Efficacy of extended ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows


Oliver, S.P.; Gillespie, B.E.; Headrick, S.J.; Moorehead, H.; Lunn, P.; Dowlen, H.H.; Johnson, D.L.; Lamar, K.C.; Chester, S.T.; Moseley, W.M.

Journal of Dairy Science 87(8): 2393-2400

2004


Little research has focused on treatment of cows with subclinical mastitis during lactation. Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use that inhibits bacterial cell wall synthesis by interfering with enzymes essential for peptidoglycan synthesis. Ceftiofur should be effective against a wide range of contagious and environmental mastitis pathogens. Objectives of the present study were to evaluate the efficacy of ceftiofur for treatment of subclinical mastitis in lactating dairy cows, and to determine if extended therapy regimens enhanced efficacy of ceftiofur. Holstein and Jersey dairy cows (n = 88) from 3 dairy research herds were used. Cows were enrolled in the study based on milk somatic cell counts >400,000/mL and isolation of the same mastitis pathogen in 2 samples obtained 1 wk apart. Cows with one or more intramammary infections (IMI) were blocked by parity and DIM and allocated randomly to 1 of 3 different ceftiofur treatment regimens: 2-d (n = 49 IMI), 5-d (n = 41 IMI), and 8-d (n = 38 IMI) treatment regimens. For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion. Eighteen cows with 38 IMI were included as an untreated negative control group. A bacteriological cure was defined as a treated infected mammary quarter that was bacteriologically negative for the presence of previously identified bacteria at 14 and 28 d after the last treatment. Efficacy of ceftiofur therapy against all subclinical IMI was 38.8, 53.7, and 65.8% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Four of 38 (10.5%) IMI in control cows were cured spontaneously without treatment. All 3 ceftiofur treatment regimens were significantly better than the negative control, and the 8-d extended ceftiofur treatment regimen treatment group was significantly better than the standard 2-d treatment group. Pathogen groups had significantly different cure rates from one another. The cure rate for the 8-d extended ceftiofur treatment regimen was 70% for Corynebacterium bovis, 86% for coagulase-negative Staphylococcus species, 36% for Staph. aureus, 80% for Streptococcus dysgalactiae ssp. dysgalactiae, and 67% for Strep. uberis.

J.
Dairy
Sci.
87:2393-2400
©
American
Dairy
Science
Association,
2004.
Efficacy
of
Extended
Ceftiofur
Intramammary
Therapy
for
Treatment
of
Subclinical
Mastitis
in
Lactating
Dairy
Cows
S.
P.
Oliver,'
B.
E.
Gillespie,'
S.
J.
Headrick,'
H.
Moorehead,'
P.
Lunn,
1
H. H.
Dowlen,
1
D.
L.
Johnson,'
K.
C.
Lamar,'
S.
T.
Chester,
2
and
W.
M.
Moseley
2
1
Department
of
Animal
Science
and
the
Food
Safety
Center
of
Excellence,
The
University
of
Tennessee,
Knoxville
37996
2
Pfizer
Animal
Health,
Kalamazoo,
MI
49001
ABSTRACT
Little
research
has
focused
on
treatment
of
cows
with
subclinical
mastitis
during
lactation.
Ceftiofur
is
a
new
broad-spectrum,
third-generation
cephalosporin
antibi-
otic
for
veterinary
use
that
inhibits
bacterial
cell
wall
synthesis
by
interfering
with
enzymes
essential
for
pep-
tidoglycan
synthesis.
Ceftiofur
should
be
effective
against
a
wide
range
of
contagious
and
environmental
mastitis
pathogens.
Objectives
of
the
present
study
were
to
evaluate
the
efficacy
of
ceftiofur
for
treatment
of
subclinical
mastitis
in
lactating
dairy
cows,
and
to
determine
if
extended
therapy
regimens
enhanced
effi-
cacy
of
ceftiofur.
Holstein
and
Jersey
dairy
cows
(n
=
88)
from
3
dairy
research
herds
were
used.
Cows
were
enrolled
in
the
study
based
on
milk
somatic
cell
counts
>400,000/mL
and
isolation
of
the
same
mastitis
patho-
gen
in
2
samples
obtained
1
wk
apart.
Cows
with
one
or
more
intramammary
infections
(IMI)
were
blocked
by
parity
and
DIM
and
allocated
randomly
to
1
of
3
different
ceftiofur
treatment
regimens:
2-d
(n
=
49
IMI),
5-d
(n
=
41
IMI),
and
8-d
(n
=
38
IMI)
treatment
regi-
mens.
For
all
groups,
125
mg
of
ceftiofur
hydrochloride
was
administered
via
intramammary
infusion.
Eigh-
teen
cows
with
38
IMI
were
included
as
an
untreated
negative
control
group.
A
bacteriological
cure
was
de-
fined
as
a
treated
infected
mammary
quarter
that
was
bacteriologically
negative
for
the
presence
of
previously
identified
bacteria
at
14
and
28
d
after
the
last
treat-
ment.
Efficacy
of
ceftiofur
therapy
against
all
subclini-
cal
IMI
was
38.8,
53.7,
and
65.8%
for
the
2-,
5-,
and
8-
d
ceftiofur
treatment
regimens,
respectively.
Four
of
38
(10.5%)
IMI
in
control
cows
were
cured
spontaneously
without
treatment.
All
3
ceftiofur
treatment
regimens
were
significantly
better
than
the
negative
control,
and
the
8-d
extended
ceftiofur
treatment
regimen
treatment
group
was
significantly
better
than
the
standard
2-d
treatment
group.
Pathogen
groups
had
significantly
dif-
Received
January
30,
2004.
Accepted
April
2,
2004.
Corresponding
author:
S.
P.
Oliver;
e-mail:
soliver@utk.edu.
ferent
cure
rates
from
one
another.
