Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
(N/A)
Non Technical Summary
Vaccines are one of the best tools to prevent infectious diseases, but sometimes they do not work as well as expected, leaving animals at risk. This is especially important for horses, which travel frequently for competitions and breeding, making disease outbreaks costly and disruptive. At the same time, veterinarians often use corticosteroid injections in joints to treat pain and inflammation. These drugs can weaken the immune system, and research in humans shows that similar treatments may reduce vaccine effectiveness. The goal of this project is to find out whether giving a joint corticosteroid injection at the same time as a booster vaccine makes the vaccine less effective and increases the chance of illness. To do this, we will compare how horses respond to vaccines when they also receive a corticosteroid injection versus when they do not and whether this makes them more likely to get sick when exposed to influenza. We expect to learn whether this common practice reduces protection from disease. If it does, veterinarians can adjust treatment schedules to keep horses healthier. This research will help prevent costly outbreaks, protect horse welfare, and guide best practices for the equine industry. It may also lead to new recommendations for other livestock, improving animal health and supporting a strong agricultural economy.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
We hypothesize that intra-articular corticosteroid administration at the time of booster vaccination (equine influenza and equine herpes virus-1) will negatively impact the immunological response to that vaccine and increase subsequent susceptibility to an important infectious pathogen. While previous work in veterinary and equine medicine have sought to examine side effects of intra-articular corticosteroids, no investigations have been undertaken to understand the relationship between this common treatment and infectious disease susceptibility. Therefore, the specific objectives of this seed grant proposal are:To evaluate the immunologic response to booster vaccination when administered concurrently with an intra-articular corticosteroidTo determine whether concurrent booster vaccination and intra-articular corticosteroid administration leads to increased susceptibility to infectious disease challenge
Project Methods
Objective 1 - Evaluate the immunologic response to booster vaccination when administered concurrently with an intra-articular corticosteroidHorse selection:All horses will have a prior history of primary vaccination for the diseases listed below, as well as normal thyrotropin releasing hormone levels and oral sugar test results to confirm that they are free from equine endocrinopathies. Pre-study samples for equine influenza hemagglutinin inhibition (HI) antibody levels will be analyzed and used to block horses based on antibody concentrations, in addition to age and sex. A total of 20 horses will be used in this Objective, with 10 horses in each intra-articular injection treatment group. An additional 6 horses will be utilized as non-vaccinated controls, while being co-housed with the other horses and subjected to the same sampling regimen listed below.Sampling and IACs treatment:Immediately prior to intra-articular treatment and concurrent vaccination, blood samples will be collected for baseline antibody concentration determination and peripheral blood mononuclear cell (PBMC) isolation (see below). Horses will then be vaccinated using a commercially available, combined equine influenza/equine herpes virus product. Immediately following vaccination, individual horses will be bilaterally administered either 9 mg of triamcinolone acetonide or an equivalent volume of saline into both front metacarpophalangeal (fetlock) joints; this dose (18 mg total) and injection site mimics a common clinical scenario.Those team members responsible for sample analysis will be blinded to treatment assignments for individual horses. Subsequent blood samples for antibody titers (HI and serum neutralizing (SN) titers) and PBMC isolation (see below) will be collected weekly for the first month post-vaccination, followed thereafter with sampling every two weeks until challenge with equine influenza virus (Objective 2). Blood samples for EHV-1 virus neutralizing (VN) antibody titers will be collected every two weeks post-vaccination for 3 months.Assessment of the immunologic response to vaccination:Serum samples will be utilized to monitor the antibody titer response following vaccination to equine influenza (HI and SN tests - University of Kentucky) and equine herpes virus-1 (virus neutralizing antibody test).The test antigen for both tests will be an equine influenza Florida clade 1 strain, as this is similar to the vaccine strain and what is currently in circulation in the U.S.While the focus of this project is on equine influenza, EHV-1 remains a disease of significant impact to the equine industry and incorporation of VN titers for EHV-1 will provide additional value and information. PMBCs will be isolated and stimulated with inactivated, gradient-purified Florida clade 1 EIV or live EHV-1. Concanavalin A (ConA) will be used as a positive control and phosphate-buffered saline as a negative control. Following a 48 hour incubation period, PBMCs will be lysed in RNEasy-Plus RNP buffer (Qiagen Inc.) and total RNA will be isolated using the MagMAX Core Kit (ThermoFisher) and KingFisher Flex (ThermoFisher). Following isolation, mRNA will be reverse transcribed, and the resulting cDNA analyzed via qPCR (QuantStudio 7, ThermoFisher) with equine-specific, exon-spanning primer/probe sets for relative quantity differences in expression of granzyme-B, interferon-gamma, interleukin-1b (IL-1b), IL-10, and Perforin as markers of cell-mediated immune function. Each horse will act as its own internal control with their baseline samples being used to calculate the Relative Quantity (RQ) for each gene and time point.Objective 2 - Determine whether concurrent booster vaccination and intra-articular corticosteroid administration leads to increased susceptibility to infectious disease challengeEquine influenza virus challenge:Four months following vaccination, 5 horses from each group will be randomly selected and moved to the University of Kentucky's Biosafety Level 2 barn. Three, randomly selected, non-vaccinated control horses will also be used to ensure the challenge model and virus perform as expected. Horses will be challenged with a Florida clade 1 strain (influenza A/equine/Kentucky/14) using a face mask (Flexi-Neb II) for aerosol inhalation of 5 x 107EID50 units. To evaluate the effects of IACs on vaccination, all challenged horses will be observed daily for clinical signs of influenza for 10 days. Respiratory tract viral shedding will be measured starting the day of challenge until 7 days post infection, after which virus shedding has typically ceased. To measure virus shedding, nasopharyngeal swabs will be collected as described (Chambers et al. 2020b). Viral loads in nasopharyngeal swabs will be assessed daily by quantitative RT-PCR as described (Balasuriya et al. 2014) as well as by virus culture on days 2 and 3 post-challenge (maximum shedding) to determine the amount of infectious viral particles present. Daily clinical examinations by the same, treatment-blinded veterinarian will include rectal temperature, heart rate, respiratory rate and effort, auscultation of lung and gut sounds, palpation of submandibular and parotid lymph nodes, presence and consistency of nasal discharge, spontaneous coughing, and signs of depression or anorexia. Severity of clinical signs will be graded and recorded for statistical analysis.Clinically ill horses will be treated per IACUC guidelines.Two-way, repeated measures analysis of variance will be used to examine continuous parameters for differences by treatment, timepoint, and treatment by timepoint interactions. Days of viral shedding and individual horse clinical scores will be summed (both daily and all days) and analyzed using the Kruskal-Wallis test with post hoc, pairwise Wilcoxon tests (with correction), as needed. Additionally, the use of linear mixed-effects models will be utilized to examine relationships between parameters.