Isolation and characterization of bacteria from the feathers of wild Dark-

eyed Juncos ( Junco hyemalis)

Authors: John W. Dille, Christopher M. Rogers, and Mark A. Schneegurt
Source: The Auk, 133(2) : 155-167
Published By: American Ornithological Society
URL: https://doi.org/10.1642/AUK-15-126.1

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 Volume 133, 2016, pp. 155–167
DOI: 10.1642/AUK-15-126.1

RESEARCH ARTICLE

Isolation and characterization of bacteria from the feathers of wild Dark-

eyed Juncos (Junco hyemalis)
John W. Dille, Christopher M. Rogers, and Mark A. Schneegurt*

Department of Biological Sciences, Wichita State University, Wichita, Kansas, USA

* Corresponding author: mark.schneegurt@wichita.edu
Submitted July 8, 2015; Accepted November 22, 2015; Published February 3, 2016

ABSTRACT
We dislodged microbes from samples of composites of ventral feathers from different birds of overwintering Dark-
eyed Juncos (Junco hyemalis) after mist-net capture in south-central Kansas, USA. Bacterial loads were measured by
standard plate counts and .300 isolates were purified by repetitive streak-plating on R2A medium (þ cycloheximide).
Biochemical and physiological characterization included identification by 16S rRNA gene phylogeny. Nearly half of the
isolates grew on keratin and 80% exhibited lipase activity, suggesting that these isolates can degrade feathers and
thus may affect survival and reproduction. Individual bacterial loads from 8 juncos varied within a 3-fold range, 105–
106 colony-forming units g1 feather. At 97% DNA sequence identity (species-level), 63 operational taxonomic units
were detected among 202 sequences; the Chao1 estimate was 123. The Shannon diversity index (H; 97% identity) was
3.75, Simpson’s diversity index (1/D) was 16.1, and Good’s coverage was 82.4. Gram-positive bacteria dominated the
culture collection, balanced between low and high GþC clades. Bacillus spp. were abundant, including B. asahii, B.
cereus, B. megaterium, and B. pumilus. Lysinibacillus, Paenibacillus, and Staphylococcus also were isolated. Remarkably,
substantial numbers of Actinomycetes were isolated, including representatives of Clavibacter, Curtobacterium,
Microbacterium, and Rathayibacter, genera recognized as being populated by xylem-filling crop plant pathogens.
Apposed to these were feather isolates implicated as beneficial to host plants, Frigoribacterium and Kitasatospora,
being antagonists to plant pathogens or acting as plant growth promoters. High GþC Gram-positive bacterial isolates
included Blastococcus, Cellulomonas, Humicoccus, Nocardioides, Promicromonospora, and Rhodococcus. Proteobacteria
dominated the Gram-negative bacteria, with Alphaproteobacteria most abundant, including the potential plant
pathogens Agrobacterium and Sphingomonas, and the oligotrophs Aurantimonas, Brevundimonas, Methylobacterium,
Rhizobium, and Rhodobacter. Gammaproteobacteria included Pantoea, Pseudomonas, and Stenotrophomonas. Ours is
the first report of abundant helpful and harmful phyllosphere bacteria on wild bird feathers. The clear implication is
that free-living migratory birds may carry bacteria throughout their geographic ranges and may transmit pathogens
and beneficial bacteria to plants.
Keywords: feather bacteria, beneficial bacteria, plant pathogens, Dark-eyed Junco, cladistics, Junco hyemalis,
migration

Aislamiento y Caracterización de Bacterias provenientes de Plumas Silvestres de Junco hyemalis

Silvestres
RESUMEN
Obtuvimos microbios provenientes muestras compuestas de plumas ventrales de Junco hyemalis, luego de capturarlos
con redes de niebla en el sur-centro de Kansas, EE.UU. Las cargas bacterianas fueron medidas recuentos en placas
estándares, y .300 aislamientos fueron purificados mediante el sembrado repetido en placas en un medio de R2A
(þcicloheximida). La caracterización bioquı́mica y fisiológica incluyó la identificación por secuenciación filogenética del
gene 16S ARNr. Cerca de la mitad de los aislamientos crecieron en queratina y 80% mostró actividad lipasa, sugiriendo
que estos aislamientos pueden degradar las plumas, afectando la supervivencia y la reproducción. La carga bacteriana
proveniente de ocho individuos de J. hyemalis presentó un rango de tres veces de 105–106 unidades de colonias en
formación g1 pluma. Con un 97% de identificación de la secuencia de ADN (nivel de especie), se detectaron 63
unidades taxonómicas operativas entre 202 secuencias; el estimado de Chao1 fue 123. El ı́ndice de diversidad de
Shannon (H; 97% de identificación) fue 3.75, la diversidad de Simpson (1/D) fue 16.1 y la cobertura de Good fue 82.4.
Las bacterias Gram-positiva dominaron la colección de cultivos, con un balance entre clados GþC bajos y altos. Las
especies de Bacillus fueron abundantes, incluyendo B. asahii, B. cereus, B. megaterium y B. pumilus. También se aislaron
Lysinibacillus, Paenibacillus y Staphylococcus. Llamativamente, se aislaron números sustanciales de Actinomicetos,
incluyendo representantes de Clavibacter, Curtobacterium, Microbacterium y Rathayibacter, géneros reconocidos por
verse poblados de patógenos del xilema de las plantas de cultivo. En sentido contrario, Frigoribacterium y

