lactic acid significantly reduced new
Staph. aureus IMI by 69.3% but did not
significantly reduce new Strep. agalac-
tiae IMI (35.2% reduction) through the
full 1 I-wk study period. Teat skin condi-
tion did not change from pretrial status
after using either teat dip during the
study.
(Key words: barrier teat dip, chlorous
acid, Staphylococcus aureus, Streptococ-
cus agalactiae)
Abbreviation key: CM = clinical mastitis,
TSA = trypticase soy agar, TSB = trypticase soy broth.
Received April 15, 1994.
Accepted June 24, 1994.
‘Approved for publication by the director of the Loui-
siana Agricultural Experiment Station as manuscript num-
ber 94-80-8072.

INTRODUCTION

Physical barrier teat dips were originally
developed to prevent coliform mastitis (9). An
acrylic latex product without a germicide ef-
fectively reduced new IMI caused by
Staphylococcus aureus, Staphylococcus epider-
midis, and coliforms but did not significantly
reduce new IMI caused by Streptococcus
agalactiae or environmental streptococci (3).
The same latex teat dip (3) was subsequently
formulated with a germicide of known ef-
ficacy. Culture of bacteria from latex dips that
did not contain germicides may have prompted
the inclusion of germicides in the first latex
barrier products (9). The acrylic latex barrier
film remaining on teats between milkings must
be peeled or washed from the teats with water
immediately before the next milking.
A new class of barrier teat dips has recently
been developed in which the physical barrier is
composed of a polymer gel within which ini-
tial germicidal activity is provided by the
generation of chlorous acid. Chlorous acid pro-
vides continuous broad-spectrum germicidal
activity when the teat dip remains moist on the
teat skin surface (8, 10). The use of an a-
hydroxy organic acid as the activator for
generation of chlorous acid provides for the
continued maintenance of germicidal activity
in the residual film remaining on the teat sur-
face after drying of the product (4). The teat
dips tested during the study presented here
were of this generic type. Both dips tested
were water-soluble and contained a moistening
agent to keep the products flexible on teats.
Barrier teat dips containing chlorous acid are
formulated to kill organisms on teat skin im-
1994 J Dairy Sci 77:3192-3197 3192

EFFICACY OF CHLOROUS ACID TEAT DIP 3 193
mediately after milking and during the inter-
milking period (9).
The objective of this study was to evaluate
two postmilking teat dips containing chlorous
acid generated from lactic or mandelic acid as
the activator using an experimental challenge
procedure.

MATERIALS AND METHODS
Sampling Schedule
Bacteriologic status of mammary quarters
was determined at the initiation of the trial by
collection and culture of duplicate milk sam-
ples. A third sample was collected from
specific quarters and cultured when results
from the first two samples differed. Milk sam-
ples were collected and analyzed weekly dur-
ing 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 2 d after first
isolation. All quarters were eligible for new
IMI during the trial except 1) those infected
with organisms of the same species as chal-
lenge organisms and 2) those with deformed or
abnormal teats.

Collection of Milk Samples
Prior to sampling, udders were washed us-
ing a hand-held hose and paper towels. After
washing, udders 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 opposite side of the udder from
the technician were sanitized first, and milk
samples were collected in reverse order in
sterile, snap-cap plastic tubes and refrigerated
at 5°C.
Laboratory Cultural Procedures
Samples were mixed by shaking, and a .01-
ml aliquot was streaked on trypticase soy agar
(TSA) (Becton Dickinson, Cockeysville, MD)
containing 5% bovine blood. Plates were in-
cubated at 37°C for 48 h and examined to
identify microorganisms present. Staphylococ-
cus aureus were identified presumptively by
hemolytic pattern and confirmed by tube
coagulase test. Streptococcus agalactiae were
identified to serogroup by the Phadebact Strep-
tococcus Test (Boule Diagnostics AB, Hud-
dinge, Sweden). An MI was confirmed when
1) Staph. aureus or Strep. agalactiae were
isolated from a clinical quarter, 2) two con-
secutive samples yielded 2500 cfu/ml of the
same pathogen, or 3) three consecutive sam-
ples contained 100 to 400 cfdml of the same
pathogen (5).
Description of Experimental Teat Dips
The teat dips were provided as a base solu-
tion containing .64% sodium chlorite in a gel
formulation and an activator that contained 3%
mandelic acid or 2.64% lactic acid (UDDER-
gold; Alcide Corporation, Norwalk, CT). The
base and activator were mixed in equal
amounts immediately prior to milking and
used fresh daily.

