SHORT COMMUNICATION

The Effectiveness of Immunotherapy in Treating

Exercise-Induced Pulmonary Hemorrhage
Tammi S. Epp, DVM, PhD,a Paul McDonough, PhD,b Don E. Myers, MS,c
Danielle J. Carlin, PhD,a Brad J. Behnke, PhD,d Casey A. Kindig, PhD,y
David C. Poole, PhD, DSc,a and Howard H. Erickson, DVM, PhDa

ABSTRACT
Inflammatory airway disease has been linked to exerciseinduced pulmonary hemorrhage (EIPH), and consequently, we hypothesized that immunomodulation via
concentrated equine serum (CES) treatment would reduce EIPH as evidenced by red blood cell (RBC) concentrations in bronchoalveolar lavage fluid (BALF). Separate
trials were conducted on Thoroughbred horses treated
with either CES (n 6) or placebo (0.9% saline; n 4).
All horses completed pre-treatment and post-treatment
(2 and 4 weeks after initiating treatment) maximal exercise tests on a 10% inclined treadmill (1 m/s/min increments to fatigue) over a 10-week period (23 weeks
between tests), with bronchoalveolar lavage (BAL) performed 30 minutes after exercise. Treatment ensued 10
days after the pre-treatment exercise test, with horses receiving a series of five CES or placebo injections 24 hours
apart (20 mL intratracheal and 10 mL intravenously),
with subsequent weekly injections for 5 weeks thereafter.
After CES treatment, both EIPH (RBC in BALF) and inflammation (white blood cell concentration [WBC] in
BALF) were significantly diminished by the 4-week posttreatment run, demonstrating 46  12% and 24  11%
decreases, respectively (P < 0.05). In contrast, EIPH
was elevated significantly at the 4-week time point, and inflammation remained constant in the placebo trial. In
conclusion, these preliminary data suggest that therapeutic intervention involving immunomodulation may represent a viable approach to reducing the severity of EIPH.
Keywords: Exercise-Induced Pulmonary Hemorrhage;
Horse; Bronchoalveolar lavage; Immunomodulation;
Inflammatory airway disease

From the Department of Anatomy and Physiology, Kansas State University,

Manhattan, KSa; Department of Applied Physiology and Kinesiology, University of
Florida, Gainesville, FLb; Sera, Inc., Shawnee Mission, KSc; and the Division of
Exercise Physiology, Robert C. Byrd Health Sciences Center, West Virginia University
School of Medicine, Morgantown, WV.d
y
Deceased.
Reprint requests: Tammi S. Epp, DVM, PhD, 228 Coles Hall, Manhattan, KS 66506-5802.
0737-0806/$ - see front matter
2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jevs.2009.04.192

Journal of Equine Veterinary Science  Vol 29, No 6 (2009)

INTRODUCTION
Evidence for exercise-induced pulmonary hemorrhage
(EIPH) can be found in virtually all racehorses in training1-3
and can be a cause for poor performance4,5 as well as potential
premature athletic retirement. In addition, epidemiologic6
and post-mortem7-9 studies have identified an association between EIPH and inflammatory airway disease (IAD). Furthermore, it is often necessary for additional veterinary care,
extended breaks from training, or permanent racetrack
banishment for those horses responding poorly to treatment.
Existing therapies for EIPH (ie, furosemide and the equine
nasal strip)10,11 are successful in reducing, but not completely
abolishing, EIPH, even when used concurrently, which
suggests that EIPH has complex and multifactorial causes.
Therefore, it may be beneficial to investigate a therapy that
could either provide an alternative strategy (ie, reduce inflammatory component) or additive reductions in EIPH.
Concentrated equine serum (CES; Sera, Inc., Shawnee
Mission, KS) is a biologic serum aggregate collected from
multiple draft horse donors, containing high levels of immunoglobulins, complement, and other serum proteins.
The equine-derived CES (an intravenous and intratracheally
administered product) and a goat-derived product called
caprine serum fraction (CSF; administered intramuscularly;
Colorado Serum Co., Denver, CO), have demonstrated evidence for reducing the chronic inflammation associated
with lower airway disease (ie, IAD and EIPH) via immunomodulation in separate studies.12,13 Additional support
comes from endoscopic evidence in racetrack field trials
(Sera, Inc., Shawnee Mission, KS).14 Immunomodulation
via intravenous immunoglobulin has been successfully used
to treat asthma and systemic inflammatory conditions in humans.15-19 However, no studies have been conducted to specifically examine the effectiveness of CES as a treatment for
EIPH in racehorses. Thus, we hypothesized that CES would
reduce EIPH (RBC in BALF) and pulmonary inflammation
(decreased WBC in BALF) following maximal exercise.

