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\begin{document}

\title{Investigation of Bovine Serum Albumin specific IgE expression in horses}



\author[1]{Elizabeth Perry}%
\author[1]{Katherine  Delph}%
\author[1]{Christopher Blevins}%
\author[1]{Kaori Knights}%
\author[2]{Nora Springer}%
\author[1]{Elizabeth Davis}%
\affil[1]{Kansas State University College of Veterinary Medicine}%
\affil[2]{The University of Tennessee Knoxville College of Veterinary Medicine}%


\vspace{-1em}



  
  \date{June 06, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Neonatal foals are born essentially agammaglobulinemic and
therefore must ingest colostrum or receive immunoglobulins to maintain
health. Failure of passive transfer treatment involves administration of
equine colostrum, plasma or commercial powdered colostrum (CPC).
Anecdotal reports suggest a risk of anaphylaxis associated with plasma
transfusion in neonates that received CPC prior to gut closure. Bovine
serum albumin (BSA) in CPC may serve as a target for BSA-specific
immunoglobulin E (IgE) in donor equine plasma. Objectives: To determine
presence of BSA-specific IgE in samples collected post-routine
vaccination in healthy horses, horses experiencing adverse vaccine
reactions and commercial equine plasma. Study Design: Prospective
Observational Methods: Serum was collected from 65 healthy horses at day
0, 14, 28, 90, 180, 270 and 365 post-vaccination, 26 horses after
vaccine reaction at day 1, 180 or 270 post-vaccination, 4 horses not
vaccinated and 10 horses from a commercial plasma donor herd.
BSA-specific IgE was determined using enzyme-linked immunosorbent assay
(ELISA). Results: BSA-specific IgE was not detected in non-vaccinated
horses and was identified in all vaccinated horses. Younger horses
demonstrated higher fold changes in post-vaccination BSA-specific IgE
expression compared to older horses. No significant difference in
BSA-specific IgE levels between commercial plasma donors and healthy
horses was identified. No significant difference in post-vaccination
anti-BSA IgE levels between reactor and healthy horses at day 180 and
270 post-vaccination were identified. Main Limitations: Small number of
reactor horses at day 180 and 270 post-vaccination with most samples
being collected 24 hours. There were no healthy horse samples for 24
hours post-vaccination; therefore, it was not possible to compare the
two groups at this timepoint. Conclusions: Horses may express BSA
specific IgE following vaccination. There may be risk of
hypersensitivity type reaction when veterinarians administer commercial
plasma to neonatal foals that have consumed CPC prior to gut closure.%
\end{abstract}%



\sloppy


\emph{Original Article}

Title: \textbf{Investigation of Bovine Serum Albumin specific IgE
expression in horses}

Authors: E. R. Perry+*, K.M. Delph+, C.A. Blevins+, K. Knights++, N.L
Springer++\selectlanguage{ngerman}§, E.G. Davis\selectlanguage{english}+

\selectlanguage{english}\emph{+Kansas State University College of Veterinary Medicine and
Veterinary Health Center; ++Kansas State Veterinary Diagnostic
Laboratory, Manhattan, Kansas; SSCurrent address: Department of
Biomedical and Diagnostic Sciences University of Tennessee College of
Veterinary Medicine}

\emph{*Corresponding author email: elizabethperrydvm@gmail.com}

\textbf{Keywords:} horse; vaccine; plasma; IgE; bovine serum albumin

Summary

\textbf{Background} : Neonatal foals are born essentially
agammaglobulinemic and therefore must ingest colostrum or receive
immunoglobulins to maintain health. Failure of passive transfer
treatment involves administration of equine colostrum, plasma or
commercial powdered colostrum (CPC). Anecdotal reports suggest a risk of
anaphylaxis associated with plasma transfusion in neonates that received
CPC prior to gut closure. Bovine serum albumin (BSA) in CPC may serve as
a target for BSA-specific immunoglobulin E (IgE) in donor equine plasma.

\textbf{Objectives} : To determine presence of BSA-specific IgE in
samples collected post-routine vaccination in healthy horses, horses
experiencing adverse vaccine reactions and commercial equine plasma.

\textbf{Study Design:} Prospective Observational

\textbf{Methods:} Serum was collected from 65 healthy horses at day 0,
14, 28, 90, 180, 270 and 365 post-vaccination, 26 horses after vaccine
reaction at day 1, 180 or 270 post-vaccination, 4 horses not vaccinated
and 10 horses from a commercial plasma donor herd. BSA-specific IgE was
determined using enzyme-linked immunosorbent assay (ELISA).

