Detection of <i>Dirofilaria repens</i> and <i>Mansonella llewellyni</i> in the United States by <i>Wolbachia</i> Surveillance

In mammals, detection of <i>Wolbachia</i> bacteria can be used to diagnose filarial infection, while antibiotic treatment to eliminate <i>Wolbachia</i> can assist in eliminating filarial infections. Because <i>Wolbachia</i> are necessary for survival of several filarioids and closely related to <i>Anaplasma</i> and <i>Ehrlichia</i>, we analyzed <i>Wolbachia</i> DNA amplification by <i>Anaplasma/Ehrlichia</i> qPCR, from 39,526 domestic and wildlife animal blood samples submitted to a diagnostic laboratory between 2017 and 2023. Filarial infection was confirmed by 28S gene amplification, followed by phylogenetic analysis utilizing filarial cytochrome oxidase subunit 1 (<i>cox1</i>), myosin heavy chain (<i>myoHC</i>), and 70 kilodalton heat shock protein (<i>hsp70</i>) gene sequencing. <i>Wolbachia</i> DNA was detected in 57 domestic dogs (<i>Canis familiaris</i>) and three raccoons (<i>Procyon lotor</i>) from 23 states and Puerto Rico. A majority of the <i>Wolbachia</i> sequences from dogs were <i>Dirofilaria immitis</i>-associated (89%, 51/57), whereas DNA from other <i>Wolbachia</i> were associated with insects (9%, 5/57) or <i>Dirofilaria repens</i> (2%, 1/57). <i>D. immitis</i> infection was confirmed by 28S filarial PCR for all samples with <i>D. immitis</i>-associated <i>Wolbachia</i> available for testing (<i>n</i> = 41). <i>D. repens</i> infection was confirmed by 28S and <i>cox1</i> PCR in the dog infected with <i>D. repens</i>-associated <i>Wolbachia</i>. This dog was originally imported from Slovakia. The <i>Wolbachia</i> DNA amplified from raccoons most closely aligned with <i>Wolbachia</i> from <i>Mansonella ozzardi</i> (98.9%). 28S filarial, <i>cox1</i>, <i>myoHC</i>, and <i>hsp70</i> sequencing did not align with currently available GenBank sequences but did align with <i>Mansonella</i>. Morphologically, microfilariae from additional raccoons were consistent with <i>Mansonella llewellyni</i>. Molecular surveillance for <i>Wolbachia</i> in wildlife and domestic animals has the potential to identify novel filarial species in the United States, including zoonotic species.