and chemically synthesized nano-selenium (Nano-Se) par-
ticles were used for comparison. Characterization of BNS
particles revealed that they were monodisperse and homoge-
neous spheres with an average size of 139.43 ± 7.44 nm. In
the mouse model of intestinal oxidative stress, BNS particles
were found to protect the mouse intestinal barrier function and
preserve intestinal redox homeostasis more efficiently than
SeMet and Nano-Se. In vitro experiments with porcine jeju-
num epithelial (IPEC-J2) cells verified the stronger epithelial
barrier-protecting effect of BNS particles against oxidative
stress, with reduced cell apoptosis and an improved cell redox
state. Biogenic nano-selenium activated nuclear factor (ery-
throid-derived-2)-like 2 (Nrf2) and increased the expression
of its downstream genes, including thioredoxin reductase-1
(TXNRD-1), NADPH dehydrogenase-1, heme oxygenase-1,
and thioredoxin, in dose- and time-dependent manners. In
contrast, SeMet and Nano-Se merely enhanced the activity of
the selenoenzymes TXNRD-1 and glutathione peroxidase-1,
indicating the role of selenium donors. Moreover, knock-
down of Nrf2 significantly blocked the antioxidative effect of
BNS, confirming that BNS protects the intestinal barrier from
oxidative stress–induced damage by activating Nrf2 and its
downstream genes. Our results suggest that BNS is a prom-
ising selenium species with potential application in treating
oxidative stress–related intestinal diseases.
Key Words: biogenic nano-selenium particles,
intestinal barrier, oxidative stress
doi:10.2527/asasann.2017.041
042 Evaluating the metagenome of two sampling
locations in the nasal cavity of cattle with bovine
respiratory disease complex. T. G. McDaneld*,
L. A. Kuehn, and J. W. Keele, USDA, ARS, U.S. Meat
Animal Research Center, Clay Center, NE.
Bovine respiratory disease complex (BRDC) is a multifactor
disease, and disease incidence may be associated with an ani-
mal’s commensal microbiota (metagenome). Evaluation of the
animal’s resident microbiota in the nasal cavity may help us
to understand the impact of the metagenome on incidence of
BRDC in cattle. Our objective was to determine whether me-
tagenome populations of the nasal cavity vary based on sam-
pling location. Therefore, 2 sampling locations (upper nasal
and deep nasal pharyngeal) were evaluated. Nasal swabs from
calves were collected when the animal was diagnosed with
BRDC after weaning in the feedlot. Samples from healthy
cohorts were also collected for each time point evaluated in
the feedlot to compare metagenome profiles of healthy and
sick animals. For each animal, 1 swab was inserted into the
nasal cavity approximately 6 inches for sampling of the upper
nasal region and a second swab was inserted approximately
8 inches for sampling of the deep nasal pharyngeal region.
Samples were pooled in groups based on when the animal was
diagnosed with BRDC (1, 2, or 3 wk after weaning), type of
sample (upper nasal or deep pharyngeal), and health status
(control or diagnosed with BRDC). To evaluate and compare
the metagenome of each pooled sample, the variable region
(V1–V3) along the 16S ribosomal RNA gene was amplified
by PCR. These amplified products were sequenced using nex-
t-generation sequencing (Illumina MiSeq) and sequence reads
were analyzed by WebMGA and GreenGenes to identify the
bacterial taxa present. In order to assess the similarities or dif-
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ferences between sampling sites, we needed a measure that
would reflect changes in the metagenome as a whole, simulta-
neously accounting for differences in incidence of all detected
bacterial taxa. The Jaccard distance between samples is such
a measure. Jaccard distances between sampling sites for the
same animal group were small relative to the distribution of
intersampling site distances between animal groups generated
by permutation (P = 0.00069). In summary, bacterial popula-
tions were similar between upper nasal and deep pharyngeal
sampling locations. These results demonstrate that shorter,
less invasive nasal swabs produce results similar to those of
deep nasal pharyngeal swabs.