\textbf{Results} : BSA-specific IgE was not detected in non-vaccinated
horses and was identified in all vaccinated horses. Younger horses
demonstrated higher fold changes in post-vaccination BSA-specific IgE
expression compared to older horses. No significant difference in
BSA-specific IgE levels between commercial plasma donors and healthy
horses was identified. No significant difference in post-vaccination
anti-BSA IgE levels between reactor and healthy horses at day 180 and
270 post-vaccination were identified.

\textbf{Main Limitations} : Small number of reactor horses at day 180
and 270 post-vaccination with most samples being collected 24 hours.
There were no healthy horse samples for 24 hours post-vaccination;
therefore, it was not possible to compare the two groups at this
timepoint.

\textbf{Conclusions} : Horses may express BSA specific IgE following
vaccination. There may be risk of hypersensitivity type reaction when
veterinarians administer commercial plasma to neonatal foals that have
consumed CPC prior to gut closure.

Clinical Relevance

\begin{itemize}
\tightlist
\item
  It has been demonstrated that horses can produce BSA-specific IgE
  following vaccination and this may contribute to the presence of
  BSA-specific IgE in commercial plasma products.
\item
  Neonatal foals receiving CPC supplementation within 24 hours of birth
  may be at risk of developing an adverse reaction to plasma transfusion
  if BSA-specific IgE is present in the administered plasma product.
\item
  Veterinarians should follow all label recommendations for USDA
  licensed Ig products, specifically including avoidance of
  administering multiple products to neonatal foals.
\end{itemize}

INTRODUCTION

Prevention of infectious disease is an important component of
maintaining equine host health. American Association of Equine
Practitioner (AAEP) vaccination guidelines are available to help equine
veterinarians implement safe and effective vaccine protocols (AAEP
2020). In most cases, vaccine administration results in immunologic
protection against administered disease antigens with low risk to the
host. In select cases, adverse reactions may occur and be associated
with triggering the innate immune system and not just stimulating the
adaptive, antigen-specific arm of the immune system (Tizard 2018). In a
previous investigation, the etiology of allergic reactions among horses
following routine vaccination was explored (Gershwin \emph{et al} .
2012). Findings from this investigation revealed that vaccine-induced
hypersensitivity reactions may be related to IgE responses towards non
vaccine-target, culture-derived proteins (Gershwin \emph{et al} . 2012).
One such protein associated with this response is bovine serum albumin
(BSA), the major constituent of fetal bovine serum (Gershwin \emph{et
al} . 2012).

Fetal bovine serum has been used for decades during vaccine
manufacturing serving as an integral part of cell line cultivation.
Previous work has demonstrated that BSA is associated with both milk and
beef allergies in people, and there is evidence that vaccine-associated
hypersensitivity reactions in people occur secondary to BSA
hypersensitivities (Restani \emph{et al} . 2004, Shoenfeld \emph{et
al.}2011, Loughney \emph{et al} . 2014, Silva \emph{et al} . 2017). Due
to concerns for life-threatening anaphylaxis, the World Health
Organization requires BSA concentration to be less than 50 ng per human
vaccine dose (Loughney \emph{et al} . 2014). Although used less commonly
in veterinary vaccines, historically BSA was used during the
manufacturing process of certain vaccines for animal use without
restrictions. Evidence from the report by Gershwin \emph{et al.} (2012)
supports the suggestion that repeated annual or semi-annual vaccination
of horses with certain vaccines could enhance the likelihood that an
immune response to non-target proteins, that include BSA, could occur.

In addition to the risk for immediate hypersensitivity to non-target
proteins upon vaccination, risk can exist for adverse reactions to
biologic product administration sourced from hyperimmunized horses.
Specifically, commercial plasma manufacturers\textsuperscript{1,
2}provide a precautionary label statement that equine plasma should not
be administered to neonatal foals if they received powdered colostral or
milk replacer product prior to gut closure due to risk for possible
hypersensitivity reaction. Foals are born agammaglobulinemic and
therefore require ingestion of maternal colostrum for adequate
absorption of circulating immunoglobulins, specifically IgG
(Lewis\emph{et al.} 2008, Felippe 2016, Tizard 2018). In cases where
mare maternal colostrum is not available, commercial powdered colostral
products may be administered (Gapper \emph{et al.} 2007). Most
commercial powdered colostral products are of bovine origin containing
BSA. During the first approximately 24 hours of life, while the equine
neonatal small intestine is capable of mediating absorption of intact
immunoglobulins through pinocytosis, macromolecule protein absorption
also takes place (Felippe 2016). In a case where a neonatal foal may
ingest bovine-sourced colostral products, BSA can also be absorbed
through the small intestine and enter general circulation. While
previous studies have shown the administration of hyperimmune equine
plasma to foals to be relatively safe (Wilson \emph{et al.} 2009;
Francesca \emph{et al.} 2017), anecdotal reports among equine clinicians
provides evidence that when foals have received powdered colostral or
milk replacer products prior to gut closure and receive a subsequent
plasma transfusion, unexpected and at times fatal hypersensitivity
reactions have occurred. The current study reports such a case
demonstrating the need for providing evidence to clinical equine
veterinarians of possible adverse and even fatal outcomes in foals
receiving plasma transfusions following administration of powdered
colostral or milk replacers.

