- Email: [email protected]
Comparison of Immunologic Responses Following Intranasal and Oral Administration of a USDA-Approved, Live-Attenuated Streptococcus equi Vaccine
Accepted Manuscript Comparison of immunologic responses following intranasal and oral administration of a USDA-approved, live-attenuated Streptococcus equi vaccine K.M. Delph, E.G. Davis, N.B. Bello, K. Hankins, M.J. Wilkerson, C.L. Ewen PII:
S0737-0806(16)30364-1
DOI:
10.1016/j.jevs.2016.08.015
Reference:
YJEVS 2174
To appear in:
Journal of Equine Veterinary Science
Received Date: 16 June 2016 Revised Date:
23 August 2016
Accepted Date: 25 August 2016
Please cite this article as: Delph KM, Davis EG, Bello NB, Hankins K, Wilkerson MJ, Ewen CL, Comparison of immunologic responses following intranasal and oral administration of a USDA-approved, live-attenuated Streptococcus equi vaccine, Journal of Equine Veterinary Science (2016), doi: 10.1016/ j.jevs.2016.08.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Original Research
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Comparison of immunologic responses following intranasal and oral administration of a
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USDA-approved, live-attenuated Streptococcus equi vaccine
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K. M. Delpha*, E. G. Davisa, N. B. Bellob, K. Hankinsd, M. J. Wilkersonc, C. L. Ewenc
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Departments of aClinical Sciences, bStatistics, cDiagnostic Medicine Pathobiology, Kansas State
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University, 1800 Denison Ave., Manhattan, KS 66506, USA; dZoetis, 100 campus Drive,
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Florham Park, NJ 07932, USA.
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*
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Corresponding author email: [email protected]
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Abstract
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While there is a commercially-available vaccine for Streptococcus equi subspecies equi licensed
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for the intranasal route of administration, some equine practitioners are administering this
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vaccine orally despite a lack of evidence for its efficacy by this route of administration. The
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purpose of this study was to compare systemic and local immune responses following intranasal
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or oral administration of the USDA-approved, live-attenuated Streptococcus equi subspecies
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equi vaccine [Pinnacle IN®]1. Eight healthy horses with low Streptococcus equi M protein
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(SeM) titers (<1:1600) were randomly assigned to an intranasal or oral two-vaccine series. SeM-
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specific serum immunoglobulins G (IgG) and A (IgA) and nasal secretion IgA were assessed
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using a commercially-available ELISA2 and a novel magnetic microsphere assay utilizing
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fluorescence. A general linear mixed models approach was used for statistical data analysis. As
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expected, intranasal vaccinates showed substantial increases in both serum SeM-specific IgG and
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IgA levels post-vaccination (P=0.0006 and P=0.007, respectively). Oral vaccinates showed an
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increase in serum SeM-specific IgG post-vaccination (P=0.0150), though only one-third the
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magnitude of intranasal vaccinates. Oral vaccinates showed no evidence of change in SeM-
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specific IgA post-vaccination (P=0.15). Results indicate that intranasal or oral vaccine
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administration resulted in increased serum SeM-specific IgG, though the magnitude of response
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differed between routes.
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Keywords: Streptococcus equi subsp. equi vaccination; horse; mucosal immunity; SeM titer
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1. Introduction Streptococcus equi subspecies equi, a gram-positive, Lancefield group C streptococci, is a highly contagious upper respiratory tract bacterial pathogen of horses. In susceptible
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populations, morbidity to S. equi can reach 100% [1]. Due to its high contagiousness and its
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short and long-term equine health implications, control of Streptococcus equi is of paramount
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importance to the equine industry. Infection with S. equi begins with entry into the mouth or
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nose, where the bacteria attaches to lingual, palatine, pharyngeal, and tubal tonsils [2]. Virulence
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factors including exposed surface proteins are involved in the attachment and penetration to
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tonsils, followed by translocation to submandibular and retropharyngeal lymph nodes, and
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finally prevention of bacterial phagocytosis and destruction by neutrophils [2, 3].
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After recovery, approximately 75% of horses develop a strong, enduring immunity that
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persists for 5 years or more [2]. Optimal immunity likely relies on both systemic and mucosal
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immune responses [4]; therefore, efficacious vaccines likely need to stimulate both for
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protection. A non-encapsulated, live-attenuated strain of S. equi [Pinnacle IN®]1 has
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demonstrated efficacy against experimental challenge [5]. The intranasal administration of this
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attenuated vaccine mimics natural exposure to the pathogen and thus intends to stimulate a
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similar immune response. Adverse effects have been associated with this vaccine including
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abscess formation at remote sites, development of disease, or purpura hemorrhagica [6]. Another
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common problem associated with intranasal vaccination is removal of the attenuated strain by
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sneeze-mediated expulsion of nasal secretions. Due to these challenges, the zoonotic potential of
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S. equi [7], and difficulty with the intranasal route of administration, some equine practitioners
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have adopted the oral route of vaccine administration for their equine patients. However, to date
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there is no evidence to support that oral vaccination elicits a systemic immune response that
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could potentially confer host protection to pathogen challenge. Therefore, the primary objective of this study was to compare immune responses
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following oral administration of the attenuated vaccine strain relative to that of the licensed
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intranasal route of vaccination. We expected that the attenuated vaccine strain would come in
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contact with the pharyngeal tonsils following oral administration, as occurs with natural
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infection, thereby eliciting a measurable immune response. We monitored the systemic immune
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response by means of serum SeM-specific immunoglobulins G (IgG) and A (IgA), and the
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mucosal immune response using nasal secretion SeM-specific IgA. Based on results,
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recommendations for the alternative route of vaccine administration can be made.
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2. Materials and Methods
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2.1 Subject selection and treatment allocation
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Eight healthy adult horses belonging to the Kansas State University College of Veterinary Medicine (CVM) teaching herd were used for this study. Horses in the teaching herd were
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closed to contact with outside horses throughout the course of investigation. The decision to use
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CVM owned horses was twofold. Horses considered for study were members of the CVM
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teaching herd for a minimum of 2 years, which assured that they did not suffer from S. equi
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infection or receive vaccination prior to investigation. Additionally, it was preferred that CVM
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horses were used for to avoid the potential for adverse reaction (e.g. purpura hemorrhagica) from
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occurring in privately owned horses. Ages ranged from 5-23 years (mean = 15 years) with body
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weights between 386-603 kg (mean = 526 kg) in horses selected for investigation. These horses
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were randomly assigned to receive either Streptococcus equi subsp. equi [Pinnacle IN®]1
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vaccination by the intranasal or oral route (n = 4 horses each). Horses received initial
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vaccination on Day 0, and a booster vaccination 3 weeks later on Day 21, consistent with
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manufacturer guidelines. Horses were maintained in individual stalls for 3 days following
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vaccination treatment to reduce the potential for nose-to-nose contact with any other horses
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following vaccination. After being housed individually, horses were maintained in a stall with
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run environment.