Treatment Method
The milking herd of the Hill Farm Research
Station (n = 130) was divided into two groups
of cows of equal number. One group was used
to determine efficacy of the mandelic acid dip
and was milked first; the other group was used
to evaluate the lactic acid dip and was milked
second. At the afternoon milking, Monday
through Friday, all 4 teats of each cow were
experimentally exposed by immersion to a
depth of approximately 25 mm in a challenge
suspension containing Staph. aureus (ATCC
29740) and Strep. agalactiae (ATCC 27956)
immediately after milking machines were re-
moved. Immediately thereafter, the distal 25
mm of 2 diagonally opposed teats were dipped
with teat dip; the remaining 2 teats served as
undipped controls. Teats were exposed to chal-
lenge organisms to increase the number of
pathogens impinging on the teat apex, result-
ing in an increased rate of IMI. After 5 wk,
sufficient numbers of new Staph. aureus IMI
developed in control quarters of both groups of
cows to perform a valid statistical analysis;
thus, subsequent challenge with Staph. aureus
was discontinued, and, for the remaining 6 wk
of the study, teats were challenged with Strep.
agalactiae only.
Preparation of Challenge Suspensions
Stock suspensions of Staph. aureus were
prepared weekly. The contents of one lyophi-
Journal of Dairy Science Vol. 77, No. 10, 1994
3194 BODDIE ET AL.
lized vial of Staph. aureus were reconstituted
in 6 ml of trypticase soy broth FSB) (Becton
Dickinson) and incubated at 37°C for 5 to 7 h.
This culture was used to inoculate 500 ml of
TSB, which was incubated on a gyratory
shaker for 16 h. After incubation, bacterial
cells were pelleted by centrifugation, washed
twice with .l% proteose-peptone (Difco
Laboratories, Detroit, MI), and resuspended to
the original volume in proteose-peptone. Serial
dilutions were prepared in proteose-peptone,
and .1 ml was plated on TSA. Plates were
incubated for 24 h at 37°C and colonies were
counted to ascertain the microbial concentra-
tion of the stock suspension. This suspension
was stored at 5°C and used daily for 1 wk to
prepare challenge suspensions of Staph.
aureus.