Key Words: bovine respiratory disease complex,
metagenome, 16S sequence
doi:10.2527/asasann.2017.042
043 Metabolomics uncovers serum biomarkers that can
predict the risk of retained placenta in transition
dairy cows. E. Dervishi1, G. Zhang1, R. Mandal2,
D. S. Wishart2, and B. N. Ametaj*1, 1Department
of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, AB, Canada,
2
University of Alberta, Edmonton, AB, Canada.
A combination of direct injection and tandem mass spectrom-
etry with a reverse-phase liquid chromatography and tandem
mass spectrometry was used to detect serum metabolite signa-
tures in 20 cows that expelled their fetal membranes normally
and 6 cows that retained placenta. We aimed to identify serum
biomarkers that can predict the risk of retained placenta (RP)
in transition dairy cows. Blood samples were obtained from
the coccygeal vein once per week at 0700 h before the morn-
ing feeding at 8 and 4 wk before parturition, at the week of
RP, and at 4 and 8 wk postpartum. We studied perturbation of
serum metabolites using an AbsoluteIDQ 180 kit (BIOCRA-
TES Life Science AG, Innsbruck, Austria), which contained
metabolites related to AA, acylcarnitines, biogenic amines,
glycerophospholipids, sphingolipids, and hexose. Univariate
analysis of data was performed using Wilcoxon–Mann–Whit-
ney test provided by R (version 3.0.3; 2008). Metabolomic
data were analyzed using the MetaboAnalyst software. Statis-
tical significance was declared at P < 0.05. Results showed
that cows with RP were characterized by a decrease in lyso-
phosphatidylcholine (LysoPC) a C18:2, C20:3, C20:4, C28:0,
and C28:1 at 8 wk before parturition and during the week of
diagnosis of RP (P < 0.01). At 4 wk prior to calving, LysoPC
21
a C20:4 and C28:0 were decreased. Concentrations of phos-
phatidylcholines (PC) changed after calving, with 12 and 4
species of PC elevated at 4 and 8 wk after calving, respectively
(P < 0.05). Cows with RP were characterized by increased
concentrations of sphingomyelins in the serum at 8 wk prior
to parturition (P < 0.05), the week of RP (P < 0.05), and at 4
and 8 wk after parturition (P < 0.05). Amino acids Lys, Ile, and
Leu were greater in cows diagnosed with RP during all exper-
imental time. Biogenic amines, acetylornithine, asymmetric
dimethylarginine, total dimethylarginine, carnosine, creatin-
ine, and sarcosine were greater in RP cows during diagnosis
week (P < 0.01). It is intriguing that pre-RP, RP, and post-RP
cows showed an increased concentration of Lys, Ile, Leu, and
acetylornithine during all experimental time points studied (P
< 0.01). Moreover, LysoPC a C20:4 and C28:0, ornithine, and
Asn decreased before parturition and at the disease diagnosis
week (P < 0.05). In conclusion, RP is preceded by and associ-
ated with alterations in multiple species of AC, PC, LPC, AA,
and BA starting from 8 wk prior to parturition. Alterations of
serum Lys, ornithine, acetylornithine, LysoPC a C28:0, Asp,
Leu, and Ile might be novel serum biomarkers for prediction
of risk of RP in dairy cows.
Key Words: dairy cows, metabolomics,
retained placenta
doi:10.2527/asasann.2017.043
044 Serum metabolomics fingerprinting during the
dry off period identifies metabolite signatures
that can predict the risk of metritis. B. N. Ametaj*,
Department of Agricultural, Food and Nutritional
Science, University of Alberta, Edmonton, AB,
Canada.
The objective of this study was to screen transition dairy cows
during the dry off period for identification of metabolite sig-
natures in the serum that can be used for prediction of risk of
metritis and give insights into the pathobiology of the disease.
