Orals: Student Competition: Genetics, Immunology, Pathology

Student Competition: Genetics, Immunology, Pathology
12 Orexin is expressed in avian muscle cells and may regulate
cell bioenergetics. K. Lassiter*, E. Greene, A. Piekarski, W. Bottje,
and S. Dridi, Department of Poultry Science, Center of Excellence for
Poultry Science, University of Arkansas, Fayetteville, AR.
The orexigenic peptide orexin or hypocretin is mainly expressed in mammalian brain and has been shown to increase wakefulness, food intake,
and lipid metabolism. In birds, however, orexin has been confined to the
brain, testis, and ovary and its role is still unknown. In the present study,
we found that prepro-orexin and its receptors are expressed (mRNA
and protein) in quail muscle (QM7) cells using PCR and Western blot
analysis, respectively. In addition, immunofluorescence staining showed
that orexin and its receptors are mainly localized in the cytoplasm,
which has been confirmed by Western blot analysis using cytoplasmic
and nuclear protein fractions. These data indicate that orexin may have
paracrine/autocrine signaling in avian muscle cells. Administration of
recombinant orexin A or B alters the expression of orexin and its receptors in QM7 cells. Orexin-treated cells also showed significantly higher
levels (P < 0.05) of mitochondrial-related genes (UCP, ANT, NRF1, and
SKI) compared with untreated cells, indicating a potential role of orexin
in mitochondrial bioenergetics. Further examination of orexin’s effect
on QM7 cellular bioenergetics using the XF24 flux analyzer (Seahorse
Biosciences) showed that mitochondrial oxygen consumption rate
(OCR) attributed to ATP synthesis increases in response to orexin treatments (that may be dose or isoform dependent). Cells provided orexin
A (100 nM) or orexin B (10 nM) exhibited higher glycolytic activity
following inhibition of ATP synthase compared with untreated control
cells. Together, our data showed for the first time that the orexin system
is expressed in avian (non-mammalian) muscle cells and that it may
regulate cellular bioenergetics and ATP synthesis. Research supported in
part by funding from Arkansas Biosciences Institute (Little Rock, AR).
Key Words: orexin, quail muscle (QM7) cells, mitochondrial bioenergetics, immunofluorescence
13 Molecular staging based on temporal myosin heavy chain
transcription. J. R. Griffin*, M. S. Lilburn, and M. Wick, The Ohio
State University, Columbus, OH.
Numerous factors can influence avian embryonic development and this
results in a relatively low correlation between chronological age (incubation days) and physiological stage of development. The prevailing staging system is based on visual morphological embryonic characteristics
to establish developmental stages, independent of chronological age
(incubation days) and size. In addition to morphological staging, it is
fundamentally important to define the temporal transcriptional events
to better understand the fundamental molecular biological mechanisms
responsible for embryonic myogenesis. The developmental fast skeletal
myosin heavy chains (MyHC), the predominant proteins in the pectoralis major (PM) during myogenesis, are expressed as a cadre of highly
specific temporal and spatial developmental isoforms. The hypothesis
is that the temporal transcription of MyHC isoforms are correlated
with the transcription of muscle-specific genes that are critical to PM
muscle growth and development and can be used as a molecular staging
mechanism of development. The goal of this study was to use a novel
molecular method, based on a quantitative analysis of the transcriptome,
to characterize the developmental stages in embryonic PM in the Single
Comb White Leghorn (SCWL) during myogenesis. Tissue samples from
the embryonic PM were collected daily from d 5 through 19. RNA was
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isolated and gene transcription analysis was quantified using NanoString,
which digitally detects and quantifies selected target genes. Data were
analyzed using the LOESS smoothing function at a 95% confidence
level. Results confirmed that the temporal transition of MyHC isoforms
transcription we obtained in the SCWL were consistent with the literature
at greater resolution. These data established a “molecular clock” for
embryonic fast skeletal muscle growth and development.
Key Words: pectoralis major (PM), myosin, isoforms, myogenesis,
transcriptome
14 Transcriptional events underlying avian myogenesis. J. R.
Griffin*, M. S. Lilburn, and M. Wick, The Ohio State University,
Columbus, OH.
