1. Fecal and Preen Oil Sampling
We obtained fecal samples from a total of 25 individual birds across 15 species (14 genera, 12 families, and 7 orders (Table 2). To capture all but one of the Passerines used in this study, (excluding the American crow (see Table 2)), we used mist nets at feeders in Northampton and Lehigh Counties in Pennsylvania, USA (U.S.G.S. Federal Banding Permits 23810 to AMR and 24256 to AVH; Pennsylvania Banding Permits 103 to AMR and 49864 to AVH). Fecal collections require careful handling to ensure the preservation of DNA. We briefly held each bird individually in a cage containing a clean cage liner until they defecated, which usually took no longer than several minutes. We then immediately collected the feces using tweezers. We also collected fecal samples from two budgerigars (Melopsittacus undulatus ) housed in a local pet store (PetSmart in Bethlehem, Pennsylvania, USA). Tweezers were cleaned with ethanol before and after each use. We transferred the feces into a 1.5 mL microcentrifuge tube containing 100% ethanol and stored the samples in a -80 °C freezer until microbial DNA extraction. Although ethanol kills the living bacteria, it preserves all the DNA present in the sample so that any bacteria present can be detected even after freezer storage68.
Additionally, in cooperation with the Wildlands Conservancy Nature Preserve in Lehigh County, Pennsylvania, we obtained fecal samples from captive birds including the American crow (Corvus brachyrhynchos ), plus several species spanning four additional orders (Table 2): red-tailed hawk (Buteo jamaicensis ), eastern screech owl (Megascops asio ), turkey vulture (Cathartes aura ), and rock pigeon (Columba livia ). The Wildlands Conservancy samples were collected as the individuals were seen defecating in their enclosures. The collections were placed in fresh Ziploc® bags rather than in ethanol-filled tubes and placed in a freezer prior to extraction. To collect from the Canada goose (Branta canadensis ), we closely observed wild geese on the DeSales University campus. When we saw defecation, we used sterile tweezers to lift a small portion of the fresh fecal samples off the ground and placed them into individual tubes of ethanol. Because the Canada goose produces larger feces than the other species we sampled, we were careful to avoid any white portions of these fecal samples, with the goal of reducing the amount of uric acid in the samples.
We collected preen oil from a total of 12 individual birds, from 12 passerine species, spanning 10 genera, 8 families and 1 order (Table 3). All these birds were captured in mist nets at bird feeders in Lehigh County, Pennsylvania, USA (U.S.G.S Federal Banding Permit 24256 and PA Game Commission Banding Permit 49864). We cleaned the uropygial gland of each bird and temporarily cleared away any nearby feathers by gently applying 100% ethanol with a cotton ball . We then used small, sterile forceps to gently squeeze the gland. Once preen oil was secreted from the uropygial gland, we used a capillary tube to collect a small oil sample (~1-2 mg). After collection, the bottom of the capillary tube was placed in a 1.5 mL microcentrifuge tube and stored in a -80 °C freezer prior to microbial DNA extraction.
All capture and sampling procedures were approved either by Lehigh University’s Institutional Animal Care and Use Committee (Protocol #237) or by DeSales University’s Institutional Animal Care and Use Committee (Protocol #1).