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grass hay daily with water available free choice and concentrate supplementation as needed. No
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feeding changes were made as a component of this investigation; horses were fed and maintained
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following routine management of the teaching herd of horses at Kansas State University College
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of Veterinary Medicine. Approval for this project was granted by the KSU Institutional Animal
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Care and Use Committee (IACUC #3285.1).
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2.2 Inclusion criteria and sample collection
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Consistent with CONSORT vaccine trial guidelines, thirteen CVM teaching herd horses were examined for eligibility. Inclusion criteria for this study included horses younger than 25
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years of age, determined to be healthy based on physical examination and baseline blood work
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(complete blood count and serum biochemistry), and that had a Streptococcus equi M protein
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(SeM) titer <1:1600 considered to be immunologically naïve with no history of previous
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vaccination or exposure/infection in at least the previous 24 months. The decision for selecting
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horses that had a SeM protein titer of <1:1600 was based on the current standard of practice in
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accordance with the American College of Veterinary Internal Medicine S. equi consensus
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statement [2]. Eight out of thirteen horses were selected to be included in the vaccine trial and
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were randomly allocated to receive either intranasal or oral vaccination. Host immunity was
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assessed via systemic and local SeM-specific humoral immune responses measured prior to and
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after vaccination. Recall that vaccine was administered by 2 different mucosal routes; consistent
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with previous equine studies aimed at detection of humoral immune activation, a detectable level
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of antibody secretion present in nasal secretions was expected by 2 weeks following vaccination
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[8]. Also, based on known kinetics of primary and anemnestic host immune responses, a 4 week
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sampling interval following booster vaccination was selected to measure a serum
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immunoglobulin response [8; 9]. Therefore, at two and four weeks post-booster vaccination,
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nasal secretion and serum samples were obtained from each horse. Whole blood and serum were
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collected via jugular venipuncture. Samples were centrifuged (3700 rpm for 10 minutes),
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aliquoted, and frozen at -80 degrees C until analysis.
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In an added effort to ensure that there was no potential for natural, environmental
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exposure to S. equi subsp. equi, three additional horses out of the thirteen examined for eligibility
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were used for analysis following intranasal saline administration. Timing of saline vaccination
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(2 dose, 21 day interval), sample collection, and sample processing were identical to vaccinate
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groups; however, due to CVM teaching horse availability, this surveillance experiment was
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performed at a separate time point one month following the vaccinates’ experiment. During this
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time there were no changes in teaching herd members or management. Analysis was performed
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separately for these three horses. This observational strategy was performed to provide absolute
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certainty that any immunologic changes identified in vaccinated horses were specific to S. equi
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vaccination and not environmental exposure. For collection of nasal secretion samples, horses were sedated with xylazine3 (0.5 mg/kg
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IV). This procedure was adapted from that described in horses [8] and cattle [10] to collect
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undiluted nasal secretions. A 4x4 gauze square was prepared as the nasal secretion collection
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device by rolling it cylindrically and applying an adhesive strip for detainment. The gauze was
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placed in the ventral nasal meatus of each horse, approximately 2 inches from the nostril opening
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and left in place for 20 minutes to allow for absorption of nasal secretions. The gauze square
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was then placed in a 50 mL conical tube. Once transported back to the laboratory, samples were
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spun down (2000 rpm for 30 minutes) to concentrate secretions. The collected nasal secretions
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were aliquoted and frozen at -80 degrees C until analysis.
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pathology laboratory at Kansas State University Veterinary Diagnostic Laboratory, and titers for
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serum IgG against SeM were quantified using an ELISA assay2.
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Serum and nasal secretion samples were tested for SeM-specific IgA immunologic response to vaccination using magnetic microspheres in a modified sandwich ELISA method
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described below.
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2.3 Protein coupling to magnetic microspheres
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Carboxylated magnetic microspheres (microsphere bead 48) were coated with
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Streptococcus equi subsp. equi M protein via a carbodiimide reaction. The protocol was
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supplied by Luminex®4 laboratories, as previously reported [11] and used 5 µg of recombinant
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protein (kindly provided courtesy of Dr. John Timoney, Gluck Equine Research Center,
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University of Kentucky, Lexington, Kentucky) per 1x106 microspheres. The SeM protein-specific IgA assay as described below was validated by using a
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negative control of bovine serum and a positive control of a horse with a robust response to
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vaccination (serum IgG SeM titer of 1:6400). Mean fluorescence intensities (MFI) were
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measured and considered relative SeM-specific IgA quantifications. Despite background MFI in
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the pre, 2-week, and 4-week post vaccination serum or nasal secretions samples, assessing both
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pre and post MFI measurements for each sample allowed for appropriate comparison and
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statistical analysis. MFI is not meant to be a measurement of absolute immunoglobulin
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concentration and cannot be correlated to immunoglobulin concentration at this point.
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2.4 Quantitation (in MFI) of SeM protein-specific IgA
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A 96 well plate map was prepared for replicate dilutions of experimental serum and nasal
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secretion samples. Dilutions used for both nasal secretion and serum samples were optimized at
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approximately 1:50 for SeM protein-specific IgA detection. A working solution of microspheres
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was prepared in phosphate-buffered solution (PBS) to allow for 5000 microspheres per well and
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50 µL per well. SeM protein-coated Region 48 microspheres were incubated with serum or
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nasal secretion samples.
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Serum or nasal secretion samples, including a blank negative control, and microsphere
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solution were aliquoted into each of the appropriate wells at 50 µL per well each. The plate was
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then sealed, protected from light, placed on a plate shaker, and allowed to incubate overnight at 4
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degrees C. The plate was then washed by placing it in the plate magnet for 1 minute, decanting
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the supernatant, and resuspending the microspheres in each well with 100 µL PBS. The wash
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step was repeated twice. The microspheres were resuspended out of the plate magnet with 50 µL
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PBS per well. A secondary antibody solution was prepared to allow for 50 µL per well using
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goat anti-horse IgA5 at a concentration of 4 µg/mL. The plate was again sealed, protected from
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light, placed on a plate shaker, and allowed to incubate for 1 hour. The plate was washed again 3
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times. A detection antibody solution was prepared to allow for 50 µL per well using
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pycoerythrin-conjugated donkey anti-goat IgG6 at a concentration of 4 µg/mL. The plate was
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incubated similarly for 30 minutes. The plate was washed three times and the microspheres were
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resuspended one final time with 100 µL of PBS per well.
