J. Dairy Sci.

107:2390–2405
https://doi.org/10.3168/jds.2023-23684
© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Outcomes after treatment of nonsevere gram-negative clinical mastitis with

ceftiofur hydrochloride for 2 or 5 days compared with negative control
D. R. Bruno,1* R. M. Cleale,2 G. Jardon,3 T. Short,2 B. Mills,4 and J. R. Pedraza2
1
University of California Agriculture and Natural Resources, Cooperative Extension, Fresno, CA 93710
2
Zoetis, Parsippany, NJ 07054
3
Department of Animal Science, South Dakota State University, Brookings, SD 57007
4
Cattle Services, Mooresville, NC 28115

ABSTRACT INTRODUCTION

A study was conducted at 3 commercial dairies in Bovine mastitis is a common and costly disease that
California to compare outcomes of treating nonsevere negatively affects dairy farms due to production losses,
(mild and moderate) gram-negative (GN) clinical mas- increased health costs, decreased milk quality, negative
titis (CM) with intramammary (IMM) ceftiofur HCl effects on cow welfare, and increased culling and death
(125 mg of ceftiofur HCl per tube) in either 2-d (SP2) rates (Cha et al., 2011). Clinical mastitis (CM) can be
or 5-d (SP5) treatment programs compared with non- classified as mild, moderate, or severe according to its
treatment (CON). In addition, we contrasted results clinical presentation, which can vary from just abnormal
from cases classified as mild and moderate. Four hun- milk to the presence of systemic signs (International
dred fifteen cases were included in the final dataset, Dairy Federation, 1999). Inflammation, characterized
including 135 CON, 133 SP2, and 147 SP5. Milk from by redness, swelling, heat, and pain, is triggered by
quarters with CM was sampled for on-farm culture various factors, including pathogens. The degree and
(OFC) to differentiate gram-positive (GP) and GN nature of immune responses are likely proportional to
bacteria, with results known within 24 h. Those with infection severity (Sharma et al., 2011). Although exac-
GN infections were randomly assigned to experimental erbated in severe cases, cows with mild and moderate
groups, while those with GP, mixed infections, and cases of mastitis can also experience pain (Leslie and
contaminated samples did not continue in the study Petersson-Wolfe, 2012), which affects animal welfare.
and received standard farm therapy. For cows with Various pathogens, including environmental and
GN infections, a sample was submitted for MALDI- contagious microorganisms, can cause mastitis. Gram-
TOF assay. Only nonsevere cases were enrolled, and all negative (GN) bacteria, especially coliforms (Esch-
quarters yielded monocultures of GN species. Clinical erichia coli, Klebsiella spp., and Enterobacter spp.), are
scores were obtained 0, 1, 2, 3, 4, 5, 14, 21, and 28 ± responsible for about 40% of all IMI (Schukken et al.,
3 d relative to enrollment. Milk samples were collected 2012; Oliveira et al., 2013) and can be a risk to a herd’s
from quarters 14, 21, and 28 ± 3 d after enrollment, productivity. If left untreated, mild and moderate GN
and submitted for routine culture and, when appropri- cases can become severe and toxic and have a nega-
ate, submitted to MALDI-TOF evaluation. For many tive effect on cows (Erskine et al.,1993). Milk SCC and
response criteria, there were significant interactions microbiological cultures are used as diagnostic tools for
between treatments and CM severity scores at the time mastitis and to guide treatment choices best suited to
of enrollment, with effectiveness of ceftiofur HCl treat- affecting bacteriological and clinical cures (Williamson
ment being more beneficial compared with CON as et al., 2022).
mastitis clinical severity increased. While most treat- Antibiotics are frequently used on lactating cows
ment responses were significant for animals with mild presenting mild and moderate cases, while additional
or moderate GN mastitis, the largest responses were support therapy is typically administered to severe
noted among cows with moderate CM cases. cases. The intent of treating CM with antibiotics is
Key words: gram-negative mastitis, bovine mastitis, to eliminate microorganisms from the infected quarter,
ceftiofur hydrochloride preventing further damage to mammary tissue, avoid-
ing a decrease in future milk production, reducing re-
covery time, and returning the quarter to healthy status
(Tomazi et al., 2018). Furthermore, antimicrobial usage
Received May 19, 2023.
Accepted October 9, 2023. has significantly improved the well-being of animals,
*Corresponding author: dfbruno@​ucanr​.edu resulting in better health and higher productivity (Hao
2390
 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2391

et al., 2014). Despite the benefits, there is criticism of MATERIALS AND METHODS
antibiotic use due to the risk of promoting antimicro-
bial resistance (AMR), which has prompted govern- Study Design and Herd Details
ment and public health officials to increase control of
antimicrobials used for food animals (FAO, 2021). This A randomized, negative-controlled field trial was
has motivated researchers to provide justification for conducted from December 2020 to August 2022 using
the use of antimicrobials to treat specific infections. dairy cattle at 3 commercial dairies in California oper-
Moreover, studies have suggested that selective therapy ated under routine management practices according to
based on on-farm culture (OFC) results provides an 7 USC 54 and FASS (2010) following a protocol ap-
opportunity to reduce antimicrobial use on dairy farms proved by the University of California Merced Institu-
(Lago et al., 2011a,b). tional Animal Care and Use Committee (Protocol #
Various antimicrobials are available to treat IMI AUP-20–0014). This protocol was replicated at each
in dairy herds (Ruegg, 2017). Ceftiofur hydrochloride participating dairy, and data were pooled across sites
(ceftiofur HCl), a third-generation, β-lactamase-stable, for final analysis. Herd details are described in Table
broad-spectrum cephalosporin, has been used in vet- 1. To be eligible for the study, selected dairies were
erinary medicine worldwide (Hornish and Katarski, required to remain in compliance with the study pro-
2002) and is approved for treatment of E. coli infections tocol, routinely perform OFC, have good record keep-
(Schukken et al., 2011; Truchetti et al., 2014). A recent ing, trained personnel, individual animal identification,
review (Ruegg, 2021) on the use of antimicrobials for treatment facilities, appropriate drug storage facilities,
the treatment of CM identified a limited number of and refrigeration and freezer capacity. The research
field trials evaluating antimicrobial efficacy exclusively team visited dairies each week throughout the study to
against GN infections involving nonsevere (Schukken et perform animal observations, collect milk samples, and
al., 2011; Fuenzalida and Ruegg, 2019) or acute (Suoja- monitor record-keeping practices by farm personnel. All
la et al., 2010; Persson et al., 2015) cases. Other studies dairies used DairyComp 305 (DC305; Valley Agricul-
also evaluated the efficacy of intramammary therapy tural Software, Tulare, CA) to maintain herd records.
with ceftiofur HCl against GN infections; however, the
proportion of GN infections was low (9–25%) and did Cow Enrollment and Treatment Groups
not include a negative (nontreated) control (Schukken
et al., 2013; Truchetti et al., 2014; Cortinhas et al., 2016; Lactating cows of all parities without prior cases of
Vasquez et al., 2016; Viveros et al., 2018). Research CM in the current lactation and without severe teat
has shown an increased bacteriological cure (BC) rate lesions were eligible for enrollment in the study. Before
when comparing treated versus untreated quarters in- beginning the study, researchers trained farm person-
fected with E. coli (Schukken et al., 2011) and Klebsiella nel to identify CM, to aseptically collect milk samples,
spp. (Fuenzalida and Ruegg, 2019). Although negative and to use a previously described clinical scoring (CS)
controls were not included in other studies, differences system (Vasquez et al., 2016). Briefly, CM cases were
in BC were shown to be dependent on the number of classified as mild (CS = 1, milk visually abnormal
d cows were treated with ceftiofur HCl (Truchetti et [watery, flakes, clots, or bloody] and no other local or
al., 2014); others did not find a significant difference systemic signs of inflammation), moderate (CS = 2,
when comparing different antimicrobials (Schukken et milk abnormal and udder had signs of inflammation,
al., 2013; Vasquez et al., 2016; Viveros et al., 2018). including swelling, redness, heat, hardness, or pain),
Results for clinical cure (CC) and other outcomes also or severe (CS = 3, milk grossly abnormal, udder had
varied among the studies mentioned above. In addition, signs of inflammation and signs of systemic disease
only a few studies evaluated long-term outcomes of GN were present; e.g., fever, dehydration, or depression).
infections. Hence, there is mixed evidence regarding the Only nonsevere (mild and moderate) GN cases of CM
benefits of treating nonsevere GN mastitis cases with were eligible for enrollment in this study. Severe cases
ceftiofur HCl. were not included in the study and were treated ac-
Objectives of the present study were (1) to evaluate cording to farm standard operating procedures (SOP).
the outcomes of 2 IMM ceftiofur HCl therapy regimens In addition, cows entering the postpartum period with
compared with nontreatment of nonsevere CM cases evidence of injuries and illnesses other than mastitis
caused by GN bacteria, (2) to contrast the outcomes that would adversely affect the likelihood of completing
for mild and moderate cases; and (3) to assess overall the study and any cow undergoing systemic antibiotic
treatment outcomes according to severity at the time therapy at the time of initiation of a mastitis event
of diagnosis. were not eligible.