The
cure
rate
for
the
8-d
extended
ceftiofur
treatment
regimen
was
70%
for
Corynebacterium
bovis,
86%
for
coagulase-negative
Staphylococcus
species,
36%
for
Staph.
aureus,
80%
for
Streptococcus
dysgalactiae
ssp.
dysgalactiae,
and
67%
for
Strep.
uberis.
(Key
words:
mastitis,
Streptococcus
uberis,
ceftiofur,
extended
therapy)
INTRODUCTION
Mastitis
is
an
inflammation
of
the
udder
that
affects
a
high
proportion
of
dairy
cows
throughout
the
world.
Clinical
and
subclinical
mastitis
are
the
2
major
forms
of
the
disease.
Clinical
mastitis
results
in
alterations
in
milk
composition
and
appearance,
decreased
milk
production,
elevated
body
temperature,
and
swelling,
redness,
or
heat
in
infected
mammary
quarters.
It
is
readily
apparent
and
easily
detected.
However,
detec-
tion
of
mammary
quarters
with
subclinical
mastitis
is
more
difficult
because
signs
are
not
readily
apparent.
Consequently,
subclinical
mastitis,
which
is
the
most
prevalent
form
of
the
disease,
often
goes
undetected.
Many
subclinical
IMI
tend
to
persist,
resulting
in
an
elevated
milk
SCC
and
decreased
milk
production,
which
may
lead
to
development
of
clinical
mastitis
and
the
opportunity
for
certain
mastitis
pathogens
to
spread
from
infected
mammary
quarters
to
uninfected
mam-
mary
quarters.
In
addition
to
the
different
forms
of
the
disease,
sev-
eral
diverse
mastitis
pathogens
are
able
to
invade
the
mammary
gland,
multiply
there,
and
produce
harmful
substances
that
result
in
an
inflammatory
response.
Mastitis
pathogens
are
categorized
as
either
contagious
or
environmental.
Contagious
pathogens
live
and
mul-
tiply
on
and
in
the
cow's
mammary
gland
and
are
spread
from
cow
to
cow
primarily
during
milking.
Contagious
pathogens
include:
Staphylococcus
aureus,
Streptococ-
cus
agalactiae,
Mycoplasma
species,
and
Corynebacte-
rium
bovis
(Smith,
1983;
Bramley
and
Dodd,
1984).
Environmental
pathogens
reside
in
the
environment
where
cows
live.
The
most
frequently
isolated
environ-
2393
2394
OLIVER
ET
AL.
mental
pathogens
are
streptococci
other
than
Strep.
agalactiae,
commonly
referred
to
as
environmental
streptococci,
and
gram-negative
bacteria
(Bramley
and
Dodd,
1984;
Smith
et
al.,
1985).
Environmental
Strepto-
coccus
species
involved
in
bovine
mastitis
include
Strep.
uberis,
Strep.
dysgalactiae
ssp.
dysgalactiae,
Strep.
equinus,
Strep.
equi,
and
the
Enterococcus
species
(Oli-
ver
et
al.,
1997;
Watts,
1988).
Among
the
environmental
streptococci,
Strep.
uberis
appears
to
be
the
most
preva-
lent
(Hillerton
et
al.,
1995;
Oliver
et
al.,
1997;
Jayarao
et
al.,
1999).
Gram-negative
bacteria
involved
in
bovine
mastitis
include
Escherichia
coli,
Klebsiella
pneumon-
iae,
K
oxytoca,
Enterobacter
spp.
and
Citrobacter
spp.
Among
the
gram-negative
mastitis
pathogens,
E.
coli
and
Klebsiella
spp.
appear
to
be
the
most
prevalent
(Hogan
et
al.,
1999).
Acceptance
and
application
of
mastitis
control
pro-
grams
implemented
in
the
1960s,
including
teat
disin-
fection,
antibiotic
therapy,
and
culling
of
chronically
infected
cows,
has
led
to
considerable
progress
in
con-
trolling
contagious
mastitis
pathogens.
However,
post-
milking
teat
disinfection
and
antibiotic
dry
cow
therapy
is
less
effective
against
environmental
mastitis
patho-
gens.
Studies
have
shown
that
as
the
prevalence
of
contagious
mastitis
pathogens
is
reduced,
the
propor-
tion
of
IMI
by
environmental
pathogens
increases
(Smith,
1983;
Bramley
and
Dodd,
1984;
Smith
et
al.,
1985).
Therefore,
environmental
mastitis
has
become
a
major
problem
in
many
well-managed
dairy
farms
that
have
successfully
controlled
contagious
pathogens
(Oliver
et
al.,
1997;
Jayarao
et
al.,
1999).
Ceftiofur
is
a
new
broad-spectrum,
third-generation
cephalosporin
antibiotic
for
veterinary
use.
Ceftiofur
inhibits
bacterial
cell
wall
synthesis
by
interfering
with
enzymes
essential
for
peptidoglycan
synthesis,
which
results
in
lysis
of
the
bacterial
cell
and
accounts
for
the
bactericidal
nature
of
this
antibiotic
(Hornish
and
Kotarski,
2002).
Consequently,
ceftiofur
should
be
effec-
tive
against
a
wide
range
of
contagious
and
environ-
mental
mastitis
pathogens.
To
date,
little
research
has
focused
on
treatment
of
cows
with
subclinical
mastitis
during
lactation.
One
objective
of
the
present
study
was
to
evaluate
efficacy
of
ceftiofur
for
treatment
of
naturally
occurring
subclin-
ical
mastitis
in
lactating
dairy
cows
caused
by
a
variety
of
mastitis
pathogens.
Another
objective
was
to
deter-
mine
if
extended
ceftiofur
therapy
regimens
for
subclin-
ical
mastitis
enhanced
efficacy
based
on
recent
reports
that
have
shown
that
lengthening
treatment
duration
enhanced
antibiotic
efficacy
(Deluyker
et
al.,
2000;
Gil-
lespie
et
al.,
2002;
Oliver
et
al.,
2003).