MATERIALS AND METHODS

Animals and Preparation
Ten Thoroughbred geldings (412 years old;
470620 kg) with a history of EIPH were divided into

527

528

placebo (n 4) and treatment (n 6 CES) groups in this

preliminary investigation. The horses were housed on dry
lots with loafing sheds on the Kansas State University campus, and had ad libitum access to water and salt. They were
fed alfalfa and grass hay, as well as concentrate (Strategy,
Purina Mills Inc., St. Louis, MO) twice daily. All procedures were approved by the Kansas State University Animal
Care and Use Committee. Horses were instrumented with
a Millar pressure transducer (for measurement of pulmonary artery pressure [Ppa; Millar Model SPC-471A, Millar
Instruments, Incorporated, Houston, TX]) and a thermistor (for measurement of core body temperature and temperature correction of blood gases and pH; Model 08407
Thermal Dilution Catheter, Columbus Instruments, Columbus, OH) that were calibrated and placed in the pulmonary artery through jugular vein introducer catheters and
verified by cardiac wave form visualization. Arterial blood
gases and plasma lactate samples were collected via an arterial catheter placed in a previously elevated carotid artery.
Administration of CES or Placebo
Dosage was based on field studies completed by Sera, Inc.,
Shawnee Mission, KS. Twenty milliliters CES or placebo
(0.9% saline) was administered by intratracheal injection
with an 18-G 1.5-inch needle in the proximal one third
of the trachea (between tracheal rings) after aseptic preparation. This was followed by slow intravenous administration of 10 mL of CES or placebo. The horses head was
elevated for 10 minutes after injection to allow gravitydependent flow into the lungs and to minimize mucociliary
clearance of CES or placebo. The procedure was repeated
every 24 hours for 5 days with weekly injections (both
the intravenous and intratracheal) given for 5 weeks thereafter. On the week of an exercise test, the CES or placebo
was administered 24 hours before exercise. No adverse
effects were noted in any of the experimental animals.
Experimental Protocol
A timeline of the experimental protocol is provided in
Figure 1. The horses were conditioned on a high-speed
treadmill (Sato, Inc, Uppsala, Sweden) using a moderate-to-heavy intensity exercise regimen (%10 m/s on
flat; %7 m/s on inclined treadmill) 3 days/week before
and throughout the study. On experimental days, horses
performed a maximal exercise test on a 10% inclined
treadmill, consisting of a warmup at 3 m/s for 4 minutes,
followed by progressive increases in speed (1 m/s  1minute increments) until volitional fatigue, and were recovered at 3 m/s for 4 minutes. Pulmonary gas exchange
was measured with an open-flow system, and cardiorespi_ 2], carbon dioxide
ratory variables (ie, oxygen uptake [VO
elimination, heart rate, respiratory rate, and Ppa), blood
gases, plasma lactate, and time-to-fatigue were measured

TS Epp et al  Vol 29, No 6 (2009)

at rest, throughout the exercise test, and during recovery

as described previously by Epp et al.20
Bronchoalveolar Lavage
Baseline (resting; with no exercise 710 days prior), 30 to
60 minutes post-exercise, and 1 week post-exercise BALs
were performed (seven in total, as numbered in Fig. 1) as previously described10,20 as a means to establish a nonexercising
baseline RBC concentration in BALF, quantify the effects of
CES on the severity of EIPH immediately post-exercise, and
to confirm a return of RBC concentration in BALF to baseline before the next exercise test. The horses were tranquilized using detomidine hydrochloride (Dormosedan, Pfizer
Animal Health, Exton, PA; 510 mg/kg intravenously)
and butorphanol tartrate (Torbugesic, Fort Dodge Animal
Health, Fort Dodge, IA; 510 mg/kg intravenously).
A BAL tube (Bivona, Smiths Medical, Philadelphia, PA;
3 m long, 10-mm OD) was advanced blindly through the
right naris, wedged in the caudodorsal lung lobe, and sealed
with an inflatable cuff located at the end of the tube. A total of
300 mL (50-mL aliquots) of 0.9% saline was infused and aspirated with gentle suction. The BALF was centrifuged
(Beckman TJ-6), the supernatant decanted, and the pellet resuspended in 0.9% saline. RBC and WBC concentrations
were determined via a hemocytometer (No. 02-671-5,
Fisher Scientific, Pittsburgh, PA) and presented as RBC/mL
of recovered BALF, accounting for tube dead space.
Statistical Methods
All data are presented as mean  standard error (SE). Data
were analyzed via repeated measures analysis of variance
using SAS PROC MIXED with treatment and time as fixed
effects and horse as a random effect (SAS Program 9.1.2 statistical package, SAS Institute, Incorporated, Cary, NC).
The relationship between variables was determined via
a Pearson product-moment correlation analysis (Sigma
Stat 3.0, SPSS, Inc., Chicago, IL). Significance was accepted at P < 0.05, and a one-tailed test was used for a priori
directional hypotheses involving EIPH and inflammation.

