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Published in final edited form as:

J Dairy Sci. 2019 March ; 102(3): 2686–2697. doi:10.3168/jds.2018-15595.

A model of clinical endometritis in Holstein heifers using

pathogenic Escherichia coli and Trueperella pyogenes
Rachel L. Piersanti1, Roney Zimpel1, Paula C. C. Molinari1, Mackenzie J. Dickson1,
Zhengxin Ma1, KwangCheol C. Jeong1, José E. P. Santos1, I. Martin Sheldon2, and John J.
Bromfield1,*
1Department of Animal Sciences, University of Florida, Gainesville 32611
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2Institute of Life Science, Swansea University Medical School, Swansea, SA2 8PP, United
Kingdom

Abstract
Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy
cows and reduces fertility, even after the resolution of disease. However, it is difficult to
disentangle the mechanisms linking reduced fertility with endometritis because cows have
multiple confounding postpartum conditions. The aim of the present experiment was to develop an
in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and
Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol,
and subsequently received exogenous progesterone to elevate circulating progesterone at the time
of uterine infusion. Endometrial scarification was performed before uterine infusion of live
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pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were
evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the
vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial
infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d
4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6.
Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers
following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal
mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal
temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and
appetite did not differ between groups. As indicated by increased vaginal mucus grade after
bacterial infusion and absence of systemic signs of illness, this model successfully induced
symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the
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isolation of effects of uterine disease on fertility from confounding factors that can occur during
the postpartum period in dairy cows.

Keywords
animal model; dairy cow; clinical endometritis; inflammation; uterine infection

*
Corresponding author: jbromfield@ufl.edu.
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INTRODUCTION
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Clinical endometritis is characterized by inflammation resulting from bacterial infection of

the endometrium that causes a purulent uterine discharge that is present 21 d or more
postpartum. Endometritis has a lactation incidence of 15 to 20% and is important because,
even after resolution of the clinical signs, cows have delayed conception and are more likely
to be culled for reproductive failure than cows without endometritis (LeBlanc et al., 2002;
Gernand et al., 2012; Ribeiro et al., 2013). However, understanding how uterine disease
affects fertility is complicated because endometritis is associated with other peripartum
problems as well as metabolic challenges associated with the onset of lactation, which also
impair fertility (LeBlanc, 2012; Ribeiro et al., 2013). Developing an experimental model of
clinical endometritis in heifers would help disentangle the mechanisms of how uterine
disease affects reproductive physiology.
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Cows with clinical endometritis do not have systemic signs of disease, and diagnosis is
based on the presence of a purulent uterine discharge detectable in the vagina 21 d or more
postpartum (LeBlanc et al., 2002; Sheldon et al., 2006). The vaginal contents can be
examined using a gloved hand, a Metricheck tool (Simcro, Hamilton, New Zealand), or a
speculum, and the severity of clinical endometritis can be graded by the amount of pus in the
mucus. There is debate about whether purulent vaginal discharge may also reflect cervicitis
or vaginitis because not all cows with purulent vaginal discharge have infiltration of
polymorphonuclear neutrophils detectable by endometrial cytology (Dubuc et al., 2010).
Nevertheless, bacteria can be isolated from the uterus of cows with endometritis, and the
presence of pus in the uterus or a slightly enlarged uterus can also be visualized using
transrectal ultrasonography (Sheldon et al., 2002).
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Clinical endometritis is associated with gram-negative endometrial pathogenic Escherichia

coli, gram-positive Trueperella pyogenes, and several anaerobes (Sheldon et al., 2002;
Bicalho et al., 2012). Both E. coli and T. pyogenes can be detected in the uterus of cows that
develop uterine disease as early as 1 to 3 DIM based on culture and PCR-based technologies
(Dohmen et al., 2000; Bicalho et al., 2012). Lipopolysaccharide from gram-negative bacteria
and lipopeptides from gram-positive bacteria activate the innate immune system in the
endometrium, leading to inflammation and increased concentrations of circulating acute
phase proteins (Sheldon et al., 2001; LeBlanc, 2012; Turner et al., 2014). Pus forms in the
uterus when neutrophils phagocytize bacteria or endometrial cells damaged by cytolysins,
such as the pore-forming toxin pyolysin secreted by T. pyogenes (Sheldon et al., 2010;
Bicalho et al., 2012; Amos et al., 2014). A notable feature of the pathogenesis of
endometritis is that the adhesion of E. coli to endometrial cells and the cytolysis caused by
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pyolysin is greatest in the stroma, which is exposed when the epithelium is lost during the
peripartum period (Sheldon et al., 2010; Amos et al., 2014). Furthermore, peripartum
problems that traumatize the endometrium, such as dystocia and retained fetal membranes,
increase the risk of development of endometritis (Dubuc et al., 2010; Potter et al., 2010;
Ribeiro et al., 2013).