In a previous study we reported the use of a novel quantitative transcriptomic method to establish the temporal transcription of the developmental fast skeletal muscle myosin heavy chains as a molecular staging
mechanism. The hypothesis is that the temporal expression of MyHC
isoforms are correlated with the expression of muscle-specific genes
that are critical to PM muscle growth and development. In a test of this
hypothesis we used a control Single Comb White Leghorn (SCWL)
line and an intermediate dystrophic line (LSN) reported to exhibit
breast muscle anomalies post-hatch. The previously established staging
mechanism was used to investigate the relationship between temporal
MyHC isoform transcription and the correlated transcription of known
myogenic transcription and regulatory factors in the embryonic PM
in the 2 unique poultry lines. Tissue samples from the embryonic PM
were collected at d 5 through 19 from SCWL and LSN embryos. RNA
was isolated and transcription was quantified using NanoString, which
digitally detects and quantifies selected target genes. Data were analyzed
using the LOESS smoothing function at a 95% confidence level. Our
results reveal differences in the quantitative temporal transcription of
the MyHC isoforms between the SCWL and LSN with shifts in peak
transcription being different. Transcription of the first embryonic
MyHC isoform, Cemb1, and MyoD, a known myogenic regulator factor
(MRF) had similar transcription patterns. The second MyHC isoform,
Cemb2, and Myf5, another MRF, also had similar transcription patterns.
Transcription of MyoD and Cemb1 in the SCWL was upregulated on
d 15 and 16 over LSN followed by upregulated expression of LSN
over SCWL on d 17 and 18. Myf5 and Cemb2 in the SWCL showed
a decrease and subsequent increase in transcription, while at the same
time expression peaked in the LSN and subsequently decreased. These
data allowed us to describe temporal transcription of regulatory factors
at identical development stages, aiding in informed genetic selection and
ultimately assisting in the identification of biomarkers for the selection
of breed stock.
Key Words: pectoralis major (PM), myosin, isoforms, myogenesis,
transcription
15 Implications of incubation temperature modulation on type
X collagen expression in embryonic duck skeletal development.
A. L. Prickett*1, M. S. Lilburn2, and M. P. Wick1, 1The Ohio State
University, Columbus, OH, 2The Ohio State University, Wooster, OH.
Optimal temperature regulation is an important component of avian
incubation and the process of embryonic development can be accelerated by even small increases in incubation temperature. In the current
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study as well as previous studies in our laboratory, it was shown that an
increase in temperature from 37.5°C to 38.5°C during early incubation
(0 to 10 d) increased embryonic body weight during mid-incubation
(approximately 10 to 18 d) in duck embryos. The aim of this project
was to specifically study the effects of increased incubation temperature
on skeletal development in duck embryos. The femur and tibiotarsus
of White Pekin ducks were collected at various stages of embryonic
development. RNA was isolated from these tissues and used to make
corresponding complementary DNA. This cDNA was then used for a
polymerase chain reaction (PCR) with a primer for type X collagen
found in the literature and the amplicon produced was consistent with the
220 base pair fragments reported in the literature. Type X collagen is a
marker specific to hypertrophic chondrocytes, which are associated with
the terminal maturation of cartilage before the onset of mineralization. It
was observed that type X collagen was expressed in embryos collected
from both temperature treatments as early as d 4 of incubation. These
preliminary results suggest that increasing the incubation temperature
early in embryogenesis was not specific to cartilage maturation as
determined by type X collagen expression.
Key Words: incubation, temperature, duck, skeleton, cartilage
16 Genetic analysis of leg problems and growth in a random
mating meat-type population. F. González-Cerón*1, A. B. Karnuah1, R. Rekaya2, N. B. Anthony3, and S. E. Aggrey1, 1Department
of Poultry Science, University of Georgia, Athens, GA 2Department
of Animal and Dairy Science, University of Georgia, Athens, GA
3Department of Poultry Science, University of Arkansas, Fayetteville,
AR.
