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).