Cultures of Strep. agalacriae were prepared
by resuspension of a lyophilized vial of Strep.
agalactiae in 6 ml of TSB, and a .Ol-ml
aliquot was streak-plated on each of five TSA
plates. Plates were incubated at 37°C for 16 h
and stored at 5°C to serve as stock cultures for
1 wk. 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
about 15 h at 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 to approximately
146 ml of pasteurized milk to adjust the con-
centration of Strep. agalactiae to approxi-
mately 5 x 107 cfdml.
An aliquot of the Staph. aureus stock sus-
pension was added to the Strep. agalactiae
suspension to obtain a concentration of ap-
proximately 5 x 107 cfu/ml of Staph. aureus.
This bacterial suspension was taken immedi-
ately 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 quar-
ters becoming infected in treatment groups
were tested as described by Hogan et al. (5)
using an approximated t statistic defined as t =
= number of new IMI in control quarters, x2 =
number of new IMI in treated quarters, nl =
[(XlInl) - (X2/"2)1/[(Xl + x2Y(nln2)P9 whel-e x1
(number of control quarters) (time unit), and n2
= (number of treated quarters) (time unit). The
denominators nl and n2 were expressed as the
summation of quarter days. A quarter was
eligible for only one IMI during the study. The
percentage of 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 are considered
to be efficacious when mean percentage of
reduction of new IMI is 240% and the lower
confidence limit of the mean is 225% (5).
Scoring of Teat Skin Condition
Characteristics of teat skin surface in both
teat dip groups were scored immediately be-
fore the trial was initiated and at the conclu-
sion of the trial to determine effects of the teat
dip products on teat skin. Teat skin was
characterized as 1) normal; 2) abnormal: ex-
hibiting a cracked, sloughed, or chapped sur-
face; 3) associated with a nonfunctional quar-
ter; 4) exhibiting mechanical injury; or 5)
displaying warts.
RESULTS AND DISCUSSION
The IMI data collected during the study are
summarized in Table 1. The lactic acid dip
reduced the number of new Staph. aureus IMI
by 69.3% (P I .001), and the number of new
Strep. agalactiae IMI was reduced 35.2% (P I
.l) over the full 11 wk of the study. The
mandelic acid dip significantly reduced new
IMI by both organisms with efficacies of
68.7% (P I .001) for Staph. aureus and 56.4%
(P I .01) for Strep. agalactiae. Reduction in
new Strep. agalactiae IMI for wk 1 through 7
for the lactic acid dip was 65% (P I .Ol), but,
during wk 8, 6 new Strep. agalactiae IMI in
dipped quarters and one new IMI in control
quarters were confirmed (data not shown).
Thus, during wk 8 through 11, efficacy
decreased to 35.2% against Strep. agalactiae
for this product. This IMI rate with Strep.
agalactiae was not observed for the first 7 wk
of the study or for the last 3 wk of the study,
but only during wk 8. The 6 new IMI in
dipped quarters during wk 8 occurred in 5
cows with normal teat skin and teat meatus
condition. The atypical, increased IMI rate was
not related to weather or herd management.
The increase in IMI rate during wk 8 was not
Journal of Dairy Science Vol. 77. No. 10, 1994
EFFICACY OF CHLOROUS ACID TEAT DIP 3 195
found for Staph. aureus in the same dip group
or for Staph. aureus and Strep. agalacriae in
the mandelic acid dip group.
Incidence of clinical mastitis (CM) for con-
trol and dipped quarters, respectively, was
similar for Sraph. aureus (0 vs. 0%) and Srrep.
agalactiae (1.36 vs. 1.34%) in the lactic acid
dip group. In the mandelic acid dip group, the
percentage of eligible control quarters that be-
came clinically infected with Staph. aureus
was higher than that of dipped quarters (4 vs.
2.9%). In contrast, the percentage of eligible
quarters with Strep. agalactiae CM was greater
for quarters dipped in the mandelic acid dip
than for control quarters (1.41 vs. .71%). The
incidence of CM was too small to test statisti-
cally.
Teats in both dip groups were scored for
chapping, sloughing, cracks, and other injuries
immediately prior to the study and at the end
of the study (Table 2). At least 97% of teats
were characterized as normal before and after
the trial for both groups. In the group treated
with lactic acid, 1 undipped control teat of 1
cow (.67%) that was characterized as normal
before the trial was characterized as abnormal
after the trial. In the group treated with man-
delic acid, 2 teats of 2 cows among the dipped
quarters that were characterized as abnormal
(.69%) or having mechanical injuries (.69%)
before the mal improved in condition score
after the trial. Thus, teat imtation or abnormal-
ities could not be attributed to use of either
product.

Few studies involving experimental ex-
posure to bacteria that cause mastitis tested
chlorous acid teat dips using the protocol
recommended by the National Mastitis Council
(5). Drechsler et al. (1) evaluated a chlorous
acid barrier teat dip with lactic acid as the
activator during experimental challenge ex-
posure and obtained efficacies of 78.9 and
52.9% against Staph. aureus and Strep.
agalacriae, respectively, after results from two
research herds were combined. The same dip
was more effective than a positive control teat
dip containing 1% iodine against major masti-
tis pathogens in a natural exposure study (1). In
another natural exposure study, a chlorous acid
barrier dip with lactic acid reduced new Staph.
aureus IMI by 67.4% in a research dairy herd
(8).

A natural exposure study of five commer-
cial herds compared a chlorous acid barrier teat
dip containing lactic acid against a positive
control product containing .5% iodophor and
found a reduction of 18.8% in new MI caused
by all pathogens and a reduction of 13.6% in
major pathogen IMI for the chlorous acid dip
group (10). Efficacy of the experimental dip
TABLE 1. Efficacy data of barrier teat dips containing chlorous acid against experimental challenge with Staphylococcus
aureus and Streptococcus agalactiae.
Organism Quarters Quarter days New IM1 per
and eligible for New at risk for 100 quarter
treatment new MI IMI new MI days at risk Reduction
(no.) (W
Staph. aureus
Lactic acid dip 138 10 4939 ,202
Control 134 29 4397 ,660 69.3***
Lactic acid dip 149 la 928 1 .I94
Control 147 26 8691 ,299 35.2*
Mandelic acid dip 136 8 5183 ,154
Control 125 22 4465 ,493 68.7***
Strep. agalactiae
Staph. aureus
Strep. agalactiae
Mandelic acid dip 142 13 9791 .133
Control 141 28 9186 ,305 56.4**
*P 5 .lo.
**P 5 .01.
***P 5; ,001.
Journal of Dairy Science Vol. 77, No. 10, 1994
3 196 BODDIE ET AL.
TABLE 2. Frequency of teat surface characteristic scores before and after the trial for barrier teat dips containing
chlorous acid.
Lactic acid dip Mandelic acid dip
Before After Before After
score Dipped Control Dipped Control Dipped Control Dipped Control
Normal 93.33 98 99.33 97.33 97.22 91.92 98.61 97.92
Abnormal 1 0 0 0 ,137 69 0 0 0
Nonfunctional .67 2 67 2 1.40 1.39 1.39 1.39
Mechanical injury 0 0 0 0 .69 0 0 0
warts 0 0 0 0 0 .69 0 .69