Uterine disease is associated with prolonged luteal phases, slowed rate of growth of ovarian
follicles post-partum, and impaired ovarian endocrine function (Opsomer et al., 2000;

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Sheldon et al., 2002). However, the mechanisms linking endometritis with reproduction are
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not fully established because fertility is also perturbed by many of the risk factors for
endometritis, including peripartum problems and the inability to adapt to the shifts in
metabolism to accommodate lactation (Chagas et al., 2007; LeBlanc, 2012). Animal models
of clinical endometritis have been reported using mature cows infused with T. pyogenes
(Rowson et al., 1953; Ayliffe and Noakes, 1982; Farin et al., 1989; Amos et al., 2014).
However, an experimental model of endometritis in heifers would be attractive for exploring
how uterine infection affects reproduction. Using heifers as the basis for the model
circumvents many of the confounding effects of parturition and lactation observed in
lactating cows. The aim of the present experiment was to develop a defined in vivo infection
model of clinical endometritis in Holstein heifers using pathogenic E. coli and T. pyogenes.

MATERIALS AND METHODS

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The University of Florida Institutional Animal Care and Use Committee approved all
procedures with heifers under protocol number 201508884. The experiment was conducted
from June to October 2017 at the University of Florida Dairy Unit.

Establishment of Uterine Infection in Heifers

Nine virgin Holstein heifers aged between 11 and 13 mo were enrolled in the experiment.
All animals were free of general health conditions and tested negative for Brucella abortus,
Neospora caninum, and Leptospirosa. Heifers were vaccinated against bovine viral diarrhea,
infectious bovine rhinotracheitis, parainfluenza, bovine respiratory syncytial virus, and
multiple serovars of Leptospira (Bovi-Shield Gold FP 5 VL5 HB, Zoetis, Parsippany, NJ)
and dewormed using moxidectin (Cydectin, Bayer HealthCare LLC, Animal Health
Division, Shawnee Mission, KS). Heifers were fed a diet as TMR that was offered once a
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day in addition to pasture access and provided ad libitum access to water.

The experiment followed a randomized complete block design with heifer as the
experimental unit. Heifers were blocked by age and weight, and randomly assigned to 1 of 2
infusion treatments: intrauterine infusion of sterile vehicle medium alone (n = 5) or
intrauterine infusion of live bacteria (n = 4; details below).

Estrous cycles were synchronized using a modified 5-d Co-synch protocol (Lima et al.,
2013). Briefly, heifers received 100 mg i.m. of GnRH (gonadorelin diacetate tetrahydrate;
Ovacyst, Bayer) followed by 25 mg i.m. of PGF2α (dinoprost tromethamine; Prostamate,
Bayer) administered 5 and 6 d later (Figure 1A). Eight days following initial GnRH, heifers
received a final dose of 100 mg of GnRH i.m. Starting on the day following the final GnRH,
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heifers received 200 mg i.m. of progesterone (P4) in corn oil (50 mg/mL; Sigma-Aldrich, St
Louis MO) daily for 7 d. Exogenous P4 was administered to mimic diestrus and ensure
elevated circulating P4 at the time of bacterial infusion.

Uterine infusion of treatments were performed 72 h following final GnRH administration

and designated as d 0 relative to treatment (Figure 1A). Endometrial scarification was
implemented before intrauterine infusion to disrupt the endometrial epithelium. Heifers were
restrained and received a caudal epidural injection of 60 mg of lidocaine hydrochloride 2%

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(Aspen Veterinary Resources, Greeley, CO). The perineum and vulva were cleaned and
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disinfected with povidone iodine followed by 70% ethanol, and a sterile metal scarification
tool enclosed by a metal catheter covered in a sanitary sheath (IMV Technologies,
Normandy, France) was introduced through the vagina and cervix. The scarification tool
consisted of a stainless-steel rod with a 39-mm-long, 6-mm-diameter threaded component
on the tip, similar to a threaded bolt (Figure 1B). The scarification tool was manipulated
through the cervix and into the body of the uterus by transrectal palpation. Once inside the
body of the uterus, the sanitary sheath was retracted, and the scarification tool was placed in
direct contact with the endometrium. The scarification tool was then rotated once to disrupt
the endometrial lining before removal from the reproductive tract. Inspection of the
scarification tool upon retraction confirmed tissue disruption by the presence of small pieces
of tissue. Following endometrial scarification, a metal infusion catheter enclosed in a
sanitary sheath was introduced transcervically into the body of the uterus. The sanitary
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sheath was retracted, and treatments were infused using 10-mL syringes. Bacterial infusion
consisted of 10 mL of 4.64 × 107 cfu/mL of E. coli MS499, 10 mL of 3.38 × 107 cfu/mL T.
pyogenes MS249 followed by 10 mL of sterile Luria-Bertani (LB) broth to flush the catheter
(Goldstone et al., 2014a,b). Vehicle infusion consisted of 30 mL of LB broth. Heifers were
returned to pasture and monitored for clinical signs for 24 h.