Q 2016 American Ornithologists’ Union. ISSN 0004-8038, electronic ISSN 1938-4254

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 156 Dark-eyed Junco feather bacteria J. W. Dille, C. M. Rogers, and M. A. Schneegurt

Kitasatospora fueron aislados de las plumas, que son reconocidos como benéficos para las plantas hospederas, siendo
antagonistas de los patógenos de las plantas o actuando como promotores del crecimiento de las plantas. Aisalmiento
bacterias Gram-positiva de GþC alto incluyeron a Blastococcus, Cellulomonas, Humicoccus, Nocardioides, Promicromo-
nospora y Rhodococcus. Proteobacteria dominó las bacterias Gram-negativa, siendo Alphaproteobacteria la más
abundante, incluyendo los patógenos potenciales de las plantas Agrobacterium y Sphingomonas, y los oligotróficos
Aurentimonas, Brevundimonas, Methylobacterium, Rhizobium y Rhodobacter. Gammaproteobacteria incluyó a Pantoea,
Pseudomonas y Stenotrophomonas. Esta es la primera cita de abundantes bacterias filósferas útiles y perjudiciales en
plumas de aves silvestres. Una clara implicancia es que las aves migratorias silvestres pueden transportar bacterias a
través de sus rangos geográficos y transmitir bacterias patógenas y benéficas a las plantas.
Palabras clave: bacterias de plumas, bacterias beneficiosas, patógenos de plantas, cladistica, Junco hyemalis,
migración

INTRODUCTION Kowalska 1999, Saranathan and Burtt 2007). Feather

Plumage is a key point of interaction between birds and the
microbial world. Feathers filter air, capturing particles and
bacteria, and frequently contact soil and plants. Our
limited knowledge of the diversity of avian microbial
communities diminishes our ability to include microbial
community interactions in discussions of avian survival
and intersexual selection. Previous studies on the bacterial
community of feathers have focused mainly on isolates
that degrade keratin, rather than specifically on describing
diversity (Shawkey et al. 2003, 2009, Lucas et al. 2005,
Whitaker et al. 2005, Peele et al. 2009, Bach et al. 2011,
Saag et al. 2011, Verea et al. 2014). Measurements of
bacterial loads on feathers also have been focused mainly
on keratinolytic bacteria (Burtt and Ichida 1999a, 2004,
Møller et al. 2009, Giraudeau et al. 2010, Czirják et al.
2013). We counted and isolated bacteria from feathers of
the Dark-eyed Junco (Junco hyemalis) and captured a
more diverse microbial community than has been
described previously.
Feathers are critical not only to flight and protection,
they also play a central role in sexual displays, sexual
selection, and reproductive success (Freeland 1976,
Hamilton and Zuk 1982, Hamilton 1990, Hill 1991, Møller
1991, Swaddle et al. 1996, Leclaire et al. 2014). Bacteria can
influence avian ecology by degrading feathers, thereby
altering coloration and physical properties, or feather-
associated bacteria may be pathogenic to birds (Burtt and
Ichida 2004, Peele et al. 2009). Bird–microbe interactions
have broad implications for the conservation of birds and
for the protection of domesticated birds and the food
supplied through aviculture.
Preen oils, sanitation behaviors, social interactions, and
environmental conditions likely play central roles in
shaping microbial community structure on plumage (Pugh
1971, 1972, Jacob and Ziswiler 1982, Altizer et al. 2004,
Kulkarni and Heeb 2007, Archie and Theis 2011). Feathers
may be a challenging environment for microbes, present-
ing low-quality nutrients, high UV irradiation, and
relatively low moisture (Mathison 1964, Korniłłowicz-
microbial communities are under substantial selection
pressures, and feathers may have microbial assemblages
distinct from the microbial communities of the soil and the
phyllosphere with which birds regularly interact.
Our work characterized the bacterial community on the
feathers of wild Dark-eyed Juncos, migratory songbirds
(18–28 g) that are common and widespread across North
America (Nolan et al. 2002). Juncos spend considerable
time during their annual cycle in diverse habitats, ranging
from boreal forest in the breeding season to forest edges,
old fields, and suburban landscapes during migration and
winter. In addition, this species is easily captured at winter
feeding stations in Kansas, USA, and responds well to
capture and banding. Junco ecology and behavior are well
studied, providing a useful background for the interpreta-
tion of new findings. Bacterial isolates from juncos were
identified by 16S rRNA gene sequencing and characterized
through biochemical and physiological assays that address
the potential of these isolates to degrade feathers and
hence influence reproductive success and sexual selection.
Our surveys of bacterial abundance and the large bacterial
isolate collection that we generated provide the best
analysis of a feather microbial community to date.
METHODS
Feather Sampling and Processing
Adult Dark-eyed Juncos were haphazardly captured using
mist nets at winter feeding stations in the Ninnescah
Reserve of the Wichita State University Biological Field
Station (37831 0 N, 97842 0 W) in Kansas. Sterile gloves were
used to grip birds from the dorsal side. Ventral contour
feathers (9) were collected from each bird and placed in
sterile Whirl-Pak bags (Nasco, Fort Atkinson, Wisconsin,
USA) held at ambient temperature. Ventral feathers are
easy to collect, relatively consistent in size, and removal
has little effect on the bird. The area near the cloaca was
avoided, since fecal bacteria could then dominate the
microbial collection. Samples were transported and
processed within 90 min. Microbes were dislodged from