Anaphylaxis is a serious and potentially life-threatening reaction
mediated predominately by IgE (Kalina \emph{et al.} 2003; Achatz\emph{et
al.} 2008). Although many factors can contribute to the development of
immediate hypersensitivity reactions, the primary objectives of the
current study were 1) to determine if BSA-specific IgE expression
occurred in healthy horses following routine annual vaccination and 2)
if BSA-specific IgE expression differed in horses with reported vaccine
reactions. Additionally, because commercial plasma harvested from
hyperimmunized horses may contain BSA-specific IgE and commercial plasma
is a commonly administered therapeutic agent in equine neonates, a
secondary aim of the study was to determine BSA-specific IgE expression
in commercial plasma products through testing of plasma samples from
commercial plasma donors used for plasma transfusion.

MATERIALS AND METHODS

\emph{Index case supporting the need to identify BSA-specific IgE levels
in commercial plasma.}

A colt of unknown gestational age was born to a malnourished dam with
little to no udder development nor colostrum/milk production. The owner
administered a commercial powdered bovine/multi-species colostrum
supplement\textsuperscript{3} orally within the first 2 hours of life.
Subsequently, the colt was examined and evaluated for transfer of
passive immunity status. An IgG test\textsuperscript{4} was performed at
12 hours of age, which revealed complete failure of transfer of passive
immunity (0 mg/dL IgG). The owners elected to continue supplementing the
foal on the farm with the powdered colostrum product despite
recommendations that it was unlikely to result in adequate transfer of
passive immunity. At 24 hours of age a recheck IgG
test\textsuperscript{4} measured an IgG level of 400 mg/dL IgG,
consistent with ongoing partial failure of transfer of passive immunity.
Once these results were provided, the owners elected to have the
attending clinician perform a plasma transfusion with 1L (22 ml/kg) of
commercial high immunoglobulin equine plasma.\textsuperscript{1} Plasma
was thawed according to the label instructions while an intravenous
catheter was placed into the left jugular vein. Initial physical
examination performed was within normal limits. Plasma administration
was initiated at a very slow rate initially of 0.05 ml/kg/minute (1
drop/2-3 seconds). Temperature, heart and respiratory rates were
measured at 3-5 minute intervals. Signs of colic, dyspnea and urticaria
were monitored for continuously. With no evidence of an adverse
reaction, the administration rate was slowly increased to 0.15
ml/kg/minute (1 drop/second) over the next 5-10 minutes. At 15 minutes
the rate was increased to approximately 0.38 ml/kg/minute (2-3
drops/second), at this point the foal quickly began to show colic signs.
The plasma infusion was stopped immediately. Crackles were auscultated
in all lung fields and pink-tinged foamy nasal discharge became evident
bilaterally suggestive of pulmonary edema. The foal became apneic after
one minute. Intravenous epinephrine\textsuperscript{5} (0.02 mg/kg) was
administered along with furosemide\textsuperscript{6} (1 mg/kg) and
dexamethasone\textsuperscript{7} (0.15 mg/kg). The foal did not respond
to emergency medications or attempts at field cardiopulmonary
resuscitation and died within 3 minutes following discontinuation of the
plasma transfusion.

Based on the history and response of this foal to plasma transfusion, it
was suspected that this foal suffered from BSA-associated
hypersensitivity. This outcome was consistent with previous anecdotal
reports of foals consuming powdered colostrum followed by IV commercial
plasma infusion, which has been reported to result in fatal reactions.
The remaining commercial plasma administered to this foal was saved at
-80\selectlanguage{english}@ C for further analysis.

\textbf{Sample Collection}

All procedures performed on horses during the study were approved by
Kansas State University Institutional Animal Care and Use Committee
(IACUC \#4003).