RESULTS AND DISCUSSION

The novel finding of the current study is that CES significantly reduced EIPH and associated inflammation after
maximal exercise in Thoroughbred horses after 4 weeks of
therapy. This was evidenced by a 46  12% decrease in
RBC concentration in the BALF between the pre-treatment and the 4-week post-CES treatment runs (Fig. 2). Inflammation after maximal exercise also declined 24  11%,
as evidenced by the WBC concentration in the BALF
(Fig. 3). In comparison, EIPH significantly increased by
245  113%, whereas no change was demonstrated in
WBC concentration in the BALF (Fig. 3) for horses under
placebo conditions.

TS Epp et al  Vol 29, No 6 (2009)

529

2-week posttreatment exercise

test
&
post-exercise
BAL #4

pre-treatment
exercise test
&
post-exercise
BAL #2
Day 0

14

27 28

17 18 19 20 21

34 35

48

41 42

CES or PL
injections

CES or PL
injections
Baseline
(resting)
BAL #1

4-week post-treatment
exercise test
&
post-exercise
BAL #6

CES or PL
injections

CES or PL
injections

BAL #3
BAL #5

daily administration

BAL #7

of CES or PL

CES Treated
PL Treated

245 113% in EIPH

during PL Treatment

400
300
200

46 12 % in EIPH
during CES Treatment

100
0
1

Run

Inflammation (Percent of WBC/ml

BALF from Pre-Treatment Run)

500

EIPH (Percent of RBC/ml

BALF from Pre-Treatment Run)

Figure 1. Timeline of experimental protocol. BAL, bronchoalveolar lavage; CES, concentrated equine serum; PL,
placebo (0.9% saline). Both CES and PL were administered at a dose of 20 mL intratracheally and 10 mL intravenously
at each time point.

160
CES Treated
PL Treated
120

80

*
24 11 % in Inflammation
during CES Treatment

40

0
1

Figure 2. Exercise-induced pulmonary hemorrhage

(EIPH) after maximal exercise before initiation of
concentrated equine serum (CES; n 6) or placebo
(PL; n 4) treatment (run 1) and after (runs 2 and 3).
Data are expressed as a percentage (mean  SE) of the
red blood cells (RBC)/mL bronchoalveolar lavage fluid
from the control (baseline) pretreatment run (run 1). A
46  12% (*P < 0.05) decrease from the pretreatment
run was observed in EIPH after the 4-week treatment
with CES, whereas a 245  113% (yP < 0.05) increase
was found in horses receiving PL.

Figure 3. Inflammation, represented by white blood

cells (WBC)/mL bronchoalveolar lavage fluid (BALF)
after maximal exercise before initiation of concentrated
equine serum (CES; n 6) or placebo (PL; n 4)
treatment (run 1) and after (runs 2 and 3). Data are
expressed as a percentage (mean  SE) of the WBC/mL
BALF from the control (baseline) pretreatment run
(run 1). A 24  11% decrease in WBC/mL BALF was
observed after the 4-week treatment with CES, whereas
no change was observed in horses receiving PL. *
indicates that mean is significantly different (P < 0.05)
from the pretreatment CES run.

In addition, no differences were detected in differential

counts (data not shown) for either pre-run or post-run lavages of the placebo or CES-treated horses. Baseline and 1
week post-run lavages illustrated that RBC and WBC concentrations returned to baseline by 1 week after maximal
exercise, eliminating confounding effects of elevated baseline RBC concentrations in subsequent runs for horses
given either placebo or CES. No differences existed

between or within placebo or CES groups, regarding the

percent BALF recovered, WBC concentration, RBC concentration, or differential counts (data not shown) for
any of the nonexercising lavage periods. No differences
were noted for any of the cardiorespiratory, metabolic, or
performance variables within the placebo and CES groups
or as a result of CES treatment for any of the runs, and the
values fell within normal published ranges10,20 (Table 1).

