Detection of Dirofilaria repens and Mansonella llewellyni in the United States by Wolbachia Surveillance

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