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The data acquisition protocol was analyzed using the Luminex MagPix®4. The MFI reported by the Luminex MagPix®4 were used as a measure of relative IgA quantification for
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each sample.
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2.5 Statistical Analysis
Each of the responses, namely serum IgG SeM titers, as well as serum and nasal secretion
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SeM-specific IgA (measured in MFI) was expressed in the natural log scale and fitted with a
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general linear mixed model. The linear predictor in the model included the fixed effects of
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treatment (oral vs. intranasal administration), time (pre-vaccination and post-vaccination time
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point) and their 2-way interaction. The random effect of horse nested within treatment was fitted
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to the model to identify the experimental unit for treatment and the unit of repeated observations
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over time.
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Kenward Roger’s procedure was used to estimate degrees of freedom and adjust estimated standard errors. Model assumptions were evaluated using externally studentized
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residuals and were considered to be reasonably met.
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Statistical models were fitted using the GLIMMIX procedure of SAS7 implemented using Newton-Raphson with ridging as the optimization technique. Estimated least square means and
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95% confidence intervals are presented in the original scale of the data. Relevant pairwise
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comparisons were conducted using Bonferroni adjustment, as appropriate in each case to avoid
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inflation of Type I error rate due to multiple comparisons.
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3. Results
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Immediately prior to the start of the study, there was no evidence for differences in serum IgG SeM titers2 between horses assigned to receive intranasal or oral vaccination (Figure 1;
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P=0.83); neither was there any evidence for differences in relative SeM-specific IgA expression
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(in MFI) in serum or nasal secretions between groups (Figures 2 and 3; P=0.93 and P=0.37,
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respectively). This is consistent with the expectation that horses recruited for this pilot study
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were previously unvaccinated and immunologically naïve to S. equi.
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Two weeks following vaccination, an increase in serum SeM-specific IgA (in MFI) was
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apparent for intranasally-vaccinated horses only (Figure 2; P=0.0068); in turn, horses vaccinated
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through the oral route showed no evidence of changes in serum SeM-specific IgA (Figure 2;
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P=0.1504). Moreover, serum SeM-specific IgA levels by 2 weeks after vaccination were greater
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for horses vaccinated via the intranasal route than those vaccinated by the oral route (Figure 2;
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P=0.03). Regarding nasal secretion of SeM-specific IgA, neither group showed any evidence for
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changes from baseline to 2 weeks after the vaccine series (Figure 3; P=0.627). By 4 weeks following completion of the 2-dose vaccination series, all vaccinated horses
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showed a significant increase in serum IgG SeM titer2 regardless of route of vaccination (Figure
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1; P=0.0132); however, the increase was substantially greater for horses vaccinated by the
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intranasal route (P=0.0006) compared with those vaccinated orally (Figure 1; P=0.0150). The
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magnitude of the increase in IgG SeM titer relative to baseline was estimated at approximately
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10-fold for the intranasally-vaccinated horses, and at approximately 3-fold for the orally
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vaccinated horses.
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4. Discussion
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In the current report, horses were vaccinated with a USDA-approved, live-attenuated S. equi vaccine [Pinnacle IN®]1 either via the licensed intranasal route or via the oral route.
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Results confirmed that horses vaccinated using the licensed intranasal route had a substantial
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increase in both serum SeM-specific IgG and IgA antibody levels post-vaccination. Although a
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significant mean increase in IgG SeM titers were observed, it was noted that a considerable
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amount of variability was observed amongst horses, probably due to individual host immune
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responsiveness. Horses that were vaccinated using the oral route also showed an increase in the
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serum IgG SeM protein antibody level post-vaccination, but not to the same magnitude as
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intranasally-vaccinated horses. Orally-vaccinated horses also did not show evidence for any
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response in serum SeM-specific IgA following vaccination. Horses vaccinated with intranasal
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saline did not show evidence of change in serum IgG SeM titer, serum SeM-specific IgA, or
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nasal secretion SeM-specific IgA (Supplementary Figures 1, 2, and 3, respectively) from pre- to
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post-vaccination indicating the source of immunologic response in experimental horses was
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vaccine administration.
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Convalescent horses have been shown to develop a strong serum IgGb response to
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specific virulence factors of S. equi, in particular surface proteins (SeM), as well as strong SeM-
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specific mucosal IgA and IgGb responses [12; 13]. Further, IgA in convalescent horses was first
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detected at 3 weeks post-challenge [13]. However, neither the intranasal nor the oral routes of
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vaccination elicited significant changes in nasal secretion SeM-specific IgA response in this
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study. These results contrast with those of Timoney [4], who provided evidence that the
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attenuated intranasal S. equi vaccine induced both systemic and mucosal antibody responses.
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Changes in mucosal antibody responses may not have been identified in this study because of
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small changes in antibody response or timing of sample collection (i.e. if samples were collected
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before detectable IgA production). Also the method of nasal secretion collection used in this
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study may not have yielded as significant amounts of IgA as compared to other sampling
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methods like nasopharyngeal washes [13]; however, the adapted method of nasal secretion
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collection used in this study has been proven to collect undiluted nasal secretion samples
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previously [8, 10].
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The oral route of administration is aimed at exposing the pharyngeal tonsils to live-
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attenuated bacteria, which is expected to effectively enhance local mucosal antibody expression.
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Yet, previous work has demonstrated that different routes of immunization evoke immunologic
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responses in different systems of the body [14]. Oral administration is known to evoke antibody
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responses in the intestines, mammary and salivary glands in humans; whereas, nasal
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immunization results in antibody response in the upper airway mucosa and regional salivary and
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nasal secretions [14]. A previous study evaluated oral administration of a killed S. equi vaccine
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compared to intraperitoneal administration to control horses after challenge [15]. Both
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vaccinated groups developed submandibular abscesses but were not as clinically ill (i.e. did not
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develop fever, anorexia, malaise) as control horses following challenge. Further, horses
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vaccinated intraperitoneally developed IgG and IgA systemic and mucosal responses, but the
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horses vaccinated orally did not [15]. The oral route of administration is appealing for several
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practical reasons, even though it may not provide consistently superior immune activation and
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protection. In support of the utility to this route, it is plausible to suggest that the lack of
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systemic immune activation reported by Wallace, et al [15] may reflect inactivated vaccine
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preparation rather than the oral route of administration.
