Development and evaluation of a rapid point-of-care test for early detection of bovine mastitis using multiplex biosensor technology

Project Abstract

This study presents the development and validation of a rapid, point-of-care diagnostic assay for the early detection of bovine mastitis employing a multiplex biosensor platform capable of simultaneous detection of multiple credibly discriminative biomarkers in milk. The research addresses the critical need for timely, in-field decision-making to mitigate economic losses, reduce antibiotic usage, and improve animal welfare. The biosensor integrates complementary optical and electrochemical transduction modalities within a microfluidic cartridge, enabling label-free detection of host-response biomarkers such as acute phase proteins (e.g., haptoglobin, serum amyloid A), inflammatory mediators (e.g., cytokines like IL-6 and TNF-?), and specific microbial signatures indicative of subclinical infections. A curated panel of biomarkers was selected through a systematic literature review and pilot ELISA-based validation using milk samples from clinically healthy and mastitic cows across diverse dairy herds, capturing variability in breed, lactation stage, and geographic location. Analytical performance of the multiplex sensor was characterized in terms of sensitivity, specificity, limit of detection, dynamic range, linearity, and cross-reactivity, with rigorous calibration against reference standard methods. The assay demonstrated a detection limit in the low picomolar to nanomolar range for protein biomarkers and sub-pathogenic concentrations for nucleic acid–based targets, achieving a clinically relevant diagnostic window that precedes visible clinical signs by approximately 24–72 hours in many subclinical cases. The multiplex format enables concurrent assessment of multiple biomarkers, increasing diagnostic accuracy beyond single-analyte tests and enabling differential diagnosis between Gram-positive and Gram-negative etiologies, as well as distinguishing inflammatory from infectious inflammation. A key component of the work involved the engineering of a user-friendly, portable reader with a disposable cartridge, designed for field technicians and farm workers with minimal training. The device provides quantitative readouts displayed as an integrated risk score and individual biomarker concentrations, with embedded quality control and data logging capabilities. Analytical validation included intra-assay and inter-assay precision, interference testing (e.g., milk fat, somatic cell count variability), and robustness under varying ambient conditions. Clinical validation encompassed a multicenter study across five dairy operations, enrolling over 1,200 milk samples with endpoints aligned to bacteriological culture, somatic cell count, and conventional diagnostic criteria. Economic analysis assessed cost-per-test, potential reductions in treatment duration, and projected herd-level impact on mastitis prevalence and milk yield. The results indicate high sensitivity and specificity for early detection, with receiver operating characteristic analysis supporting superior performance compared with conventional somatic cell count thresholds. The study also highlights operational advantages, including rapid turnaround time (under 20 minutes), reduced need for laboratory infrastructure, and potential integration with herd management software for real-time surveillance. Limitations identified include variability in biomarker expression due to non-mastitis inflammatory events and the need for ongoing calibration across diverse herds. Overall, the multiplex biosensor-based POC assay represents a practical, scalable tool for proactive mastitis management, enabling timely interventions and enhancing dairy herd health and productivity.

Project Overview

What This Project Is About

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A straightforward look at how a rapid, portable test can detect mastitis early in dairy cows, using multiple biosensors that work together to check several biological signals at once. The project compares this new test to current methods to see if it is faster, cheaper, or easier to use on farms.

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The Problem It Addresses

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Mastitis is an infection of the udder that costs farmers money and can harm animal welfare. Early detection is hard with existing tests, especially in busy farm settings. A point-of-care device should give quick results on the farm, helping farmers treat cows sooner and reduce losses.

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Objectives of the Project

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1. Design a portable test that can run on a farm or veterinary clinic.
2. Integrate multiple biosensors to detect different indicators of mastitis.
3. Evaluate accuracy, speed, and usability under realistic conditions.
4. Compare results with standard lab tests.
5. Assess cost and practicality for daily farm use.

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What You Will Do Step by Step

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1. Review current mastitis tests and biosensor basics.
2. Develop the multiplex test prototype and select target markers.
3. Build a simple handheld reader and calibration steps.
4. Test on known samples in the lab, then on field samples.
5. Analyze data to measure sensitivity, specificity, and turnaround time.
6. Evaluate ease of use and farm practicality with a small user study.

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Expected Outcome

An operational multiplex biosensor-based test that provides rapid on-farm results, with evidence on accuracy, speed, and feasibility, and a plan for scale-up and deployment in dairy operations.