Veterinary Microbiology 134 (2009) 192–198

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Efficacy of vaccination against staphylococcal mastitis:

A review and new data
John R. Middleton a,*, Christopher D. Luby b, D. Scott Adams c
a
Department of Veterinary Medicine and Surgery, University of Missouri, 900 East Campus Drive, Columbia, MO 65211, United States
b
Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK S7N 5E3, Canada
c
Veterinary Medical Research and Development, Inc., P.O. Box 502, Pullman, WA 99163, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Infection of the heifer mammary gland with common mastitis pathogens, particularly
Staphylococcus aureus staphylococci, prior to calving is well documented. Efforts to eliminate pre-partum
Coagulase-negative staphylococci intramammary infections (IMI) in heifers have focused primarily on intramammary
Mastitis antibiotic therapy shortly before or at the time of calving. Few studies have evaluated
Vaccination
vaccination of heifers against staphylococcal mastitis. The objectives of the present study
Humoral immunity
were to evaluate the efficacy of a commercially available Staphylococcus aureus bacterin in
protecting against staphylococcal IMI (S. aureus and coagulase-negative staphylococci
(CNS)), to study the effect of vaccination on milk SCC, and to evaluate the milk antibody
isotype response to vaccination using a lactating cow model. Ninety Holstein–Friesian
lactating dairy cows of various parities were systematically assigned to a vaccinated
(n = 44) or control (n = 46) group. Vaccinates received two 5 ml doses of the bacterin 14
days apart starting on day 0. Quarter milk samples for bacterial culture were collected
prior to each vaccination and approximately monthly thereafter for 6 months. Composite
milk samples were collected on days 0, 14, 28, 49 and 70 for IgA, IgG1, IgG2, and IgM
determinations and somatic cell count. No animals in either group developed a new S.
aureus IMI after vaccination. The numbers of mammary quarters that developed a new CNS
IMI, time to new CNS IMI, milk somatic cell count, and milk antibody isotype sample-to-
positive ratio did not significantly differ between groups (P > 0.05). In a herd with a 3%
prevalence of S. aureus IMI and a 30% prevalence of CNS IMI, the vaccine did not reduce the
new staphylococcal IMI rate. There may be insufficient vaccine-induced opsonizing
antibody in milk to facilitate phagocytosis and clearance of staphylococci from the
mammary gland.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction heifers have focused primarily on intramammary anti-

Infection of the heifer mammary gland with common
mastitis pathogens, particularly staphylococci, prior to
calving is well documented (Oliver and Mitchell, 1983;
Oliver and Sordillo, 1988; Trinidad et al., 1990a; Pankey
et al., 1991; Oliver et al., 1992; Fox et al., 1995; Oliver et al.,
1997). Efforts to eliminate pre-partum infections in
* Corresponding author. Tel.: +1 573 882 6857; fax: +1 573 884 0173.
E-mail address: middletonjr@missouri.edu (J.R. Middleton).
biotic therapy shortly before or at the time of calving
(Trinidad et al., 1990b; Owens et al., 1991; Oliver et al.,
1992, 1997, 2003, 2004; Middleton et al., 2005; Borm
et al., 2006; Oliver et al., 2007). While periparturient
intramammary antibiotic therapy has been shown effi-
cacious at reducing intramammary infection (IMI) rates
after calving, particularly those IMIs caused by coagulase-
negative staphylococci (CNS), economic benefit following
such treatment has not been uniformly demonstrated
(Oliver et al., 2003; Middleton et al., 2005; Borm et al.,
2006).