Animals did not receive any additional treatments or medication during the experimental
period.

Bacterial Culture and Preparation of Inoculants

Escherichia coli MS499 and T. pyogenes MS249 were collected and isolated from cows with
metritis and characterized previously (Goldstone et al., 2014a,b).
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Escherichia coli was cultured from frozen glycerol stocks on LB agar. The day before
infusion, a single colony was picked from the plate and inoculated into LB broth containing
1% tryptone, 0.5% yeast extract, and 1% sodium chloride. The culture was incubated
overnight at 37°C with shaking at 200 rpm. Growth was monitored by measuring optical
density at 600 nm (OD600 = 5.0). A final preparation of 4.64 × 107 cfu/mL E. coli was
diluted in sterile LB broth and loaded into 10-mL syringes for infusion.

Trueperella pyogenes MS249 was grown from frozen glycerol stocks on Trypticase Soy
Blood agar at 37°C for 48 h. The day before infusion, a single colony was selected and
inoculated into Bacto Brain Heart Infusion broth (BHI, Fisher Scientific, Pittsburgh, PA)
supplemented with 5% fetal bovine serum (FBS, Fisher Scientific) and cultured overnight at
37°C with shaking at 200 rpm. Growth was monitored by measuring optical density at 600
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nm (OD600 = 0.2). A final preparation of 3.38 × 107 cfu/mL T. pyogenes was diluted in
sterile BHI and loaded into 10-mL syringes for infusion. Syringes were loaded with sterile
LB broth for flushing catheters and vehicle infusions. Inoculants were transported to the
farm on ice for infusion.

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Blood Sampling and Hematology

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Blood was collected from the coccygeal vessels into evacuated tubes (Vacutainer, Becton
Dickson, Franklin Lakes, NJ) containing sodium heparin for plasma separation or potassium
EDTA for hematology. Blood was sampled every other day from d −2 to 18 relative to
treatment. Blood was placed on ice until further processing. Tubes containing heparin were
centrifuged and plasma was collected, aliquoted, and stored at −20°C. Whole blood was
transported to the laboratory on ice within 2 h of collection and used for hematology
analysis performed using an automated hematology analyzer (ProCyte Dx Hematology
Analyzer, IDEXX Laboratories, Westbrook ME). Hematology analysis included total and
differential leukocyte counts (neutrophils, lymphocytes, and eosinophils), red blood cells,
hematocrit, and hemoglobin. Hematology was evaluated on d −1, 1, 3, and 7 relative to
treatment (Figure 1A).
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Plasma haptoglobin (Life Diagnostics Inc., West Chester, PA) and P4 (DRG International
Inc., Spring-field, NJ) were measured using commercially available ELISA according to the
manufacturer’s instructions. Plasma haptoglobin was evaluated on d 0, 7, and 13 relative to
treatment, and P4 was evaluated on d −2, 1, 4, 11, and 17 relative to treatment (Figure 1A).
The P4 ELISA is human specific and was validated for bovine plasma using spike-in/
recovery performance based on actual and expected recovery of P4 supplied as standard with
the kit. The intraassay coefficient of variation was calculated at 6.5%, and recovery of spike-
in P4 was 89 to 101.8% of expected P4.

Examination and Grading of Vaginal Mucus, Transrectal Ultrasonography, and Rectal

Temperature
Vaginal mucus was collected and examined using a clean Metricheck tool (Simcro). Vaginal
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mucus was scored as grade 0, no mucus or clear or translucent mucus; grade 1, mucus
containing flecks of white or off-white pus; grade 2, mucus containing ≤50% white or off-
white mucopurulent material; and grade 3, mucus containing >50% purulent material
(Sheldon et al., 2009). Evaluation of vaginal mucus was performed daily from d −1 to 7
relative to treatment (Figures 2A–2C).