Blood samples were collected from coccygeal vein at 8 and 4
wk prepartum, disease diagnosis week, and 4 and 8 wk post-
partum. Gas chromatography mass spectrometry was used to
identify and quantify 29 metabolites in the serum of 20 healthy
control cows (CON) and 6 cows that were diagnosed with me-
tritis. Data were analyzed using univariate and multivariate
analysis. Results showed that 16, 12, 14, 15, and 10 metabo-
lites were significantly altered at −8 and −4 wk, at disease diag-
nosis, and at +4 and +8 wk around calving in cows diagnosed
with metritis versus healthy CON. The multivariate analyses
indicated consistent disease-dependent clustering with fairly
similar set of metabolites distinguishing premetritis cows
from healthy controls at −8 and −4 wk. The utility of these me-
tabolites as biomarkers of risk of disease was assessed by the
area under the curve (AUC), and AUC values of 1.0 and 0.969
were observed at −8 and −4 wk, respectively. Overall, results
of this study indicated that selected metabolites can be used
22
to early predict the risk of metritis in transition dairy cows.
Results indicated significant (P < 0.05) metabolite alterations
at −8 and −4 wk and at disease diagnosis in premitritis cows
and those that developed metritis compared with healthy
CON. The multivariate analyses also demonstrated consistent
disease-dependent clustering with fairly similar set of metab-
olites distinguishing premetritis cows from healthy controls
at −8 and −4 wk. Among the metabolites that distinguished
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premetritic cows from healthy CON at −8 wk, oxalate, orni-
thine, pyroglutamic acid, glutamic acid, and d-mannose were
ranked as the top 5 in variable importance in the projection. A
similar set of metabolites (except oxalate substituted by phos-
phoric acid) were ranked as the top 5 at −4 wk, indicating that
those top 5 metabolites can be used as predictive biomarkers
at −8 and −4 wk before the incidence of postpartum metritis.
Intriguingly, multiple metabolites, such as galactose, phos-
phoric acid, oleic acid, urea, and oxalate, were identified to
be different (P < 0.5) even at 4 and 8 wk after parturition.
The significant alterations of serum metabolite concentrations
and disease-dependent clustering around parturition indicate
the potential of these metabolites to track the progression and
development of metritis in dairy cattle.
Key Words: dairy cows, metabolomics, metritis
doi:10.2527/asasann.2017.044
045 Genomic characterization of intrauterine
pathogenic Escherichia coli from cows with
metritis. Z. Ma*1,2, A. Ginn1,2, R. Mir1,2, M. Kang1,2,
K. N. Galvão3, and K. Jeong1,2, 1Department of
Animal Sciences, University of Florida, Gainesville,
2
Emerging Pathogens Institute, University of Florida,
Gainesville, 3Department of Large Animal Clinical
Sciences, College of Veterinary Medicine, University
of Florida, Gainesville.
Metritis is a major disease in dairy cows causing animal death,
decrease of birth rate and milk production, and economic loss.
Antibiotic treatment is generally used to treat such disease,
but it is associated with a high failure rate (23–35%). The rea-
son for the treatment failure is not clear. Our hypothesis is that
pathogens in the uteri carry extended spectrum b-lactamases
(ESBL), which give resistance to ceftiofur, the common anti-
biotic used to treat the disease. The objective was to investi-
gate the prevalence of ESBL in cows with metritis and char-
acterize the isolated ESBL carriers. Our study investigated the
prevalence of ESBL-producing bacteria in uterine samples of
cows with metritis (n = 24), and whole genomic sequencing of
the isolated intrauterine pathogenic Escherichia coli (IUPEC)
was conducted by Illumina MiSeq for further genomic char-
acterization. We found that the IUPEC-causing metritis had
a high prevalence of ESBL (70.8%), which may explain the
failure to the treatment. The pathogenicity of these IUPEC iso-
lates was investigated by invasion assay, minimal inhibitory
concentration (MIC) test, and antimicrobial susceptibility test.