1. Fecal and Preen Oil Sampling
We obtained fecal samples from a total of 28 individual birds across 15
species (plus hybrids between black-capped chickadees (Poecile
atricapillus ) and Carolina chickadees (P. carolinensis )), 14
genera, 12 families, and 7 orders (Table 1). To capture all but one of
the Passerines used in this study, (excluding the American crow (see
Table 1)), 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. 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 1): 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 Ziplock bags rather than in ethanol filled tubes and placed in a
freezer prior to extraction. Fecal samples were collected from two
budgerigars (Melopsittacus undulatus ) housed in a local pet store
(PetSmart in Bethlehem, Pennsylvania, USA) using sterile tweezers and
placed in microcentrifuge tubes containing ethanol as described above.
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 the fresh fecal samples off
the ground and placed them into individual tubes of ethanol.
We collected preen oil from a total of 12 passerine species, spanning 10
genera, and 8 families (Table 2). 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 used 100% ethanol to clean the uropygial gland of each bird,
and to temporarily clear away any nearby feathers. 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 (~1uL). After collection, 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).