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 J. W. Dille, C. M. Rogers, and M. A. Schneegurt
feathers in 10 mL of 0.1% Na pyrophosphate (a chaotropic
agent), with shaking on a rotary platform at 150 rpm for 35
min before plating (Caton et al. 2004).
Three distinct sets of feather samples were taken for
different experiments in this study. Feathers from 8
individual birds were used for measuring bacterial loads.
Separate composite feather samples from different groups
of individual birds were used for measuring growth
tolerances and for microbial isolation. A third set of feather
samples was used for determining average feather weight in
order to quantitate bacterial abundance per gram of feather.
This third set of feathers was collected because it was
important for cultivation experiments to control contam-
ination introduced through manipulation of feather samples
and sufficient numbers of feathers (at least 10; more than
could be safely taken from an individual bird) were needed
to obtain a reliable average weight. We did not record the
age or sex of the birds sampled for any part of this study;
however, wintering juncos are mainly female and are of
similar size. Of the juncos (n ¼ 221) captured during recent
winters (2010–2013), 60% were female, 28% were older than
1 yr, and only 5 of 141 birds were outside the range of 17.5
to 22.0 g (C. M. Rogers personal observation). Since feather
size scales with body size, the feathers were expected to be
within 25% of each other in weight.
Bacterial Load and Growth Tolerances
Microbes were separately dislodged from 9 feathers
collected from each of 8 individual juncos, and 100-lL
aliquots were spread onto triplicate plates of Reasoner’s 2A
agar (R2A) supplemented with 0.2% cycloheximide. This
medium is commonly used for soil microbiology and
suppresses fast-growing organisms that can dominate
mixed cultures. Cycloheximide was used to inhibit the
growth of fungi. Colonies were counted after incubations
of 1, 3, 7, and 14 days. Blank controls with no feathers were
not performed, however all materials used were sterile.
Surveys of thermotolerance and halotolerance used
composite samples of 3 feathers from each of 3 birds.
Triplicate R2A plates (with cycloheximide) were main-
tained at 4, 25, and 378C to measure temperature
tolerance. R2A medium (with cycloheximide) was supple-
mented with 1, 5, 10, 15, and 20% NaCl for survey counts
of halotolerant bacteria on triplicate plates. Individual
feather weights were very low, so batches of 10 feathers
each were carefully weighed using a closed scale (on a
marble block) to determine an average feather weight of
5.6 3 104 6 3.7 3 105 g per feather (n ¼ 20 batches of 10
feathers each) to use for calculations. Counts are expressed
as colony-forming units (CFU) g1 feather 6 SD.
Microbial Isolation
A composite sample of 3 ventral feathers from each of 3
birds (9 feathers total) was processed to dislodge microbes
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Dark-eyed Junco feather bacteria 157
as above and aliquots (100 lL) were spread-plated on R2A
medium (with cycloheximide) at 25 or 378C. Over a 2-
week period, isolated colonies were collected sequentially
as they arose, without discrimination by colony morphol-
ogy. Isolated colonies were purified by 6 rounds of
repetitive streak-plating on R2A medium. All isolates were
maintained on R2A plates (with cycloheximide) and
archived as 20% glycerol stocks at 808C.
Identification and Characterization of Bacterial
Isolates
Bacterial isolates were identified by 16S rRNA gene
sequence analysis. Pure cultures grown in R2A broth were
harvested by centrifugation and lysed by 6 freeze–thaw
cycles in liquid N2 and 908C water as previously described
(Caton et al. 2004). Crude DNA extracts were subjected to
PCR amplification with TaKaRa Ex Taq polymerase (Takara
Bio, Kusatsu, Shiga, Japan) using universal bacterial
primers that give nearly full-length 16S rRNA gene
sequences: pA 5 0 -AGAGTTTGATCCTGGCTCAG-3 0
and pH 0 5 0 -AAGGAGGTGATCCAGCCGCA-3 0 (Edwards
et al. 1989). PCR was performed as described previously
(Kilmer et al. 2014), with an annealing temperature of
538C. Amplicons were confirmed by 2% agarose gel
electrophoresis, with visualization after ethidium bromide
staining. PCR products were cleaned with the Promega
Wizard SV Gel and PCR Cleanup System (Promega
Corporation, Madison, Wisconsin, USA) before Sanger
sequencing of 202 isolates at the University of Kansas
Biodiversity Institute (Lawrence, Kansas, USA) using the
pA primer.
DNA sequences were aligned using CLUSTAL W
(Thompson et al. 1994), including contextual sequences
selected with BLAST from the GenBank database (Altschul
et al. 1990). Aligned sequences were trimmed to equal
length in MacClade 4.08 (Sinauer Associates, Sunderland,
Massachusetts, USA) and realigned in CLUSTAL W.
Phylogenetic analysis was performed with PAUP* 4.0 b10
(Swofford 1998) through distance analysis by neighbor-
joining using Jukes-Cantor rules, giving ~350 positions
suitable for analysis after removing missing and ambiguous
bases. Jackknife resampling was used to assess the relative
support for each branch, with a total of 100 bootstrap
replicates conducted heuristically using the distance-based
neighbor-joining algorithm and the nearest-neighbor-
interchange algorithm in PAUP*. No putative chimeras
were identified using Pinwheel within Sequin (http://www.
ncbi.nlm.nih.gov/Sequin/). Sequences appear in GenBank
with accession numbers KM513853–KM514055. Diversity
indices, Chao estimators, and rarefaction curves were
generated using the mothur statistical package (Schloss et
al. 2009).
Individual isolates were assayed for halotolerance on
R2A medium supplemented with 1, 5, 10, 15, and 20%
 158 Dark-eyed Junco feather bacteria
FIGURE 1. Bacterial loads of ventral feathers from 8 individual
Dark-eyed Juncos. Bacteria were dislodged from feather samples
and serially diluted onto triplicate R2A plates. Values are colony-
forming units (CFU) g1 feather 6 SD. The SD includes variance
due to errors in microbial counting and batch feather weight
measurement.
NaCl. The water activity of each medium was measured
using an AquaLab Series 3 water activity meter (Decagon
Devices, Pullman, Washington, USA) calibrated with