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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2392
Table 1. Herd description upon entering the study

Item Herd A Herd B Herd C

Housing type Freestall Freestall Freestall
Bedding Compost Compost Compost
Number of cows 5,800 2,800 5,400
Breed Jersey Holstein Holstein
Milkings Mid and late lactation 2×; 3× 2×
fresh and high 3×
DHIA testing Monthly Monthly No–automated daily milk-yield
recording systems
Milk production1 (kg) 10,614 13,485 13,962
SCC2 (cells/mL) 96,000 213,000 190,000
Gram-negative core antigen vaccine No Yes Yes
Total clinical3 mastitis cases (n) 914 1,859 2,214
No culture3 (n) 16 861 10
No growth3 (%) 63.3 20.2 42.5
Gram negative3 (%) 7.1 23.6 9.2
Gram positive3 (%) 29.6 52.4 42.8
Mixed3 (%) — 0.5 5.5
Other3 (%) — 3.3 —
Mastitis treatment protocol Treat GP; do not treat NG Treat all CM cases Treat GP and GN; do not treat NG
before study 4 and GN
1
Milk production annual rolling herd average.
2
Mean bulk tank milk SCC for 1 yr preceding study initiation.
3
Based on dairy records (DairyComp 305; Valley Agricultural Software, Tulare, CA): Herd A, December 2019 to November 2020; herd B,
January to December 2020; herd C, April 2020 to March 2021 (total cases/year before the beginning of the study). If a clinical mastitis was
observed 14 d or more after the previous case it was considered a new infection. Dashes indicate data was not collected.
4
GP = gram positive; GN = gram negative; NG = no growth.

Cows identified with CM were assigned an enrollment were also eligible to participate in the study. In these
clinical score (ECS), and pre-enrollment milk samples cases, quarters were enrolled to the same experimental
were collected from the affected quarter(s) the day CM group. After enrollment, CM cases previously identified
was identified (d 0) using standard aseptic techniques as GN by OFC but not confirmed by MALDI-TOF
(NMC, 1999). Milk samples were subjected to OFC for were excluded from the study and treated by the dairy
differentiation of GP and GN organisms and used for following their SOP. For cows started on ceftiofur HCl
pre-screening cows for enrollment. Two of the 3 dairies that MALDI-TOF showed not to be infected by GN
had a long history of using OFC before the study. The bacteria, the dairy modified the prescribed treatment
third dairy, not previously using OFC, was methodi- in DC305 to follow an appropriate GP treatment pro-
cally trained before study implementation. Moreover, tocol. If a cow was initially enrolled in the CON group
all dairies followed an OFC previously described (Lago and not confirmed to be a GN case, the dairy immedi-
et al., 2011a). Briefly, aseptically collected milk samples ately started the protocol according to their SOP for
from the affected quarters were plated on a bi-plate that specific pathogen. The final determinant for the
(Minnesota Easy Culture System, University of Min- inclusion of a CM case in the study was based on the
nesota, St. Paul) at dairies A and C, or on Accumast confirmation of GN infection in the pretreatment milk
plates (FERA Diagnostics and Biologicals LCC, Col- sample by MALDI-TOF. After enrollment, cows were
lege Station, TX) at Dairy B. Plates were placed in evaluated daily for clinical signs of mastitis by trained
on-farm incubators and incubated at approximately farm personnel, and cows that developed severe symp-
37°C for 18 to 24 h. After inoculation onto OFC plates, toms were treated according to the dairy’s SOP. These
milk samples were frozen at −18°C and later submitted cows were included in final data and included in the
to a diagnostic laboratory for bacteria confirmation by overall treatment success analysis.
MALDI-TOF testing. Samples were also cultured for Randomization was performed using a previously
Mycoplasma spp. Cases of CM with OFC plates yield- prepared spreadsheet using the RAND function of Ex-
ing no growth (NG), GP bacteria, or containing more cel software (Microsoft Office Corporation, Redmond,
than one bacterial species (mixed culture or contamina- WA). Weekly visits were made by research personnel,
tion) were not enrolled in the study and were treated and the spreadsheet for treatment enrollment was in-
per dairy’s SOP. Quarters were only enrolled once in spected to ensure protocol compliance. Cows meeting
the study during a lactation, but cows with CM identi- enrollment criteria were randomly assigned to one of 3
fied in more than one-quarter in the same lactation treatment groups: (1) SP2, IMM treatment with ceft-
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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2393

