Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare . Song sparrow ( Melospiza melodia ) blood infected with a low P . homopolare parasitemia was inoculated into a naïve domestic canary ( Serinus canaria forma domestica ). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P . homopolare , both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P . homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P . homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P . homopolare DNA. This pattern of mixed Pcat6 and P . homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P . cathemerium lineage (Pcat8); none developed PCR detectable P . homopolare infections. These observations suggest that the original P . homopolare -infected songbird also had two un-detectable P . cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.