standard NaCl solutions and run at room temperature.
Lipase was assayed using agar plates infused with 2%
tributyrin oil, noting zones of clearing. Starch hydrolysis
was tested on agar plates infused with 0.4% soluble starch.
Two days after inoculation, plates were flooded and
incubated with Gram’s iodine for 5 min. The stain solution
was removed and colonies were examined for zones of
clearing. Gelatin stab tubes were inoculated with isolates
and incubated for 48 hr. A liquefied mixture indicated that
the isolate was positive for protein hydrolysis. Keratin
utilization was determined by growth on BSM plates (33.3
mM KH2PO4; 33.3 mM K2HPO4; 15.1 mM (NH4)2SO4;
0.79 mM MgCl26 H2O; 0.18 mM CaCl2; 9.67 lM
NaMoO42 H2O; 7.98 lM MnCl24 H2O; 6.58 lM FeSO4)
supplemented with 1% (w/v) feather meal (Down to Earth
organic feather meal 12-0-0 fertilizer; Everwood Farm,
Brooks, Oregon, USA), as compared with growth on BSM
plates without an exogenous carbon source.
RESULTS
Survey of Microbial Abundance
The abundance of aerobic bacteria dislodged from the
feathers of overwintering Dark-eyed Juncos, as determined
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J. W. Dille, C. M. Rogers, and M. A. Schneegurt
FIGURE 2. Abundance of feather bacteria after cultivation at
different temperatures. Bacteria dislodged from composite Dark-
eyed Junco feather samples were serially diluted and plated in
triplicate onto R2A medium incubated at 4, 25, or 378C. Values
are colony-forming units (CFU) g1 feather 6 SD. The SD
includes variance due to errors in microbial counting and batch
feather weight measurement.
by standard plate counts on R2A medium (with cyclohex-
imide), was in the range of 105 to 106 CFU g1 feather
(Figure 1). Values for the 8 individual birds varied within a
3-fold range (compare bird B with bird H; Figure 1).
Variation in feather batch weights was too small to explain
the differences observed in bacterial loads.
Salinity testing found that microbial growth (1.2 3 106
CFU g1 feather with no added salt) was inhibited as
salinity increased. However, 17% of the cultivable bacteria
grew at 5% NaCl, twice the salt concentration of seawater.
Approximately 2% of the bacteria grew at 10% NaCl, a
medium with relatively low water activity (aw ¼ 0.92). Only
6.0 3 103 CFU g1 feather grew at 15% NaCl (aw ¼ 0.88),
and no growth was observed at 20% NaCl. The cultivable
bacteria were predominantly mesophilic. Growth was best
at 258C, with lower counts observed at 378C (Figure 2).
Colonies developed most slowly at 48C, but reached nearly
40% of the counts observed at 258C.
Identification of Bacterial Isolates
All separated colonies were collected from plates of
microbes dislodged from composite samples of junco
feathers, so that community analysis would not be biased
by colony morphology. More than 300 bacterial isolates
were obtained by repetitive streak-plating of clonal
colonies, although some expired before the completion
 J. W. Dille, C. M. Rogers, and M. A. Schneegurt
FIGURE 3. Phylogenetic tree for Gram-positive bacteria from
Dark-eyed Junco feathers, based on 16S rRNA gene sequences.
Bootstrap values greater than 50% are shown. A full tree with
GenBank accession numbers can be found in Appendix Figure 4.
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Dark-eyed Junco feather bacteria 159
of the study and were not identified by 16S rRNA gene
sequencing. Results from the 202 isolate sequences
accepted by GenBank are given here.
Isolates from genera within the Gram-positive bacteria
were more abundant in the collection than those of other
clades and were nearly balanced between high GþC
Actinomycetes and low GþC Firmicutes (Figure 3 and
Appendix Figure 4). In the low GþC Gram-positive clade,
isolates were predominantly from the genus Bacillus,
mainly clustering with B. pumilus. Bacillus species isolated
from juncos included B. asahii, B. cereus, B. drentensis, B.
megaterium, B. pumilus, and B. subtilis. Closely related
Lysinibacillus, Paenibacillus, and Staphylococcus were
recovered as well.
Isolates in the high GþC Gram-positive cluster were
predominantly Actinomycetes. Microbacteriaceae were
abundant, including Clavibacter, Curtobacterium, Micro-
bacterium, and Rathayibacter. These genera are known to
be predominantly or entirely populated by plant patho-
gens, affecting crop plants such as wheat and ryegrass
(Bergey’s Manual Trust 2011). Other high GþC Gram-
positive isolates were from genera (e.g., Frigoribacterium
and Kitasatospora) implicated as being beneficial to plant
hosts, acting as antagonists to fungal and bacterial plant
pathogens or as plant growth promoters (Sessitsch et al.
2004, Haesler et al. 2008). Isolates of Cellulomonas,
Promicromonospora, and Rhodococcus were identified,
along with representatives of Blastococcus, Humicoccus,
and Nocardioides.
Isolates within the Proteobacteria dominated the
collection of Gram-negative bacteria (Figure 5 and
Appendix Figure 6). Alphaproteobacteria were most
abundant and included representatives of the genera
Agrobacterium, Aurantimonas, Brevundimonas, Methylo-
bacterium, Rhizobium, Rhodobacter, and Sphingomonas,
oligotrophic organisms observed in the phyllosphere and
soils (Bergey’s Manual Trust 2011). The Gammaproteo-
bacteria included Pantoea, Stenotrophomonas, and Pseu-
domonas, the latter clustering with Pseudomonas syringae,
considered a plant pathogen (Bergey’s Manual Trust 2011).
Variovorax was the only Betaproteobacteria genus ob-
served. The 2 isolates from the Bacteroidetes group were
related to Pedobacter and Spirosoma.
Diversity indices determined from the 202 rRNA gene
sequences are given in Table 1. At the species level (97%
identity), 63 operational taxonomic units (OTUs) were
observed. Chao1 estimates suggest that this represents
approximately half of the species present in the sample.
Good’s coverage suggests somewhat higher recovery (82%).
At the 88% sequence identity level, it appears that nearly
all of the bacterial diversity on feathers was observed
within the collection. This is supported by the rarefaction
curves for different levels of identity (Appendix Figure 7).
Only the 88% sequence identity curve appears to be
 160 Dark-eyed Junco feather bacteria J. W. Dille, C. M. Rogers, and M. A. Schneegurt

