Development of a multiplex serological assay to detect immunoreactivity against a novel Bartonella species in military working dogs

Bartonella pose a significant health risk to military working dogs (MWDs), and these zoonotic organisms may also cause disease in humans. According to the U.S. Army zoonotic disease surveillance program, there was a 47.4% seroprevalence for Bartonella spp. among feral dogs in Iraq. This surveillance program identified infection with a novel species Candidatus Bartonella merieuxii (CBm) in 37% of the dogs by whole blood Bartonella DNA amplification and sequencing for the gltA and rpoB genes and intergenic spacer region. This bacterium has not been successfully cultured to date. The objectives of the present study were to (i) develop an assay to serologically detect infection with CBm in dogs, and (ii) to define the prevalence and Bartonella species acquired by MWD during deployment to Iraq. To establish a multiplex serological surveillance panel capable of assessing infection with Bartonella spp., with special emphasis on CBm, we identified in silico 20 peptides representing potential linear B cell epitopes. The newly developed serosurveillance panel was used to test paired samples (pre- and post-Iraq deployment) from 52 MWD. Conventional Bartonella diagnostic testing (PCR and indirect immunofluorescence assays [IFA]) was also performed. Twenty-seven percent (14/52) of the paired samples had statistically significant antibody differences between pre- and post-Iraq deployment, suggesting seroconversion during deployment. In conclusion, the newly developed multiplex serologic assay, with its advantages of high throughput testing, requirement of low sample volumes, and ability to adapt new Bartonella species to the panel, has the potential to serve as a new diagnostic tool and will be useful for serosurveillance.