TS Epp et al  Vol 29, No 6 (2009)

530

Numerous investigations have linked IAD with EIPH as

either a contributing or a consequential factor,1,6-9,21 and
all horses in the current investigation exhibited evidence
of IAD: specifically, higher than normal WBC concentrations (>200 cells/mL), mild neutrophilic inflammation
(520% neutrophils), and lymphocytosis in BALF,22 which
may exacerbate future EIPH episodes. CES-related reductions in WBC and RBC concentrations in BALF in the current study were substantiated by subjective reductions in
both inflammation (decreased airway mucus) and EIPH
(endoscopic grading) observed after field use of CES in
187 horses at three racetracks (Fonner Park, NE; Prescott,
AZ; and Marshall, VA) from 1995 to 1999 (Sera, Inc.,
Shawnee Mission, KS).14 Moreover, preliminary data
from our laboratory (n 4) suggest at least an equivalent
reduction in EIPH with CES (68%) versus furosemide
(58%), the nasal strip (42%), or the concurrent use of the nasal strip and furosemide (46%) in the same maximally exercising horses. These data suggest that mitigation of IAD
may address a vital component of EIPH that is at least
equivalent in terms of importance to the reduction of

intravascular (furosemide)10 and extravascular (equine nasal strip)10,11 pressures and that can be modulated independent of changes in these pressures, because no alterations
_ 2 in the current study (Table 1).
were seen in Ppa or VO
One major difference between acute treatments (eg, equine
nasal strip and furosemide) and CES is the 4 weeks necessary
to evoke a reduction in EIPH. The delayed time-dependent
mechanism of CES may be consequent to mitigation of the
prolonged inflammatory response to blood in the alveoli
and altered compliance and resistance during breathing
that ultimately predisposes a greater number of capillaries
to stress failure with each succeeding EIPH episode.21,23,24
The placebo trial, although conducted with somewhat lighter bleeders and at a different time, showed a much different
pattern, in that EIPH increased over time, which is in agreement with the current literature.3,25
The BAL technique as used herein has several distinct advantages. Specifically, it samples that region of the lung
where EIPH primarily occurs (ie, dorsocaudal lobes)26
and, unlike endoscopy, is sensitive enough to detect
EIPH without the necessity for the blood to track up the

Table 1. Pre- and post-placebo (PL) and concentrated equine serum(CES) treatment comparisons for cardiorespiratory and metabolic variables during maximal exercise and bronchoalveolar lavage variables post-exercise
Variable
_ 2max (L/min)
VO
_
VCO
2max (L/min)
PaO2 (mmHg)
PaCO2 (mmHg)
HR max (beats/min)
RRmax (breaths/min)
[La-] (mM)
Ppa (mmHg)
pH
Hematocrit
Time-to-fatigue (s)

Treatment

Pre-treatment Run
(Baseline Run)

2-Week Post-treatment
Run

4-Week Post-treatment
Run

PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES
PL
CES

72.9  2.0
73.7  1.9
82.8  3.3
81.0  2.4
63.4  1.3
64.9  2.7
61.7  3.9
64.2  4.1
218  2
219  4
115  3
121  2
21.9  3.3
25.3  3.2
87.3  7.3
96.1  6.2
7.22.02
7.18.04
59  1
63  1
672  25
711  14

75.4  2.6
67.7  5.6
86.5  1.3
76.5  6.1
65.0  3.0
64.1  2.0
62.0  4.2
63.8  3.7
219  5
224  8
115  2
120  2
29.6  1.7
28.8  3.2
91.4  3.1
95.7  6.9
7.22.02
7.21.03
61  1
63  1
661  24
679  9

76.5  2.3
71.9  3.1
85.9  2.2
81.0  3.7
60.9  1.5
62.4  1.7
62.0  2.6
67.5  4.0
214  4
216  4
115  2
121  2
26.5  4.6
32.6  1.7
89.2  3.9
97.0  4.6
7.18.02
7.19.04
61  1
63 1
673  15
682  12

Values are means  SE.

_ 2, oxygen uptake at STPD; VCO2, carbon dioxide elimination at STPD; PaO2, partial pressure of oxygen in the arterial blood; PaCO2, partial
VO
pressure of carbon dioxide in the arterial blood; HR, heart rate; RR, respiratory rate; [La-], plasma lactate concentration; Ppa, mean peak pulmonary
artrial pressure.
No significant differences were found between the pretreatment and either of the posttreatment runs for any of these variables.

