Development and validation of enzyme-linked immunosorbent assays for the serodiagnosis of canine bartonelloses

Bartonella species, emerging vector-borne pathogens of dogs, are increasingly associated with severe, chronic sequelae, as well as potentially life-threatening diseases, such as endocarditis and myocarditis. Diagnosis of bartonelloses is mainly based on PCR, culture, and serological assays. Despite molecular and biotechnological advances, serological assays employing immunofluorescence antibody (IFA), Western blotting, and enzyme-linked immunosorbent assay (ELISA) technologies have encountered diagnostic limitations, primarily due to poor sensitivity. Using sera from Bartonella-infected and naïve dogs, we applied an immunoproteomic approach to develop a reliable ELISA assay for the diagnosis of bartonelloses in dogs. Five recombinant Bartonella henselae immunodominant proteins (rATP-β, rGroEL, rLemA, rSucB, and rVirB5) were tested in an ELISA format. Sensitivity and specificity of each protein were calculated based on an imperfect reference IFA assay. Dogs comprised Group I: 36 Bartonella spp. naturally infected dogs (all B. henselae IFA seroreactive) and Group II: 34 Bartonella spp. PCR-negative and IFA-negative dogs. Based upon the ELISA seroreactivity results, rATP-β and rGroEL represented the most sensitive and specific candidate peptide targets for utilization in a canine diagnostic ELISA assay. rGroEL resulted in the sensitivity of 83% and specificity of 94% at an optical density (OD) cutoff value of 0.439 and area under curve (AUC) score of 0.93 (95% CI 0.87-0.99), while the sensitivity and specificity of rATP-β were 69% and 94%, respectively, at a cutoff value of 0.565. The combination of rATP-β with rGroEL resulted in an improved sensitivity of 88% and specificity of 92% at an OD cutoff value of 0.505. A receiver operating characteristic curve analysis for the rATP-β plus rGroEL yielded an AUC score of 0.899 (95% CI 0.809-0.989). Combining rATP-β with rGroEL could potentially further improve both the diagnostic sensitivity and specificity of an ELISA assay for the diagnosis of canine bartonelloses.IMPORTANCEBartonella species are associated with a wide spectrum of clinical signs and life-threatening diseases in dogs. There is an increased risk of Bartonella transmission from dogs to dogs, and from dogs to other animals and humans via vectors, such as ticks, fleas, or direct contact with infected clinical specimens. Due to the poor sensitivity of currently available molecular and serological assays, the diagnosis, treatment, and prevention of Bartonella infection in dogs remains challenging. Developing a reliable serodiagnostic assay is essential for the clinical management of canine bartonelloses, a group of infections caused by Bartonella species in dogs. Rapid diagnosis and timely treatment of canine bartonelloses could save the lives of thousands of dogs worldwide each year. This study provides key insights into the design of diagnostic tools utilizing Bartonella henselae proteins that show promise as serological markers to improve the diagnosis of canine bartonelloses.