MATERIALS
AND
METHODS
Lactating
dairy
cows
from
3
dairy
research
herds
(The
University
of
Tennessee
Middle
Tennessee
Experi-
ment
Station,
Spring
Hill,
TN;
Dairy
Experiment
Sta-
tion,
Lewisburg,
TN;
and
The
University
of
Tennessee
Knoxville
Experiment
Station,
Knoxville,
TN)
were
in-
cluded
in
this
study.
All
lactating
cows
in
each
herd
with
a
milk
SCC
.400,000/mL
based
on
Dairy
Herd
Improvement
herd
test
data
were
considered
for
inclu-
sion.
Quarter
foremilk
samples
from
these
cows
were
obtained
for
microbiological
analysis
on
2
occasions
1
wk
apart.
Cows
(n
=
88)
were
enrolled
in
the
study
based
on
milk
SCC
>400,000/mL
and
isolation
of
the
same
mastitis
pathogen
in
the
2
samples
obtained
1
wk
apart.
Infected
cows
in
each
herd
were
blocked
by
parity
(1st
and
2nd
lactation,
3rd
or
greater
lactation)
and
DIM
(100,
101
to
200,
>200),
and
allocated
randomly
to
1
of
4
treatment
groups.
Ceftiofur
hydrochloride
was
formulated
as
an
oil-based
suspension
in
a
plastet
can-
nula
designed
for
intramammary
delivery
(Pfizer
Ani-
mal
Health,
Kalamazoo,
MI).
The
manufacturer
recom-
mends
infusion
of
one
syringe
of
ceftiofur
hydrochloride
into
each
affected
mammary
quarter,
which
should
be
repeated
at
least
once
after
a
24
h
interval.
Infected
mammary
quarters
of
cows
in
the
standard
2-d
ceftiofur
treatment
regimen
group
(n
=
23
infected
cows,
49
IMI)
were
treated
with
125
mg
of
ceftiofur
hydrochloride
at
24-h
intervals.
Infected
mammary
quarters
of
cows
in
the
5-d
extended
ceftiofur
treatment
regimen
group
(n
=
23
infected
cows,
41
IMI)
were
treated
once
daily
with
125
mg
of
ceftiofur
per
quarter
for
5
consecutive
days.
Infected
mammary
quarters
of
cows
in
the
8-d
extended
ceftiofur
treatment
regimen
group
(n
=
24
infected
cows,
38
IMI)
were
treated
once
daily
with
125
mg
of
ceftiofur
per
quarter
for
8
consecutive
days.
Eighteen
cows
with
38
IMI
were
included
as
an
untreated
negative
control
group.
Infected
mammary
quarter(s)
of
each
cow
in-
cluded
in
the
study,
except
for
cows
in
the
untreated
negative
control
group,
were
infused
intramammarily
with
ceftiofur
via
full
insertion
of
the
plastet
cannula
into
the
teat
end.
Milk
was
discarded
for
14
d
from
all
cows
in
the
study.
Mammary
quarter
foremilk
samples
were
obtained
14
and
7
d
before
treatment,
immediately
prior
to
treat-
ment,
and
14
and
28
d
after
the
last
treatment
for
microbiological
evaluation.
All
samples
were
collected
immediately
before
regular
milking
using
standard
pro-
cedures
described
by
the
National
Mastitis
Council
(Harmon
et
al.,
1990;
Hogan
et
al.,
1999).
Before
sample
collection,
teats
were
dipped
in
a
premilking
teat
disin-
fectant,
cleaned
thoroughly,
dried
with
paper
towels,
and
teat
ends
were
sanitized
with
swabs
containing
70%
isopropyl
alcohol.
Milk
samples
were
examined
following
procedures
recommended
by
the
National
Mastitis
Council
(Hogan
et
al.,
1999)
and
as
described
by
Oliver
et
al.
(1994)
Journal
of
Dairy
Science
Vol.
87,
No.
8,
2004
EXTENDED
CEFTIOFUR
THERAPY
2395
with
modifications.
Briefly,
foremilk
samples
(10
/./L)
from
each
quarter
were
plated
onto
one
quadrant
of
a
trypticase
soy
agar
plate
supplemented
with
5%
defi-
brinated
sheep
blood.
Plates
were
incubated
at
37°C
and
bacterial
growth
was
observed
and
recorded
at
24-
h
intervals
for
3
d.
Bacteria
on
primary
culture
medium
were
identified
tentatively
according
to
colony
morpho-
logic
features,
hemolytic
characteristics,
and
catalase
test.
Isolates
identified
presumptively
as
staphylococci
were
tested
for
coagulase
by
the
tube
coagulase
method.
Isolates
identified
presumptively
as
streptococci
were
evaluated
initially
for
growth
in
6.5%
NaCl,
hydrolysis
of
esculin,
and
Christie,
Atkins,
and
Munch-Peterson
(CAMP)
reaction.
Streptococci
were
identified
to
the
species
level
using
the
API
20
Strep
System
(bioMer-
ieux,
Inc.,
Hazelwood,
MO)
upon
initial
isolation
of
the
organism
from
infected
mammary
quarters.
Streptococ-
cal
organisms
isolated
subsequently
were
identified
by
sodium
hippurate
hydrolysis
for
Strep.
uberis,
and
a
rapid
latex
test
for
streptococcal
grouping
(Streptex,
Remel,
Inc.,
Lenexa,
KS)
for
Strep.
dysgalactiae
ssp.
dysgalactiae.
Bacteria
identified
presumptively
as
gram-negative
were
plated
on
MacConkey's
agar
(Bec-
ton
Dickinson
Microbiology
Systems,
Franklin
Lakes,
NJ)
and
evaluated
by
the
following
biochemical
tests:
triple
sugar
iron,
urea,
oxidase,
motility,
indole,
and
ornithine
decarboxylase.
Gram-negative
organisms
were
identified
to
the
species
level
using
the
API
20E
System
(bioMerieux,
Inc.)
upon
initial
isolation
of
the
organism
from
infected
mammary
quarters.