TABLE 1. Diversity analyses of the bacterial assemblage isolated

from Dark-eyed Junco feathers.
Level of sequence identity
Parameter 99% 97% 94% 88%
Operational taxonomic units 78 63 52 29
Chao1
Average 143 123 90 33
95% CI 108–217 88–207 66–155 30–47
Good’s coverage 76.9 82.4 87.4 96.0
Shannon diversity (H) 4.10 3.75 3.49 2.59
Shannon evenness (E) 0.94 0.91 0.88 0.77
Simpson’s diversity (1/D) 19.2 16.1 14.6 6.60

isolates were more balanced between bacilli and cocci,

with some coccobacilli (Figure 8B). The only filamentous
organism was F64, related to Kitasatospora, a Streptomy-
cete. Neither catalase nor oxidase was detected in the
majority of the isolates (Figure 8A). Catalase was
somewhat more prevalent in the Gram-negative isolates
than in the Gram-positive isolates. Approximately a third
of all isolates were positive for amylase or gelatinase
(Figure 8C). More than 80% of isolates were lipase-positive
(Figure 8C). Nearly half of the isolates could use keratin as
a sole carbon and energy source (Figure 8C). Isolates in the
collections had time to acclimate to the laboratory
environment during streak-plate isolation and showed
more robust halotolerance than microbes from the direct
survey. Most of the isolates (77%) grew at 5% NaCl, and
20% of the isolates grew at 10% NaCl. Nine isolates (F33,
F107, F116, F126, F154, F168, F233, F303, and F314) grew
at 15% NaCl, and 1 isolate (F107) grew at 20% NaCl.