‘Teat skin that was cracked, sloughed, or chapped.
against coagulase-negative staphylococci was
33.3%, and, because these organisms colonize
the teat canal, Poutrel et al. (10) theorized that
the barrier dip was able to penetrate the teat
canal and to kill organisms colonizing this
area.

In vitro studies (6) to evaluate germicidal
activity of a chlorous acid product showed that
addition of growth medium or neutralizing
broth did not affect the reduction in number of
Staph. aureus colonies after 30 s of exposure
to the dip. Similarly, effectiveness of the dip
against Staph. aureus was not affected by time
up to 48 h or by the presence of organic matter
(6). A chlorous acid disinfectant with a lactic
acid activator was 100% effective in killing
Staph. aureus after exposure of 30 min even
when it was diluted 1:l (voVvol) with
phosphate-buffered saline (1 1).
The pharmacological activity of chlorous
acid that is primarily responsible for the de-
struction of microorganisms derives from the
partial conversion of the chlorite ion (CIO;) to
its corresponding acid form, chlorous acid
(HC102) (4). This reaction occurs when sodium
chlorite is combined with an organic acid un-
der appropriate conditions of pH and chlorite
concentration. Chlorous acid is an unstable
entity that acts as a source of a series of rapid
and highly efficient cidal oxidants as it under-
goes a degradation cascade. Chlorous acid can,
however, be maintained in a metastable
equilibrium under the appropriate conditions of
acidity and chlorite concentration, as for the
teat dip formulations tested in this study. At
the pH achieved in the lactic acid product from
ionization of the lactic acid component, ap-
Journal of Dairy Science Vol. 77, No. IO, 1994
proximately 7% of the chlorite ion that is
initially present in the formulation has been
transformed to the chlorous acid form. The
latter undergoes a series of disproportionation
reactions on contact with organic matter (e.g.,
microorganisms) and on evaporation that lead
to higher, more unstable concentrations of
chlorous acid. The cidal oxidant species that
are produced on disproportionation include
C1202, HC10, Cl02, and C1,Oi (4).

As chlorous acid is depleted, the system
reequilibrates by combining newly ionized or-
ganic acid with residual chlorite, thereby
replenishing the consumed chlorous acid and
continuing the cidal activity. Upon evaporation
and eventual drying of the barrier formulation
(approximately 10 min), the cidal action from
the chlorous acid cascade becomes virtually
exhausted. However, the antimicrobial activity
during that period has resulted in a minimum
reduction of 6 log in surface microorganisms
(G. K. Kemp, 1994, Alcide Corp., Norwalk,
CT, personal communication).

Once dried, the organic acid that was ini-
tially present as the activator of the chlorite
species is reduced to about 62% of its original
concentration to exist eventually as a lactic
acid and lactate buffer. The residual lactic acid,
although of less bactericidal ability than the
chlorous acid cascade, remains on the teat
skin, thereafter to provide continued bacterio-
static and bactericidal activity. The AOAC
testing of lactic acid against Escherichia coli
and Staph. aureus has shown population reduc-
tions of up to 4 logs and 2 logs, respectively,
within 15 s of exposure (G. K. Kemp, 1994,
personal communication).