Gram-neg-
ative
organisms
isolated
subsequently
were
plated
on
MacConkey's
agar
and
evaluated
by
the
following
bio-
chemical
tests:
triple
sugar
iron,
urea,
oxidase,
motility,
indole,
and
ornithine
decarboxylase.
The
milk
SCC
was
determined
by
the
Dairy
Herd
Improvement
Association
Laboratory
(Knoxville,
TN).
Milk
samples
(-25
mL)
for
SCC
determination
were
collected
before
cows
were
milked
and
after
conducting
the
strip
cup
evaluation
and
collection
of
milk
for
micro-
biological
evaluation.
A
bacteriological
cure
was
defined
as
an
infection
that
was
negative
for
the
presence
of
previously
identified
bacteria
at
14
and
28
d
after
last
treatment.
The
per-
centage
of
subclinical
IMI
cured
in
mammary
quarters
receiving
the
standard
2-d
therapy
regimen
was
com-
pared
with
mammary
quarters
receiving
no
treatment.
The
percentage
of
subclinical
IMI
cured
in
quarters
receiving
extended
therapy
regimens
(5-d
and
8-d)
was
compared
with
the
percentage
cured
in
the
standard
2-
d
therapy
regimen
group
and
in
untreated
controls.
The
percentage
of
subclinical
IMI
cured
by
the
8-d
extended
therapy
regimen
was
also
compared
with
the
percent-
age
of
subclinical
IMI
cured
in
the
5-d
extended
therapy
regimen
group.
Data
were
analyzed
by
multidimensional
contin-
gency
table
methodology,
which
compared
cure
rates
between
treatments
by
use
of
Pearson
x
2
(release
8.02,
SAS
Inst.,
Inc.,
Cary,
NC).
Design
factors,
such
as
herds,
cows,
and
pathogen
groups,
were
examined
to
determine
whether
they
could
be
eliminated
and
data
pooled
without
a
loss
of
information.
For
example,
to
test
the
hypothesis
that
there
was
no
cow
effect,
a
x
2
lack-of-fit
test
statistic
was
used
to
compare
estimated
cure
rates
with
cows
in
the
model
against
estimated
cure
rates
with
cows
dropped
from
the
model
and
their
results
summarized
within
each
herd
x
treatment
group.
A
significant
lack-of-fit
test
result
suggests
that
cows
were
different
enough
that
collapsing
across
them
resulted
in
lost
information,
whereas
a
nonsignificant
lack-of-fit
test
means
that
there
was
no
evidence
that
a
cow
contributed
unique
information,
and
thus
no
in-
formation
was
lost
if
results
were
combined
across
animals.
Contingency
table
analysis
used
for
this
study
treated
all
factors
as
fixed
except
for
the
cure/fail
re-
sponse.
This
differed
from
approaches
such
as
the
gen-
eralized
linear
mixed
models
analysis,
which
consid-
ered
herds,
cows,
and
quarters-within-cows
random
ef-
fects.
The
fundamental
difference
between
these
2
approaches
had
to
do
with
inference.
When
herds
were
fixed,
analysis
results
reflected
only
those
herds
and
cows
enrolled
in
the
study
and
provided
inference
im-
plicitly
to
comparable
herds
and
cows
outside
the
study.
When
herds
were
random,
the
analysis
reflected
the
universe
of
herds
and
cows
from
which
the
experimen-
tal
units
came.
However,
the
generalized
linear
mixed
models
analysis
was
a
large
sample
analysis
and
its
conclusions
might
not
be
reliable
when
data
were
as
sparse
as
they
were
when
analyzing
separate
pathogen/
pathogen
groups.
This was
the
primary
reason
for
the
methodology
used.
The
basic
structure
of
data
incorporated
herds,
treat-
ments,
cows
within
herds
x
treatments,
quarters
within
cows,
pathogens
within
quarters,
and
cure/fail
re-
sponses
of
the
pathogen
infections
within
quarters.
Of
the
166
IMI
identified
in
cows
included
in
the
study,
only
11
quarters
had
more
than
one
IMI
per
infected
quarter;
all
other
IMI
had
only
one
pathogen
isolated
from
the
infected
mammary
quarter.
It
was
assumed
that
pathogen
infections
within
a
cow
were
independent
of
one
another
(i.e.,
the
intracow
correlation
was
negli-
gible)
and
the
cure/fail
responses
were
defined
at
the
cow
level.
For
a
particular
pathogen/pathogen
group,
the
re-
sulting
3-factor
contingency
table
was
herd
x
treatment
x
response.
To
test
whether
there
was
a
herd
effect,
a
2-step
analysis
was
conducted.
The
first
hypothesis
tested
was
that
there
was
no
herd
x
treatment
interac-
Journal
of
Dairy
Science
Vol.
87,
No.
8,
2004
2396
OLIVER
ET
AL.
tion.
If
this
hypothesis
was
not
significant,
then
the
second
hypothesis,
that
there
was
no
herd
effect,
was
tested.
If
the
second
hypothesis
was
not
significant,
then
herds
were
conditionally
independent
of
treat-
ments.
In
this
case,
treatment
by
response
information
could
be
pooled
over
herds,
and
the
Pearson
x
2
statistic
for
2-way
tables
was
the
appropriate
statistic
to
com-
pare
treatments.
When
there
was
no
herd
x
treatment
interaction,
but
there
was
a
significant
herd
effect,
treatment
comparisons
needed
to
be
made
adjusting
for
herds.
In
this
case,
the
Mantel-Haenszel
x
2
statistic
was
used
with
herds
as
strata.
When
there
was
a
significant
herd
x
treatment
inter-
action,
treatment
differences
were
not
always
compara-
ble
across
herds.
Tests
to
analyze
design
effects
in-
cluded
all
4
treatment
groups.
Thus,
when
there
was
a
significant
herd
x
treatment
interaction,
the
strategy
was
to
let
the
statistic
used
to
compare
each
pair
of
treatments
depend
upon
the
nature
of
the
herd
effect
for
those
2
treatments
alone.