iofur HCl (125 mg in 10 mL, Spectramast LC, Zoetis, Outcomes

Parsippany, NJ) at study enrollment and 24 h later for
a total of 2 d; (2) SP5, IMM treatments with ceftiofur Outcomes evaluated in this study included BC,
HCl (125 mg in 10 mL, Spectramast LC, Zoetis, Parsip- CC, days to clinical cure (DCC), mastitis recurrence
pany, NJ) beginning on the day of enrollment and for (MR), new intramammary infections (NIMI), MC,
the next 4 d at 24 h intervals; or (3) CON, no treat- SCC, need for supplemental IMM or systemic therapy,
ment (control group). Treatment was administered by culling or death, and overall treatment success.
trained farm personnel following aseptic techniques A BC was achieved if milk samples collected at 14
(NMC, 1999), and cows treated with antibiotics were ± 3 d postenrollment were negative for the infecting
marked using a leg band for easier identification per GN species present at enrollment, and if there was no
farm SOP. Milk from all enrolled cows was discarded additional IMM or systemic antibiotic therapy. If GN
until it returned to normal (nontreated group) or until bacteria, regardless of species, were isolated in the 14 ±
the end of the 72-h milk-withholding period after the 3 d samples, the case was considered a treatment fail-
end of antimicrobial therapy. Days in milk and parity ure. If no bacteria were recovered 14 d after enrollment
number (lactation ≤2 or ≥3) were documented. but were recovered 21 or 28 d after enrollment along
Given the nature of the study and treatment struc- with CM signs, it was considered a treatment success,
ture, blinding was not possible, as individuals treat- but incurred a NIMI. If a cow received supplemental
ing cows needed to know which cows received IMM IMM or systemic antibiotic therapy before 14 ± 3 d
therapy to follow appropriate milk and meat withhold- after enrollment, the 14 d sample was considered com-
ing times. However, laboratory personnel were blinded promised, and bacteriological data were not included in
to treatment and details of the study, and the farm the final analysis. If a cow received IMM or systemic
personnel were unaware of study outcomes until study antibiotic therapy after d 14 for mastitis due to a GP
completion. organism but had a NG culture result on d 14, it was
considered a BC; this therapy may have affected d 21
Post-Treatment Assessments and Sample Collection or d 28 milk culture results but did not affect d 14
milk culture results. If cows died or were culled before
Postenrollment milk samples were collected from 14 ± 3 d after enrollment due to GN mastitis-related
enrolled quarters 14, 21, and 28 ± 3 d after enroll- reasons, data were excluded from BC analysis, but were
ment into 2 vials: one for assessment of bacteriological accounted for in the culling and death analysis and
outcomes and the second for assessment of milk compo- considered a treatment failure.
nents. Samples were identified with cow number, quar- Quarters were considered clinically cured when milk
ter, date, and dairy. Samples for bacterial culture and and udder appearance returned to normal within 14
MALDI-TOF were chilled on ice or frozen at −18°C un- d after enrollment. Days until CC was defined as the
til sent to the laboratory for microbiological evaluation. number of d from first identification of CM until milk
Milk samples for quantification of components (milk returned to normal, along with the absence of clinical
composition; MC); fat, protein, lactose, SNF, MUN, signs. Any abnormality at follow-up evaluations within
and SCC were collected in vials containing Bronopol 14 ± 3 d was considered a treatment failure with re-
(Microtabs II, D&F Control Stems Inc., Norwood, MA) spect to clinical assessment. Cases enrolled based on
according to International Dairy Federation (1999), and CS, treated (or not) according to the study protocol,
evaluated by the Dairy Herd Improvement Laboratory declared CC, then returned to being milked with the
within 24 to 48 h. herd but later observed with abnormal milk and or/
Clinical assessments were performed by farm person- signs of inflammation within 14 d of enrollment with or
nel or the research team for the first 5 d after enroll- without culture and re-treated (or not) or culled, were
ment, then at 14, 21, and 28 ± 3 d after enrollment. considered treatment failures. Mastitis recurrence was
Cows were examined during milking, and the same CS defined when CM was identified in the same quarter
used at enrollment was used for post-treatment clinical at least 14 d after enrollment. Information was docu-
assessments. A CS 0 was included to classify cows as mented in DC305, including the date of detection, OFC
normal (normal milk appearance and absence of signs results when available, and treatment.
of inflammation). Cows were monitored for up to 90 Other observations recorded included the date a cow
d after enrollment or until dried off or culled or died. was declared clinically well, date of reinfection after
General cow behavior was also observed by the hospital d 14, d between initial mastitis and reinfection, OFC
barn manager. DairyComp 305 was used to document results of the reinfecting organism (if performed/docu-
all clinical observations, treatments, and follow-up mented), remarks in the DC305 cowcard, supplemental
measurements. IMM or systemic therapy, including drug names, dates,
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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2394

and length of treatment. All this information was as- of farm (A, B, C), cow breed (Holstein, Jersey), and
sessed as part of the process of determining whether a season of the year (spring, summer, fall, winter). ECS
BC or CC was affected. Clinical outcome success was was used as a covariate. Treatment differences were
declared if CS improved from 1 to 0 or 2 to 0 within evaluated with a 2-tailed t-test to determine whether
14 d. responses by cows treated with SP2 or SP5 differed
Milk composition was evaluated at the quarter level from CON, and whether SP2 and SP5 were different
during post-treatment assessments 14, 21, and 28 ± from each other. Treatment effects were considered
3 d after enrollment and at a composite (cow) level significant if P ≤ 0.05; if 0.05 > P ≤ 0.10, the treat-
monthly in dairies A and B up to 90 d after enrollment. ment effect was considered a tendency. Adjusted odds
Milk SCC were converted to linear scores (LSSCC) ratios and respective 95% confidence intervals were
for statistical analysis by the formula: LSSCC = log2 calculated.
(SCC/100) + 3 (Schukken et al., 2003). In addition, Because they are ordinal data with a multinomial
data from DHIA test dates relative to when animals distribution, CS were analyzed using PROC GLIMMIX
were diagnosed with GN mastitis, DIM on each test to evaluate fixed effects of treatment, parity, treatment
day, test day FCM yield (kg/d), and LSSCC were ex- by parity, ECS, and treatment by ECS; and random
tracted from DC305 for the test day preceding mastitis effects of dairy, breed, and season of the year. Data
diagnosis and 4 test dates following mastitis treatment. were fit to a cumulative logit proportional-odds model,
Data pertaining to death and culling were retrieved treatment effects were assessed, and odds ratios were
from DC305. Duration animals remained in the study computed to provide an interpretation of effects. The
was calculated based on the time elapsed between en- LIFETEST procedure of SAS was used to calculate
trance into the study and exit from the study due to survival probabilities for time-to-event outcomes, where
death or culling as previously described (Schukken et time was defined as the number of d until the event of
al., 2011) up to 90 d following enrollment. interest (culling or death due to GN CM) occurred.
Overall treatment success was declared for each cow Censoring was used for cows that completed the study
that had BC at d 14, had CC by d 14, was not culled, through 90 d postenrollment (maximum survival was
and did not die during study evaluation (<28 ± 3 d 90 d). GraphPad Prism 8 software (GraphPad Software
after enrollment), had no MR, and did not receive Inc., LaJolla, CA) was used to create a Kaplan-Meier
other IMM or systemic antibiotic treatment before 14 survival plot to visualize time cow remained in the
d. Treatment failure was declared for cows that never study in each treatment group.
improved clinically, went on to develop a severe case
of mastitis, required antibiotic or supportive therapy RESULTS
other than experimental treatment, developed severe
clinical signs, died or were culled due to the mastitis Descriptive Data
case, lost a teat due to the mastitis event, had an-
other case of GN mastitis before 14 d after enrollment, A total of 423 quarters that exhibited signs of mild
or did not experience a BC at 14 d after enrollment. to moderate CM and were infected with GN organ-
Cows that developed signs of severe mastitis during isms were randomized to treatments between December
the study were considered treatment failures, returned 2, 2020, and July 3, 2022. Of these 423 quarters (135
to standard dairy management practices, and treated CON, 133 SP2, 155 SP5), 8 quarters from SP5 cows
by dairy personnel according to established veterinary were excluded from the final analysis because of devia-
treatment protocols. tions from the treatment protocol. Final enrollment in
data analysis included 135 CON, 133 SP2, and 147 SP5
Statistical Analysis quarters. Details of enrollments to treatments at each
study site are summarized in Table 2. Because Site A
Data analysis was facilitated using a recognized bio- was a Jersey herd and Sites B and C were Holstein
metrics program (SAS Release 9.4, SAS Institute, Cary, herds there is confounding of breed and study site, but
NC), and quarter was the experimental unit. Descrip- these factors were considered random site effects in the
tive statistics were performed using PROC FREQ. Bi- statistical model and are appropriately accounted for.
nomially distributed categorical data were analyzed by Across farms, animals in treatment and control groups
PROC GLIMMIX, and continuous data were analyzed did not differ significantly in parity, DIM, milk produc-
by PROC MIXED to evaluate fixed effects of treatment tion, or SCC at the time of enrollment. No significant
(CON, SP2, SP5), parity (lactation ≤ 2 or lactation ≥ difference was present among groups at the onset of
3), treatment by parity interaction, ECS (1 or 2), ECS treatments. Table 3 summarizes the herd-wide distri-
by parity interaction, mastitis DIM; and random effects bution of CM cases (nonsevere vs. severe) occurring
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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2395
Table 2. Summary of enrollments to treatments at each study site1

Item CON SP2 SP5 Total, n (% of column)