DISCUSSION

Estimating the bacterial load of feathers is challenging

FIGURE 5. Phylogenetic tree for Gram-negative bacteria from
Dark-eyed Junco feathers, based on 16S rRNA gene sequences.
Bootstrap values greater than 50% are shown. A full tree with
GenBank accession numbers can be found in Appendix Figure 6.
flattening. Shannon indices suggest a diverse community,
giving a value near 4 at the species level. Community
diversity appears to be relatively even, especially at higher
sequence identity levels.
Characterization of Bacterial Isolates
The isolate collection from junco feathers was dominated
by Gram-positive bacteria. In all cases, Gram staining
results agreed with phylogenetic placement based on 16S
rRNA gene sequences (Figure 8A). Most of the Gram-
negative bacteria were cocci, while the Gram-positive
because cells may be firmly attached to or inside feather
structures, potentially leading to underestimates of micro-
bial abundance. It is difficult to compare estimates
published previously with our current results as different
methods were used, e.g., using feather presses on agar or
standardizing values on a per-feather basis (Lucas et al.
2005, Shawkey et al. 2009, Saag et al. 2011, Leclaire et al.
2014). A study of captive male House Sparrows (Passer
domesticus) found 2–4 3 106 CFU g1 feather (Czirják et
al. 2013). On Mallard (Anas platyrhynchos) feathers, 105
CFU g1 feather were reported (Giraudeau et al. 2010).
Bacterial loads on junco feathers, 105–106 CFU g1 feather,
were in a similar range. However, it should be noted that
R2A medium is not suitable for all bacteria and our
conditions selected for aerobic heterotrophs. We also
examined only feather washes, thus our results likely
underestimated bacterial loads.

