# A fluorescent bead-based multiplex assay for the detection of Brucella sp. specific antibodies in canine serum

## TL;DR

A new multiplex assay detects antibodies against Brucella canis in dog blood, offering a more accurate diagnostic tool for brucellosis.

## Contribution

A novel fluorescent bead-based assay using two antigens for detecting Brucella canis antibodies in canine serum is developed.

## Key findings

- The assay achieved 91.6% sensitivity and 94.9% specificity in detecting B. canis antibodies.
- Seroprevalence in diagnostic submissions was 16.1%, while export samples showed 0.1%.
- Adding a third antigen further improved assay sensitivity.

## Abstract

The zoonotic pathogen, Brucella canis, causes brucellosis in dogs. Infection with B. canis is usually diagnosed by serological testing. We developed a fluorescent bead-based multiplex assay for detection of B. canis specific antibodies in canine serum. The assay consists of two antigens detected simultaneously by canine serum antibodies. One antigen, BP26, was selected from a set of immunodominant proteins identified through western blot and proteomics analysis. The second antigen, PO1, is a 17 amino acid peptide derived from B. canis Omp31.

Dog sera from diagnostic submissions were tested in parallel with a reference assay consisting of a rapid slide agglutination test (2ME-RSAT) and an agar gel immunodiffusion test (AGID II). A Bayesian latent class model (BLCM) was utilized to determine sensitivity and specificity of both assays. For the model to be identifiable, two groups with differing prevalence were included; one group was composed of 1,192 diagnostic submissions, and the second group was composed of 390 samples submitted for export purposes.

The seroprevalence of B. canis specific antibodies in these two groups was estimated to be 16.1% (95% CI, 12.6–19.3%) and 0.1% (95%, 0.0–0.6%), respectively. Diagnostic sensitivity and specificity of the two-antigen assay for detecting B. canis specific antibodies were 91.6% (95% CI, 85.2–98.0%) and 94.9% (95% CI, 92.3–96.9%), respectively.

The addition of a third cytoplasmic antigen further increased assay sensitivity. The Canine Brucella Multiplex assay is a novel and quantitative diagnostic tool for detecting B. canis antibodies in canine serum to aid in the diagnosis of brucellosis in dogs.