Farm
Site A, n (% of column) 51 (37.8) 49 (36.8) 40 (27.2) 140 (33.7)
Site B, n (% of column) 37 (27.4) 22 (16.5) 48 (32.7) 107 (25.8)
Site C, n (% of column) 47 (34.8) 62 (46.6) 59 (40.1) 168 (40.5)
Total, n (% of row) 135 (32.5) 133 (32.0) 147 (35.4) 415 (100.0)
Quarters infected
LF, n (% of column) 35 (25.9) 32 (24.1) 50 (33.3) 116 (28.0)
RF, n, (% of column) 37 (27.4) 36 (27.1) 38 (22.4) 106 (25.5)
LR, n (% of column) 30 (22.2) 32 (24.1) 37 (24.5) 98 (23.6)
RR, n (% of column) 33 (24.4) 33 (24.8) 30 (19.7) 95 (22.9)
Severity score
Mild, n (% of column) 81 (60.0) 64 (48.1) 74 (50.3) 219 (52.8)
Moderate, n (% of column) 54 (40.0) 69 (51.9) 73 (49.7) 196 (47.2)
Season of year at enrollment
Spring, n (% of column) 50 (37.1) 45 (33.9) 40 (27.2) 135 (32.5)
Summer, n (% of column) 23 (17.0) 24 (18.0) 29 (19.7) 76 (18.3)
Fall, n (% of column) 22 (16.3) 29 (21.8) 32 (21.8) 83 (20.0)
Winter, n (% of column) 40 (29.6) 35 (26.3) 46 (31.3) 121 (29.2)
Parity group
First and second lactation, n (% of column) 63 (46.7) 64 (48.1) 68 (46.3) 195 (47.0)
≥Third lactation, n (% of column) 72 (53.3) 69 (51.9) 79 (53.7) 220 (53.0)
Breed
Holstein, n (%) 84 (62.2) 82 (61.7) 107 (72.8) 274 (66.0)
Jersey, n (%) 51 (37.8) 50 (37.6) 40 (27.2) 141 (34.0)
1
Control = nontreated negative controls; SP2 = ceftiofur HCl once daily for 2 d; SP5 = ceftiofur HCl once daily for 5 d.

during the study period at each site. Not all cases of noted. Mycoplasma spp. were not isolated from any
GN CM were enrolled in the study because they did not milk sample.
present as nonsevere.
The number of quarters infected with each genus of Bacteriological Cures
GN bacteria was relatively uniformly distributed across
treatments (Table 2). Escherichia coli was the most The overall effect of treatments on BC rates is
common GN organism identified in CM cases (362/415; represented in Table 4. Due to the number of cases
CON = 117, SP2 = 116, SP5 = 129) followed by Klebsi- enrolled in the study, there was adequate replication
ella spp. (K. oxytoca, K. pneumonia, and K. aerogenes; within treatment by ECS combinations for meaningful
26/415; CON = 11; SP2 = 8, SP5 = 7). Other isolates analysis. Thirty-seven cows (CON = 18; SP2 = 15; SP5
found in low numbers were combined into an “others” = 4) received supplemental IMM or support therapy
category (27/415; CON = 7, SP2 = 9, SP5 = 11). before 14 ± 3 d after enrollment, were removed from
Importantly, a single species was cultured from each BC analysis, and were considered treatment failures.
of the quarters enrolled, and no mixed cultures were Unfortunately, OFC was not performed for all cases

Table 3. Summary of all clinical mastitis (CM) culture results1 and distribution of severity (nonsevere or
severe cases) of GN2 cases for each herd

Item Herd A Herd B Herd C

CM cases, n 3,309 2,732 1,828
Cultured, n (%) 3,295 (99.7) 2,373 (86.9) 1,774 (97.0)
GN,2 n (%) 277 (8.4) 225 (9.5) 215 (12.1)
GP,2 n (%) 1,308 (39.7) 1,607 (67.7) 750 (42.3)
Mixed, n (%) 0 (0.0) 62 (2.6) 153 (8.6)
Other, n (%) 0 (0.0) 2 (0.1) 0 (0.0)
NG,2 n (%) 1,713 (52.0) 477 (20.1) 656 (37.0)
CM severity of GN at time of detection
Nonsevere (mild and moderate), n (%) 140 (50.6) 107 (47.5) 168 (78.3)
Severe, n (%) 137 (49.4) 118 (52.5) 47 (21.7)
1
CM cases over the study period.
2
GP = gram positive; GN = gram negative; NG = no growth.

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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2396

(CON = 7: 3 GN and 4 NG; SP2 = 4: 2 NG,1 GP and 28. Odds that SP5 quarters would have lower CS than
1 mixed; SP5 = 4: 2 NG, 1 GP, 1 mixed). CON quarters were significant at 2, 5, and 14 d after
Results showed significant effects of treatment the start of treatment. There was no significant likeli-
(P = 0.0008) and parity (P = 0.0333), and a tendency hood of lower scores by SP5 quarters compared with
for ECS (P = 0.0937). Bacteriological cure rate was SP2 quarters.
higher for treated groups compared with controls Odds ratios comparing ECS1 to ECS2 are also shown
(P < 0.05), and higher (P < 0.05) for quarters of older in Table 5. The odds that quarters with ECS of 2 have
(or lactation ≥ 3) than for quarters of younger cows lower CS than quarters with ECS of 1 were significant
(lactation ≤ 2). There was also a BC response differ- on d 2, 5, and 14 (P < 0.05). Overall, ECS scores ac-
ence between ECS1 and ECS2 quarters within the same counted for a significant proportion of the variation
group. For ECS 1 quarters there were no differences in data through 14 d following the start of treatment.
between treatments. Among ECS 2 quarters, however, Thereafter, CS progressed lower across both ECS lev-
BC rates for both SP2 and SP5 were higher (P < 0.05) els, and the effect that ECS had on CS was negligible.
than CON and not different from each other. Figure 1 Perhaps a better indicator of clinical treatment out-
presents the treatment by ECS interaction on BC rates. comes is the success rate on CS associated with treat-
Table 6 summarizes main effects of treatment on BC ment, defined as the proportion of quarters in each
rates across ECS. Quarters treated with SP2 or SP5 group that experienced a reduction in CS from 2 to 0
responded with higher BC than CON (P < 0.05). or 1 to 0 by d 14 (Table 4). By this standard, there was
Statistical analysis was not possible to differentiate a significant effect of treatment (P < 0.0001) and ECS
BC responses by etiology because the majority of cases (P < 0.0001), and the treatment by ECS interaction
were caused by E. coli. Among 26 quarters enrolled was not significant. Thus, treatments behaved similarly
with Klebsiella spp. (CON: ECS 1 = 5, ECS 2 = 6; SP2: among quarters with ECS of either 1 or 2. As with
ECS 1 = 4, ECS 2 = 4; SP5: ECS 1 = 2, ECS 2 = 5), 10 other measures of treatment success, the response to
(38%) experienced BC (CON: ECS 1 = 2, ECS 2 = 1; treatments was more robust when ECS was 2 rather
SP2: ECS 1 = 4, ECS 2 = 1; SP5: ECS 1 = 0, ECS 2 = than 1. Regarding clinical outcome success, CON quar-
2), and among the 27 quarters enrolled with other GN ters with ECS of 1 had a lower success rate than SP2
bacteria, (CON: ECS 1 = 6, ECS 2 = 1; SP2: ECS 1 = and SP5 (P < 0.05). Among quarters with ECS of 2,
6, ECS 2 = 3; SP5: ECS 1 = 5, ECS 2 = 6), 22 (81%) CON cows had a treatment success rate inferior (P <
experienced BC (CON: ECS 1 = 4, ECS 2 = 1; SP2: 0.05) to the success rate among quarters treated with
ECS 1 = 4, ECS 2 = 1; SP5: ECS 1 = 5, ECS 2 = 5). SP2 or SP5; SP5 was numerically higher than SP2 but
was not significantly different. Table 6 summarizes
Clinical Assessments clinical outcome success data across parity groups and
ECS. In all instances, quarters treated with SP2 or SP5
Clinical assessment of quarters from the time of responded with higher clinical outcome success than
enrollment through 28 ± 3 d postenrollment is summa- CON (P < 0.05).
rized in Table 4. With respect to mean CS at individual Average DCC was significantly affected by treatment
observation points, significant treatment effects were (P < 0.0001) and ECS (P = 0.0127), but there was no
noted at observation points on d 2, 5, and 14 (P < 0.05) treatment by ECS interaction (Table 4). Summarized
but not on d 21 or 28. Similarly, significant effects of across ECS (Table 6), DCC observed by SP2 quarters
ECS were noted at observation points on d 2, 5, and 14 was not different from CON quarters, but both treat-
but not on d 21 or 28. The interaction of treatment and ments were less than the average for SP5 quarters (P <
ECS was significant only on d 14. Figure 2 is a graphic 0.05). Milk from cows treated with ceftiofur HCl must
presentation of these mean CS by treatment and ECS. be withheld for 72 h following the last treatment, while
Table 5 presents treatment comparisons based on odds milk for untreated cows must be withheld as nonsaleable
ratios derived from analysis of the multinomial CS until clinical signs, including abnormal milk, have re-
data, including exponentiated odds ratio estimates for solved. Given that clinical signs for both CON and SP2
treatment and ECS comparisons. An example of how quarters resolved after d 5 from study enrollment, there
to interpret data follows. At CS2, the comparison of is essentially no difference regarding the required milk
CON to SP2 had an odds ratio of 2.32, which indicates discard duration between these treatments. Although
that the odds of SP2 quarters having lower CS than clinical signs among SP5 quarters were not resolved
CON quarters was increased by 2.32-fold, and this ef- until just over 7 d from enrollment, milk withholding
fect was significant (P = 0.0161). Examination of all was necessary for 8 d to meet product label compliance.
comparisons of CON to SP2 showed increased odds of Similar to BC, it was not possible to evaluate CC
lower scores for SP2 at d 5 but not for d 14, 21, and by etiology. However, our data showed that among 26
Journal of Dairy Science Vol. 107 No. 4, 2024
 Table 4. Results by treatment group and enrollment clinical score (ECS) groups (LSM ± SE)1