For
each
pair
of
treat-
ments,
the
herd
x
treatment
interaction
and
herd
effect
were
tested.
If
neither
was
significant,
then
the
Pearson
x
2
statistic
was
used.
If
there
was
a
significant
herd
effect
but
no
herd
x
treatment
interaction,
then
the
Mantel-Haenszel
x
2
statistic
was
used
with
herds
as
strata.
When
there
was
a
significant
herd
x
treatment
interaction
for
the
2
treatments,
the
Pearson
x
2
statistic
was
used
to
compare
them.
In
one
analysis,
the
underlying
model
included
not
only
herds,
treatments,
and
responses,
but
also
patho-
gen
groups.
In
this
case,
the
analysis
of
what
effect
these
design
factors
had
was
determined
using
a
back-
wards
stepwise
approach,
starting
with
the
full
model
including
all
the
terms,
and
sequentially
dropping
fac-
tors
(Agresti,
1990).
The
significance
level
used
depended
upon
the
test.
For
pairwise
treatment
comparisons,
a
(one-sided)
5%
significance
level
was
used.
For
testing
design
factors
(herds
and
pathogen
groups
along
with
their
interac-
tions),
the
Bonferroni
adjustment
was
used
to
keep
the
overall
type
I
error
at
10%.
Thus,
when
herd
was
the
only
design
factor,
since
testing
the
herd
effect
required
2
tests,
each
test
was
conducted
at
a
0.10/2
=
0.05
sig-
nificance
level.
When
both
herd
and
pathogen
group
were
factors
in
the
model,
each
test
was
conducted
at
a
0.10/5
=
0.02
significance
level
since
there
were
5
tests
or
steps
in
the
backwards
stepwise
algorithm
to
analyze
the
design
effect.
RESULTS
There
were
166
subclinical
IMI
in
the
88
cows
en-
rolled
in
this
study.
Most
subclinical
IMI
were
due
to
Staph.
aureus,
Corynebacterium
bovis,
CNS
and
Strep-
Table
1.
Distribution
of
pathogens
causing
subclinical
mastitis
across
herds.
1
Pathogen
Herd
Total
A
B
C
Corynebacterium
bovis
1
0
36
37
CNS
2
12
13
8
33
Staphylococcus
aureus
18
27
5
50
Streptococcus
dysgalactiae
5
8
1
14
Streptococcus
uberis
2
3
15
20
Other
environmentals
3
0
4
1
5
Other
4
2
3
2
7
Total
40
58
68
166
1
Mastitis
pathogens
causing
>10
infections
were
listed
separately.
Otherwise
pathogens
were
grouped.
2
Coagulase-negative
Staphylococcus
species.
3
Enterococcus
faecalis,
Enterococcus
faecium,
and
Aerococcus
yiri-
dans.
4
Bacillus
species,
Corynebacterium
species,
Enterobacter
cloacae,
Escherichia
coli,
Nocardia
species,
Klebsiella
oxytoca,
and
yeast.
tococcus
species
(Table
1).
If
a
mastitis
pathogen
caused
10
or
more
infections,
they
were
listed
separately.
Oth-
erwise,
pathogens
were
pooled
with
other
infrequently
isolated
pathogens.
The
first
data
analysis
compared
treatments
over
all
subclinical
IMI
(Table
2).
Efficacy
of
ceftiofur
therapy
against
all
subclinical
IMI
was
38.8,
53.7,
and
65.8%
for
the
2-,
5-,
and
8-d
ceftiofur
treatment
regimens,
respectively.
All
3
ceftiofur
treatment
regimens
were
significantly
better
than
the
negative
control,
and
the
8-d
extended
ceftiofur
treatment
regimen
treatment
group
was
significantly
better
than
the
standard
2-d
treatment
group.
Since
there
was
no
herd
x
treatment
interaction,
the
listed
cure
rates
were
representative
of
the
3
herds
used
in
this
study.
In
the
first
analysis
(Table
2),
there
was
no
differenti-
ation
among
infections
for
the
pathogen
causing
the
infection.
In
the
second
analysis,
pairwise
comparison
of
the
different
treatment
regimens
over
all
infections
adjusting
for
pathogen/pathogen
group
was
performed.
When
pathogen/pathogen
group
was
put
in
the
model
as
strata,
all
3
ceftiofur
treatment
regimens
were
sig-
nificantly
better
than
the
negative
control,
and
both
the
5-
and
8-d
ceftiofur
extended
therapy
treatment
regimens
had
significantly
higher
bacterial
cure
rates
than
the
standard
2-d
ceftiofur
treatment
regimen
(Ta-
ble
3).
Pathogen
groups
had
significantly
different
cure
rates
from
one
another.
It
was
also
of
interest
to
investigate
treatment
differ-
ences
with
respect
to
each
pathogen/pathogen
group
separately.
Results
of
subclinical
mastitis
cure
rates
and
pairwise
comparisons
among
treatments
for
C.
bovis,
CNS,
Staph.
aureus,
Strep.
dysgalactiae
ssp.
dys-
galactiae,
Strep.
uberis,
and
the
combined
environmen-
tal
streptococci
are
shown
in
Table
4.
Pathogen
groups
Journal
of
Dairy
Science
Vol.
87,
No.
8,
2004
EXTENDED
CEFTIOFUR
THERAPY
2397
Table
2.
Pairwise
comparison
of
subclinical
mastitis
cure
rates
following
different
ceftiofur
treatment
regimens.
1
Ceftiofur
treatment
regimen
Cured
(%)
2
P-values
3
2
d
5
d
8
d
Control
2d
19/49
(39%)
. . .
0.079
0.006
0.002
5d
22/41
(54%)
0.079
. . .
0.136
<0.001
8d
25/38
(66%)
0.006 0.136
. . .
<0.001
Negative
control
4/38
(11%)
0.002
<0.001 <0.001
. . .
1
Data
were
analyzed
using
PROC
FREQ
and
PROC
GENMOD
of
SAS
(version
8.2,
SAS
Inst.,
Inc.,
Cary,
NC).