\emph{Blood collection procedure}

After cleansing of the haired skin surface with 70\% isopropyl alcohol,
20 mL of blood was collected through an 18 g vacutainer needle directly
into a red topped tube via jugular venipuncture. Blood collection was
performed on days 0 (baseline), 14, 30, 90, and 180 after vaccination. A
subset of horses also had blood collected on either or both days 270 and
360 after vaccination. Blood samples were stored at
4\textsuperscript{o}C immediately after collection for less than 48
hours prior to be being centrifuged at 2200-2500 RPM for 15 minutes.
Serum was removed and frozen at -18\textsuperscript{o}C for a maximum of
14 days. Serum was thawed at 4\textsuperscript{o}C for 24 hours, after
which it was aliquoted into 2 mL plastic collection tubes and stored at
-80\textsuperscript{o}C. Serum samples were batched for testing and were
only thawed once on the morning of testing to avoid freeze thaw
degradation, thus maintaining protein integrity for testing.

\emph{Enrollment of healthy horses following routine vaccination}

Horses were selected based on health status and historical information
that they had been on an AAEP-approved vaccine protocol for annual
vaccinations. Enrollment criteria included that the owners reported
routine annual vaccines had been administered and confirmed no previous
adverse vaccine reactions observed. Information was collected via a
client questionnaire for all enrolled animals (supplemental
information). Horses were determined to be healthy and able to receive
routine annual vaccinations following physical examination by a
veterinarian (CAB). All horses received the same type of vaccine that
were administered by the same veterinarian (CAB). Vaccines administered
included a combination against Eastern equine encephalitis (EEE),
Western equine encephalitis (WEE), tetanus, West Nile virus (WNV),
equine influenza, and equine herpesvirus-1 and -4
(EHV-1/-4)\textsuperscript{8} and a separate vaccine against
rabies.\textsuperscript{9} All vaccines were administered
intramuscularly following label instructions and site of administration
was recorded in individual medical records.

Sixty-five healthy horses were enrolled in the study. Horses were either
client-owned with client consent or part of the Kansas State University
College of Veterinary Medicine (KSU-CVM) teaching herd horses. A
convenience sample of client-owned horses and all KSU-CVM teaching herd
horses that were due for their annual booster vaccinations during the
months of March through July were enrolled into the study. Healthy
horses were comprised of Quarter horses (n=39), Thoroughbreds (n=9),
Warmbloods (n=5), Appaloosas (n=3), Paints (n=3), Morgan (n=1),
Miniature (n=1), Mustang (n=1), Draft (n=1), Haflinger (n=1), and a
Tennessee Walking Horse (n=1). All horses were between 3 and 32 years of
age. Thirty-three were mares, 31 were geldings and 1 was a stallion.

All 65 healthy horses had blood samples obtained on days 0, 14, 30, 90
and 180 after vaccination. Nineteen of the 65 horses also had blood
samples obtained on day 270 after vaccination. Twenty-one of the 65
horses also had blood samples obtained on day 365 after vaccination.
Eight of the 65 horses had blood samples collected at both 270 and 365
days after vaccination in addition to the 0, 14, 30, 90, and 180 day
time points.

In an effort to determine if there was an age-associated response to
BSA-specific IgE expression following vaccination, horses were grouped
by age. Subject grouping included horses as young adult (3-9 years),
adult (10-14 years), middle aged (15-19 years) and geriatric (20-32
years) as detailed in Table 1.

\emph{Enrollment of horses that demonstrated severe adverse vaccine
reactions (reactors)}

A group of 21 horses were accepted into the study for sample submission
purposes. These samples were defined as a horse that had experienced a
severe adverse reaction after vaccination either previously or during
the course of the current study. A questionnaire was completed either by
the owner or primary veterinarian (supplemental information). Clinical
signs exhibited by these 21 horses included severe swelling at the
injection site, fever, colic, laminitis, lethargy, and markedly stiff
neck. Blood was collected and processed as previously described.
Fourteen horses had blood drawn within 24 hours, two at 180 days, three
at 270 days and two at unknown times after vaccination. Horses ranged
from 2 to 15 years of age while age was not documented for 3 horses.
There were 13 Warmbloods, 4 Quarter horses and 3 horses did not have
their breed reported. Thirteen were geldings and 8 were mares.

\emph{Enrollment of unvaccinated horses}

A group of 4 Quarter horses between 6-12 months of age were utilized in
the study that had never been vaccinated previously. Blood was collected
at a single time-point, processed and stored as described above. These
samples were used as negative controls.

\emph{Utilization of plasma samples from donor horses from a commercial
plasma manufacturer\textsuperscript{1}}

Based on the reaction of the neonatal foal to a commercial plasma
product, the decision was made to test additional aliquots of samples
from commercial plasma donor horses. Plasma samples were provided from
10 horses among the donor herd. Two 10 mL plasma samples from each donor
horse were collected and shipped to Kansas State University. These
samples were aliquoted into 2 mL vials and stored in a
-80\textsuperscript{o}C freezer. No information on the plasma donors was
provided other than that they were routine donor horses used for
commercial biologic product collection.