Treatment group2 P-value

CON SP2 SP5

Item ECS 1 ECS 2 ECS 1 ECS 2 ECS 1 ECS 2 Treatment (T) Parity (P) ECS
Quarters enrolled, n (% of treatment 81 (60.0) 54 (40.0) 64 (48.1) 69 (51.9) 74 (50.3) 73 (49.7) — — —

Journal of Dairy Science Vol. 107 No. 4, 2024

total)
Bacteriological cure,3 % 78.7 ± 7.4 52.4 ± 12.6x 86.2 ± 5.8 79.8 ± 7.5y 86.9 ± 5.5 89.3 ± 4.9y 0.0008 0.0333 0.0937
Clinical score4
Enrollment 1.0 2.0 1.0 2.0 1.0 2.0 — — —
Day 2 0.96 2.07 0.88 1.90 0.89 1.82 0.01785 0.2611 <0.0001
Day 5 0.33 1.74 0.14 1.02 0.19 0.99 <0.0001 0.4739 <0.0001
Day 14 0.17 0.57 0.13 0.07 0.06 0.23 0.0371 0.0294 0.03356
Day 21 0.08 0.36 0.13 0.08 0.09 0.10 0.2325 0.5225 0.3261
Day 28 0.09 0.08 0.13 0.09 0.09 0.05 0.4738 0.3365 0.5262
Clinical outcome,7 % success 75.8 ± 10.4a 22.1 ± 10.4x 95.1 ± 3.4b 74.8 ± 10.8y 94.4 ± 3.6b 80.9 ± 9.0y <0.0001 0.0896 <0.0001
Days to clinical cure 4.6 ± 0.6a 6.6 ± 0.8xy 5.0 ± 0.6a 6.0 ± 0.6x 7.4 ± 0.6b 7.2 ± 0.6y <0.0001 0.1306 0.0127
Overall treatment outcome,8 % 63.4 ± 10.6a 21.1 ± 8.7x 86.5 ± 6.2b 66.7 ± 10.5y 83.8 ± 6.8b 72.3 ± 9.5y <0.0001 0.0215 <0.0001
success
Odds ratio of overall treatment — — 3.69 7.51 2.99 9.76 — — —
outcome
a,b
Within ECS 1, means in the same row with different superscripts are different (P < 0.05).
x,y
Within ECS 2, means in the same row with different superscripts are different (P < 0.05).
1
Categorical data were analyzed by PROC GLIMMIX and continuous data were analyzed by PROC MIXED in SAS 9.4 (SAS Institute Inc., Cary, NC) to evaluate fixed effects of
treatment, parity, treatment by parity interaction, ECS, ECS by parity interaction, mastitis DIM, and random effects of farm, breed, and season of year.
2
CON = nontreated negative controls; SP2 = ceftiofur HCl once daily for 2 d; SP5 = ceftiofur HCl once daily for 5 d.
3
Proportion of quarters that experienced bacteriological cure at 14 ± 3 d.
Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS

4
CS of 0 = normal milk and no local or systemic signs; 1 = abnormal milk and no local or systemic signs; 2 = abnormal milk, abnormal udder with signs of inflammation, and one
clinical sign (rectal temperature ≥39.5°C, moderate to marked enophthalmos, or marked depression defined as inappetence or not able to stand, or both); 3 = milk grossly abnormal,
abnormal behavior, and at least 2 systemic signs.
5
Treatment × parity interaction is significant (P < 0.05).
6
Treatment × ECS interaction is significant (P < 0.05).
7
Clinical outcome success declared if clinical score improved from 1 to 0 or 2 to 0 within 14 ± 3 d.
8
Defined as a cow with bacteriological and clinical cures without additional antimicrobial intervention or support therapy, and no culling or death as consequence of the gram-
negative clinical mastitis, respectively.
2397
 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2398

Figure 2. Interactions between enrollment clinical scores (ECS,

score of 1 or 2) and treatment over the first 28 ± 3 d of the study on
post-treatment clinical scores.

Figure 1. Bacteriological cure rates for cows that received no sup-

plemental IMM or systemic antibiotic treatments. Bars with x and y are presented in Table 6. The proportion of quarters
superscripts are different (P < 0.05). ECS = clinical score at enroll-
ment.
with MR among CON was greater (P < 0.05) than
either SP2 or SP5; SP2 and SP5 were not different from
each other. Treatment differences were not observed for
quarters enrolled with Klebsiella spp. Twelve (46%) quarters that incurred a NIMI >14 d after enrollment
experienced CC (CON: ECS 1 = 3; ECS 2 = 1; SP2: (P = 0.7346).
ECS 1 = 4, ECS 2 = 2; SP5: ECS 1 = 1, ECS 2 = The proportion of quarters that required supplemen-
1), and among the 27 quarters enrolled with other GN tal IMM antibiotic intervention within 28 ± 3 d from
bacteria, 22 (81%) experienced BC (CON: ECS 1 = 6; the onset of experimental treatments was significantly
ECS 2 = 1; SP2: ECS 1 = 4, ECS 2 = 1; SP5: ECS 1 affected by treatment (P = 0.0068; Table 7) but not by
= 5, ECS 2 = 6). parity or ECS. Examining mean comparisons of main
effects of treatments across ECS levels (Table 6), the
MR and New IMI proportion of CON quarters that required supplemen-
tal IMM antibiotic treatment showed a tendency to be
Proportions of quarters with MR were calculated, higher (P = 0.0998) than SP2 quarters but was sig-
and effects of both treatment and ECS were significant nificantly higher (P = 0.0025) than SP5 quarters. The
(P < 0.0224; Table 7), but there was no treatment by difference between SP2 and SP5 quarters approached
ECS interaction; as such, the main effects of treatment significance (P = 0.0557).

Table 5. Comparisons of clinical score (CS) odds ratios between treatments and enrollment clinical score (ECS)1,2

CON vs. SP2 CON vs. SP5 SP2 vs. SP5 ECS1 vs. ECS2
3
Item Odds ratio P-value Odds ratio P-value Odds ratio P-value Odds ratio P-value
CS2 2.32 0.0161 2.46 0.0088 1.06 0.8542 0.003 <0.0001
CS5 3.55 <0.0001 3.16 <0.0001 0.89 0.6934 0.058 <0.0001
CS14 3.05 0.0916 2.44 0.0478 0.80 0.6488 0.447 0.0335
CS21 1.83 0.2067 2.11 0.1097 1.15 0.7757 0.690 0.3261
CS28 0.70 0.5250 1.30 0.6581 1.85 0.2266 1.325 0.5262
1
Categorical data with a multinomial distribution were analyzed by PROC GLIMMIX to evaluate fixed effects of treatment, parity, treatment
by parity interaction, ECS, ECS by parity interaction, mastitis DIM, and random effects of farm, breed, and season of year.
2
ECS = clinical mastitis severity score at the time of enrollment; 1 = mild, 2 = moderate.
3
CS2, CS5, CS14, CS21, CS28 are clinical scores at 2, 5, 14, 21, and 28 d after the start of treatment.