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 J. W. Dille, C. M. Rogers, and M. A. Schneegurt
FIGURE 8. Characterization of bacterial isolates from Dark-eyed
Junco feathers by (A) Gram stain and catalase and oxidase
assays (solid ¼ positive; open ¼ negative); (B) cell morphology
(solid ¼ Gram-positive isolates; open ¼ Gram-negative isolates);
and (C) catabolic enzyme activities (solid ¼ positive; open ¼
negative).
Most previous studies of bird feather microbial com-
munities were not focused on diversity and did not detect
the diversity of bacteria that we recorded from juncos
(Muza et al. 2000, Shawkey et al. 2003, 2005, 2009, Lucas et
al. 2005, Whitaker et al. 2005, Bisson et al. 2007, 2009,
Peele et al. 2009, Bach et al. 2011, Saag et al. 2011, Kilgas et
al. 2012a, 2012b, Verea et al. 2014). These earlier studies
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Dark-eyed Junco feather bacteria 161
detected low diversity for a variety of methodological
reasons, including selective growth conditions and quan-
tification schemes (Lopez-Velasco et al. 2011, Schneegurt
2013). For instance, a study of American Redstarts
(Setophaga ruticilla) reported a Shannon diversity index
of ,1, with nearly 80% of the isolates within the
Pseudomonads, after storage of feathers at 48C before
cultivation (Bisson et al. 2007, 2009). Our study gave a
Shannon diversity index near 4 for the bacterial assem-
blage on fresh junco feathers. Cultivation inherently selects
for certain populations of microbes, which in our case
were aerobic, heterotrophic bacteria. The feather microbial
community is surely more diverse than what was observed
here using a single culture medium and growth conditions.
R2A is an oligotrophic medium commonly used for soil
microbiology, as it slows rapidly proliferating microbes
that overgrow on plates. Cycloheximide was needed to
suppress the copious growth of the fungi that are common
on feathers (Pugh 1971, 1972). While cultivation yields
microbial isolates that can be characterized biochemically
and physiologically, culture-independent metagenomic
techniques can reveal greater diversity through next-
generation sequencing of rRNA genes (Epstein 2013).
Ours is the first report of abundant culturable
recognized plant pathogens and beneficial bacteria on
the feathers of wild migratory birds. Although juncos are
ground-feeding birds, bacteria found in the phyllosphere
were abundant on their feathers. Common in both the soil
and the phyllosphere, bacteria related to Bacillus were
abundant, as expected from previous work (Burtt and
Ichida 1999a, 2004, Whitaker et al. 2005, Hasegawa et al.
2006, Izhaki et al. 2013, Singh et al. 2014). Most surprising
was the abundance of Actinomycetes, specifically Micro-
bacteriaceae, which are predominantly populated by
recognized plant pathogens of crops that are widely
cultivated in Kansas and across North America. Note that
pathogenicity and transmission were not directly assayed
in our study. While previous work suggests some degree of
taxon specificity of keratinolytic fungi on wild bird feathers
(Hubálek 1976), it seems unlikely that juncos are the only
common birds carrying putative plant pathogens and
beneficial bacteria on their feathers. Sample contamination
is possible and difficult to detect; however, our isolate
collection did not include human-associated bacteria such
as certain Staphylococcus species. It is possible that minute
particles of plant debris or soil were caught in feathers and
not casually observed. We did not fractionate feathers to
determine the location of microbes dislodged for isolation.
The Actinomycetes recovered from juncos clustered
with recognized xylem-filling plant pathogens that cause
gumming disease in wheat and annual ryegrass. Winter
wheat is a primary agricultural crop in Kansas. All reported
Clavibacter isolates are plant pathogens, and transmission
is believed to be by contaminated seeds, asymptomatic
 162 Dark-eyed Junco feather bacteria
seedlings, and plant debris (Bergey’s Manual Trust 2011).
Curtobacterium, Okibacterium, and Plantibacter have
been isolated primarily from the phyllosphere, with the
latter reported to be transmitted by nematodes (Jurkevitch
and Shapira 2000, Behrendt et al. 2002, Bergey’s Manual
Trust 2011). Rathayibacter transmission also appears to be
by nematodes. Rathayibacter toxicus is a U.S. Department
of Agriculture (USDA) select agent, serious enough to be
reportable as a severe threat to agriculture. Agrobacterium
and Sphingomonas include members that are recognized
plant pathogens (Bergey’s Manual Trust 2011). Frank bird
or animal pathogens were not found in the junco bacterial
collection, with the exception of Lysinibacillus, a reported
insect pathogen (Berry 2012, Yang et al. 2012). However,
only ventral feathers were sampled, while enteric patho-
gens have been associated with cloaca (Scullion 1989,
Lombardo et al. 1996).
Migratory birds previously have been found to carry
pathogenic microbes on their feathers, including avian
pathogens (Lombardo et al. 1996, Corry and Atabay 2001,
Bengis et al. 2004, Abulreesh et al. 2007, Tsiodras et al.
2008). Interest in avian hosts as potential vectors and
reservoirs for the transmission of human disease has arisen
from serious concerns about emerging infections and the
spread of pathogens. It is not surprising that birds similarly
may carry plant pathogens and potentially act as vectors.
Bashan (1986) included birds as carriers of the plant
pathogens Pseudomonas syringae and Xanthomonas cam-
pestris, but concluded that their low abundance on feathers
may preclude transmission. We found a variety of
abundant culturable bacteria on feathers that are impli-
cated as plant pathogens.
A number of junco feather isolates clustered with
bacterial groups known to be beneficial to plants.
Lysinibacillus, Lysobacter, Stenotrophomonas, and several
Bacillus spp. include members shown to be antagonistic
to fungal pathogens (e.g., Fusarium) in the rhizosphere
and phyllosphere (Hayward et al. 2010, Kavroulakis et al.
2010, Izhaki et al. 2013, Singh et al. 2014). Frigoribacte-
rium and Labedella have few cultured representatives, are
associated with the phyllosphere, and are suggested to be
beneficial bacteria (Sessitsch et al. 2004, Lee 2007,
Yashiro et al. 2011). Kitasatospora is antagonistic to
Phytophthora (Haesler et al. 2008). Cellulomonas de-
grades cellulose, but also has been shown to promote
growth in the wheat rhizosphere (Egamberdiyeva and