Herds
were
conditionally
independent
of
treatments.
(P
=
0.315
for
test
of
no
herd
by
treatment
interaction
with
6
degrees
of
freedom,
and
P
=
0.071
for
the
test
of
no
herd
effect
with
2
df.
Each
test
used
a
=
0.05).
Treatments
were
compared
using
Pearson
x
2
statistic.
Number
of
cured
infections
divided
by
total
number
of
infections.
3
One-sided
P-value
comparing
treatment
listed
in
row
with
treatment
listed
in
the
column.
had
significantly
different
cure
rates
from
one
another.
None
of
the
treatments
was
different
from
one
another
for
Strep.
uberis.
The
2-d
standard
ceftiofur
therapy
treatment
regimen
was
significantly
greater
than
the
negative
control
for
C.
bovis
and
Strep.
dysgalactiae
ssp.
dysgalactiae.
For
all
other
pathogen/pathogen
groups,
the
8-d
ceftiofur
extended
therapy
treatment
regimen
was
significantly
better
than
the
negative
con-
trol,
and
except
for
Staph.
aureus,
the
5-d
ceftiofur
ex-
tended
therapy
treatment
regimen
was
significantly
greater
than
the
negative
control
as
well.
The
cure
rate
for
Staph.
aureus
was
36%
in
the
8-d
ceftiofur
extended
therapy
treatment
regimen
and
considerably
less
in
the
other
ceftiofur
treatment
regimens.
When
all
of
the
environmental
Streptococcus
species
were
grouped
to-
gether,
the
5-
and
8-d
ceftiofur
extended
therapy
treat-
ment
regimens
were
significantly
greater
than
the
neg-
ative
control
but
not
different
from
the
2-d
standard
ceftiofur
therapy
treatment
regimen.
DISCUSSION
Efficacy
of
ceftiofur
therapy
against
all
subclinical
IMI
was
38.8,
53.7,
and
65.8%
for
the
2-,
5-,
and
8-d
ceftiofur
treatment
regimens,
respectively.
Significant
differences
in
efficacy
were
detected
between
all
ceftio-
fur
treatment
regimens
and
the
untreated
negative
con-
trol
group,
and
between
the
8-d
ceftiofur
extended
ther-
apy
group
and
the
standard
2-d
treatment
group.
When
pathogen/pathogen
group
was
put
in
the
statistical
model,
all
3
ceftiofur
treatment
regimens
were
signifi-
cantly
better
than
the
negative
control,
and
both
the
5-d
and
8-d
ceftiofur
extended
therapy
treatment
regi-
mens
had
significantly
higher
bacterial
cure
rates
than
the
standard
2-d
ceftiofur
treatment
regimen.
Results
of
this
study
indicate
that
ceftiofur
therapy
was
effec-
tive
in
eliminating
subclinical
IMI
in
lactating
dairy
cows
caused
by
several
different
mastitis
pathogens,
and
that
extended
ceftiofur
therapy
significantly
en-
hanced
treatment
efficacy.
Table
3.
Pairwise
comparisons
of
different
ceftiofur
treatment
regimens
over
all
subclinical
infections
adjusting
for
pathogens
groups.
1,2
Ceftiofur
treatment
regimen
Cured
(%)
3
P-values
4
2
d
5
d
8
d
Control
2d
19/49
(39%)
. . .
0.035
0.005
0.002
5d
22/41
(54%)
0.035
. . .
0.216
<0.001
8d
25/38
(66%)
0.005
0.216
<0.001
Negative
control
4/38
(11%)
0.002
<0.001
<0.001
. . .
1
Pathogen
groups
were
C.
bovis,
S.
aureus,
CNS,
S.
uberis,
environmentals
(including
S.
dysgalactiae
ssp.
dysgalactiae),
and
"other"
(refer
to
Table
1).
2
Data
were
analyzed
using
PROC
FREQ
and
PROC
GENMOD
of
SAS
(version
8.2,
SAS
Inst.,
Inc.,
Cary,
NC).
The
full
model
consisted
of
herds
by
treatments
by
pathogens
by response.
The
backwards
stepwise
hierarchical
test
series
showed
that
herds
were
conditionally
independent
of
treatments,
but
that
there
was
a
significant
pathogen
group
effect
(P
=
0.310,
P
=
0.035,
P
=
0.033,
P
=
0.225,
and
P
<
0.001
for
the
test
of
no
herd
x
group
x
treatment
interaction,
no
herd
x
treatment
or
herd
x
group
interactions,
no
herd
effect,
no
group
x
treatment
interaction,
and
no
group
effect,
respectively,
associated
with
9,
15,
2,
15,
and
5
df,
respectively.
Each
test
used
a
=
0.02).
Treatments
were
compared
using
the
Mantel-Haenszel
statistic
with
pathogen
groups
as
strata.
3
Number
of
cured
infections
divided
by
total
number
of
infections.
4
0ne-sided
P-value
comparing
treatment
listed
in
row
with
treatment
listed
in
the
column.
Journal
of
Dairy
Science
Vol.
87,
No.
8,
2004
2398
OLIVER
ET
AL.
Table
4.
Subclinical
mastitis
cure
rates
and
pairwise
comparisons
among
different
ceftiofur
treatment
regimens
for
pathogen
groups.
1
Pathogen
2,3,4
Ceftiofur
treatment
Staphylococcus
Streptococcus
Streptococcus
Environmental
regimen
C.
bovis
CNS
aureus
dysgalactiae
uberis
Streptococci
5
2d
4/10
(40%)a
7/10
(70%)a
b
1/15
(7%)a
3/4
(75%)a
1/6
(17%)a
6/12
(50%)a
b
5d
3/3
(100%)
b
8/13
(62%)a
2/12
(17%)
ab
2/2
(100%)a
5/9
(56%)a
8/12
(67%)a
8d
7/10
(70%)a
b
6/7
(86%)a
4/11
(36%)
b
4/5
(80%)a
2/3
(67%)a
7/9
(78%)a
Negative
control
0/14
(m)a
1/3
(33%)
b
0/12
(0%)a
0/3
(0%)
b
1/2
(50%)a
1/6
(17%)"
1
For
the
analyses
within
individual
pathogen
groups,
data
were
analyzed
using
PROC
FREQ
and
PROC
GENMOD
of
SAS
(version
8.2,
SAS
Inst.,
Inc.,
Cary,
NC).