0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.09.053
 J.R. Middleton et al. / Veterinary Microbiology 134 (2009) 192–198
A common practice in the control of many infectious
diseases is vaccination. In very few studies vaccination of
heifers to prevent mastitis either before or after calving has
been evaluated. Commercially available R-mutant Gram-
negative mastitis bacterins are widely used on dairy farms to
significantly reduce the severity of clinical mastitis in the
immediate post-partum period. However, research specifi-
cally focused on the efficacy of these vaccines in heifers is
lacking. Furthermore, heifer mammary glands are most
commonly infected with staphylococcal organisms around
the time of calving (Fox et al., 1995). A commercially
available Staphylococcus aureus mastitis bacterin (Lysigin,
Boehringer Ingelheim Vetmedica, Inc.) has been evaluated
in heifers (Nickerson et al., 1999; Middleton et al., 2006;
Luby et al., 2007). In one study, the efficacy of commercially
available Lysigin was compared with two experimental
formulations and unvaccinated controls in primiparous
heifers (Middleton et al., 2006). Heifers were vaccinated
twice, 28 days apart in late gestation with either a 3-isolate
experimental bacterin (Group I; n = 11), a 5-isolate experi-
mental bacterin (Group II; n = 11), or commercially available
Lysigin (Group III; n = 14). Group IV consisted of 11
unvaccinated control cattle. All groups (vaccinates and
controls) were challenged with a heterologous strain of S.
aureus by intramammary infusion on days 6–8 of lactation in
a single infection-free mammary quarter. All cattle became
infected with S. aureus after challenge. While three cattle in
Group I, and one cow in Group III, cleared their S. aureus IMI
by the end of the study, there were no differences in S. aureus
clearance rates between groups. Cattle vaccinated with
Lysigin had a lower mean duration of clinical mastitis and
lower total mastitis score post-challenge than controls.
Overall, there was no evidence that any of the vaccinated
groups had a lower mean somatic cell count (SCC) than
control, and no evidence that vaccinates had greater milk
yield than controls post-challenge. Heifers vaccinated with
Lysigin had higher mean serum IgG1 and IgG2 sample-to-
positive (S:P) ratios than controls against S. aureus strains of
polysaccharide serotypes 5, 8, and 336. Milk total anti-S.
aureus IgG S:P ratios were only different from controls
against strains of serotype 8 and 336, and there were no
differences between groups for milk IgG1, IgG2, and IgM
(Luby et al., 2007). These data suggest that there may be
insufficient vaccine-induced opsonising antibody in milk to
facilitate clearance of S. aureus from the mammary gland. In
contrast to our work, Nickerson et al. (1999) vaccinated
heifers with commercially available Lysigin at 6 months of
age followed by a booster dose 2 weeks later and subsequent
vaccinations every 6 months until calving. Vaccinates had a
45% reduction in both new S. aureus IMI during pregnancy
and new S. aureus IMI at calving relative to controls. In
addition, vaccinates had a 30% reduction in new CNS IMIs
which became chronic and a 31% reduction in new CNS IMI
at calving relative to controls, thus providing evidence that
Lysigin may be of use in reducing staphylococcal mastitis in
periparturient heifers (Nickerson et al., 1999).
In order to further examine the efficacy of Lysigin against
S. aureus, and more specifically CNS, under field conditions,
we conducted a trial using a lactating dairy cow model. In
our previous studies (Middleton et al., 2006; Luby et al.,
2007), primiparous heifers were vaccinated in late gestation
193
based on expected calving date. However, because the
heifers were bull bred, time from vaccination to calving
varied. Hence, time from vaccination to intramammary
challenge with S. aureus and time to characterization of the
immune response in milk varied between heifers. To
eliminate this variability, the present study was performed
in lactating cattle so that the sampling intervals were the
same in all cattle. The objectives of the study were to
evaluate (1) the efficacy of a Lysigin in protecting against
staphylococcal mastitis (S. aureus and CNS), (2) the effect of
vaccination on milk SCC, and (3) the milk antibody isotype
response to vaccination when administered at the labeled
dose interval in a lactating cow model.
