Development and Evaluation of a Rapid Diagnostic Kit for Equine Influenza Detection

Thesis Abstract

Equine influenza remains a major infectious disease affecting performance, economic stability, and animal health within the equine industry globally, largely due to limitations of existing diagnostic methods that often require specialized laboratories, extended turnaround times, and are cost-prohibitive for widespread field application. This study aims to develop and evaluate a rapid, accurate, and cost-effective diagnostic kit for detecting equine influenza virus (EIV) suitable for use in field conditions. The specific objectives are to design a lateral flow immunoassay (LFIA) that targets conserved epitopes of the hemagglutinin (HA) and nucleoprotein (NP) of EIV, optimize the assay’s analytical performance, and assess its diagnostic accuracy, sensitivity, specificity, and practicality compared to standard laboratory-based methods such as reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation. The research adopts an experimental diagnostic test development design grounded in the theoretical framework of the Health Belief Model, emphasizing the importance of perceived susceptibility, severity, benefits, and barriers to prompt diagnostic testing. The study population comprises 200 domestic horses from equine facilities within the region, stratified into symptomatic and asymptomatic groups. A purposive sampling technique is used to select samples ensuring representation across different clinical stages. Blood, nasal swab, and serum samples are collected from each subject, with nasal swabs subjected to both the newly developed LFIA and reference standards (RT-PCR and virus culture). Data collection instruments include validated immunoassay components, while the assay performance is quantified through analytical validation procedures such as limit of detection (LOD), reproducibility, and cross-reactivity testing. The evaluation employs statistical techniques including receiver operating characteristic (ROC) curve analysis, calculation of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and Cohen’s kappa for agreement assessment. Data analysis is performed using SPSS version 26, with results interpreted within the context of diagnostic accuracy standards. The anticipated findings suggest that the developed LFIA will demonstrate a sensitivity exceeding 85%, specificity above 90%, and an overall diagnostic accuracy comparable to RT-PCR, with a markedly reduced turnaround time of less than 20 minutes. The assay's ease of use and cost-effectiveness are also expected to favor its field deployment for early detection and control. The study’s contribution to knowledge lies in innovating a field-deployable diagnostic tool tailored specifically for equine influenza, filling a significant gap in rapid diagnosis at the animal owner and field veterinarian levels. It advances the diagnostic toolkit by providing an affordable, reliable, and scalable option that can facilitate timely intervention, containment strategies, and epidemiological monitoring. The findings will elucidate the assay’s operational utility under varied environmental and field conditions, thereby informing policy on infectious disease surveillance. The main conclusion indicates that the newly developed LFIA exhibits high diagnostic accuracy and operational feasibility for equine influenza detection outside laboratory settings. Recommendations include further validation in different geographic regions, integration into existing disease control programs, and training of veterinary personnel. Future research is suggested to explore multiplexed versions that can simultaneously detect multiple equine respiratory pathogens, thus enhancing disease management strategies. Overall, this research advances preparedness and response capacities against equine influenza outbreaks, contributing significantly to both veterinary diagnostics and disease control paradigms.