Development and validation of a point-of-care diagnostic assay for early detection of bovine brucellosis using recombinant Brucella spp. antigens

Project Abstract

Early detection of bovine brucellosis remains a critical challenge for animal health, public safety, and international trade, necessitating rapid, accurate, and field-deployable diagnostic tools. This study reports the development and validation of a point-of-care diagnostic assay leveraging recombinant Brucella spp. antigens to enable sensitive and specific detection of Brucella infections in cattle at the farm or clinic level. We undertook a multi-phase approach combining antigen engineering, assay design, analytical validation, and clinical performance evaluation under real-world conditions. Recombinant antigens were selected from Brucella outer membrane proteins and virulence-associated factors optimized for immunodiagnostic recognition across diverse Brucella spp. strains. A lateral flow immunoassay (LFIA) format was engineered to achieve rapid visual readouts within 15–20 minutes, suitable for use by veterinarians and para-veterinary staff without specialized equipment. The assay incorporates an optimized conjugate pad, nitrocellulose membrane, and signal amplification strategies to enhance sensitivity while preserving specificity. Analytical characterization demonstrated a limit of detection consistent with clinically relevant antibody titers observed in acute and subclinical bovine brucellosis, with minimal cross-reactivity to antigenically related Gram-negative bacteria and vaccination-induced antibodies, thereby reducing false positives. We conducted a rigorous diagnostic performance assessment using a well-characterized reference panel comprising sera from Brucella-infected cattle, recovered animals, vaccinated individuals, and brucellosis-free controls across multiple geographic regions. Receiver operating characteristic (ROC) analysis established a predefined cut-off achieving sensitivity in the 90–95% range and specificity exceeding 95%, with stable performance across age groups, infection stages, and circulating Brucella spp. variants. Repeatability and lot-to-lot consistency tests yielded coefficients of variation within acceptable limits, confirming assay reliability for deployment in diverse field settings. Field validation involved prospective sampling from dairy and beef herds subjected to routine brucellosis surveillance, comparing the new LFIA against established laboratory assays such as ELISA and Rose Bengal test. Agreement metrics, including Cohen’s kappa, demonstrated substantial concordance, while the point-of-care test offered substantially faster turnaround times and reduced logistic burdens associated with centralized testing. Importantly, the assay demonstrated robustness against sample matrices encountered in on-farm collection and required minimal operational training. Economic and practical feasibility analyses indicated favorable cost per test relative to standard diagnostics, with a clear impact on herd-level management decisions, including rapid quarantine, exposure tracing, and vaccination strategies. The study also explored user-centered design considerations, result interpretation guidelines, and post-market surveillance frameworks to ensure sustained diagnostic accuracy in diverse epidemiological contexts. Collectively, the validated assay presents a viable, scalable solution for early detection of bovine brucellosis, supporting timely disease control actions, mitigating zoonotic risk, and enhancing animal health surveillance infrastructure.

Project Overview

What This Project Is About

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A straightforward exploration of a new, quick test that can be used directly in farms to check if cows have brucellosis early. The project looks at making a small, easy-to-use test using special proteins from the Brucella bacteria to detect infection.

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The Problem It Addresses

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Brucellosis is a contagious disease that can cause illness in cattle and can spread to people who handle animals or dairy products. Current tests can be slow, require lab facilities, or need trained personnel. This project aims to create a rapid, reliable test that farmers can use on-site, reducing delays and improving animal and public health.

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Objectives of the Project

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1. Design a point-of-care test using recombinant Brucella proteins.
2. Evaluate the test’s accuracy against standard lab methods.
3. Assess how easy the test is to use by non-lab personnel.
4. Test stability under typical farm conditions.
5. Identify any limitations and suggest improvements for field deployment.

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What You Will Do Step by Step

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1) Review existing tests and select suitable Brucella proteins. 2) Develop a prototype test format suitable for on-site use. 3) Collect milk or blood samples from cattle with known infection status. 4) Compare results with established laboratory tests. 5) Test performance under different temperatures and handling conditions. 6) Analyze data to determine sensitivity and specificity. 7) Document limitations and propose practical recommendations.

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Expected Outcome

Anticipated to produce a functional, user-friendly on-site test with proven accuracy comparable to standard methods, plus guidelines for field use and suggestions for further refinement.