2. Materials and methods
2.1. Animals and treatments
The study was conducted in the University of Missouri
Foremost dairy research facility that milks approximately
180 cattle. The project was approved by the institutional
animal care and use committee. Approximately 3% of
mammary quarters (7% of cows) were infected with S.
aureus and 30% of mammary quarters (58% of cows) were
infected with CNS at the beginning of the study. Ninety
lactating Holstein–Friesian cattle free of S. aureus IMI were
systematically assigned to one of the two groups based on
ear tag number such that even-numbered cattle were
vaccinated (n = 44) and odd-numbered cattle served as
unvaccinated controls (n = 46). Cattle were considered free
of S. aureus IMI based on the absence of S. aureus on three
sequential weekly mammary quarter foremilk cultures,
and a negative result on the S. aureus Antibody Test Kit
(SAATK, VMRD, Inc.) performed according to the manu-
facturer’s instructions. All study animals were at least 30
days in milk and producing greater than 14 kg (30 lbs) milk
per day during the first 70 days of the study. Vaccinates
received two doses of Lysigin 14 days apart (study days 0
and 14) subcutaneously in the neck. Control cattle were
not vaccinated. Composite samples containing milk from
all quarters of each cow were collected via an in-line
sampler (Westphalia Surge, Inc.) during the morning
milking on days 0, 14, 28, 49 and 70 for antibody
determinations and SCC enumeration. Quarter foremilk
samples were aseptically collected using standard techni-
ques (Hogan et al., 1999) immediately prior to each
vaccination and at approximately 1-month intervals
thereafter for 6 months for bacterial culture. Milk samples
for culture and antibody determinations were stored at
20 8C without preservative until they were analyzed.
Milk samples for SCC enumeration were collected into
non-sterile vials containing a 2-bromo-2-nitropropane-
1,3-diol preservative tablet (D & F Control Systems, Inc.),
stored at 4 8C, and shipped overnight to an external
laboratory (Mid-South Dairy Records) within 72 h of
sample collection.
2.2. Milk bacteriology and somatic cell count determination
Stored milk samples were thawed at room temperature
and cultured using standard methods (Hogan et al., 1999).
194 J.R. Middleton et al. / Veterinary Microbiology 134 (2009) 192–198
Briefly, approximately 10 ml milk was applied to half a
Columbia blood agar plate containing 5% sheep blood
(Remel) using a cotton-tipped swab. Plates were incu-
bated at 37 8C for 24 h followed by further 24 h incubation
at room temperature (22 8C). Plates were examined for
bacterial growth at 24 and 48 h. Staphylococci were
identified based on colony morphology, hemolytic pat-
tern, catalase test, and tube coagulase test. A S. aureus IMI
was defined as the recovery of a hemolytic coagulase-
positive Staphylococcus on at least two out of three
consecutive milk samples (Middleton et al., 2001). A CNS
IMI was defined as the recovery of CNS colonies of similar
morphology from a quarter not previously infected with a
CNS in one of the following quantities: (1) >10 colonies/
plate on a single culture, (2) 2–10 colonies/plate on two
out of three consecutive cultures or (3) 1 colony/plate on
three consecutive cultures (adapted from Zadoks et al.,
2001).
Milk SCC enumerations were performed by a commer-
cial laboratory (Mid-South Dairy Records) using an
automated counter (SomatoCount 300, Bentley Instru-
ments). Milk samples were tempered at 39–40 8C prior to
analysis to facilitate homogenization of the sample.
2.3. Detection of different antibody isotypes
Antibody isotype (IgA, IgG1, IgG2, and IgM) S:P ratios
against S. aureus in milk were determined using an
antibody-capture ELISA on composite milk samples
collected on days 0, 14, 28, 49, and 70. The ELISA was
adapted from a commercially available ELISA (SAATK,
VMRD, Inc.). Microtiter plates coated with partially
purified exoprotein antigens from the Wood 46 strain
of S. aureus were obtained from the manufacturer
(VMRD, Inc.). Assay positive control samples differed
according to antibody isotype being detected. For the
milk IgG1 and IgG2 assays, the positive control was the
SAATK positive control. For the milk IgA and IgM assays,
the positive control was milk from a cow with a S. aureus
IMI which had been experimentally infected with S.