Improvement in growth has been widely reported as the cause of leg
problems in broiler chickens. We report herein the genetic relationship
between growth and leg problems in a random mating broiler control
population. The traits studied were valgus (VL), varus (VR), and tibial
dyschondroplasia (TD), which were expressed on a binary scale of 0
(normal) and 1 (abnormal) and growth rates from 0 to 4 (BWG 0–4),
from 0 to 6 wk of age (BWG 0–6), and residual feed intake from 5 to 6
wk of age (RFI 5–6). We used a threshold-linear mixed model for the
joint analysis of the categorical and linear traits. Incidences of VL, VR,
and TD were 26, 4, and 2%, respectively. Heritability of leg problems
ranged from 0.11 to 0.13. Phenotypic correlations pointed to an unfavorable relationship between growth and leg problems; however, the
genetic relationship between growth and leg problems was extremely
weak, ranging from −0.02 to 0.08. There is, therefore, a basis for genetic
improvement in leg problems; however, improved management practices would also go a long way to reduce incidence of leg problems in
broiler chickens.
Key Words: varus, valgus, tibia dyschondroplasia, growth, feed
efficiency
17 Using RNA-seq to characterize the biological basis of variation in feed efficiency in broiler chickens. N. Zhou*1, W. R. Lee2,
and B. Abasht1, 1University of Delaware, Newark, DE, 2Heritage
Breeders, Princess Anne, MD.
Despite tremendous progress in improving feed efficiency (FE) in chickens, a significant portion of feed—a highly valuable commodity—is
still wasted because of poor efficiency of nutrient utilization. A further
consequence is that an excess of manure is produced, causing environmental concerns in regions with intense poultry production. Therefore,
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from both economic and environmental standpoints, efficient use of feed
is vital for sustainable poultry production. Although different selection
criteria have been used for improving FE in broilers, biological basis
of differences in chicken FE is not well understood. To develop more
efficient selection strategies and to foresee potential long-term issues
that may arise by selection for high FE, a more profound understanding of this highly complex trait is needed. Using a high-throughput
RNA sequencing approach and breast muscle samples from chickens
with extreme high and low FE, our research aims to characterize the
biological basis of variation in FE in broiler chickens. Total RNA was
isolated from breast muscle samples harvested from 10 high and 13
low FE chickens at 7 wk of age. Using Truseq Stranded RNA Prep kit
(Illumina, San Diego, CA), each sample was converted to a uniquely
indexed cDNA library, and the resulting cDNA libraries were pooled
and sequenced on an Illumina Hiseq 2000 sequencer. On average, about
34 million short sequence reads (2 × 75 nucleotides) were produced for
each sample. We analyzed the sequence data using bioinformatics tools
Tophat and Cufflinks and detected 1,059 genes differentially expressed
between the high and low FE chickens. Network analysis of these genes
suggests that anabolic pathways; for example, growth hormone and
insulin receptor signaling pathways, are more activated in the high FE
chickens compared with the low FE birds, partly explaining the feed
efficiency advantage of the high FE birds. Overall, results suggest that
many genes and biological pathways in breast muscle contribute to FE
in broilers, and provide important insights into the molecular basis of
variation in broilers’ FE.
Key Words: chicken, feed efficiency, gene expression, breast muscle,
RNA-seq
18 Sperm-mediated transgenesis in chicken using a PiggyBac
transposon system. E. S. Quansah*1,2, J. A. Long2, D. M. Donovan2, S. Becker2, J. Foster-Frey2, B. P. Telugu2, and N. A. R. Urwin1,
1Charles Sturt University and Graham Center for Agricultural
Innovation, Wagga Wagga, New South Wales, Australia, 2Beltsville
Agricultural Research Center, ARS, USDA, Beltsville, MD.