Transrectal ultrasonography with a linear 5.0 MHz probe (Aloka SSD-500, Hitachi
Healthcare Americas, Twinsburg, OH) was performed to visualize fluid and pus in the
uterus. Ultrasonography was performed every other day from d −1 to 10 relative to
treatment. Rectal temperature (AG-102 thermometer, AG-Medix, Mukwonago, WI) was
measured daily between 0700 and 0900 h from d −11 to 10 relative to treatment. A rectal
temperature of >39.5°C was classified as fever.
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Quantification of Total 16S rRNA Isolated From Vaginal Mucus

Vaginal mucus was stored in sterile bijou bottles at −20°C until processing. Total DNA was
isolated from vaginal mucus using the DNeasy Power Soil Kit according to the
manufacturer’s instructions with modification (Qiagen, Hilden, Germany). Briefly, samples
were thawed on ice and vortexed and 250 mg of vaginal mucus was used for DNA extraction
using a bead beater tissue homogenizer (Precellys 24, Bertin Technologies SAS, Montigny-
le-Bretonneux, France). The mucus was added to guanidine thiocyanate and homogenized

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with garnet particles using 3 bead beater cycles (30 s at 6,000 × g, 60 s pause, 30 s at 6,000
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× g) with a 5 min incubation on ice between each cycle. Following homogenization,

supernatants were applied to the DNeasy Power Soil spin columns for DNA purification.

Total 16S rRNA was quantified using the Femto Bacterial DNA Quantification Kit
according to the manufacturer’s instructions (Zymo Research, Irvine, CA). Thermocycling
conditions included initial denaturation at 95°C for 10 min, 40 cycles of amplification
consisting of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C
for 1 min, followed by a final extension at 72°C for 7 min. A total of 2 μL of extracted total
DNA sample was applied to each PCR reaction. Quantification of 16S rRNA was analyzed
based on a standard curve performed in parallel with mucus samples. Data for total 16S
rRNA are described as nanograms of 16S rRNA per milligram of vaginal mucus. The
extraction of 16S rRNA in mucus samples was validated using spike-in/recovery using
known quantities of purified bacteria. Intraassay coefficient of variation was calculated at
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0.3%, interassay coefficient of variation was calculated at 2.2%, and recovery of 16S rRNA
following extraction of bacteria spike-in pus was 100.5% of expected 16S rRNA content.

Statistical Analysis
All data were analyzed using SAS v. 9.4 (SAS Institute Inc., Cary, NC). Vaginal mucus
grade was reported as the median for each treatment group and analyzed using the
GLIMMIX procedure following a Poisson distribution. Cow within treatment was
considered a random effect, and fixed effects of treatment and day were analyzed.
Haptoglobin, P4, hematology, and DNA concentration for the 16S rRNA gene were
analyzed using the MIXED procedure of SAS and the models included the fixed effects of
treatment (bacterial infusion), day (repeated measure), and their interaction. Heifer nested
within treatment was considered as a random effect. First order autoregressive covariance
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structure AR (1) was used as the covariate structure. Values are reported as least squares
means ± standard error of the mean. Differences with P ≤ 0.05 were considered statistically
significant.

RESULTS
Intrauterine Bacterial Infusion Increased Vaginal Mucus Grade
Vaginal discharge of pus was visually evident in heifers that were infused with live bacteria,
but not in control heifers (Figure 2A). Vaginal mucus collected by Metricheck was graded
and compared between treatments (Figure 2B–2C). An increase (P < 0.02) was observed in
the vaginal mucus grade of heifers treated with bacteria (Figure 3). Heifers in both infusion
groups had a median mucus grade of 0 before uterine infusion (Figure 3). Intrauterine
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infusion of live bacteria resulted in a median mucus grade of 2 on d 3 relative to treatment,

whereas control heifers maintained a median mucus grade of 0 (Figure 3). On d 4 to 6
relative to treatment, bacteria-infused heifers had a median mucus grade of 3, whereas
control heifers presented a median mucus grade ≤1. The presence of echogenic fluid in the
uterus of bacteria-infused heifers was confirmed by transrectal ultrasound, and control
heifers had no evidence of increased fluid, or echogenic material present in the uterus
(Figure 2D–2E).

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Intrauterine Bacterial Infusion Increased 16S rRNA Concentration in Vaginal Mucus

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Total vaginal 16S rRNA was increased (P < 0.05) in bacteria-infused heifers compared with
control heifers on d 5 (Figure 4A). In addition, total vaginal 16S rRNA was greater (P <
0.05) on d 5 in heifers infused with bacteria compared with other sampled days (Figure 4A).
No interaction was observed between treatment and day relative to treatment (P = 0.14).

Effect of Uterine Bacterial Infusion on Hematology and Rectal Temperature

Treatment had no effect on any hematological parameters measured (Table 1). Day relative
to treatment had an effect (P < 0.05) on the number of red blood cells (M/μL), number of
lymphocytes (K/μL), number of platelets (K/μL), and percent of hematocrit and hemoglobin
(g/dL). A tendency was observed for an effect of treatment (P = 0.08) on rectal temperature
with bacteria-infused heifers showing elevated temperature on d 1 compared with control
heifers (Table 1). Two bacteria-infused heifers had rectal temperatures above 39.5°C on d 1
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relative to treatment (40.0 and 40.2°C). No overt signs of systemic disease were observed in
any heifers.