\emph{Utilization of serum from index case prior to plasma
administration}

A single serum sample was obtained from the one-day old neonatal colt
that received commercial powdered colostrum and experienced a fatal
anaphylactic reaction within the first 15 minutes of plasma
administration. The serum sample that was collected during the first 12
hours of life was stored at -80\textsuperscript{o}C prior to analysis.

\emph{Utilization of commercial plasma administered to the index case}

An aliquot of the commercial plasma that had been administered to the
index case described previously, which apparently contributed to a fatal
anaphylactic reaction was available for analysis. This sample was
aliquoted into 10 mL and 2 mL vials and stored at
-80\textsuperscript{o}C.

\emph{Indirect ELISA to detect BSA-specific IgE}

This protocol was adapted from Gershwin \emph{et al.} 2012 with
modifications. ELISA plates were coated with 1 ug/well BSA in
bicarbonate-carbonate buffer pH 9.6 overnight. ELISA wells were blocked
with 0.5\% rabbit serum albumin (RSA) in coating buffer. Each well in a
96 well plate was filled with 200 \selectlanguage{greek}µ\selectlanguage{english}L of 0.5\% RSA for blocking. The
plates were incubated at 4\textsuperscript{o}C overnight. After
incubation concluded, the wells were washed with 300 \selectlanguage{greek}µ\selectlanguage{english}L of PBSt (PBS +
0.1\% Tween-20) by an electronic plate washer 3 times. Plates were
coated and blocked in bulk and stored at 4@ \selectlanguage{english}C until use within one week.
The commercial plasma that was administered to the index case was used
as a positive control. Unvaccinated horse serum was used as a negative
control. PBS was used as a blank to establish background absorbance. Two
wells were designated as the positive control, two as the negative
control and two as the blank. Undiluted serum samples (100 \selectlanguage{greek}µ\selectlanguage{english}L) were
added to each remaining well in duplicate and incubated at room
temperature for 2 hours. A standard curve was not available and optical
density (OD) values were used to analyze the data.

Plates were washed as described above. Anti-horse IgE horse radish
peroxidase (HRP)\textsuperscript{10} was diluted out to 1:500 with wash
buffer, added to each well (100 \selectlanguage{greek}µ\selectlanguage{english}L) and incubated for 1 hour at room
temperature. The plates were washed again. Tetramethylbenzidine (TMB)
(100 \selectlanguage{greek}µ\selectlanguage{english}L) was added to each well and left to incubate for 30 minutes at
room temperature protected from the light. After 30 minutes 100 \selectlanguage{greek}µ\selectlanguage{english}L of
sulfuric acid (2 M SO2H4) was added to each well to terminate the
reaction. Plates were read with an electronic plate reader at 450 nm and
recorded to obtain the optical density.

All horse samples including 0, 14, 30, 90, 180, 270, and 365 day
post-vaccination, if available, were tested by way of ELISA for the
presence of BSA-specific IgE. All horses classified as a reactor were
tested by ELISA. Plasma from the plasma donor samples from commercial
source were all tested by ELISA. All plates contained the same positive
control, negative control and blanks. The OD value for blank wells was
averaged for each plate and then subtracted from each sample well to
correct for background absorbance. The adjusted duplicate sample OD
values were then averaged together for each horse.

\subsubsection*{Statistical analysis}

{\label{statistical-analysis}}

Statistical analysis was completed with Prism (v.8) on the BSA-specific
IgE ELISA data. Horses in the healthy vaccine group had their
post-vaccine Ig OD measurements compared with baseline values. Means
were determined for each timepoint for all age groups. Differences
between groups at specific timepoints were determined with t-tests.
Significance was defined as a \emph{p} -value \textless{}0.05. Data was
then compiled in Microsoft Excel as a spreadsheet function.

RESULTS

\subsubsection*{ELISA BSA-specific IgE}

{\label{elisa-bsa-specific-ige}}

The OD value for the positive control mean across all plates was 0.340.
The OD value for the negative control mean across all plates was 0.062.
The OD value for the blank control mean across all plates was 0.071. The
OD value for the positive control mean was significantly different from
the negative control mean (\emph{p} value \textless{}0.001). The OD
value for the negative control mean was significantly different from the
OD value blank control mean (\emph{p} value = 0.008).

\subsubsection*{Healthy Horses}

{\label{healthy-horses}}

The mean OD value +/- standard deviation for day 0 BSA-specific IgE in
all healthy horses (0.108 +/- 0.089) was not significantly different
when compared to the mean OD value +/- standard deviation (SD) for 14
days post-vaccination BSA-specific IgE in all healthy horses (0.124 +/-
0.118) (\emph{p} value = 0.43) (Figure 1). The mean OD value +/- SD for
day 0 BSA-specific IgE in all healthy horses (0.108 +/- 0.089) was
significantly higher when compared to the mean OD value +/- SD for 180
days post-vaccination BSA-specific IgE in all healthy horses (0.073 +/-
0.069) (\emph{p} value = 0.007) (Figure 1).