EFFICACY OF CHLOROUS ACID TEAT DIP 3197
The physical kinetics of destruction of
microorganisms following application of a ger-
micidal system of chlorous acid to teat skin is
not directly measurable. The mechanism of
cidal action is hypothesized to be oxidative,
involving the rapid and nonspecific oxidation
of labile bonds (e.g., S-S, S-CH3, phenyl-OH)
on amino acid moieties of microbial surface
proteins (7). This oxidation disrupts the surface
potentials of the organisms, leading to electro-
lyte leakage and cellular death. The anti-
microbial activity of chlorous acid includes an
extremely wide range of Gram-positive and
Gram-negative bacteria, fungi, mycoplasma,
and lipophilic and hydrophilic viruses.
The use of a-hydroxy organic acids other
than lactic acid, e.g., mandelic acid, as activa-
tors of generation of chlorous acid has no
measurable effect on initial germicidal activity.
This result is to be expected, because efficacy
of the chlorous acid antimicrobial system is
based on the generation of comparable
amounts of chlorous acid in both formulations.
Both lactic acid and mandelic acid at the same
pH and in the presence of sodium chlorite
initiate the generation of chlorous acid to much
the same degree; therefore, no differences in
initial efficacy would be anticipated.
The teat dips tested in this study compared
favorably with other barrier teat dips contain-
ing a chlorous acid germicide against the same
mastitis organisms (1, 8, 10, 12). These dips
offer antimicrobial activity that is greater than
or equal to that of chlorine, with greater
residual activity (2), plus the effect of a barrier
film, which may seal the teat end.

ACKNOWLEDGMENTS
We express appreciation to Nancy Boddie,
Patty Harville, Corinne Ray, and the Hill Farm
Research Station dairy personnel for technical
assistance and to Frances Huff for secretarial
support. The Alcide Corporation is also grate-
fully acknowledged for partial support of the
experimental exposure trial.
REFERENCES
1 Drechsler, P. A,, E. E. Wildman, and J. W. Pankey.
1990. Evaluation of a chlorous acid-chlorine dioxide
teat dip under experimental and natural exposure con-
ditions. J. Dairy Sci. 73:2121.
2Dychdala, G. R. 1991. Chlorine and chlorine com-
pounds. Page 142 in Disinfection, Sterilization, and
Preservation. 4th ed. S. S. Block, ed. Lea and Febiger,
Philadelphia, PA.
3 Famsworth, R. J., L. Wyman, and R. Hawkinson.
1980. Use of a teat sealer for prevention of intramam-
mary infections in lactating cows. J. Am. Vet. Med.
Assoc. 177:441.
4Gordon. G. 1972. The chemistry of chlorine dioxide.
Page 201 in Progress in Organic Chemistry. S. J.
Lippard, ed. Wiley Interscience, New York, NY.
5 Hogan, J. S., D. M. Galton, R. J. Harmon, S. C.
Nickerson, S. P. Oliver, and J. W. Pankey. 1990.
Protocols for evaluating efficacy of postmilking teat
dips. J. Dary Sci. 73:2580.
6Langlois, B. E., R. J. Harmon, and K. Akers. 1992. In
vitro study of the germicidal activity of a chlorine
dioxide teat dip. Page 237 in Proc. Natl. Mastitis
Counc., Arlington, VA.
7 Masschelein, W. J. 1979. Chemistry and environmen-
tal impact of oxychlorine compounds. Page 79 in
Chlorine Dioxide. R. G. Rice, ed. Ann Arbor Press,
Ann Arbor, MI.
8 Oliver, S. P., S. H. King, P. M. Torre, E. P. Shull, H.
H. Dowlen, M. J. Lewis, and L. M. Sordillo. 1989.
Prevention of bovine mastitis by postmilking teat dis-
infectant containing chlorous acid and chlorine diox-
ide in a soluble polymer gel. I. Dairy Sci. 72:3091.
9 Pankey, J. W., R. J. Eberhart, A. L. Cuming, R. D.
Daggea, R. J. Farnsworth, and C. K. McDuff. 1984.
Update on postmilking teat antisepsis. J. Dairy Sci.
67: 1336.
10Poutrel. B., F. Serieys, and M. Ducelliez. 1990. Ef-
ficacy of a germicidal post milking barrier-type teat
dip in preventing intramammary infections. Vet. Rec.
126:638.
11 Redding, W. R., and L. C. Booth. 1991. Effects of
chlorhexidine gluconate and chlorous acid-chlorine
dioxide on equine fibroblasts and Stuphylococcus
uureus. Vet. Surg. 20:306.
12 Schmidt, A. L., S. P. Oliver, and M. E. Fydenkevez.
1984. Evaluation of experimental teat dip containing
sodium chlorite and lactic acid by excised teat assay.
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Journal of Dairy Science Vol. 77. No. IO. 1994

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