Journal of Dairy Science Vol. 107 No. 4, 2024

 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2399
Table 6. Results by treatment group across parity and enrollment clinical score (ECS) groups (LSM ± SE)1

Treatment group2 P-value

Item CON SP2 SP5 Treatment Parity ECS

Quarters enrolled, n 135 133 147
Bacteriological cure,3 % 66.8 ± 9.1a 83.3 ± 5.8b 88.2 ± 4.4b 0.0008 0.0333 0.0937
Days to clinical cure 5.6 ± 0.6a 5.5 ± 0.6a 7.3 ± 0.5b <0.0001 0.1306 0.0127
Clinical outcome,4 % success 48.5 ± 13.5a 88.4 ± 6.0b 89.4 ± 5.4b <0.0001 0.0896 <0.0001
Overall treatment outcome,5 % success 40.5 ± 10.6a 78.2 ± 7.7b 78.6 ± 7.4b <0.0001 0.0215 <0.0001
Odds ratio of overall treatment outcome — 5.27 5.40 — — —
Mastitis recurrence ≤14 d from enrollment,6 % 26.0 ± 6.2a 4.8 ± 2.3b 8.1 ± 2.3b 0.0002 0.7069 0.0224
New IMI >14 d from enrollment,7 % 8.7 ± 3.7 11.7 ± 3.6 12.3 ± 3.6 0.7346 0.3155 0.3835
Supplemental intramammary therapy <28 ± 3 d, % 11.3 ± 8.2a 6.1 ± 4.9ab 1.8 ± 1.7b 0.0068 0.5747 0.1069
Mastitis culls or deads as a percent of culls and deads 81.2 ± 4.9a 48.6 ± 7.5b 52.5 ± 6.0b 0.0009 0.9295 0.0326
Culled or dead, %
<30 d after enrollment 21.4 ± 6.6a 8.5 ± 3.6b 9.3 ± 3.6b 0.0066 0.3497 <0.0001
<60 d after enrollment 30.6 ± 6.6a 16.1 ± 4.6b 16.9 ± 4.5b 0.0103 0.7220 <0.0001
<90 d after enrollment 35.9 ± 5.3a 19.1 ± 4.0b 22.8 ± 3.9b 0.0131 0.3151 <0.0001
Days on the study at cull, removal, or at 90 d 62.8 ± 3.8a 79.3 ± 3.8b 77.9 ± 3.7b <0.0001 0.3558 <0.0001
a,b
Means in the same row with different superscripts are different (P < 0.05).
1
Categorical data were analyzed by PROC GLIMMIX and continuous data were analyzed by PROC MIXED to evaluate fixed effects of treat-
ment, parity, treatment by parity interaction, ECS, ECS by parity interaction, mastitis DIM, and random effects of farm, breed, and season of
year.
2
CON = nontreated negative controls; SP2 = ceftiofur HCl once daily for 2 d; SP5 = ceftiofur HCl once daily for 5 d.
3
Proportion of quarters that experienced bacteriological cure at 14 ± 3 d.
4
Clinical outcome success declared if clinical score improved from 1 to 0, 2 to 1, or 2 to 0 within 14 d.
5
Defined as case that had a combination results: bacteriological and clinical cures without additional antimicrobial intervention or support
therapy, no culling or death as consequence of the gram-negative clinical mastitis.
6
Qualified as clinically cured based on clinical score, but then developed clinical mastitis again before 14 d after the start of treatment in the
same quarter.
7
Defined as new infections identified at d 21 and d 28 after the quarter was considered a bacteriological cure at d 14.

Culling and Death reasons related to GN CM was higher than proportions

of SP2 or SP5 cows that left by 30 d, 60 d and 90 d
Proportions of cows that left herds due to death or postenrollment (Table 6). The average d cows remained
culling were an important measure of treatment effec- in the study was evaluated after right-censoring d on
tiveness in this study. No trial animals were euthana- study to a maximum fixed value of 90 d (any cow that
tized during the study. Forty-nine cows were culled or remained in the study through 90 d was assigned a
died due to GN CM before 14 ± 3 d (CON: ECS 1 = 4, value of 90 d). Main effects of both treatment and ECS
ECS 2 = 24; SP2: ECS 1 = 0, ECS 2 = 10; SP5: ECS were significant (P < 0.0001), and the interaction of
1 = 0, ECS 2 = 11). Animals that were culled or died treatment and ECS was also significant (P < 0.05).
due to GN CM, expressed as a proportion of all culled For quarters with an ECS of 1, treatment means for
or dead cows in each treatment, are shown in Table 7 CON (80.0 d), SP2 (85.5 d), and SP5 (80.7 d) were
(by treatment and ECS) and Table 6 (by treatment). not different. However, among quarters with ECS of 2,
For this metric, the overall effect of treatment was sig- the number of d cows remained in the study was 45.5
nificant (P = 0.0009), as was ECS (P = 0.0326), but for CON, which was less (P < 0.0001) than either SP2
there was no treatment by ECS interaction. Main ef- (73.0) or SP5 (75.1). Figure 3 is a graphic representa-
fects of treatment showed that the proportion of dead tion of the cumulative proportion of cows that did not
and cull cows that left the study for reasons related to depart the herd through culling or death due to mas-
mastitis among CON cows was higher (P < 0.0007) titis for each treatment over the 90 d after enrollment
than for either SP2 or SP5. for ECS 1 and 2.
Proportions of cows in each treatment that left the
herds due to GN CM-related death or culling through MC and Milk Production
30, 60, and 90 d postenrollment were also summarized
as a percent of cows enrolled in each treatment group; Results of milk samples harvested at 14, 21, and 28 ±
in each case, the effect of treatment was significant 3 d following enrollment showed no effects of treatment,
(P < 0.0131), as was the effect of ECS (P < 0.0001). parity, ECS, or interactions on MC treatment means,
The proportion of CON cows that left the herd for except for percent protein on d 21 which is likely a
Journal of Dairy Science Vol. 107 No. 4, 2024
 Table 7. Outcomes as influenced by treatment and enrollment clinical score (ECS, score of 1 or 2; LSM ± SE)1

Treatment group2 P-value

CON SP2 SP5

Item ECS 1 ECS 2 ECS 1 ECS 2 ECS 1 ECS 2 Treatment Parity ECS
Quarters enrolled, n (% of treatment total) 81 (60.0) 54 (40.0) 64 (48.1) 69 (51.9) 74 (50.3) 73 (49.7) — — —
Mastitis recurrence ≤ 14 d from enrollment,3 % 16.4 ± 4.9a 38.4 ± 11.4x 3.1 ± 2.3b 7.4 ± 3.8y 5.7 ± 2.9b 11.6 ± 4.5y 0.0002 0.7069 0.0224
New IMI >14 d from enrollment,4 % 13.7 ± 4.7 5.4 ± 4.0 10.0 ± 4.1 13.6 ± 5.0 14.0 ± 4.8 10.7 ± 4.3 0.7346 0.3155 0.3835
Supplemental intramammary therapy < 28 ± 3 d, % 9.9 ± 7.6a 12.9 ± 9.7x 3.8 ± 3.4a 9.8 ± 7.6x 1.3 ± 1.5b 2.4 ± 2.4y 0.0068 0.5747 0.1069
Mastitis culls or deads, % 69.5 ± 7.7a 89.1 ± 5.1x 35.3 ± 10.5b 62.0 ± 8.5y 55.9 ± 8.1ab 49.0 ± 8.5y 0.0009 0.9295 0.0326
Culled or dead, % of enrolled
<30 d after enrollment 8.4 ± 3.8 44.6 ± 10.3x 3.9 ± 2.6 17.3 ± 6.3y 6.5 ± 3.3 13.1 ± 5.2y 0.0066 0.3497 <0.0001
<60 d after enrollment 15.0 ± 4.9 52.3 ± 9.1x 11.5 ± 4.6 22.1 ± 6.4y 13.5 ± 4.8 20.8 ± 6.1y 0.0103 0.7220 <0.0001
<90 d after enrollment 16.8 ± 4.4 60.9 ± 7.2x 12.5 ± 4.3 28.1 ± 6.0y 20.9 ± 5.0 24.9 ± 5.4y 0.0131 0.3151 <0.0001