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In this investigation, no adverse effects to vaccination were found in any horses,
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consistent with other studies [5; Fort Dodge Animal Health licensing report TIA number:
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22740]. There has been work on the live-attenuated strain to remove the hasA and hasB genes
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making the microorganism acapsular and incapable of reversion to an encapsulated and more
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virulent form [16; 5]. In a study to assess the safety of the vaccine used in our study, it was
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determined that the vaccine was safe and efficacious in adult horses with low antibody titers but
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may cause clinical disease in young, naïve ponies [6]. Another study evaluated adverse
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behavioral responses to intranasal vaccine administration; while horses did not show any adverse
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responses, a few ponies did show substantial adverse reactions through avoidance behavior [17].
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On a separate note, we highlight that a novel microsphere assay was adopted here to measure immunologic response to S. equi vaccination. Potential advantages of the microsphere
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assay over the standard ELISA include smaller volumes of both protein and sample that are
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needed to perform the assay, as well as the potential to multiplex the assay to measure multiple
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analytes in the same sample [11]. Thus this microsphere assay could also be modified for the
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quantitation of individual immunoglobulins (in ng/ml) in the sample, versus serum titers, which
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may be useful for determining immunologic response to vaccination or immunologic status
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against S. equi with further development.
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4.1 Conclusions
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In conclusion, we identified responses in serum IgG SeM titer elicited by both intranasal
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and oral vaccination for S. equi using the USDA-approved, live-attenuated vaccine [Pinnacle
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IN®]1. However, the magnitude of the response was considerably greater following the
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intranasal route of vaccination. It is unknown whether the levels of serum IgG identified in
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orally vaccinated horses confer protection against S. equi. Further work is warranted to
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definitively determine the potential utility of oral vaccination in horses. Until that time,
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manufacturer guidelines for the USDA-approved, live-attenuated S. equi vaccine [Pinnacle
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IN®]1 should be followed to ensure a maximal immune response.
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Manufacturers’ addresses
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1
Pinnacle IN®, Zoetis, Florham Park, New Jersey
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2
SeM ELISA, Equine Diagnostics Solutions, LLC, Lexington, Kentucky
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3
AnaSed LA®, Lloyd Laboratories Inc, Quezon City, Philippines
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4
Luminex MagPix®, Luminex Corp., Austin, Texas
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5
Goat anti-horse IgA, Bethyl Laboratories Inc., Montgomery, Texas
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6
Pycoerythrin-conjugated donkey anti-goat IgG, Thermo Fisher Scientific Inc., Rockford, Illinois
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7
SAS Version 9.4, SAS Institute, Cary, NC
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Acknowledgements
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The authors thank Dr. John Timoney, Gluck Equine Research Center, University of Kentucky,
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Lexington, Kentucky for provision of S. equi M protein; Dr. Jennifer Morrow, Equine Diagnostic
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Solution, LLC, Lexington, Kentucky for performing SeM protein titer assays; and Kara Smith
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for technical assistance.
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Sources of funding
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This work was supported by funding from Zoetis Inc., Florham Park, NJ.
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Conflict of interest statement
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K. Hankins is employed by Zoetis Inc.
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References
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isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated
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horses. Vet Immunol Immunopathol 1997;59:239-251.
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[14] Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005;11:S45-S53.
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[15] Wallace FJ, Emery JD, Cripps AW, Husband AJ. An assessment of mucosal immunization
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in protection against Streptococcus equi (‘Strangles’) infections in horses. Vet Immunol
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Immunopathol 1995;48:139-154.
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[16] Waller AS, Jolley KA. Getting a grip on strangles: Recent progress towards improved
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diagnostics and vaccines. Vet J 2007;173:492-501.
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[17] Grogan EH, McDonnell SM. Behavioral responses to two intranasal vaccine applicators in
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horses and ponies. J Am Vet Med Assoc 2005;226:1689-1693.
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Figure legends:
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Figure 1: Least square mean estimates (and corresponding 95% confidence intervals) of serum
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IgG SeM titer (Equine Diagnostic Solutions, LLC)2 at baseline prior to vaccination (week 0) and
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4 weeks following a 2-dose vaccine series (week 7) for horses vaccinated via intranasal (purple
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diamond symbols) or oral (red cross symbols) routes. Arrows indicate time of vaccine
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administration.
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the study (i.e. 4 weeks post-booster) within each vaccination group.
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difference (P< 0.05) between intranasal and oral vaccinates at 7 weeks into the study. Point
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estimates were jittered horizontally to allow for clear visualization.
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Figure 2: Least square mean estimates (and corresponding 95% confidence intervals) of serum
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SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2 weeks following a 2-
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dose vaccine series (week 5) for horses vaccinated via the intranasal (purple diamond symbols)
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or oral (red cross symbols) routes. Arrows indicate time of vaccine administration.
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letters indicated difference (P< 0.05) between intranasal and oral vaccinates at 5 weeks into the
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study. Point estimates were jittered horizontally to allow for clear visualization.
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Figure 3: Least square mean estimates (and corresponding 95% confidence intervals) of nasal
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secretion SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2 weeks
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following a 2-dose vaccine series (week 5) for horses vaccinated via the intranasal (purple
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diamond symbols) or oral (red cross symbols) routes. Arrows indicate time of vaccine
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administration. Point estimates were jittered horizontally to allow for clear visualization.
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a,b
Different
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Supplementary Information Items
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Supplementary Figure 1
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Supplementary Figure 2
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Supplementary Figure 3
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Highlights
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There is current use of the licensed, intranasal (IN) S. equi vaccine orally
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Compared immunologic responses induced by IN S. equi vaccine to oral administration
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Serum SeM-specific IgG titers rose in response to both intranasal and oral vaccine
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Higher magnitude immunologic response with intranasal compared to oral vaccination
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Supplementary Information
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Supplementary Figure 1:
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Individual serum IgG SeM titers (Equine Diagnostic Solutions, LLC)2 at baseline prior to
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vaccination (week 0) and 4 weeks following a 2-dose vaccine series (week 7) for horses
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vaccinated with intranasal saline. Arrows indicate time of vaccine administration.