When comparing the fold change between day 0 BSA-specific IgE OD values
and 14 days post-vaccination BSA-specific IgE OD values, 63\% of horses
between the ages of 3 and 9 years had a fold increase greater than 1.5
(Figure 2 and Table 1). The percentage of horses displaying a fold
change greater than 1.5 decreased with age across age groups. Only 29\%
displayed a fold change greater than 1.5 in the 10-14 year old age
group, 0\% in the 15-19 year old age group and 5.6\% in the 20 -32 year
old age group (Table 1).

The mean BSA-specific IgE OD values +/- SD at time points 0, 14, 30, 90,
180, 270 and 365 days after vaccination were compared across age groups
(Table 1). The mean BSA-specific IgE OD value +/- SD for horses 3-9
years of age at day 0 was 0.072 +/- 0.086 and at 14 days
post-vaccination had increased to 0.125 +/- 0.09 (p-value = 0.013)
(Table 1). The mean BSA-specific IgE OD value +/- SD decreased to 0.070
+/- 0.070 at 180 days post-vaccination with days 0 and 180 being
significantly different (p-value = 0.035) (Table 1). All other
timepoints compared to day 0 within this age group were not
significantly different. When comparing all other age groups' mean
BSA-specific IgE OD values at 14 days after vaccination and day 0, the
differences were not significant. When comparing all other age groups'
mean BSA-specific IgE OD values at day 0 and 180 days after vaccination,
the differences were significant for age groups 10-14 years (p value =
0.001) and 20-32 years (p value = 0.028). In all of these age groups,
the mean BSA-specific IgE OD values decreased at 180 days
post-vaccination compared to time 0 and 14 days post-vaccination (Table
1). No other time points in any age group were significantly different
when compared to day 0.

Anamnestic response of BSA-specific IgE expression in response to annual
vaccination in healthy horses was most common in younger horses, aged
horses rarely demonstrated a \textgreater{} 1.5 fold change in
BSA-specific IgE expression within 14 days of annual booster vaccination
(Table 1).

Among young adult horses that were evaluated over the course of a year,
two-thirds of the horses demonstrated a greater than 1.5 fold increase
in BSA-specific IgE expression without adverse event (Figure 2). Among
horses greater than 10 years of age, it was uncommon to have a marked
increase in BSA-specific IgE expressed following booster vaccination
(Table 1).

\textbf{Horses demonstrating adverse vaccine reaction (Reactors)}

Horses with a history of severe adverse vaccine reaction were evaluated
for BSA-specific IgE expression at the time of evident vaccine reaction
(Figure 3). At the time of initial IgE assessment following a vaccine
reaction, mean BSA-specific IgE OD +/- SD for all reactors was 0.286 +/-
0.08. Comparing the mean BSA-specific OD values for reactors and healthy
horses at similar time points, there was a trend for higher OD values,
but the differences were not statistically significant (Figure 3). ELISA
measurement effectively allowed for an assessment of immunoglobulin
expression.

\textbf{Comparison among all samples}

Figure 4 provides a comparison of mean OD values for BSA-specific IgE
among all samples that were tested. The index case serum at 12 hours of
age and unvaccinated horses were among the lowest values measured.
Healthy horses and commercial plasma donor horses' values were
intermediate. The highest values were observed from reactor horses and
the commercial plasma sample administered to the index case.

DISCUSSION

Data from the current investigation as well as from previous work
demonstrate that horses express BSA-specific IgE following routine
vaccination. These findings are consistent with post-vaccination studies
in both humans (Hardefeldt \emph{et al.} 2010; Silva \emph{et al.} 2017)
and horses (Loughney \emph{et al.} 2014). As expected, horses that had
never been vaccinated did not demonstrate notable levels of BSA-specific
IgE. Among horses tested, the younger age group (3-9 years of age) had
more horses displaying a greater than 1.5-fold increase in BSA-specific
IgE at 2 weeks post-vaccination compared to baseline. This suggests that
younger horses develop a greater immune response to BSA than older
horses. Forty-six of the 65 healthy horses demonstrated an increase in
BSA-specific IgE during at least one time point after vaccination
compared to baseline. Sixteen of the 65 healthy horses showed a greater
than 1.5-fold increase in BSA-specific IgE at 14 days post-vaccination
compared to baseline. Six of these 16 horses had a greater than 4-fold
increase in BSA-specific IgE at 14 days post-vaccination. All of these 6
horses were between 3 and 11 years of age. Interestingly, none of these
65 horses developed clinical signs associated with an adverse vaccine
reaction. This is suggestive that the presence of BSA-specific IgE or
the rise in BSA-specific IgE does not directly correlate with the
development of an adverse vaccine reaction, which is consistent with the
previous report from Gershwin \emph{et al.} (2012).