aureus. Milk from this cow had a significantly higher
optical density when tested for IgA and IgM than the
negative control sample (P < 0.001). Positive controls
differed between assays due to differing reactivity for
each antibody isotype ELISA. For all assays, the negative
control was the SAATK negative control. All samples
were run in duplicate. Fifty microliters of each control or
test sample were added to each well and incubated for
20 min at room temperature (22 8C). All milk samples
were run undiluted. Following incubation, each well was
manually washed four times with 250 ml of the SAATK
wash solution (0.01 M potassium phosphate, 0.35 M
sodium chloride, 0.065% Tween 20 and 0.02% Thimer-
osal). Subsequently, 50 ml of a 1:1000 dilution in
phosphate buffered saline (pH 7.2) of either sheep
anti-bovine IgA, IgG1, IgG2, or IgM horseradish perox-
idase conjugate were added to each well and incubated
and washed as before. Fifty microliters of tetramethyl-
benzidine substrate (BioFx) were then added to each well
and plates were incubated for 20 min at room tempera-
ture (22 8C). Following incubation, 50 ml of SAATK stop
solution (1.5% sodium fluoride, pH 11.5) was added to
each well and optical densities (OD) were determined
using an automated plate reader (Thermolab Systems) at
650 nm. Antibody S:P ratios were calculated using the
following formula: S:P ratio = (mean test sample
OD mean negative control sample OD)/(mean positive
control sample OD mean negative control sample OD).
Each antibody isotype was run on its own plate with an
approximately even distribution of samples from vacci-
nates and controls on each plate. Each sample period
required a total of three plates per antibody isotype
measured. Sample-to-positive ratios were calculated
within plate such that mean test sample and positive
and negative control ODs in the S:P ratio calculation
represented the mean of two wells tested on each plate.
Hence, all assays were controlled within plate to
eliminate plate-to-plate variation in assay results.
2.4. Data analysis
Positive and negative control sample ODs for each
antibody isotype were compared using a Wilcoxon Rank
Sum test. Baseline data (milk antibody isotype S:P ratios
and SCC) collected prior to first vaccination were
investigated as a covariate for post-vaccination data
using the Kruskal–Wallis one-way analysis of variance
(ANOVA). No differences were noted between groups for
any antibody isotype (P  0.5) or for composite milk SCC
(P = 0.12) at baseline. Therefore, repeated measures
ANOVAs were performed on data collected after first
vaccination, as per Ludbrook (1994). Several attempts
were made to transform the data to meet the assumption
of normality, but all attempts were unsuccessful. There-
fore, since all data following baseline in its raw form met
the equality of variances assumption, raw data were used
in the analyses, as per Ludbrook (1994). The statistical
model included group, time and the interaction of group
and time as fixed effects, and cow was nested within
group. The dependent variables were antibody isotype
(IgA, IgG1, IgG2, and IgM) S:P ratio and milk SCC.
Regardless of F-test result, pair-wise comparisons of
means were conducted to test for improvement of
vaccinates over control. Separate analyses were per-
formed for each antibody isotype.
Incidence of new staphylococcal IMI during the
follow-up period was calculated as the number of new
infections per quarter month at risk. A quarter was
considered at risk for a new staphylococcal IMI if it was
not infected with staphylococci at the start of the study
and was considered at risk until it became infected.
Staphylococcal IMI definitions are stated above in the
milk bacteriology section. The proportions of quarters
which developed a new staphylococcal IMI were com-
pared between groups using the Chi-square test. Time to
new staphylococcal IMI was compared between groups
using a Kaplan–Meier survival analysis on quarters at risk
of new staphylococcal IMI at the time of vaccination. A
quarter was censored from the data if it ceased to lactate
during the follow-up period. Data for S. aureus and CNS
IMIs were analyzed separately. Significance for each
test was P < 0.05. All analyses were performed using
 J.R. Middleton et al. / Veterinary Microbiology 134 (2009) 192–198
computer software (Excel, Microsoft, Inc.; NCSS; Sigma-
Stat 3.1).
3. Results
3.1. New staphylococcal intramammary infections
All quarters (176 vaccinates and 184 controls) were free
of S. aureus at the start of the study. S. aureus was isolated
from 11 quarters (vaccinates, n = 7; controls, n = 4) during
the follow-up period. However, each of the isolations only
occurred on one occasion in a given quarter and therefore
did not meet the definition of a new S. aureus IMI. The
proportion of S. aureus isolations did not differ between
groups (P  0.5). None of the single isolations occurred in
the same cow more than once.