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Supplementary Figure 2:
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Individual serum SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2
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weeks following a 2-dose vaccine series (week 5) for horses vaccinated with intranasal
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saline. Arrows indicate time of vaccine administration.
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Supplementary Figure 3:
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Individual nasal secretion SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0)
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and 2 weeks following a 2-dose vaccine series (week 5) for horses vaccinated with intranasal
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saline. Arrows indicate time of vaccine administration.
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S0737-0806(16)30364-1
DOI:
10.1016/j.jevs.2016.08.015
Reference:
YJEVS 2174
To appear in:
Journal of Equine Veterinary Science
Received Date: 16 June 2016 Revised Date:
23 August 2016
Accepted Date: 25 August 2016
Please cite this article as: Delph KM, Davis EG, Bello NB, Hankins K, Wilkerson MJ, Ewen CL, Comparison of immunologic responses following intranasal and oral administration of a USDA-approved, live-attenuated Streptococcus equi vaccine, Journal of Equine Veterinary Science (2016), doi: 10.1016/ j.jevs.2016.08.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Original Research
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Comparison of immunologic responses following intranasal and oral administration of a
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USDA-approved, live-attenuated Streptococcus equi vaccine
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K. M. Delpha*, E. G. Davisa, N. B. Bellob, K. Hankinsd, M. J. Wilkersonc, C. L. Ewenc
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Departments of aClinical Sciences, bStatistics, cDiagnostic Medicine Pathobiology, Kansas State
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University, 1800 Denison Ave., Manhattan, KS 66506, USA; dZoetis, 100 campus Drive,
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Florham Park, NJ 07932, USA.
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*
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Corresponding author email: [email protected]
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Abstract
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While there is a commercially-available vaccine for Streptococcus equi subspecies equi licensed
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for the intranasal route of administration, some equine practitioners are administering this
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vaccine orally despite a lack of evidence for its efficacy by this route of administration. The
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purpose of this study was to compare systemic and local immune responses following intranasal
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or oral administration of the USDA-approved, live-attenuated Streptococcus equi subspecies
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equi vaccine [Pinnacle IN®]1. Eight healthy horses with low Streptococcus equi M protein
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(SeM) titers (<1:1600) were randomly assigned to an intranasal or oral two-vaccine series. SeM-
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specific serum immunoglobulins G (IgG) and A (IgA) and nasal secretion IgA were assessed
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using a commercially-available ELISA2 and a novel magnetic microsphere assay utilizing
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fluorescence. A general linear mixed models approach was used for statistical data analysis. As
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expected, intranasal vaccinates showed substantial increases in both serum SeM-specific IgG and
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IgA levels post-vaccination (P=0.0006 and P=0.007, respectively). Oral vaccinates showed an
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increase in serum SeM-specific IgG post-vaccination (P=0.0150), though only one-third the
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magnitude of intranasal vaccinates. Oral vaccinates showed no evidence of change in SeM-
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specific IgA post-vaccination (P=0.15). Results indicate that intranasal or oral vaccine
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administration resulted in increased serum SeM-specific IgG, though the magnitude of response
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differed between routes.
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Keywords: Streptococcus equi subsp. equi vaccination; horse; mucosal immunity; SeM titer
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1. Introduction Streptococcus equi subspecies equi, a gram-positive, Lancefield group C streptococci, is a highly contagious upper respiratory tract bacterial pathogen of horses. In susceptible
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populations, morbidity to S. equi can reach 100% [1]. Due to its high contagiousness and its
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short and long-term equine health implications, control of Streptococcus equi is of paramount
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importance to the equine industry. Infection with S. equi begins with entry into the mouth or
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nose, where the bacteria attaches to lingual, palatine, pharyngeal, and tubal tonsils [2]. Virulence
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factors including exposed surface proteins are involved in the attachment and penetration to
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tonsils, followed by translocation to submandibular and retropharyngeal lymph nodes, and
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finally prevention of bacterial phagocytosis and destruction by neutrophils [2, 3].
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After recovery, approximately 75% of horses develop a strong, enduring immunity that
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persists for 5 years or more [2]. Optimal immunity likely relies on both systemic and mucosal
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immune responses [4]; therefore, efficacious vaccines likely need to stimulate both for
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protection. A non-encapsulated, live-attenuated strain of S. equi [Pinnacle IN®]1 has
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demonstrated efficacy against experimental challenge [5]. The intranasal administration of this
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attenuated vaccine mimics natural exposure to the pathogen and thus intends to stimulate a
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similar immune response. Adverse effects have been associated with this vaccine including
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abscess formation at remote sites, development of disease, or purpura hemorrhagica [6]. Another
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common problem associated with intranasal vaccination is removal of the attenuated strain by
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sneeze-mediated expulsion of nasal secretions. Due to these challenges, the zoonotic potential of
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S. equi [7], and difficulty with the intranasal route of administration, some equine practitioners
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have adopted the oral route of vaccine administration for their equine patients. However, to date
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there is no evidence to support that oral vaccination elicits a systemic immune response that
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could potentially confer host protection to pathogen challenge. Therefore, the primary objective of this study was to compare immune responses
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following oral administration of the attenuated vaccine strain relative to that of the licensed
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intranasal route of vaccination. We expected that the attenuated vaccine strain would come in
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contact with the pharyngeal tonsils following oral administration, as occurs with natural
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infection, thereby eliciting a measurable immune response. We monitored the systemic immune
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response by means of serum SeM-specific immunoglobulins G (IgG) and A (IgA), and the
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mucosal immune response using nasal secretion SeM-specific IgA. Based on results,
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recommendations for the alternative route of vaccine administration can be made.
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2. Materials and Methods
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2.1 Subject selection and treatment allocation
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Eight healthy adult horses belonging to the Kansas State University College of Veterinary Medicine (CVM) teaching herd were used for this study. Horses in the teaching herd were
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closed to contact with outside horses throughout the course of investigation. The decision to use
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CVM owned horses was twofold. Horses considered for study were members of the CVM
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teaching herd for a minimum of 2 years, which assured that they did not suffer from S. equi
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infection or receive vaccination prior to investigation. Additionally, it was preferred that CVM
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horses were used for to avoid the potential for adverse reaction (e.g. purpura hemorrhagica) from
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occurring in privately owned horses. Ages ranged from 5-23 years (mean = 15 years) with body
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weights between 386-603 kg (mean = 526 kg) in horses selected for investigation. These horses
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were randomly assigned to receive either Streptococcus equi subsp. equi [Pinnacle IN®]1
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vaccination by the intranasal or oral route (n = 4 horses each). Horses received initial
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vaccination on Day 0, and a booster vaccination 3 weeks later on Day 21, consistent with
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manufacturer guidelines. Horses were maintained in individual stalls for 3 days following
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vaccination treatment to reduce the potential for nose-to-nose contact with any other horses
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following vaccination. After being housed individually, horses were maintained in a stall with
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run environment.