Horses that had either historically or currently experienced an
immediate severe adverse vaccine reaction all had detectable levels of
BSA-specific IgE on ELISA. The majority of these horses had blood
samples collected within 24 hours post-vaccination. Ten out of the 12
reactor horse samples at 24 hours post-vaccination had an BSA-specific
IgE OD value greater than 0.200 (83.3\%). Whereas only 9 out of the 65
healthy horses had an BSA-specific IgE OD value greater than 0.200 at 14
days post-vaccination (13.8\%). Twenty-four-hour post-vaccination blood
samples from horses not experiencing a vaccine reaction could be
investigated by ELISA to determine a reference interval for BSA-specific
IgE in healthy horses. A very small subset of these reactor horses had
blood samples from 180- and 270-days post-vaccination. While their
BSA-specific IgE levels appeared to be higher than the healthy horses in
this study, these values were not statistically different from healthy
horses. A larger sample of horses is needed in order to further
investigate whether reactor horses have a significantly increased
BSA-specific IgE level at different time points post-vaccination. With
this information, horses developing a vaccine reaction could be tested
in order to determine if they have elevated BSA-specific IgE levels.
With reference intervals available, it could be possible to screen
horses prior to vaccination to determine if they are at a higher risk
for developing a vaccine reaction specifically against BSA.

Horses used as commercial plasma donors had BSA-specific IgE levels
similar to healthy, non-reactor horses. The history of these horses was
unknown. Important information would include the age of these horses,
how frequently they were vaccinated, when their last vaccine was
administered and how long they had been in the program for plasma
collection.

The index foal had serum collected at 12 hours of life which was
analyzed for BSA-specific IgE and was similar to a non-vaccinated horse.
Commercial plasma administered to the index foal was analyzed for
BSA-specific IgE and was found to have higher BSA-specific IgE compared
with healthy horses, commercial donor plasma or vaccine reactor horses.
Based on this finding, it appears that at least an individual commercial
plasma sample had elevated BSA-specific IgE levels and this could occur
in other donor horses although it appears uncommon. In the described
index case, the authors presume interaction of the high BSA-specific IgE
administered in plasma interacted with circulating BSA absorbed from the
powdered colostral product, which led to an immediate hypersensitivity
and anaphylactic reaction due to mast cell degranulation and acute
release of histamine and serotonin with resulting effects on the shock
organs of horses: respiratory and gastrointestinal tracts.

Two commercial plasma products administered to equine neonatal patients
are USDA-licensed for administration in equine neonates for the
treatment of failure of transfer of passive immunity. USDA-licensed
plasma\textsuperscript{1} is guaranteed to have an IgG concentration of
at least 2800 mg/dL. When plasma is administered to a foal that has been
exposed to BSA in commercial powdered colostrum, it has the possibility
to lead to a fatal, idiosyncratic reaction as described above. Among
available commercial plasma products,
manufacturers\textsuperscript{1,2}explicitly state that the product
should not be administered to neonatal foals that have received any
commercial powdered colostral or milk replacer product for this specific
reason.

Further investigation is indicated to determine the frequency of
elevated BSA-specific IgE among commercial plasma donor horses as well
as the kinetics of administered BSA-specific IgE in foals and when or if
it may be safe to administer plasma products to foals that have received
powered colostral products in their first 24 hours of life. Further
investigation is also warranted in larger number of reactor horses to
determine if BSA-specific IgE may be predictive of adverse vaccine
reactions. This study additionally brings up the practice of vaccine
manufacturing using BSA and its safety.

This study supports the theory that horses can mount an immune response
to BSA in vaccines by producing BSA-specific IgE. Based on the current
report, there is an indication to use caution upon administration of
commercial plasma to a neonate that has consumed powdered colostrum
supplementation prior to complete gut closure. Collectively, USDA
licensed plasma manufacturer guidelines should be followed to avoid
administration of greater than any one commercial IgG supplement to
neonatal foals.

\textbf{Author's declaration of interests}

No conflicts of interest have been declared

\textbf{Ethical animal research}

Written consent was obtained from each owner prior to participation in
the study which was approved by Kansas State University Institutional
Animal Care and Use Committee (IACUC \#4003).

