Appendix 1: Ring-billed and Franklin's Gull AIV rT-PCR and ELISA Results

Abstract

The natural reservoirs for avian influenza virus (AIV) are wild waterbirds such as ducks and gulls. In Minnesota, gulls are abundant and known to visit poultry farms, yet are underrepresented in AIV wild bird surveillance efforts. The purpose of this research was to fill part of this surveillance gap by studying AIV in Ring-billed Gulls (Larus delawarensis) (RBGU) and Franklin’s Gulls (Leucophaeus pipixcan) (FRGU) in August 2017. Results from previous work showed that RBGUs in Minnesota have the highest AIV prevalence levels in the fall, when gulls migrate from their breeding colonies, through areas with poultry farms, to their wintering grounds. This finding, coupled with the reported presence of gulls of all species loafing and foraging near poultry dense areas of Minnesota, warranted further targeted surveillance efforts of other related species, in particular the FRGU. Due to their slow and widely dispersed fall migration pattern, the FRGU may have even more opportunities than the RBGU to become infected with and transmit AIV to other species. This study evaluated 120 FRGU sera, oropharyngeal swabs and cloacal swabs from a wild bird surveillance project conducted in Minnesota in 2017. These samples were tested to determine the AIV sero- and viral prevalence of the FRGU, and subsequently compared their prevalence rates to RBGU. In addition to performing surveillance to determine AIV prevalence in FRGU and RBGU in Minnesota, the swabs that tested positive for AIV from the RBGU and FRGU samples were subjected to whole genome sequencing to determine subtype and lineage. Analyses of each virus segment showed there was no virus movement between the gulls in this study and the 2015 H5N2 highly pathogenic AIV that infected domestic poultry in Minnesota. In other words, the AIV from these gulls in Minnesota did not share a common ancestry with the domestic HPAI H5 viruses of 2014-2015. The RBGU and FRGU H13N2, H13N6, and H13N8 AIV detected were reassortants with AIV genes of North American and Eurasian lineage primarily from other gull species but also commonly containing gene segments from North American duck species. The implications for not finding H5 AIV by rRT-PCR could mean that gulls do not harbor or circulate H5 AIV or that the timing and geographic location are imperative for detection of H5 AIV in wild birds during outbreaks in domestic birds. Finally, to add support to the genetic evidence that AIV in RBGU are a result of RBGU mixing with other species from different geographic locations during migration, we explored the ecological drivers of these AIV infections to see how they affect viral genetic diversity. We investigated gull movements by collecting resighting events from banded RBGU and from satellite location tracking of a small subset of RBGU from one breeding colony on Interstate Island in Duluth, Minnesota. The predominant resightings and recorded movements of Minnesota RBGU were along the Great Lakes, crossing both the Mississippi and Atlantic flyways during fall migration, and overwintering along the Atlantic coast of the Eastern United States. This study determined that the geographic dissemination of the gulls and the geographic lineage of their viruses were in agreement with RBGU having ample opportunity during their life stages to interact and mix with other avian species and become infected with AIV from those other species. Overall, these studies determined that targeted surveillance of gulls over the migration and breeding seasons allows for the successful identification of AIV and estimates of AIV prevalence in gulls. Although there are populations of gulls in Minnesota with reassortant AIV containing gene segments from AIV of multiple avian species and of varying geographic lineage, there is no evidence that exchange of viruses or gene segments of viruses between domestic poultry and gulls has occurred in Minnesota. Through the satellite monitoring of RBGUs from a single breeding colony, we were able to investigate gull movement to and from Minnesota which will facilitate timing and location of future AIV surveillance efforts.