Journal of Dairy Science Vol. 107 No. 4, 2024

Mean days on the study at cull, removal, or 90 d 80.0 ± 4.2 45.5 ± 4.8x 85.5 ± 4.5 73.0 ± 4.4y 80.7 ± 4.4 75.1 ± 4.4y <0.0001 0.3558 <0.00015
a,b
Within ECS = 1, means in the same row with different superscripts are different (P < 0.05).
x,y
Within ECS = 2, means in the same row with different superscripts are different (P < 0.05).
1
Categorical data were analyzed by PROC GLIMMIX and continuous data were analyzed by PROC MIXED to evaluate fixed effects of treatment, parity, treatment by parity
interaction, ECS, ECS by parity interaction, mastitis DIM, and random effects of farm, breed, and season of the year.
2
CON = nontreated negative controls; SP2 = ceftiofur HCl once daily for 2 d; SP5 = ceftiofur HCl once daily for 5 d.
3
Qualified as clinically cured based on clinical score, but then developed clinical mastitis again before 14 d after the start of treatment in the same quarter.
4
Defined as new infections identified at d 21 and d 28 after the quarter was considered BC at d 14.
5
Treatment by ECS interaction is significant (P < 0.05).

was possible.
Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS

Overall Treatment Outcomes

ment; SP5 = ceftiofur HCl 5-d treatment.

with an ECS of 2. Table 6 summarizes overall treat-

with ECS of both 1 and 2, the largest responses were
The definitive measure of treatment success reflects

rized in Table 4 for treatment by ECS combinations.

overall treatment success rates, and data are summa-
computed to demonstrate the effect of data related to
to that of the SP2 and SP5 groups. Odds ratios were
noted among cows with ECS of 2. The overall treatment
supplemental IMM in the same quarter, systemic an-
milk yield or milk quality; therefore, no further analysis
spurious statistical observation and not a biological ef-

even more pronounced for quarters that were enrolled

success rate for the CON group was inferior (P < 0.05)
(P < 0.0215), and there were no interactions. As before,
ment outcome data are presented in Table 4. Main ef-
tibiotic treatments, and could not be the subject of
outcomes. To be considered an overall treatment suc-
collected to evaluate pre- and postinfection MC and
fect of treatment with ceftiofur HCl. The DHIA data

ment success across ECS scores. The overall treatment

experience overall treatment success than CON quar-
fects of treatment, parity, and ECS were all significant
cess, in addition to BC and CC, cows could not receive
opportunity to reliably assess the effect of GN CM on
milk production was inconsistent and offered a limited
2400

ters in mild (ECS 1) cases. The effect of treatment was

mastitis through 90 d post-treatment. SP2 = ceftiofur HCl 2-d treat-
Figure 3. Survival curve of culling and death events related to

Quarters treated with SP2 and SP5 were more likely to

while treatment responses were significant for animals
responses in terms of both bacteriological and clinical

either culling or death due to mastitis. Overall treat-

 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2401

success rate for CON was inferior to both SP2 and SP5 is important because our results showed differences
(P < 0.05); and SP2 and SP5 were not different from in outcomes according to case severity at the time of
each other. Computation of odds ratios revealed that enrollment.
SP2 and SP5 cases were more likely to experience an Although our study was not designed to evaluate
overall treatment success than CON cases. efficacy of GN core vaccine and its association with
mastitis severity, it is important to highlight that im-
DISCUSSION munization is a tool to control mastitis in dairy cows
and minimize disease severity and duration (Wilson et
Mastitis caused by GN bacteria has been a subject al., 2007). However, vaccination does not appear to be
of discussion leading to multiple clinical trials to inves- a factor affecting severity at enrollment in this study
tigate the need to treat mild and moderate cases with as herds A and B had similar proportions of severe
antimicrobials. The present study was designed to de- and nonsevere cases, even though herd A does not vac-
termine if there was a benefit to treating mild to moder- cinate, and herd B does. Herd C, which uses GN core
ate GN CM with ceftiofur HCl and included treatment vaccine, had a lower proportion of severe cases. More-
outcomes from 415 CM cases from 3 commercial dairies over, because of the possible confounding of the use of
in California. Although pre-study power calculations the vaccine and its effect on the outcomes evaluated,
were based on data from previously published studies, herd factor was accounted for in the statistical analysis,
the sample size per treatment was greater than in prior and results showed no difference in outcomes among
negative-controlled randomized clinical trials intended herds.
to assess IMM treatment effect on nonsevere GN mas- Several researchers have proposed that BC should be
titis (Schukken et al., 2011; Fuenzalida and Ruegg, the goal of antimicrobial therapy, but most mild and
2019). It was suggested that 40% of CM cases in dairy moderate GN cases do not require antimicrobial treat-
herds are caused by GN bacteria, and case severity is ment as they can be quickly cleared by the cow's im-
equally distributed (Oliveira et al., 2013). However, the mune system (Leininger et al., 2003; Lago et al., 2011a;
proportion of GN cases in herds enrolled in this study Fuenzalida and Ruegg, 2019). In fact, Leininger et al.
was lower, varying between 8.4% and 12.1%. Although (2003) showed that 85% of CM infected with E. coli
OFC systems are reliable and widely used to identify experienced spontaneous BC within 7 d of CM onset.
mastitis pathogens, they are not 100% accurate and In our study, the proportion of quarters experiencing
can result in false negatives (Ferreira et al., 2018). This spontaneous cure by 14 d after mild or moderate CM
study relied on OFC for pre-screening cases for study onset was lower (70.1%) than BC on quarters treated
enrollment, and we acknowledge that there could have for either 2 or 5 d with ceftiofur HCl (88.3 and 84.0%,
been some samples that were false negatives for GN respectively). However, when we separate results by se-
infections. Escherichia coli was the dominant species verity, mild cases had a higher rate of spontaneous cure
cultured, followed by Klebsiella spp. This is not un- (77.5%) compared with moderate cases (68.3%). These
expected, as cows on all 3 dairies were housed in free results intuitively make sense insofar as mild cases of
stalls bedded with recycled manure solids, which is a GN mastitis appear to largely cure spontaneously, but
known source of E. coli and Klebsiella spp. (Rowbo- in moderate cases, the application of IMM antibiotic in
tham and Ruegg, 2016). Previous studies evaluating the form of ceftiofur HCl significantly improved health
the efficacy of ceftiofur HCl therapy on GN infections outcomes.
found a proportion of Klebsiella spp. infections (Schuk- Researchers discourage antimicrobial therapy for GN
ken et al., 2011; Fuenzalida and Ruegg, 2019), and this infections due to the lack of improvement in BC rates,
could be the result of differences in the prevalence of especially E. coli infections (Pyörälä et al., 1994; Suoja-
mastitis pathogens in different geographies. la et al., 2010). However, these studies did not consider
Previous studies that evaluated outcomes of GN case severity when making recommendations. Results
mastitis therapy have unequal severity distributions of the present study showed that across herds, severity
on the enrolled cases, which could have contributed to scores (ECS1-mild or ECS2–moderate), and bacterial
no better or more favorable outcomes when comparing species, BC was higher for treatment groups compared
treated and untreated quarters. For instance, the dis- with the CON group. These results agree with previous
tribution of mild and moderate cases was different in a studies showing higher BC rates for quarters treated for
study comparing outcomes of ceftiofur HCl treatment, 2, 5, and 8 d with ceftiofur HCl (Schukken et al., 2011;
which may have affected the outcomes (Fuenzalida Fuenzalida and Ruegg, 2019). However, when evaluat-
and Ruegg, 2019). In contrast, our study had mild and ing mild and moderate cases separately, we found that
moderate cases equally distributed among groups. This the proportions of quarters with BC in mild cases in