\textbf{Source of funding}

MCAT College of Veterinary Medicine Intramural Grant Program Kansas
State University Grant

\textbf{Acknowledgements}

The authors would like to acknowledge Dr. Ann Rowlands, Ph. D. and
President of Lake Immunogenics, Inc. for providing plasma samples from
donor horses used for harvesting plasma products. Dr. Robert Larson,
KSU-CVM, was extremely helpful in directing the statistical analysis of
the data. We would also like to acknowledge Kara Smith (KSU-CVM) for
help procuring experimental supplies and Misty Bear (KSU-CVM) for
laboratory assistance with running ELISA plates. Finally, we would like
to acknowledge Dr. Jason Grady and Dr. Laurie Beard for assistance with
sample acquisition in the field.

\textbf{Authorship}

\textbf{ERP} was involved in experimental design, grant proposal, sample
collection, experimental data collection and analysis, and manuscript
drafting and revisions. \textbf{KMD} and \textbf{CAB} were involved in
experimental and study design, grant proposal, sample collection, data
analysis, and manuscript drafting and revisions. \textbf{KK}
,\textbf{NLS} , and \textbf{EGD} were all involved in experimental and
study design, grant proposal, experimental data collection and analysis,
and manuscript revisions. All authors reviewed the manuscript and
approved the final version.

\textbf{Manufacturers' addresses}

\begin{enumerate}
\tightlist
\item
  HiGamm Plasma, Lake Immunogenics, Inc. 348 Berg Rd, Ontario, NY 14519,
  USA
\item
  MG Biologics, Inc. 2366 270\textsuperscript{th} St, Ames, IA 50014,
  USA
\item
  Manna Pro Products LLC, 707 Spirit 40 Park Dr, Chesterfield, MO 6300,
  USA
\item
  Snap Foal IgG test, IDEXX Laboratories, Inc. 1 IDEXX Drive, Westbrook,
  Maine 04092, USA
\item
  Adrenaline, Par Pharmaceutical, 1 Ram Ridge Rd., Chestnut Ridge, NY
  10977
\item
  VetOne, MWI Animal Health, 3041 Pasadena Dr., Boise, ID 83705
\item
  VetOne, Sparhawk Laboratories Inc., 12340 Santa Fe Trail Dr., Lenexa,
  KS 66215
\item
  Vetera Gold, Boehringer-Ingelheim Pharmaceuticals, Inc. 900 Ridgebury
  Rd., Ridgefield, CT 06877, USA
\item
  EquiRab, Merck \& Co., Inc. 2000 Galloping Hill Road, Kenilworth, NJ
  07033
\item
  Anti-EQ IgE -- HRP, Bio-Rad Laboratories, 1000 Alfred Nobel Dr.,
  Hercules, CA 94547
\end{enumerate}

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\textbf{Table Legends}

Table 1: BSA-specific IgE fold changes according to age group. Within
each age group average fold change (Day 0 compared to 14 day
post-vaccine and 180 day post-vaccine) is provided. The percentage of
horses in each age group that displayed a fold change greater than 1.5
(Day 0 compared to 14 day post-vaccine) is also provided.

Table 2: BSA-specific mean OD values at day 0, 14 and 180 days after
vaccination according to age group. The difference between these time
points is listed as a p-value. A * indicates a significant difference
between the two timepoints (p value \textless{} 0.05)

\textbf{Figure Legends}

Figure 1: BSA-specific IgE OD values for all healthy horses for a 1-year
sampling period. Mean OD at each time point is represented by red X and
standard deviation by red horizontal line. Each individual horse is
represented by a circle in gray scale. Symbol * represents a time point
that differed significantly from day 0 (p value \textless{}0.05).

Figure 2: BSA-specific IgE values for healthy horses aged 3-9 years.
Each individual horse is labeled A-L, and colored bars represent time
points of BSA-specific IgE measurement after vaccination. A blue star
indicates post-vaccination time point where the fold change in anti-BSA
IgE was greater than 1.5 when compared to Day 0.

Figure 3\textbf{:} BSA-specific IgE for reactor horses. Individual
reactor horses are represented as blue circles for each of the time
points when collected. Purple triangles depict the mean ELISA OD
BSA-specific IgE value for the reactor horses at each time point. Red X
depict the mean ELISA OD BSA-specific IgE value for healthy horses at
each similar time point.

Figure 4: BSA-specific IgE mean OD values of the index case at 12 hours
of life (purple bar), unvaccinated horses (blue bar), healthy horses at
Day 0 (green bar), commercial donor horses (N=10) (yellow bar), reactor
horses at \textasciitilde{} 24 hours post-vaccination (red bar), and the
commercial plasma treatment administered to the index case (orange bar).\selectlanguage{english}
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