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grass hay daily with water available free choice and concentrate supplementation as needed. No
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feeding changes were made as a component of this investigation; horses were fed and maintained
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following routine management of the teaching herd of horses at Kansas State University College
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of Veterinary Medicine. Approval for this project was granted by the KSU Institutional Animal
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Care and Use Committee (IACUC #3285.1).
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2.2 Inclusion criteria and sample collection
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Consistent with CONSORT vaccine trial guidelines, thirteen CVM teaching herd horses were examined for eligibility. Inclusion criteria for this study included horses younger than 25
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years of age, determined to be healthy based on physical examination and baseline blood work
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(complete blood count and serum biochemistry), and that had a Streptococcus equi M protein
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(SeM) titer <1:1600 considered to be immunologically naïve with no history of previous
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vaccination or exposure/infection in at least the previous 24 months. The decision for selecting
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horses that had a SeM protein titer of <1:1600 was based on the current standard of practice in
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accordance with the American College of Veterinary Internal Medicine S. equi consensus
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statement [2]. Eight out of thirteen horses were selected to be included in the vaccine trial and
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were randomly allocated to receive either intranasal or oral vaccination. Host immunity was
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assessed via systemic and local SeM-specific humoral immune responses measured prior to and
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after vaccination. Recall that vaccine was administered by 2 different mucosal routes; consistent
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with previous equine studies aimed at detection of humoral immune activation, a detectable level
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of antibody secretion present in nasal secretions was expected by 2 weeks following vaccination
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[8]. Also, based on known kinetics of primary and anemnestic host immune responses, a 4 week
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sampling interval following booster vaccination was selected to measure a serum
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immunoglobulin response [8; 9]. Therefore, at two and four weeks post-booster vaccination,
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nasal secretion and serum samples were obtained from each horse. Whole blood and serum were
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collected via jugular venipuncture. Samples were centrifuged (3700 rpm for 10 minutes),
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aliquoted, and frozen at -80 degrees C until analysis.
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In an added effort to ensure that there was no potential for natural, environmental
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exposure to S. equi subsp. equi, three additional horses out of the thirteen examined for eligibility
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were used for analysis following intranasal saline administration. Timing of saline vaccination
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(2 dose, 21 day interval), sample collection, and sample processing were identical to vaccinate
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groups; however, due to CVM teaching horse availability, this surveillance experiment was
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performed at a separate time point one month following the vaccinates’ experiment. During this
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time there were no changes in teaching herd members or management. Analysis was performed
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separately for these three horses. This observational strategy was performed to provide absolute
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certainty that any immunologic changes identified in vaccinated horses were specific to S. equi
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vaccination and not environmental exposure. For collection of nasal secretion samples, horses were sedated with xylazine3 (0.5 mg/kg
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IV). This procedure was adapted from that described in horses [8] and cattle [10] to collect
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undiluted nasal secretions. A 4x4 gauze square was prepared as the nasal secretion collection
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device by rolling it cylindrically and applying an adhesive strip for detainment. The gauze was
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placed in the ventral nasal meatus of each horse, approximately 2 inches from the nostril opening
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and left in place for 20 minutes to allow for absorption of nasal secretions. The gauze square
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was then placed in a 50 mL conical tube. Once transported back to the laboratory, samples were
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spun down (2000 rpm for 30 minutes) to concentrate secretions. The collected nasal secretions
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were aliquoted and frozen at -80 degrees C until analysis.
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pathology laboratory at Kansas State University Veterinary Diagnostic Laboratory, and titers for
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serum IgG against SeM were quantified using an ELISA assay2.
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Serum and nasal secretion samples were tested for SeM-specific IgA immunologic response to vaccination using magnetic microspheres in a modified sandwich ELISA method
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described below.
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2.3 Protein coupling to magnetic microspheres
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Carboxylated magnetic microspheres (microsphere bead 48) were coated with
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Streptococcus equi subsp. equi M protein via a carbodiimide reaction. The protocol was
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supplied by Luminex®4 laboratories, as previously reported [11] and used 5 µg of recombinant
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protein (kindly provided courtesy of Dr. John Timoney, Gluck Equine Research Center,
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University of Kentucky, Lexington, Kentucky) per 1x106 microspheres. The SeM protein-specific IgA assay as described below was validated by using a
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negative control of bovine serum and a positive control of a horse with a robust response to
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vaccination (serum IgG SeM titer of 1:6400). Mean fluorescence intensities (MFI) were
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measured and considered relative SeM-specific IgA quantifications. Despite background MFI in
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the pre, 2-week, and 4-week post vaccination serum or nasal secretions samples, assessing both
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pre and post MFI measurements for each sample allowed for appropriate comparison and
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statistical analysis. MFI is not meant to be a measurement of absolute immunoglobulin
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concentration and cannot be correlated to immunoglobulin concentration at this point.
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2.4 Quantitation (in MFI) of SeM protein-specific IgA
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A 96 well plate map was prepared for replicate dilutions of experimental serum and nasal
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secretion samples. Dilutions used for both nasal secretion and serum samples were optimized at
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approximately 1:50 for SeM protein-specific IgA detection. A working solution of microspheres
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was prepared in phosphate-buffered solution (PBS) to allow for 5000 microspheres per well and
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50 µL per well. SeM protein-coated Region 48 microspheres were incubated with serum or
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nasal secretion samples.
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Serum or nasal secretion samples, including a blank negative control, and microsphere
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solution were aliquoted into each of the appropriate wells at 50 µL per well each. The plate was
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then sealed, protected from light, placed on a plate shaker, and allowed to incubate overnight at 4
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degrees C. The plate was then washed by placing it in the plate magnet for 1 minute, decanting
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the supernatant, and resuspending the microspheres in each well with 100 µL PBS. The wash
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step was repeated twice. The microspheres were resuspended out of the plate magnet with 50 µL
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PBS per well. A secondary antibody solution was prepared to allow for 50 µL per well using
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goat anti-horse IgA5 at a concentration of 4 µg/mL. The plate was again sealed, protected from
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light, placed on a plate shaker, and allowed to incubate for 1 hour. The plate was washed again 3
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times. A detection antibody solution was prepared to allow for 50 µL per well using
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pycoerythrin-conjugated donkey anti-goat IgG6 at a concentration of 4 µg/mL. The plate was
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incubated similarly for 30 minutes. The plate was washed three times and the microspheres were
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resuspended one final time with 100 µL of PBS per well.