Effect of Bacterial Infusion on Circulating Concentrations of Haptoglobin and

Progesterone in Plasma
Plasma concentration of haptoglobin did not differ (P = 0.70) between treatments and there
was no effect of day (P = 0.36) or interaction between day and treatment (P = 0.41). A
numerical 3.5-fold increase in haptoglobin concentration on d 13 relative to treatment was
observed in bacteria-infused heifers compared with control heifers (Figure 4B; P > 0.05).

Plasma concentrations of P4 increased in both treatment groups as a result of exogenous P4

supplementation (effect of day, P < 0.001); however, infusion with bacteria did not affect
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concentrations of P4 in plasma (P > 0.05, Figure 4C). The peak concentration was observed
on d 4 relative to treatment, and once exogenous P4 supplementation was concluded on d 4,
concentrations decreased in both treatments.

DISCUSSION
The present experiment developed an in vivo model of clinical endometritis in Holstein
heifers using pathogenic E. coli and T. pyogenes. Heifers that received intrauterine infusion
of pathogenic bacteria developed purulent vaginal mucus, accumulation of echogenic fluid
in the uterus, and increased bacterial load in vaginal mucus. In parallel, bacteria-infused
heifers displayed no systemic signs of illness, such as altered hematology or general
sickness. Control heifers did not display any of the clinical signs of clinical endometritis.
Based on the criteria previously described for the disease, these results recapitulate the
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symptoms of clinical endometritis observed in the postpartum dairy cow (LeBlanc et al.,
2002; Sheldon et al., 2006, 2009).

Previous experimental models of clinical endometritis have focused on the use of mature
cows and intrauterine infusion of T. pyogenes (Rowson et al., 1953; Ayliffe and Noakes,
1982; Amos et al., 2014). These studies were capable of generating active infection of the
uterus, evident by the presence of mucopurulent vaginal contents, using a single uterine

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pathogen. Although these models induce clinical symptoms of disease, it may be that the use
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of a second pathogen, E. coli, better reflects the molecular profile of endometritis to evaluate
the consequences of disease on fertility. The dual pathogen model of Del Vecchio et al.
(1992) used repeated infusions of both E. coli and T. pyogenes over the course of 3 d (Del
Vecchio et al., 1992); however, the strain and pathogenicity of bacteria used may also be
important. Strains used by others include β-hemolytic E. coli or T. pyogenes sourced from
cows with severe endometritis (presumably from the uterus). Additionally, Farin et al.
(1989) used intrauterine infusion of T. pyogenes and the gram-negative anaerobes,
Fusobacterium necrophorum and Bacteroides melaninogenicus (now Prevotella
melaninogenica), to induce “pyometria” in lactating multiparous cows. The model describes
the implementation of tissue damage using an intrauterine infusion of iodine solution, and
intravenous administration of hCG to ensure elevated circulating P4, 2 factors recapitulated
in our model but using different approaches. The model of Farin et al. (1989) also reports the
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consistent isolation and culture of uterine T. pyogenes during an extended window of active
disease lasting up to 30 d postinfusion in some cows. The presence of E. coli was not
reported in these studies, and isolation and culture of the anaerobes, F. necrophorum and P.
melaninogenica, were inconsistent between cows with disease (Farin et al., 1989).
Pathogenic strains of E. coli and T. pyogenes used in the current model were sourced from
the uterus of cows with endometritis and have subsequently been sequenced (Goldstone et
al., 2014a,b), allowing a detailed understanding of virulence factors and host-pathogen
interactions involved in disease. These 2 species of pathogens were specifically chosen to
establish infection as they are readily detectable within days of parturition in the uterus of
cows that develop spontaneous uterine disease (Dohmen et al., 2000; Bicalho et al., 2012).
The pore-forming toxin produced by T. pyogenes causes cellular damage to the
endometrium during active infection, although little is known about its actions on ovarian
function, especially after the clearance of disease. Conversely, LPS derived from gram-
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negative bacteria is present in follicular fluid after the resolution of uterine disease. Indeed,
studies have demonstrated that the presence of LPS negatively effects oocyte development
and alters the follicular environment of the growing oocyte in vitro (Bromfield and Sheldon,
2011).

Species other than the cow have been used to develop models of uterine infection, including
the sheep and mouse (Regassa et al., 2002; Sheldon and Roberts, 2010; Bromfield and
Sheldon, 2013). However, when attempting to determine the mechanisms by which
endometritis affects fertility of the dairy cow, it is imperative to use the target species in
question. Rodents are relatively inexpensive and convenient for such a model, but the
reproductive physiology and immune function of the mouse is considerably different from
those of the cow. Mice have been shown to be 1 million-fold less sensitive to LPS than
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humans (Seok et al., 2013). More appropriately, the human and bovine genomes share a high
degree of AA sequence homology, limited species-specific orthologs, and a similar
chromosomal organization, suggesting the bovine as a useful experimental model for human
physiology (The Bovine Genome Sequencing and Analysis Consortium et al., 2009). In
addition, bovine reproductive biology is closer to that of humans than mice; cows and
humans are both monotocous with similar hormonal profiles over a 3- to 4-wk ovarian cycle,
compared with the polytocous mouse with a 4-d estrous cycle. The current model of

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endometritis may even serve as a tool to study the effects of uterine infections on human
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fertility.

