Stambaugh el al. • INNATE IMMUNITY IN NESTLING TREE SWALLOWS 
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transported to the laboratory within 60 min of 
collection for the best results during immune 
assays (Millet et al. 2007). House Sparrow 
[Passer domeslicus) whole blood showed greater 
in vitro killing of E. coli (American Type Culture 
Collection [ATCC] #8739) than did plasma (Liebl 
and Martin 2009). 
laboratory Methods .—We followed Millet et 
al. (2007) to evaluate in vitro ability of whole 
blood to kill E. coli. The in vitro microbicidal 
ability of whole blood provides a measure of 
constitutive innate immune function (e.g.. Tiele- 
man etal. 2005, Matson cl al. 2006, Millet et al. 
2007) and shows individual variation and high 
repeatability within individuals in some bird 
species (Tieleman et al. 2010, Wileoxen et al. 
2010). Male Florida Scrub-Jay (Aphelocoma 
coerulescens) survivorship during a suspected 
eastern equine encephalitis epidemic was posi¬ 
tively correlated with the in vitro ability of their 
"hole blood to kill E. coli suggesting this measure 
of innate immunity may be an indicator of 
individual quality (Wileoxen et al. 2010). E. coli 
is a potentially pathogenic Gram negative bacte¬ 
rium that has been isolated from the cloacae of 
adults (Lombardo et al. 1996) and nestlings (Mills 
etal. 1999), and in semen (Lombardo and Thorpe 
2000) in Tree Swallows. 
£ coli was supplied as I0 7 organisms per 
l.vophilized pellet (ATCC #8739. Microbiologies 
Inc,. St. Cloud, MN, USA). This assay was 
developed to be a rapid, simple, and reliable in 
v ''ro measure of the effectiveness of the innate 
■niniune system to kill microbes. It integrates 
many ol the important components of the innate 
immune system (e.g.. white blood cells, natural 
md specific antibodies, lysozymes, and opsonins, 
molecules that facilitiate phagocytes to identify 
a, id phagocytize microbes) (Millet et al. 2007). 
also tried to use this microbicidal assay to kill 
Staphylococcus aureus but our attempts al this in 
y " ro assay failed because neither adult nor 
n '. , stling whole blood killed .S', aureus (TS et al.. 
Un publ, data). 
All laboratory work occurred inside laminar 
llow hoods. Microbe pellets were reconstituted 
following manufacturer’s instructions in 40 mL ot 
s,er ile. endotoxin-free Phosphate Buffered Saline 
,p BS> ant i held at 4 C. The stock culture was 
dtluted with cold PBS each day that assays were 
^ to make a working culture with ■ 50.000 
Colony Forming Units (CFU)/mL. Working 
cultures were kept on ice. Whole blood was 
diluted 1:4 with pre-warmed (41° C) C0 2 -inde- 
pendent media (#18045; Gibco-Invitrogcn, Carls¬ 
bad, CA, USA) plus 4mM L-glutamine in a sterile 
1.5 mL capped tube, Ten microliters of the 
working culture (=500 CFU) were added to 
100 pL of diluted blood, vortexed, and incubated 
for 45 min at 40,5 C, samples were removed 
from the incubator and vortexed, and 50 pL 
aliquots were pipetted onto Trypticase Soy Agar 
(TSA) plates, spread, inverted, and incubated at 
37 C for 24 hrs, Samples were run in duplicates. 
E. coli colonies were counted after 24 hrs. The 
number of CFU in the initial inoculums was 
calculated by working culture in media alone (i.e., 
10 pL of working culture per 100 pL of media) 
and then immediately plated. Negative controls in 
which no E. coli were added were also prepared. 
Statistical Analyses.—We calculated the mean 
proportion of E. coli killed (hereafter. MPK) as 1 
- (mean number of experimental colonies/mean 
number of control colonies) following Millett et 
al. (2007). We calculated brood means of MPK. 
mass, and wing chord length for use in some 
analyses to avoid statistical pseudoreplication. We 
found no significant differences between adult 
males and females or between SY and ASY 
females in the microbicidal ability of their whole 
blood (all P > 0.05) (B.IH et al.. unpubl. data), 
and pooled their MPK data for these analyses. We 
estimated narrow sense heritability by regressing 
midnestling MPK on uiidparent MPK at the seven 
nests for which wc had nestling and adult male 
and female MPK data. Narrow sense heritability is 
the ratio of additive genetic variance to the total 
phenotypic variance (/r = Va/Vp) and provides an 
estimate of a trail’s ability to respond to selection 
(Lynch and Walsh 1998). Tree Swallows have a 
high level of extra pair paternity (Lifjeld et al. 
1993, Dunn et al, 1994. Barber et al. 1996. 
Laskemocn et al. 2010) that can bias estimates of 
/,- (Charmantier and Reale 2005), -and we also 
performed a midnestling MPK-mother MPK 
regression at ND18 nests. Intraspecific brood 
parasitism is rare in Tree Swallows (Robertson 
et al. 1992). 
Data were examined for normality and ana¬ 
lyzed using SPSS 12.0 for Windows (SPSS 2002). 
We performed parametric and nonparametric 
analyses where appropriate. No data transforma¬ 
tions were required. All tests were two-tailed. The 
probability level for significance was set at a = 
0.05. Unless otherwise noted, ail values are 
reported as mean ± SD. 