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The data acquisition protocol was analyzed using the Luminex MagPix®4. The MFI reported by the Luminex MagPix®4 were used as a measure of relative IgA quantification for
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each sample.
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2.5 Statistical Analysis
Each of the responses, namely serum IgG SeM titers, as well as serum and nasal secretion
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SeM-specific IgA (measured in MFI) was expressed in the natural log scale and fitted with a
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general linear mixed model. The linear predictor in the model included the fixed effects of
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treatment (oral vs. intranasal administration), time (pre-vaccination and post-vaccination time
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point) and their 2-way interaction. The random effect of horse nested within treatment was fitted
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to the model to identify the experimental unit for treatment and the unit of repeated observations
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over time.
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Kenward Roger’s procedure was used to estimate degrees of freedom and adjust estimated standard errors. Model assumptions were evaluated using externally studentized
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residuals and were considered to be reasonably met.
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Statistical models were fitted using the GLIMMIX procedure of SAS7 implemented using Newton-Raphson with ridging as the optimization technique. Estimated least square means and
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95% confidence intervals are presented in the original scale of the data. Relevant pairwise
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comparisons were conducted using Bonferroni adjustment, as appropriate in each case to avoid
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inflation of Type I error rate due to multiple comparisons.
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3. Results
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Immediately prior to the start of the study, there was no evidence for differences in serum IgG SeM titers2 between horses assigned to receive intranasal or oral vaccination (Figure 1;
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P=0.83); neither was there any evidence for differences in relative SeM-specific IgA expression
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(in MFI) in serum or nasal secretions between groups (Figures 2 and 3; P=0.93 and P=0.37,
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respectively). This is consistent with the expectation that horses recruited for this pilot study
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were previously unvaccinated and immunologically naïve to S. equi.
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apparent for intranasally-vaccinated horses only (Figure 2; P=0.0068); in turn, horses vaccinated
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through the oral route showed no evidence of changes in serum SeM-specific IgA (Figure 2;
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P=0.1504). Moreover, serum SeM-specific IgA levels by 2 weeks after vaccination were greater
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for horses vaccinated via the intranasal route than those vaccinated by the oral route (Figure 2;
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P=0.03). Regarding nasal secretion of SeM-specific IgA, neither group showed any evidence for
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changes from baseline to 2 weeks after the vaccine series (Figure 3; P=0.627). By 4 weeks following completion of the 2-dose vaccination series, all vaccinated horses
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showed a significant increase in serum IgG SeM titer2 regardless of route of vaccination (Figure
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1; P=0.0132); however, the increase was substantially greater for horses vaccinated by the
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intranasal route (P=0.0006) compared with those vaccinated orally (Figure 1; P=0.0150). The
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magnitude of the increase in IgG SeM titer relative to baseline was estimated at approximately
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10-fold for the intranasally-vaccinated horses, and at approximately 3-fold for the orally
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vaccinated horses.
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4. Discussion
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In the current report, horses were vaccinated with a USDA-approved, live-attenuated S. equi vaccine [Pinnacle IN®]1 either via the licensed intranasal route or via the oral route.
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Results confirmed that horses vaccinated using the licensed intranasal route had a substantial
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increase in both serum SeM-specific IgG and IgA antibody levels post-vaccination. Although a
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significant mean increase in IgG SeM titers were observed, it was noted that a considerable
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amount of variability was observed amongst horses, probably due to individual host immune
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responsiveness. Horses that were vaccinated using the oral route also showed an increase in the
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serum IgG SeM protein antibody level post-vaccination, but not to the same magnitude as
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intranasally-vaccinated horses. Orally-vaccinated horses also did not show evidence for any
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response in serum SeM-specific IgA following vaccination. Horses vaccinated with intranasal
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saline did not show evidence of change in serum IgG SeM titer, serum SeM-specific IgA, or
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nasal secretion SeM-specific IgA (Supplementary Figures 1, 2, and 3, respectively) from pre- to
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post-vaccination indicating the source of immunologic response in experimental horses was
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vaccine administration.
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Convalescent horses have been shown to develop a strong serum IgGb response to
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specific virulence factors of S. equi, in particular surface proteins (SeM), as well as strong SeM-
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specific mucosal IgA and IgGb responses [12; 13]. Further, IgA in convalescent horses was first
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detected at 3 weeks post-challenge [13]. However, neither the intranasal nor the oral routes of
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vaccination elicited significant changes in nasal secretion SeM-specific IgA response in this
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study. These results contrast with those of Timoney [4], who provided evidence that the
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attenuated intranasal S. equi vaccine induced both systemic and mucosal antibody responses.
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Changes in mucosal antibody responses may not have been identified in this study because of
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small changes in antibody response or timing of sample collection (i.e. if samples were collected
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before detectable IgA production). Also the method of nasal secretion collection used in this
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study may not have yielded as significant amounts of IgA as compared to other sampling
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methods like nasopharyngeal washes [13]; however, the adapted method of nasal secretion
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collection used in this study has been proven to collect undiluted nasal secretion samples
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previously [8, 10].
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The oral route of administration is aimed at exposing the pharyngeal tonsils to live-
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attenuated bacteria, which is expected to effectively enhance local mucosal antibody expression.
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Yet, previous work has demonstrated that different routes of immunization evoke immunologic
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responses in different systems of the body [14]. Oral administration is known to evoke antibody
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responses in the intestines, mammary and salivary glands in humans; whereas, nasal
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immunization results in antibody response in the upper airway mucosa and regional salivary and
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nasal secretions [14]. A previous study evaluated oral administration of a killed S. equi vaccine
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compared to intraperitoneal administration to control horses after challenge [15]. Both
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vaccinated groups developed submandibular abscesses but were not as clinically ill (i.e. did not
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develop fever, anorexia, malaise) as control horses following challenge. Further, horses
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vaccinated intraperitoneally developed IgG and IgA systemic and mucosal responses, but the
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horses vaccinated orally did not [15]. The oral route of administration is appealing for several
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practical reasons, even though it may not provide consistently superior immune activation and
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protection. In support of the utility to this route, it is plausible to suggest that the lack of
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systemic immune activation reported by Wallace, et al [15] may reflect inactivated vaccine
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preparation rather than the oral route of administration.