Herds
and
treatments
were
considered
fixed
effects.
2
For
each
pathogen
group,
none
of
the
tests
of
no
cow
x
response
interaction within
herd
x
treatment
was
significant
at
the
10%
significance
level
(results
not
shown).
Thus,
except
for
the
environmental
pathogen
group,
infections
were
pooled
across
cows
within
herd
x
treatment,
creating
a
3-way
contingency
table
of
herd
x
treatment
x
response.
For
the
environmental
pathogen
group,
infections
were
pooled
across
cows
within
herd
x
treatment
x
pathogen,
creating
a
4-way
contingency
table.
For
Staph.
aureus,
Strep.
dysgalactiae
ssp.
dysgalactiae,
and
Strep.
uberis
pathogen
groups,
treatments
were
conditionally
independent
of
herds,
thus
treatments
were
compared
using
Pearson
x
2
.
For
the
environmental
pathogen
group,
treatments
were
conditionally
independent
of
both
herds
and
pathogen
groups,
so
treatments
were
compared
using
Pearson
X
2
.
For
the
CNS
pathogen
group,
there
was
a
significant
herd
x
treatment
interaction
so
the
form
of
the
test
(Pearson
X
2
or
Mantel-Haenszel)
depended
upon
the
herd
x
treatment
interaction
for
each
pair
being
tested.
Finally,
for
the
Corynebacterium
bovis
pathogen
group,
there
were
too
few
infections
in
one
herd
so
that
no
herd
effect
could
be
tested.
Treatments
were
compared
using
Pearson
X
2
statistic.
Number
of
infections
cured
divided
by
total
number
of
infections.
4
Percentages
with
different
letters
are
significantly
different
from
one
another
at
the
(one-sided)
5%
level.
5
Includes
Strep.
dysgalactiae
ssp.
dysgalactiae,
Strep.
uberis,
Enterococcus
faecalis,
Enterococcus
faecium,
and
Aerococcus
viridans.
In
the
present
study,
2
different
analyses
that
in-
volved
all
infections
were
conducted.
Because
this
study
used
naturally
occurring
subclinical
IMI,
the
observed
distribution
of
infections
over
all
pathogens
was
as-
sumed
to
be
representative
of
the
actual
underlying
population.
Thus,
these
analyses
provided
information
on
efficacy
of
treatments
used
on
subclinical
mastitis
when
nothing
was
known
about
the
underlying
infec-
tion.
Each
analysis
provided
different
information.
The
first
analysis
summarized
in
Table
2
provided
informa-
tion
about
how
one
treatment
would
do
on
one
infection
compared
with
another
treatment
on
a
second
infection.
The
second
analysis
(Table
3)
adjusted
for
the
pathogen
causing
the
infection.
This
meant
that
comparative
cure
rates
reflected
how
they
would
perform
on
similar
pathogens.
By
adjusting
for
the
pathogen
causing
the
infection,
the
second
analysis
most
closely
reflects
the
comparative
efficacy
one
would
expect
to
see
in
the
field.
Gillespie
et
al.
(2002)
demonstrated
that
extended
pirlimycin
therapy
was
more
effective
in
eliminating
naturally
occurring
subclinical
mastitis
caused
by
envi-
ronmental
streptococci
and
Staph.
aureus
in
lactating
dairy
cows
than
the
standard
2-d
treatment
regimen.
Overall
efficacy
of
pirlimycin
therapy
against
environ-
mental
streptococci
and
Staph.
aureus
was
44.4,
61.1,
95.0,
and
0%
for
the
2-,
5-,
and
8-d
pirlimycin
treatment
regimens,
and
the
untreated
negative
control
group,
respectively.
Significant
differences
in
efficacy
were
de-
tected
between
all
pirlimycin
treatment
regimens
and
the
untreated
negative
control
group,
and
between
the
8-d
pirlimycin
extended
therapy
group
and
the
stan-
dard
2-d
treatment
group.
In
the
present
study,
signifi-
cant
differences
in
efficacy
against
all
subclinical
IMI
were
detected
between
all
ceftiofur
treatment
regimens
and
the
untreated
negative
control
group,
and
between
the
8-d
ceftiofur
extended
therapy
group
and
the
stan-
dard
2-d
treatment
group
similar
to
results
observed
by
Gillespie
et
al.
(2002).
A
study
conducted
in
Europe
evaluated
efficacy
of
pirlimycin
for
treatment
of
subclinical
mastitis
(De-
luyker
et
al.,
2000).
In
this
study,
cows
either
received
no
treatment
or
treatment
with
pirlimycin
for
2
d
or
8
d.
A
mammary
quarter
was
considered
not
cured
if
a
bacterial
species
cultured
before
treatment
was
isolated
after
treatment.
Cure
rates
for
subclinical
Staph.
aureus
infections
were
25%
for
the
standard
2-d
pirli-
mycin
treatment
group
and
51%
for
the
8-d
pirlimycin
extended
therapy
group.
Cure
rates
for
subclinical
Staph.
aureus
infections
in
the
standard
2-d
ceftiofur
treatment
group
observed
in
the
present
study
were
similar
to
the
standard
2-d
pirlimycin
treatment
group
reported
by
Deluyker
et
al.
(2000),
however,
efficacy
of
the
8-d
ceftiofur
treatment
group
was
lower
than
that
for
the
8-d
pirlimycin
extended
therapy
group
reported
by
Deluyker
et
al.
(2000).
Deluyker
et
al.
(2000)
also
evaluated
efficacy
of
pirli-
mycin
on
intramammary
infections
with
Strep.
uberis
and
Strep.
dysgalactiae
ssp.
dysgalactiae.
