Molecular Discovery of Filarial Nematode DNA in an Endangered Wild Pinniped (Galapagos Sea Lion, Zalophus wollebaeki)

Rapidly changing environments are contributing to the spread of non-native species and their associated pathogens into new and vulnerable ecosystems, such as the Galapagos archipelago. These pathogens represent a significant threat to emblematic species. The Galapagos sea lion (Zalophus wollebaeki) (GSL) is an endangered and endemic pinniped that is increasingly at risk of acquiring infectious diseases due to interactions with introduced companion animals. Previously, we reported the first detection of antigens from Dirofilaria immitis, the parasite that causes canine heartworm disease, in the GSL. To investigate further, we developed a multifilarial PCR assay and successfully detected DNA from D. immitis and the closely related Dirofilaria repens in 10.7% of our sample cohort of juvenile GSLs. This assay, based on a conserved region in the filarial 28S gene, can be used in conjunction with restriction endonuclease digestion or Sanger sequencing to identify the species of the causative nematode. Our method proved effective without nonspecific amplification in a wide host range, and highly sensitive, detecting as little as one parasite. Further, this assay can be used in cases of immature, low-worm burden, or all-male infections. Our molecular approach offers a sensitive and specific method for detecting filarial parasites in wild animals. Further investigations are necessary to confirm the pathology of filarial nematodes in the GSL and their prevalence in the general population. Our identification of Dirofilarial species in the GSL underscores the urgent need for measures to manage the risk of pathogen transmission from introduced species to native wildlife.