TS Epp et al  Vol 29, No 6 (2009)

airways and is not as time-restricted as endoscopy.11 Thus,

the extent of EIPH can be quantitatively and reproducibly
assessed across a broad range of magnitudes, affected by exercise workloads/intensities27,28 and therapeutic interventions.14,27 There is also good bilateral agreement in EIPH2
such that it is not necessary to sample from both lungs, and
a good correlation exists between BAL cytology and histopathology of horses with EIPH.29 Disadvantages of BAL
include the possibility of iatrogenic bleeding29 (which can
be avoided with careful technique as demonstrated in the
consistent RBC concentrations in nonexercising BALs of
the current study) and also the inability to measure the total
volume of blood in the lungs (correlate RBC concentration
in BALF with EIPH severity without removal of the lungs
for whole lung lavage).30 Bronchoscopy can also cause iatrogenic bleeding and, as mentioned, is less sensitive in that
it cannot assess EIPH until blood emerges into the large airways. In addition, the EIPH endoscopic score is subjective,
which may make EIPH severity discrimination between different experimental or therapeutic interventions impossible
(ie, the variable response to furosemide in numerous studies
and the nasal strip study by Manohar et al)27. Hence, despite limitations as noted, BAL, remains the only technique
available to concurrently assess the severity of EIPH and the
cytologic profile of the lungs, because transtracheal wash
cytology does not correlate well with histopathology.29
The mechanism through which CES reduces EIPH remains speculative. However, it appears to be the result of
immunomodulation, based on research by Ragland
et al12 (CES administration) and Hamm et al13 (CSF administration) demonstrating that a reduction in pulmonary
inflammation and a more rapid recovery of horses from
suppurative lower airway disease occurred as a result of
non-specific stimulation of the immune system. CES
may enhance clearance12,13,31-33 of the blood in the alveoli,
because it has been demonstrated that removing blood
from the lungs is problematic for alveolar macrophages.34-43 The current study demonstrated a reduction
in WBC concentrations and a concurrent and marked increase (102  75%) in the WBC/RBC ratio as a result of
CES treatment. Overall, macrophages decreased as inflammation decreased in CES-treated horses (Run 2; r2 0.94
and Run 3; r2 0.76, P < 0.05). In contrast, horses treated
with placebo demonstrated a decreased WBC/RBC ratio
(57  15%). In addition, the correlation between the
WBC/RBC ratio and the level of hemorrhage found pretreatment (r2 0.73, P < 0.05) disappeared post-treatment
on reduction of EIPH (r2 0.34, P > 0.05) in the horses
treated with CES, whereas the opposite occurred in the
placebo horses, and a trend for a relationship (r2 0.84;
P (.08) appeared as EIPH increased. In vitro CES treatment of alveolar macrophages obtained from BAL samples
of resting and exercising horses suggests that CES may enhance phagocytosis and oxidative burst function

531

(preliminary data). However, in vivo studies are needed

to confirm these effects. These preliminary and collective
findings suggest that CES treatment may expedite clearance of blood from the lungs, thus reducing the inflammatory cycle and its consequences.
The current data are provocative and support the imperative for performing a full-scale laboratory investigation of
CES. In particular, it would be valuable to confirm EIPH
and IAD benefits, elucidate the immunomodulatory mechanism of action, determine optimal dose and route of administration, and evaluate fully any synergistic benefits
resulting from concurrent use of CES with furosemide
and the nasal strip that have so far only been observed in
one horse treated with all three interventions.
ACKNOWLEDGMENTS
The authors thank Dr. Bonnie Rush, Dr. Beth Davis, Dr. Judy
Cox, Dani Goodband, Dr. Ann Buchanan, Joanna Thomas,
Dr. Sandy Green, Russell Green, Shanna Miller, Nancy Sproul,
Vicki Johnson, Rachel Workman, Katie Giles, Dr. Lavica
Gates, Dr. Becka Erwin, Dr. Susan Sears, Dr. Leslie Mikos,
Dr. Maureen Reynolds, Lindsey McClintock, Rachel Lindberg,
Nicole Harper, Chasity Champlin, and Matt Jones for their assistance. This research was supported by Sera, Inc., Shawnee Mission, KS, and The Kansas Racing and Gaming Commission.
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