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

<i>Bartonella</i> 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 <i>Bartonella</i> spp. among feral dogs in Iraq. This surveillance program identified infection with a novel species <i>Candidatus</i> Bartonella merieuxii (<i>C</i>Bm) in 37% of the dogs by whole blood <i>Bartonella</i> DNA amplification and sequencing for the <i>gltA</i> and <i>rpoB</i> 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 <i>C</i>Bm in dogs, and (ii) to define the prevalence and <i>Bartonella</i> species acquired by MWD during deployment to Iraq. To establish a multiplex serological surveillance panel capable of assessing infection with <i>Bartonella</i> spp., with special emphasis on <i>C</i>Bm, we identified <i>in silico</i> 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 <i>Bartonella</i> 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 <i>Bartonella</i> species to the panel, has the potential to serve as a new diagnostic tool and will be useful for serosurveillance.IMPORTANCE<i>Bartonella</i> pose a significant health risk to military working dogs (MWDs), and these organisms also cause disease in humans. <i>Bartonella</i> has not been assessed in MWD deployed to Iraq, despite a report that a novel <i>Bartonella</i>, <i>Candidatus</i> B. merieuxii (<i>C</i>Bm), was detected in almost half of the stray dogs in Baghdad. Infection in dogs is similar to that in humans, and to date, at least three MWDs have died of <i>Bartonella</i> infection. The goal of the proposed study is to develop an assay to serologically detect infection with <i>C</i>Bm in dogs and to define the prevalence and <i>Bartonella</i> species acquired by MWD during deployment to Iraq. Dogs are potential reservoirs and sentinels for vector-borne infections and are implicated in the transmission of these infections to humans. Detecting the presence of <i>Bartonella</i> in Iraq-deployed MWDs is essential to identify and prevent the introduction of a new <i>Bartonella</i> species to North America.