Nineteen quarters in vaccinated cattle and 25 quarters
control cattle were infected with a CNS IMI at the start of
the study. Eighteen new CNS IMIs developed in 157 at risk
quarters in the vaccinated group and 20 new CNS IMI
developed in 159 at risk quarters in the control group
(P  0.8). Incidence of new CNS IMI was 0.82 new CNS IMI
per quarter month for vaccinates and 0.81 new CNS IMI per
quarter month for controls. Median time to new CNS IMI
was 56 days for both vaccinates and controls (P  0.9).
3.2. Individual cow somatic cell counts
Individual cow composite milk SCC data was not
available for all cows at all time points due to samples
being lost to follow-up. Missing data included those for one
vaccinate on day 14, three vaccinates and two controls on
day 28, six vaccinates and two controls on day 49 and five
vaccinates and two controls on day 70. Data were analyzed
with missing cells. Mean SCC was not significantly
different between groups (P  0.1) or sample period
(P  0.7), and no significant group by time interaction
was detected (P  0.25; Fig. 1).
Fig. 1. Mean somatic cell counts (SCC) for vaccinated (n = 44) and control
(n = 46) cows. Vaccinates received Lysigin on days 0 and 14; milk samples
on these days were collected prior to vaccination. Baseline data (day 0)
were investigated as a covariate and no differences between groups were
detected; hence only post-vaccination data were analyzed (days 14–70).
Mean SCC did not differ between groups (P = 0.12) or study days (P = 0.71)
and no significant group by time interaction was detected (P = 0.27). Error
bars represent standard error of the mean.
195
Fig. 2. Mean IgA S:P ratios against S. aureus for vaccinated (n = 44) and
control (n = 46) cows. Vaccinates received Lysigin on days 0 and 14; milk
samples on these days were collected prior to vaccination. Baseline data
(day 0) were investigated as a covariate and no differences between
groups were detected; hence only post-vaccination data were analyzed
(days 14–70). No differences were detected between groups and no group
by time interaction was detected (P = 0.97 and P = 0.52, respectively).
Mean IgA S:P ratios differed between sample days regardless of group
(P < 0.001; denoted by different letters above the bars). Error bars
represent standard error of the mean.
3.3. Milk antibody isotype
Assay positive and negative control sample ODs
differed from each other for all antibody isotypes studied.
Specifically, median (range) positive and negative control
optical densities for each of the antibody isotypes were
0.119 (0.066–0.167) and 0.054 (0.034–0.075) for IgA
(P < 0.001), 0.227 (0.096–0.276) and 0.046 (0.033–0.099)
for IgG1 (P < 0.001), 0.143 (0.083–0.199) and 0.037 (0.030–
0.074) for IgG2 (P < 0.001), and 0.221 (0.120–0.306) and
0.038 (0.031–0.083) for IgM (P < 0.001).
Fig. 3. Mean IgG1 S:P ratios against S. aureus for vaccinated (n = 44) and
control (n = 46) cows. Vaccinates received Lysigin on days 0 and 14; milk
samples on these days were collected prior to vaccination. Baseline data
(day 0) were investigated as a covariate and no differences between
groups were detected; hence only post-vaccination data were analyzed
(days 14–70). No significant differences were detected between groups
and no group by time interaction was detected (P = 0.85 and P = 0.81,
respectively). Mean IgG1 S:P ratios differed between sample days
regardless of group (P < 0.001; denoted by different letters above
bars). Error bars represent standard error of the mean.
196 J.R. Middleton et al. / Veterinary Microbiology 134 (2009) 192–198
Fig. 4. Mean IgG2 S:P ratios against S. aureus for vaccinated (n = 44) and
control (n = 46) cows. Vaccinates received Lysigin on days 0 and 14; milk
samples on these days were collected prior to vaccination. Baseline data
(day 0) were investigated as a covariate and no differences between
groups were detected; hence only post-vaccination data were analyzed
(days 14–70). No differences were detected between groups, times, and
no group by time interaction was detected (P = 0.82, P = 0.09 and P = 0.31,
respectively). Error bars represent standard error of the mean.