Journal of Dairy Science Vol. 107 No. 4, 2024

 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2402

both treatment groups were similar to the CON group. ficacy of ceftiofur HCl. Identification of CM in the work
For moderate cases, however, results showed only half by Fuenzalida and Ruegg (2019) and in the present
of the CON quarters experienced BC, while over 70% study was done by farm personnel and documented in
and 80% of cows treated with either 2-d or 5-d courses herd record-keeping software. Therefore, differences
of ceftiofur HCl, respectively, experienced BC. Al- in results between the present study and the above-
though other studies (Todhunter et al., 1991; Gröhn et mentioned study could be due to failure of accurate CM
al., 2004; Fuenzalida and Ruegg, 2019) suggested more identification by milkers or failure to accurately enter
negative outcomes in quarters infected with Klebsiella information in herd records.
spp., it was not possible to evaluate outcomes by etiol- Monitoring milk SCC is considered a valuable indica-
ogy in the present study because most of the cases were tor of mastitis. We evaluated quarter SCC on d 14, 21,
due to E. coli infection. Even though we were not able and 28 following enrollment but found no significant
to statistically evaluate outcomes, our data showed difference in quarter SCC between groups; these results
that among the 26 quarters enrolled with Klebsiella agree with previous research (Schukken et al., 2011;
spp.,3 experienced BC, and 12 experienced CC. Hence, Fuenzalida and Ruegg, 2019). In addition, there were
no conclusions can be drawn specific to Klebsiella spp. no significant treatment differences in MC between
infections, and more studies are needed. groups and no interaction with ECS, parity, or other
Clinical outcomes are widely used by farmers and effects on outcomes. However, Williamson et al. (2022)
researchers as a visual indication of the success of IMM studied associations between pretreatment SCC values
therapy (Swinkels et al., 2014) but should not be an and BC rates following antibiotic therapy for mastitis
objective determinant of treatment efficacy because it and showed that quarters with lower SCC before an-
may take time for milk to resume normality and for tibiotic treatment are more likely to successfully cure
inflammation to resolve (Ruegg, 2021). In our study, than quarters with high SCC before treatment.
we found that the majority of mild cases across all 3 Research evaluating pathogen-specific production
groups returned to normal CS (CS = 0) by d 2, while losses following a CM case found that infections with
for moderate cases, it took an average of 5 d for treated E. coli diagnosed before peak lactation caused losses of
quarters and 14 d for nontreated quarters. It is impor- 10.6% of the 305-d milk yield (3.5 kg/d; Heikkilä et al.,
tant to mention that CS is a subjective measure and 2018). Whereas our data did not offer an opportunity
could be interpreted differently by each person, even to assess the effect of GN CM on milk yield before,
when following a defined CS system. during, or after a GN CM diagnosis, previous stud-
Previous studies also investigated DCC and reported ies have shown a milk production decrease associated
no differences in DCC in treated and nontreated quar- with a case of GN mastitis, but did not show persistent
ters (Fuenzalida and Ruegg, 2019; Schukken et al., significant differences in milk production decreases or
2011) or were higher in treated quarters (Morin et al., recovery times between treated and control groups
1998). The average DCC for the present study was sim- (Schukken et al., 2011; Fuenzalida and Ruegg, 2019).
ilar to values reported in previous studies (Fuenzalida While CM is associated with an increased risk of
and Ruegg, 2019; Schukken et al., 2011) but higher culling (Pinzón-Sánchez et al., 2011), IMM antibiotic
than the results reported by Lago et al. (2011a). No treatment positively affects cow survival and decreases
explanation for the higher average DCC for SP5 was mastitis-related milk losses (Roberson et al., 2004;
identified, but we speculate that it could be attributed Schukken et al., 2009). When comparing treated and
to a longer duration of treatment and, therefore, more untreated quarters, Schukken et al. (2011) found that
prolonged stimulation of the local epithelia by syringes cows treated for 5 d with ceftiofur HCl left the study
used to administer the IMM antibiotic. Some earlier in through culling, death, or farmer interference less fre-
vitro studies showed that antimicrobials might disturb quently (18%) compared with control animals (27%).
phagocytosis when given IMM (Nickerson et al., 1986), In the present study, cows in the CON group repre-
leading to the appearance of clinical signs. However, sented the majority of cows that died or were culled by
the clinical relevance of this finding is unknown. 30 d from enrollment. Although we evaluated up to 90
The proportion of quarters with MR differed between d postenrollment, the proportion of cows culled in this
the 3 treatment groups and was more frequent in the study was higher than those noted in previous studies
CON group. Our results do not agree with those of (Schukken et al., 2011; Fuenzalida and Ruegg, 2019).
Fuenzalida and Ruegg (2019), who found no signifi- We hypothesize that dairies were more aggressive in
cant differences between treated and untreated groups. eliminating problem cows that would not cure or re-
Because of the higher proportion of MR in the CON cover productivity. In addition, others showed that CM
group, we attributed treatment differences to the ef- caused by Klebsiella spp. leads to more significant milk

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 Bruno et al.: TREATMENT OF GRAM-NEGATIVE MASTITIS 2403

losses (Gröhn et al., 2004) and a higher risk of cull- mastitis-related death and culling, all leading to better
ing (Gröhn et al., 2004; Fuenzalida and Ruegg, 2019) overall treatment success compared with nontreated
compared with E. coli infections. However, the limited treated cases. Based on these results, there is a benefit
number of Klebsiella spp. cases in our study did not al- to investing in the development of treatment protocols
low comparisons regarding outcomes. Moreover, in this to treat nonsevere cases of GN mastitis, and the ben-
study early treatment of nonsevere GN CM infections efits outweigh the risks of not treating.
with IMM antibiotics proved significantly beneficial
compared with not treating cases and simply assuming ACKNOWLEDGMENTS
that cows would self-cure without consequence.
Clinical mastitis therapy should aim to promote ani- The authors acknowledge the support of the 3 Cali-
mal welfare. Denying or delaying therapy due to etiol- fornia dairies that participated in this study and Hilary
ogy could lead to worsening health, death, or animal Spivey, Annika Paris, Kaitlyn McFarland, Katlyn Mc-
discard. There are limited data evaluating GN CM Clellan, Jordyn Perrin, Kyleigh Shanahan, and Caitlyn
bacteria on vitro susceptibility to ceftiofur HCl (Sheedy Fagundes (California State University, Fresno, CA) for
et al., 2021), but AMR monitoring should continue. their support in data collection. This study was funded
Results of this study show the benefits of treating GN by the University of California Cooperative Extension
CM, with better outcomes for both treated groups com- and Zoetis Inc. (Parsippany, NJ). Authors RMC, TS,
pared with the nontreated control group. In addition, and JRP were Zoetis Inc. employees during the study
this paper highlights significant interactions between period. The Zoetis-affiliated authors had no role in data
treatment outcomes and severity of CM at the time of collection, sample analysis, or data entry, or influence
enrollment, with effectiveness of the treatment being on the methods for documenting animal observations,
more pronounced as clinical severity of GN mastitis animal management decisions made at the farm level,
increased. Furthermore, our results suggest that moder- or finalization of results. The remaining authors (DRB,
ate cases may benefit more from treatment than mild GJ, and BM) declare that the research was conducted
cases. Successful evaluation of treatment outcomes by in the absence of any commercial or financial relation-
severity scores (mild x moderate) was possible because ships that could be construed as a potential conflict of
research personnel were thoroughly involved to ensure interest. The authors have not stated any other con-
protocol compliance. However, in a commercial dairy, flicts of interest.
it is unlikely that farm personnel will differentiate
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