The use of the Holstein heifers in the present experiment was a deliberate choice in the
generation of the experimental model presented. The long-term goal of this model is to study
the mechanisms of endometritis-mediated reproductive failure in the cow. As uterine disease
occurs in the early postpartum period, several significant challenges can confound
experimental investigation into the causes of endometritis-mediated infertility. Specifically,
postpartum uterine damage, uterine involution, metabolic demands of lactation, negative
nutrient balance, and additional postpartum illnesses that affect almost half of all postpartum
cows (Ribeiro et al., 2016). Retained placenta and dystocia are significant risk factors for the
development of endometritis, and are both associated with damage to the endometrium
(Dubuc et al., 2010). To recapitulate postpartum endometrial damage, we performed a
scarification procedure to disrupt the endometrial epithelial layer at the time of bacterial
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infusion. This process of scarification may be critical to the establishment of the disease
model to facilitate bacterial access to the underlying stroma. Previous work has reported
differential susceptibility of epithelial and stromal endometrial cells to pathogenic E. coli,
with pathogenic E. coli binding with stronger affinity to stromal cells, and purified LPS
inducing a stronger inflammatory reaction in the endometrial stroma (Sheldon et al., 2010).
A similar phenomenon of differential cellular susceptibility has been described in the
response of endometrial epithelial and stromal cells to T. pyogenes pyolysin, in which
stroma cells are considerably more sensitive to the cytoxic effects of pyolysin (Amos et al.,
2014), suggesting the stroma is the target of pyolysin. These in vitro experiments, in
combination with the risk factors associated with endometritis, suggest that endometrial
scarification may be an important procedure in the establishment of clinical endometritis
observed in the present model.
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The administration of exogenous P4 was a calculated approach to modulate the immune

function of heifers at the time of bacterial infusion. Rowson et al. (1953) reported that
intrauterine infusion of T. pyogenes (reported as Corynebacterium pyogenes) during the
luteal phase of the estrous cycle resulted in “pyometritis”; however, no infection could be
established when bacterial infusion was performed at estrus. These experiments were
repeated using ovariectomized cows in conjunction with administration of exogenous P4 or
estradiol. Only with the administration of exogenous P4 could pyometritis be achieved
(Rowson et al., 1953). Since the 1950s, the immune modulating properties of P4 and
estrogen have been reported in immune cells and the endometrium (reviewed in detail by
Wira et al., 2015), with various studies demonstrating the immune-suppressive function of
P4 within the endometrium of ruminants (Hawk et al., 1964; Del Vecchio et al., 1992;
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Hansen, 1998; Seals et al., 2002; Lewis, 2003). The ability of steroid hormones to modulate
endometrial immune function have led to the practice of administering PGF2α as a treatment
for uterine infection to induce luteolysis in cows in diestrus, and elevate estrogen by
stimulating a new follicular phase of the estrous cycle (Lewis, 1997); however, this remains
debated. In parallel, 55% of cows with metritis display extended luteal phases (Etherington
et al., 1991; Opsomer et al., 2000). The extension of the luteal phase in cows with uterine
disease can have consequences on the calving to conception interval, negatively affecting
productivity. Exposure of endometrial cells to bacterial LPS in vitro switches prostaglandin

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synthesis from luteolytic PGF2α to luteotrophic PGE2 (Herath et al., 2006, 2009). This
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apparent LPS-mediated switch in endometrial secretion of luteotrophic PGE2 may explain

the extended luteal phase in some cows with uterine disease. In the current study we
evaluated circulating P4 until d 18 postinfusion, and did not observe any effect of treatment
at any time point. This result may be an influence of exogenous P4 administration for 7
consecutive days, or an artifact of the extensive manual manipulations performed on these
heifers during the course of the study. Future studies using this model of induced infection
should evaluate the duration of the luteal phase, monitoring follicular growth, ovulation, CL
size, peak P4, and luteal phase duration.