Fig. 5. Mean IgM S:P ratios against S. aureus for vaccinated (n = 44) and
control (n = 46) cows. Vaccinates received Lysigin on days 0 and 14; milk
samples on these days were collected prior to vaccination. Baseline data
(day 0) were investigated as a covariate and no differences between
groups were detected; hence only post-vaccination data were analyzed
(days 14–70). No differences were detected between groups and no group
by time interaction was detected (P = 0.95 and P = 0.37, respectively).
Mean IgM S:P ratios differed between sample days regardless of group
(P < 0.001; denoted by different letters above bars). Error bars represent
standard error of the mean.
Differences in mean milk S:P ratios for IgA, IgG1, IgG2
and IgM were not detected between groups (P = 0.965,
P = 0.848, P = 0.817 and P = 0.953, respectively) and no
significant group by time interactions were detected
(P = 0.524, P = 0.807, P = 0.313 and P = 0.367, respectively;
Figs. 2–5). Differences in mean milk S:P ratios were
detected between sample days for IgA, IgG1 and IgM
regardless of group (P < 0.001; Figs. 2, 3 and 5,
respectively). No differences in mean milk S:P ratios
between sample days were detected for IgG2 (P = 0.088;
Fig. 4).
4. Discussion
Based on the stated definitions of an IMI, no animals
developed a new S. aureus IMI in either the vaccinated or
non-vaccinated groups. Eleven animals had S. aureus
isolated from a single mammary quarter milk culture
during the follow-up period. However, the number of
animals from which S. aureus was isolated did not differ
between groups (P  0.5). The current study was per-
formed in a herd with a low prevalence of S. aureus IMI, and
cattle selected for the study were free of S. aureus at the
start of the study. Hence, the lack of new S. aureus IMI in
either group likely reflects a low rate of contagious
transmission in the herd, or possibly a decreased suscept-
ibility of the selected cattle to S. aureus IMI. In a previous
report (Luby and Middleton, 2005), pulsed-field gel
electrophoresis demonstrated that this herd had a diverse
population of S. aureus strains rather than a predominant
strain of S. aureus, suggesting that S. aureus mastitis in this
herd may not be a highly contagious disease.
In contrast to the study by Nickerson et al. (1999), the
results of the current study suggest that vaccination does
not prevent acquisition of new CNS IMI. However, the
previous study evaluated hyperimmunized primiparous
heifers, whereas the present study was performed in
lactating cattle which were vaccinated twice according to
the labeled dose interval of 14 days. Coagulase-negative
staphylococci are a group of several staphylococcal species
that do not express the coagulase enzyme. Neither study
speciated CNS. Hence, another potential reason for the
difference between the current study and that of Nickerson
et al. (1999) may be differences in the prevalence of
different CNS species in each herd.
As no difference in SCC was observed between groups
during the study, it did not appear that vaccination affected
individual cow composite SCC. This is consistent with
Middleton et al. (2006) who reported that dairy heifers
vaccinated with Lysigin did not have significantly different
mammary quarter milk SCC from unvaccinated controls
(P  0.1).
A final objective of the current study was to determine
whether vaccinates had different milk antibody isotype
responses from unvaccinated controls. The intended use
of the SAATK is to diagnose a S. aureus IMI. A diagnosis of
S. aureus IMI is made by comparing the OD generated by a
milk sample from a cow suspected to have a S. aureus IMI
with that generated by the test kit positive control. If the
OD generated by the milk sample is equal to the test kit
positive control, the cow or quarter is considered to have
a S. aureus IMI. If the OD generated by the milk sample
achieves 85% of the positive control OD the cow or quarter
is considered a suspect for S. aureus IMI. In the current
study, the SAATK was adapted to detect different anti-
body isotypes and determine whether there were
differences in S:P ratios between vaccinated cattle and
unvaccinated controls, and hence a cut-point for deter-
mining the presence or absence of a S. aureus IMI was not
used. The significant difference in ODs between positive
and negative control samples in all the assays demon-
strates that the modified SAATK was able to detect each
antibody isotype in the positive control samples and
 J.R. Middleton et al. / Veterinary Microbiology 134 (2009) 192–198
differentiate it from a sample containing no S. aureus
antibody.