Höflich 2002). While Bacillus licheniformis has been
isolated from feathers previously (Burtt and Ichida
1999b), it was not observed here; however, in earlier
studies samples were cultured at 508C specifically to
select for B. licheniformis strains. Various Bacillus species
were recovered from juncos, but these species are
common in soils and the phyllosphere (Bergey’s Manual
Trust 2011).
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J. W. Dille, C. M. Rogers, and M. A. Schneegurt
A majority of the bacterial isolates grew on keratin as a
sole carbon and energy source, including many of the
putative plant pathogens (Dille et al. 2011, 2012). This
could have significant consequences for bird survival,
should the integrity of feathers be compromised, and for
fecundity, should the bird appear less attractive due to
duller or damaged feathers (Møller 1991, Swaddle et al.
1996, Shawkey et al. 2009). Similarly, the high proportion
of isolates expressing lipase suggests that feather oils may
be a nutritional source for bacteria, potentially leading to
feather degradation by the removal of water-proofing (Al
Musallam and Radwan 1990). There is likely interplay
between the microbial community and antimicrobial
compounds found on feathers and in preen oils (Pugh
1971, Jacob and Ziswiler 1982, Møller et al. 2009,
Giraudeau et al. 2013). Enhanced halotolerance would be
consistent with organisms adapted to dry, oily environ-
ments, such as feathers, and suggests that the microbial
community on feathers may have some specificity for
feathers, since the halotolerance of bacteria from common
soils (Porazka et al. 2011) is substantially less pronounced
than the halotolerance of feather bacteria.
The clear implication of our study is that migratory
birds carry potential plant pathogens and beneficial
bacteria on their feathers and may transmit these
organisms to plants after dispersing long distances.
Microbial pathogens on crop plants lead to broad societal
costs, being detrimental to the production of commodities
from foods to biofuels. Demonstration of a novel vector for
phyllosphere bacteria, both helpful and harmful, especially
one with such great potential for widespread dissemina-
tion, would abrogate our current understanding of the
epidemiology of plant pathogens and beneficial bacteria. It
is an intriguing system. Birds interact with phyllosphere
and soil communities that include pathogenic and
beneficial bacteria. They use preen oils and sanitation
behaviors that presumably select for microbial communi-
ties favorable to feather health and reproductive success,
with no regard to plant pathogenicity or benefit. Then
birds potentially disseminate and transmit the resultant
microbial community over broad spatial scales.
There are major implications for avian biology and
ecology suggested by our findings. The high bacterial
species diversity on junco contour feathers suggests that
bacterial feather communities are much richer than known
from previous studies. Keratinolytic isolates that can
degrade feathers may have real impacts on feather quality,
and hence survival, mating success, and intersexual
selection. Examining bacterial assemblages on feathers
adds a dimension to microbe–host interactions, enhancing
our understanding of avian ecology and conservation.
Studying a new vertebrate host–microbe system also can
enhance our understanding of human and animal systems.
Finally, juncos may be utilizing agricultural habitats to an
 J. W. Dille, C. M. Rogers, and M. A. Schneegurt
appreciable extent, reflecting suboptimal habitat selection
caused by loss and/or degradation of historically important
winter habitats, such as forest edges, old fields, and
riparian zones. Factors reducing winter habitat quality
include decreased food supply resulting from lower plant
species diversity and elevated predator density due to
increased abundance of Cooper’s Hawks (Accipiter cooper-
ii), forcing juncos to use a broader selection of habitat
types (Ratajczak et al. 2012, Sauer et al. 2014). Ecological
habitat loss and alteration in this case may lead to greater
exposure of birds to bacterial plant pathogens and
potentially to broader spread of these pathogens to
agricultural plants.
ACKNOWLEDGMENTS
This is publication #28 in the series of reports from the
Wichita State University Biological Field Station. We are
grateful for the preliminary laboratory work performed by
Sarah Jack, Shanna Kelzenberg, and Brian Kilmer. Water
activity was kindly measured by Fadi Aramouni.
Funding statement: This work was supported by funding
from Kansas INBRE NIH NIGMS IDeA (P20 GM103418),
Kansas NSF EPSCoR (EPS-0903806), and NSF GK-12 (DGE
0537844). None of the funding agencies had any input into the
content nor required approval of the manuscript prior to
submission or publication.
Ethics statement: Ethical animal handling protocols were
approved by the Wichita State University Institutional Animal
Care and Use Committee (WSU IACUC permit #196).
Author contributions: C.M.R. and M.A.S. conceived the idea,
design, and experiment (supervised research, formulated
question or hypothesis); J.W.D., C.M.R., and M.A.S. per-
formed the experiments (collected data, conducted the
research); C.M.R. and M.A.S. wrote the paper (or substantially
edited the paper); J.W.D., C.M.R., and M.A.S. developed or
designed the methods; J.W.D., C.M.R., and M.A.S. analyzed
the data; and M.A.S. contributed substantial materials,
resources, or funding.
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 166 Dark-eyed Junco feather bacteria J. W. Dille, C. M. Rogers, and M. A. Schneegurt

APPENDIX FIGURE 4. Phylogenetic tree for Gram-positive bacteria from Dark-eyed Junco feathers, based on 16S rRNA gene
sequences. Bootstrap values greater than 50% are shown.

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 J. W. Dille, C. M. Rogers, and M. A. Schneegurt Dark-eyed Junco feather bacteria 167

APPENDIX FIGURE 7. Rarefaction curves based on 16S rRNA

gene sequences of bacteria collected from Dark-eyed Junco
feathers. The curves represent different levels of DNA sequence
identity.

APPENDIX FIGURE 6. Phylogenetic tree for Gram-negative

bacteria from Dark-eyed Junco feathers, based on 16S rRNA
gene sequences. Bootstrap values greater than 50% are shown.

The Auk: Ornithological Advances 133:155–167, Q 2016 American Ornithologists’ Union

Downloaded From: https://bioone.org/journals/The-Auk on 28 May 2019

Terms of Use: https://bioone.org/terms-of-use Access provided by Universidad de Guadalajara