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 (WB), 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. 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 OD cutoff value of 0.439 and AUC score of
0.93 (95% CI 0.87-0.99), while the sensitivity and specificity of rATP-β
was 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 ROC 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 diagnosis of canine Bartonelloses.