Based on the reported results, significant differences in
mean milk antibody S:P ratios between vaccinates and
controls were not detected for any of the antibody isotypes
(P  0.8). In contrast to the current study, Williams et al.
(1966) reported that cattle vaccinated with Lysigin had
increased total immunoglobulin against staphylococci in
milk whey compared with unvaccinated controls. Simi-
larly, in a previous study, vaccination with Lysigin resulted
in a significant rise in total anti-S. aureus IgG in milk against
two strains of S. aureus found in the vaccine, but milk IgG1,
IgG2 and IgM S:P ratios did not differ between vaccinates
and controls (Luby, 2007). The current study did not
evaluate the total anti-S. aureus IgG response in milk
following vaccination.
While the inability of this study to detect differences in
milk immunoglobulin isotypes between vaccinates and
controls may reflect the absence of the studied antibody
isotypes to the vaccine in milk, it may also reflect the fact
that the SAATK coating antigen did not detect vaccine-
induced antibody. However, a previous study using heat-
killed S. aureus Wood 46, the same strain of S. aureus from
which the SAATK coating antigens are derived, as an ELISA
coating antigen was capable of detecting significant
differences in colostral anti-S. aureus IgA and IgG1 between
vaccinates and controls when mammary quarters were
inoculated with suspension containing heat-killed S.
aureus strains of serotypes 5 and 8 (Barrio et al., 2003).
Similar to the present study, Barrio et al. (2003) did not
detect differences in colostral IgM content between
vaccinates and controls, and IgG2 could not be detected
in the colostrum of any animal (Barrio et al., 2003).
Differences in vaccine preparation and route of adminis-
tration are possible explanations for the lack of similar
results for IgA and IgG1 in the present study.
Significant differences in antibody S:P ratios were
detected between sample periods for milk anti-S. aureus
IgA, IgG1, and IgM regardless of group. Variations in serum
IgG between sample periods were similarly observed in a
previous study evaluating the same bacterin studied in the
present report (Nickerson et al., 1999). The day-to-day
variation in the absence of differences between groups
suggests that the variability was due to a factor or factors
other than vaccination. While the day-to-day variation
might be explained by assay variability from one sample
set to the next, this explanation seems unlikely because S:P
ratios were calculated within plate to eliminate plate-to-
plate variation in results. Other potential explanations for
the effect of sample period on milk antibody content
include fluctuations in milk antibody content due to
alterations in milk production or alterations in IMI status
(Charlier et al., 2005).
5. Conclusion
Results of this study suggest that two doses of Lysigin
administered per label during lactation will not reduce the
incidence of new S. aureus or CNS IMI. Additionally,
differences in milk IgA, IgG1, IgG2, IgM between vaccinates
and controls could not be detected. These data are
197
consistent with our challenge trial data in heifers
vaccinated twice in late gestation (Middleton et al.,
2006; Luby et al., 2007). Collectively, our data suggest
that there may be insufficient vaccine-induced opsonizing
antibody in milk to facilitate phagocytosis and clearance of
S. aureus from the mammary gland. In contrast to our work,
Nickerson et al.’s (1999) study suggests that early
vaccination followed by multiple immunizations before
calving may be a useful adjunct in controlling staphylo-
coccal mastitis in heifers.
Acknowledgements
This project was funded by the Missouri Institute for
Cattle. Vaccine was provided by Boehringer Ingelheim
Vetmedica, Inc., St. Joseph, Missouri, USA. Technical
assistance was provided by Julie Holle, Ryan Bader,
Jonathon Bell and Jeff Dill.
Conflict of interest
D.S. Adams is President and CEO of VMRD, Inc. who
manufactured the test kits that were the basis for the ELISA
assays used in the study. Vaccine for the study was donated
to J.R. Middleton by Boehringer Ingelheim Vetmedica, Inc.
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