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consistent with other studies [5; Fort Dodge Animal Health licensing report TIA number:
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22740]. There has been work on the live-attenuated strain to remove the hasA and hasB genes
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making the microorganism acapsular and incapable of reversion to an encapsulated and more
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virulent form [16; 5]. In a study to assess the safety of the vaccine used in our study, it was
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determined that the vaccine was safe and efficacious in adult horses with low antibody titers but
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may cause clinical disease in young, naïve ponies [6]. Another study evaluated adverse
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behavioral responses to intranasal vaccine administration; while horses did not show any adverse
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responses, a few ponies did show substantial adverse reactions through avoidance behavior [17].
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On a separate note, we highlight that a novel microsphere assay was adopted here to measure immunologic response to S. equi vaccination. Potential advantages of the microsphere
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assay over the standard ELISA include smaller volumes of both protein and sample that are
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needed to perform the assay, as well as the potential to multiplex the assay to measure multiple
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analytes in the same sample [11]. Thus this microsphere assay could also be modified for the
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quantitation of individual immunoglobulins (in ng/ml) in the sample, versus serum titers, which
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may be useful for determining immunologic response to vaccination or immunologic status
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against S. equi with further development.
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4.1 Conclusions
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In conclusion, we identified responses in serum IgG SeM titer elicited by both intranasal
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and oral vaccination for S. equi using the USDA-approved, live-attenuated vaccine [Pinnacle
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IN®]1. However, the magnitude of the response was considerably greater following the
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intranasal route of vaccination. It is unknown whether the levels of serum IgG identified in
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orally vaccinated horses confer protection against S. equi. Further work is warranted to
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definitively determine the potential utility of oral vaccination in horses. Until that time,
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manufacturer guidelines for the USDA-approved, live-attenuated S. equi vaccine [Pinnacle
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IN®]1 should be followed to ensure a maximal immune response.
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Manufacturers’ addresses
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1
Pinnacle IN®, Zoetis, Florham Park, New Jersey
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2
SeM ELISA, Equine Diagnostics Solutions, LLC, Lexington, Kentucky
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3
AnaSed LA®, Lloyd Laboratories Inc, Quezon City, Philippines
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Luminex MagPix®, Luminex Corp., Austin, Texas
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5
Goat anti-horse IgA, Bethyl Laboratories Inc., Montgomery, Texas
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6
Pycoerythrin-conjugated donkey anti-goat IgG, Thermo Fisher Scientific Inc., Rockford, Illinois
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7
SAS Version 9.4, SAS Institute, Cary, NC
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Acknowledgements
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The authors thank Dr. John Timoney, Gluck Equine Research Center, University of Kentucky,
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Lexington, Kentucky for provision of S. equi M protein; Dr. Jennifer Morrow, Equine Diagnostic
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Solution, LLC, Lexington, Kentucky for performing SeM protein titer assays; and Kara Smith
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for technical assistance.
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Sources of funding
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This work was supported by funding from Zoetis Inc., Florham Park, NJ.
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Conflict of interest statement
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K. Hankins is employed by Zoetis Inc.
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References
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pp 306-311.
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[10] McKercher DG, Kaneko JJ, Mills RJ, Wada EM. Simple method for obtaining undiluted
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[13] Sheoran AS, Sponseller BT, Holmes MA, Timoney JF. Serum and mucosal antibody
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isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated
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horses. Vet Immunol Immunopathol 1997;59:239-251.
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[15] Wallace FJ, Emery JD, Cripps AW, Husband AJ. An assessment of mucosal immunization
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Figure legends:
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Figure 1: Least square mean estimates (and corresponding 95% confidence intervals) of serum
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IgG SeM titer (Equine Diagnostic Solutions, LLC)2 at baseline prior to vaccination (week 0) and
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4 weeks following a 2-dose vaccine series (week 7) for horses vaccinated via intranasal (purple
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diamond symbols) or oral (red cross symbols) routes. Arrows indicate time of vaccine
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administration.
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the study (i.e. 4 weeks post-booster) within each vaccination group.
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difference (P< 0.05) between intranasal and oral vaccinates at 7 weeks into the study. Point
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estimates were jittered horizontally to allow for clear visualization.
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Figure 2: Least square mean estimates (and corresponding 95% confidence intervals) of serum
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SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2 weeks following a 2-
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dose vaccine series (week 5) for horses vaccinated via the intranasal (purple diamond symbols)
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or oral (red cross symbols) routes. Arrows indicate time of vaccine administration.
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letters indicated difference (P< 0.05) between intranasal and oral vaccinates at 5 weeks into the
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study. Point estimates were jittered horizontally to allow for clear visualization.
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Figure 3: Least square mean estimates (and corresponding 95% confidence intervals) of nasal
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secretion SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2 weeks
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following a 2-dose vaccine series (week 5) for horses vaccinated via the intranasal (purple
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diamond symbols) or oral (red cross symbols) routes. Arrows indicate time of vaccine
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administration. Point estimates were jittered horizontally to allow for clear visualization.
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Supplementary Information Items
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Supplementary Figure 1
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Supplementary Figure 2
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Supplementary Figure 3
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Highlights
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There is current use of the licensed, intranasal (IN) S. equi vaccine orally
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Compared immunologic responses induced by IN S. equi vaccine to oral administration
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Serum SeM-specific IgG titers rose in response to both intranasal and oral vaccine
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Higher magnitude immunologic response with intranasal compared to oral vaccination
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Supplementary Information
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Supplementary Figure 1:
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Individual serum IgG SeM titers (Equine Diagnostic Solutions, LLC)2 at baseline prior to
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vaccination (week 0) and 4 weeks following a 2-dose vaccine series (week 7) for horses
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vaccinated with intranasal saline. Arrows indicate time of vaccine administration.
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Supplementary Figure 2:
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Individual serum SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0) and 2
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weeks following a 2-dose vaccine series (week 5) for horses vaccinated with intranasal
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saline. Arrows indicate time of vaccine administration.
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Supplementary Figure 3:
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Individual nasal secretion SeM-specific IgA (in MFI) at baseline prior to vaccination (week 0)
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and 2 weeks following a 2-dose vaccine series (week 5) for horses vaccinated with intranasal
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saline. Arrows indicate time of vaccine administration.
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