Toward development of transgenic chickens without the use of viral
vectors, we are studying factors affecting sperm mediated gene transfer
(SMGT) using a PiggyBac vector. The plasmid pPBCAG-GFP contains
13-bp terminal invert repeats flanking a GFP gene driven by the CAG
promoter. A helper plasmid containing a PiggyBac transposase gene
is co-transformed to cause transposition of the GFP gene into TTAA
chromosomal sites. Lipofectamine LTX (LPX; Invitrogen) is a new
generation transfection agent with low toxicity reportedly able to infect
a wide range of cell types. Our experiments examined the effects of LPX
and DNA on sperm which had been purified from seminal fluid using
Accudenz gradient centrifugation. The effect of LPX alone (5, 10 or 15
µL) or in combination with the pPBCAG-GFP (5, 10, or 15 µg) on sperm
viability and mobility at 25°C and 41°C (chicken body temperature) was
studied by flow cytometry. Sperm viability was >90% with up to 3 h of
incubation for all treatments. Similarly, LPX alone or in combination
with pPBCAG-GFP had little effect on sperm viability (~90%) or mobility (~0.1) at 25°C for up to 3 h of incubation. A total of 2 × 108 sperm
(purified from seminal plasma) transformed with 15 µL of LPX and 15
µg of pPBCAG-GFP DNA (1 h; 25°C) was inseminated into 13 White
Leghorns hens. Additional hens were inseminated with untransformed
sperm (negative control) or with LPX treated sperm (positive control).
Egg fertility at d 6 of incubation resulting from negative control, positive control, and pPBCAG-GFP transformed sperm was 17.4, 7.7, and
14.3%, respectively. These results demonstrate that a combination of the
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plasmid and LPX did not negatively affect viability, mobility, or fertility
of chicken sperm; however, the low fertility seen over all treatments and
controls suggests that purification of the sperm compromised fertility.
Key Words: chicken, Lipofectamine LTX, PiggyBac, transformation,
viability
19 Site-specific recombination in transgenic chicken-derived
cells. H. J. Lee*, H. C. Lee, Y. S. Hwang, Y. M. Kim, Y. H. Park, T.
S. Park, and J. Y. Han, WCU Biomodulation Major, Department of
Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Site-specific recombination technology has been used a useful tool for
genetic modification as well as studying the specific gene itself by the
targeted introduction of exogenous gene cassette. In spite of the rapid
development of genetic recombination technology, few successful studies have been reported in avian species which have lots of benefits as an
animal model. Therefore, here we applied the site-specific recombination
technology in avian species, chicken. First, we produced transgenic
chickens that have the recombinase recognition target sequences mediated by transposon system and analyzed the transposon integration site.
Then, we performed the site-specific recombination in the transgenic
chicken derived cells that have one identified integration site. As a
result, the gene cassette of donor plasmid was successfully integrated
into predetermined genomic loci in the cells that co-transfected with
recombinase and donor plasmids. In conclusion, site-specific recombination could be widely applied to avian genome rearrangement, and will
facilitate the practical use of chicken as an animal model.
Key Words: site-specific recombination, transgenic chicken, avian
20 Development of a heterophil extracellular trap assay to
determine the effect of broiler chick age and sex on ex vivo innate
immunity against Escherichia coli. M. Cho* and D. R. Korver,
University of Alberta, Edmonton, AB, Canada.
Young chicks rely on innate immunity early in life as adaptive immunity matures. However, relatively little is known about immune system
development in the young chick. A novel assay was developed to assess
the involvement of the heterophil extracellular trap (HET), in which
heterophils release DNA to trap and ultimately kill invading pathogens,
in early chick innate immune function. First, an ex vivo assay to measure
HET was developed using blood from Single-Comb White Leghorn
hens. Then, an experiment was conducted to measure the effect of chick
(Ross × Ross 308) age and sex on ex vivo Escherichia coli bactericidal
activity, phagocytic capacity, phagocytic activation, and HET at 1, 4,
6, and 8 d of age. Phagocytic capacity is the proportion of phagocytic
cells that engulfed at least 1 E. coli bioparticle and phagocytic activation
is the average number of E. coli bioparticles engulfed by each cell, as
measured using flow cytometry. HET is expressed as percentage externalized DNA relative to total DNA. Proc MIXED of SAS was used for
statistical analysis; differences were considered significant at P < 0.05.