Vaginal mucopurulent content observed in heifers treated with intrauterine infusion

containing live bacteria could be associated with cervicitis or vaginitis as previously
observed in spontaneously occurring cases in postpartum cows (Dubuc et al., 2010).
Although the infusate was placed in the uterine horns, retrograde movement of the content
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could have inoculated the cervix and vagina. However, quantification of vaginal 16S rRNA
the day after infusion on experiment d 1 revealed comparable total bacterial load between
treatments. The presence of elevated 16S rRNA observed in the bacteria-infused heifers 5 d
following infusion suggests that mucopurulent vaginal discharge is derived from the infected
uterus induced by infused bacteria, and not from environmental contamination. The
accumulation of echogenic uterine fluid in bacteria-infused heifers supports these
assumptions and suggests that any cervicitis or vaginitis would likely be the result of
contamination or excessive manipulation, and would also be observed in vehicle-infused
controls. Interestingly, however, hematological evaluation of heifers suggests a possible
effect of the procedure of infusing material into the uterus itself. Although we did not
observe a treatment effect for any of the hematological parameters evaluated, we did see an
effect of day on numbers of red blood cells, lymphocytes, and platelets, and the percent of
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hematocrit and hemoglobin. Consistently, all of these parameters were elevated 24 h

following infusion and steadily declined to pre-infusion levels by d 7. This observation may
be indicative of the manual manipulation affecting systemic hematological parameters in the
heifers.

Any experimental model of infection has limitations. Here, we propose using the described
experimental model to define the mechanisms of endometritis-mediated subfertility.
However, using heifers as the experimental unit limits our understanding of the reproductive
potential of the individual animal, unlike using mature lactating cows that would normally
have uterine disease. Additionally, the use of heifers revealed experimental limitations
because of body size and ability to perform the manipulations in 12- to 13-mo-old animals,
indicated by the consistent day effect of some hematological parameters. Experimentation
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for the purposes of determining mechanisms of endometritis-mediated subfertility will

require multiple, repeat manipulations that might be difficult in small-frame heifers
compared with mature cows. The longevity of the experimental model is also an important
factor to determine in future cohorts of animals; it will be important to establish that the
infection observed in the current cohort of heifers is reminiscent of the duration of
spontaneous endometritis observed in lactating cows. Additionally, quantification of
endometrial inflammation and microbial populations present as a result of experimentally

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 Piersanti et al. Page 11

induced infection will be critical to establish the validity of the model to study endometritis-
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mediated subfertility in the dairy cow.

CONCLUSIONS
We have successfully created a model of experimentally induced clinical endometritis in
virgin Holstein heifers. This model will allow the investigation into the mechanisms of
endometritis-associated reproductive failure. The use of an experimental model of
endometritis allows investigation into endometritis-mediated sub-fertility independent of
confounding postpartum events common in lactating cows that may influence reproductive
physiology. We used a combination of interventions to facilitate uterine infection, including
disruption of the endometrial epithelial barrier, supplementation of exogenous P4, and
infusion of uterine pathogenic bacteria. We conclude that our experimental model
recapitulates clinical endometritis symptoms in virgin Holstein heifers.
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ACKNOWLEDGMENTS
The authors thank Todd Pritchard, Miguel To-rrado, and the University of Florida Dairy Research Unit staff for
their assistance. Research reported in this publication was supported by the Eunice Kennedy Shriver National
Institute of Child Health & Human Development of the National Institutes of Health (Bethesda, MD) under Award
Number R01HD084316. The content is solely the responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health.

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Figure 1.
Requirements for establishment of experimental uterine infection. (A) Timeline of
experimental procedures employed during the experimental period. Gonadotropin-releasing
hormone and PGF2α were used to synchronize estrous cycles in 9 virgin Holstein heifers
before intrauterine infusion of treatments. On d 0, intrauterine infusion of either vehicle or
live bacteria was performed. Vehicle infusion consisted of 30 mL of sterile Luria-Bertani
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broth. Bacteria infusion consisted of 10 mL of Escherichia coli MS 499 (4.64 × 107 cfu/mL),
10 mL of Trueperella pyogenes MS249 (3.38 × 107 cfu/mL), and 10 mL of sterile Luria-
Bertani broth. Progesterone (P4) was administered at 200 mg/d i.m. Days relative to
treatment for evaluation of plasma haptoglobin (hpt), P4 (P4), and hematology (hem) are
indicated in italics. (B) The tool used to enable scarification of the endometrium.
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Figure 2.
Clinical observations of induced uterine disease. (A) Vaginal mucus was visually confirmed
in heifers receiving bacterial infusion. (B) Metricheck (Simcro, Hamilton, New Zealand)
tool containing vaginal mucus of a bacteria-infused heifer. (C) Examples of vaginal mucus
samples collected from bacteria-infused heifers using the Metricheck tool. (D and E)
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Representative ultrasound images of a transverse cross-section of a uterine horn from a

control (D) and bacteria-infused (E) heifer. The arrow head denotes the uterine lumen; the
double-headed arrow denotes the uterine wall. The scale bar represents 5 mm.
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Figure 3.
Vaginal mucus grade following intrauterine infusion of bacteria. Vaginal mucus was
collected using a Metricheck (Metricheck, Simcro, Hamilton, New Zealand) tool and graded
according to Sheldon et al. (2009). Vaginal mucus was graded from 0 to 3 and each heifer is
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represented by a single circle. Control heifers are represented by open circles (A, ○), and
bacteria-infused heifers are represented by filled circles (B, ●). The vertical dotted line
denotes the day of treatment; solid horizontal lines indicate the median vaginal mucus score
for the day of observation. Data were analyzed using the GLIMMIX procedure following a
Poisson distribution to determine the effect of treatment (Trt).