Cure
rates
for
Strep.
uberis
were
21%
for
2-d
and
75%
for
8-d
pirlimycin
treatment
regimens.
Cure
rates
for
Strep.
dysgalactiae
ssp.
dysgalactiae
were
69
and
100%
for
the
2-d
and
8-d
pirlimycin
treatment
regimens,
similar
to
the
50,
83.3,
and
100%
for
the
2-,
5-,
and
8-d
pirli-
mycin
treatment
of
Strep.
uberis,
and
the
100,
83.3,
Journal
of
Dairy
Science
Vol.
87,
No.
8,
2004
EXTENDED
CEFTIOFUR
THERAPY
2399
and
100%
for
treatment
of
Strep.
dysgalactiae
ssp.
dys-
galactiae
observed
in
the
study
by
Gillespie
et
al.
(2002).
Efficacy
of
ceftiofur
against
Strep.
dysgalactiae
and
all
environmental
Streptococcus
species
in
the
present
study
is
consistent
with
that
reported
by
Deluyker
et
al.
(2000)
and
Gillespie
et
al.
(2002)
for
pirlimycin.
In
another
study
involving
extended
pirlimycin
ther-
apy,
Owens
et
al.
(1997)
reported
improved
cure
rates
against
chronic
Staph.
aureus
IMI
using
an
extended
therapy
regimen
(6
d)
when
compared
with
conven-
tional
2-d
therapy
according
to
the
manufacturers'
in-
structions.
These
researchers
reported
cure
rates
of
26,
41.5,
42,
and
86%
for
chronic
Staph.
aureus
IMI
in
4
dairy
herds
using
the
6-d
treatment
protocol.
In
their
study,
the
presence
of
one
colony
of
Staph.
aureus
was
considered
positive
for
infection
and
therefore
a
treat-
ment
failure.
Results
of
our
work
(Gillespie
et
al.,
2002;
Oliver
et
al.,
2003,
2004)
and
others
(Owens
et
al.,
1997;
De-
luyker
et
al.,
2000)
support
the
concept
that
extended
therapy
is
significantly
more
effective
at
eliminating
natural
and
experimentally
induced
IMI
than
is
stan-
dard
intramammary
treatment.
It
would
appear
that
lengthening
the
duration
of
antibiotic
therapy
increases
treatment
efficacy.
This
has
been
demonstrated
for
cef-
tiofur
and
pirlimycin
against
Strep.
uberis,
other
envi-
ronmental
Streptococcus
species,
and
Staph.
aureus.
CONCLUSIONS
A
study
involving
166
IMI
of
88
Holstein
and
Jersey
cows
was
conducted
to
investigate
the
efficacy
of
ex-
tended
ceftiofur
therapy
for
treatment
of
subclinical
mastitis
in
lactating
dairy
cows.
A
cure
was
defined
as
the
pathogen
isolated
at
enrollment
not
being
present
at
14
and
28
d
after
the
last
antibiotic
infusion.
Efficacy
of
ceftiofur
therapy
against
all
subclinical
IMI
was
38.8,
53.7,
and
65.8%
for
the
2-,
5-,
and
8-d
ceftiofur
treat-
ment
regimens,
respectively.
Four
of
the
38
(10.5%)
IMI
in
control
cows
were
cured
spontaneously
without
treatment.
All
3
ceftiofur
treatment
regimens
were
sig-
nificantly
better
than
the
negative
control,
and
the
8-d
extended
ceftiofur
treatment
regimen
treatment
group
was
significantly
better
than
the
standard
2-d
treat-
ment
group.
When
pathogen/pathogen
group
was
put
in
the
statistical
model,
all
3
ceftiofur
treatment
regimens
were
significantly
better
than
the
negative
control,
and
both
the
5-d
and
8-d
ceftiofur
extended
therapy
treat-
ment
regimens
had
significantly
higher
bacterial
cure
rates
than
the
standard
2-d
ceftiofur
treatment
regi-
men.
Pathogen
groups
had
significantly
different
cure
rates
from
one
another.
None
of
the
treatments
was
different
from
one
another
for
Strep.
uberis.
The
2-d
standard
ceftiofur
therapy
treatment
regimen
was
sig-
nificantly
greater
than
the
negative
control
for
C.
bovis
and
Strep.
dysgalactiae
ssp.
dysgalactiae.
For
all
other
pathogen/pathogen
groups,
the
8-d
ceftiofur
extended
therapy
treatment
regimen
was
significantly
better
than
the
negative
control,
and
except
for
Staph.
aureus,
the
5-d
ceftiofur
extended
therapy
treatment
regimen
was
significantly
greater
than
the
negative
control
as
well.
The
cure
rate
for
Staph.
aureus
was
36%
in
the
8-d
ceftiofur
extended
therapy
treatment
regimen
and
considerably
less
with
the
other
ceftiofur
treatment
reg-
imens.
When
all
of
the
environmental
Streptococcus
species
were
grouped
together,
the
5-
and
8-d
ceftiofur
extended
therapy
treatment
regimens
were
signifi-
cantly
greater
than
the
negative
control
but
not
differ-
ent
from
the
2-d
standard
ceftiofur
therapy
treatment
regimen.
Results
of
this
study
indicate
that
ceftiofur
therapy
was
effective
in
eliminating
subclinical
IMI
in
lactating
dairy
cows
caused
by
several
different
masti-
tis
pathogens,
and
that
extended
ceftiofur
therapy
regi-
mens
significantly
enhanced
treatment
efficacy.
ACKNOWLEDGMENTS
This
investigation
was
supported
by
Pfizer
Animal
Health,
the
Tennessee
Agricultural
Experiment
Sta-
tion,
The
University
of
Tennessee
Food
Safety
Center
of
Excellence,
and
The
University
of
Tennessee,
College
of
Veterinary
Medicine,
Center
of
Excellence
Research
Program
in
Livestock
Diseases
and
Human
Health.
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