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Figure 4.
Effect of treatment on vaginal mucus 16S rRNA and circulating concentrations of
haptoglobin and progesterone in plasma. (A) Total 16S rRNA quantification from vaginal
mucus on d −5, −1, 1, and 5 relative to treatment in either control (open bars) or bacteria-
infused (solid bars) heifers. Total 16S rRNA was normalized to the weight of vaginal mucus
processed for nucleic acid extraction (ng of rRNA per mg of mucus). Different letters denote
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differences within the bacteria-infused group; * denotes difference between treatment groups
on a given day (P < 0.05). (B) Haptoglobin plasma concentration (ng/mL) was measured in
control (○) and bacteria-infused (●) heifers on d 0, 7, and 13 relative to treatment. (C)
Progesterone plasma concentration (ng/mL) was measured in control (○) and bacteria-
infused (●) heifers on d −2, 1, 4, 11, and 15 relative to treatment. Progesterone (P4) denotes
the period of exogenous administration of 200 mg/d of P4. All data are presented as LSM ±
SEM.

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Table 1.

Effect of treatment on rectal temperature and hematology

Treatment 3
P-value
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1 2 Control Bacteria SEM Trt Day Trt × day

Variable Days
Temperature (°C) −1 38.4 38.3 0.1 0.08 0.01 0.19
1 38.7 39.5
3 38.3 38.7
7 38.5 38.5
RBC (M/μL) −1 6.78 6.36 0.31 0.3 <0.01 0.85
1 7.79 7.03
3 7.37 6.75
7 7.17 6.70
HCT (%) −1 30.04 29.60 1.50 0.68 <0.01 0.81
1 33.70 32.80
3 33.04 31.43
7 32.34 31.73
HGB (g/dL) −1 10.20 9.93 0.52 0.65 <0.01 0.96
1 11.24 10.98
3 11.00 10.55
7 10.80 10.43
WBC (K/μL) −1 12.12 11.72 0.85 0.65 0.45 0.86

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1 12.31 12.81
3 10.38 11.43
7 10.96 12.08
NEU (%) −1 30.86 22.33 3.47 0.34 0.34 0.28
1 24.82 19.18
3 24.18 13.56
7 19.24 24.08
NEU (K/μL) −1 3.88 2.72 0.59 0.66 0.23 0.22
1 3.06 2.58
3 2.53 1.42
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Treatment 3
P-value

1 2 Control Bacteria SEM Trt Day Trt × day

Variable Days
7 1.94 3.19
LYM (%) −1 44.24 54.38 4.64 0.70 0.27 0.11
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1 50.12 58.35
3 50.98 51.23
7 60.80 52.70
LYM (K/μL) −1 5.20 6.25 0.43 0.29 0.59 0.51
1 6.14 7.35
3 5.33 6.64
7 6.89 6.07
MON (%) −1 17.44 16.95 1.88 0.52 0.27 0.37
1 18.32 14.93
3 18.00 26.60
7 14.32 17.18
MON (K/μL) −1 2.13 1.95 0.20 0.84 0.32 0.47
1 2.28 2.18
3 1.83 1.84
7 1.59 2.11
EOS (%) −1 7.42 6.30 1.00 0.86 0.13 0.27
1 6.68 5.50
3 6.80 9.18

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7 5.01 6.03
EOS (K/μL) −1 0.91 0.80 0.15 0.85 0.14 0.20
1 0.81 0.69
3 0.70 0.92
7 0.53 0.72
PLT (K/μL) −1 312.2 241.00 68.98 0.45 <0.01 0.82
1 380.00 261.00
3 318.20 221.00
7 146.20 117.75
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1
Data are presented as LSM with respective pooled SEM for the interaction between treatment and day. Data quantified in whole blood included red blood cell count (RBC), hematocrit (HCT), hemoglobin
concentration (HGB), white blood cell count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MON), eosinophils (EOS), and platelets (PLT) measured as the proportion of total cells (%) and
number of cells per μL (K = 1,000; M = 1,000,000).
2
Sample days relative to infusion (d −1, 1, 3, and 7).
3
Trt = effect of treatment (control vs. bacteria); day = effect of day relative to treatment; Trt × day = interaction between Trt and day.
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