There were no interactions of age and sex. The % HET was higher in
male (36.55 ± 2.09%) than in female chicks (29.20 ± 2.29%). Surprisingly, bacterial killing was greater at 1, 4, and 6 d (64.23 ± 6.92%, 72.89
± 3.72%, 82.08% ± 3.67%, respectively) compared with that at 8 d of
age (31.61 ± 9.06%). Phagocytic capacity did not follow a clear pattern with age; phagocytic activation was greater at 4 d (17,021 ± 2,028
relative fluorescence units) than at 1 and 6 d (9,464 ± 3,642 and 10,143
± 1,967 relative fluorescence units, respectively). However, HET was
greater at d 1 (51.01 ± 6.37%) than at any other age (ranging from 23.96
to 29.76 ± 3.35 to 8.17%). This suggests that HET plays an important
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role in early immune protection. The novel HET assay is a useful tool
to measure innate immunity in chickens.
Key Words: innate immunity, heterophil extracellular trap, Escherichia coli, age, broiler
21 Effects of proinflammatory cytokine stimulation on calbindin-D28K gene and protein expression in the uterine mucosa
of hens. T. Nii*, N. Isobe, and Y. Yoshimura, Hiroshima University,
Higashi-Hiroshima, Japan.
Calbindin-D28k plays a primary role in Ca2+ transportation for eggshell
formation. Infection by avian infectious bronchitis virus (IB virus) in
the oviduct leads to disorder of eggshell formation. We have indicated
that gene expressions of proinflammatory cytokines (IL-1β and IL-6)
were increased, but that of calbindin-D28K (calbindin) was decreased
in the uterus after attenuated IB virus treatment. In addition, IL-6
receptor was expressed in the uterine glandular cell. We speculated that
proinflammatory cytokines produced in response to infection affect the
expression of calbindin gene. The aim of this study was to determine
the effects of IL-1β and IL-6 on expression of calbindin in the hen
uterine mucosa. Uterine mucosa tissues collected from White Leghorn
laying hens were cultured in TCM-199 medium with or without chicken
recombinant IL-1β or IL-6 at concentrations of 0 to 1,000 ng/mL for
1.5 or 3 h. Total RNA and protein were extracted from the cultured tissues. Gene expression of calbindin was analyzed by real-time PCR, and
protein density of calbindin was analyzed by Western blot. Calbindin
gene expression was significantly higher in the 100 or 1000 ng/ml IL-1β
group and in the 10 or 100 ng/mL IL-6 group than in the control group
at 1.5 h of culture (P < 0.05). However, no difference was observed in
the 3 h culture between control and IL-1β or IL-6 treatment groups. In
contrast, the density of calbindin protein was significantly lower in the
100 or 1000 ng/mL IL-1β or IL-6 group at 1.5 h, and IL-6 group at 3 h
of incubation (P < 0.05). These results suggest that IL-1β and IL-6 may
temporarily increase the gene expression and decrease protein synthesis
of calbindin in hen uterine mucosa at the early stage of stimulation. We
assume that these proinflammatory cytokines may affect translation of
calbindin-D28K gene to its protein synthesis.
Key Words: calbindin-D28K, IL-1β, IL-6, oviduct, uterine mucosa
22 Monitoring leukocyte recruitment in chicken blood and
feather pulp during the inflammatory response to lipopolysaccharide (LPS). K. A. Byrne*, O. Alaamri, and G. F. Erf, University of
Arkansas, Fayetteville, AR.
In chickens, intravenously injected LPS (cell wall component of gramnegative bacteria) was shown to activate monocytes/macrophages as
well as recruit leukocytes into the blood and organs (Bowen et al.,
2009). We established intradermal (pulp) injection of growing feathers
as a minimally invasive test-site to monitor the in vivo tissue effects of
various substances. The purpose of this study was to monitor qualitative
and quantitative aspects of leukocyte recruitment into feather pulp and
blood after intradermal injection of LPS into growing feathers. Growing
feathers of twelve 16-wk-old, layer-type roosters were injected with
either 10 µL of sterile, endotoxin-free PBS or 10 µL of 1 mg/mL LPS
per feather (18 feathers/chicken). Blood and 3 feathers were collected
at each time point: 0 (before injection), 2, 4, 8, and 24 h post-injection.
For each time point, blood leukocyte concentrations were determined
using an automated hematology analyzer. Feather pulp cell suspensions were immunofluorescently stained to determine recruitment of
heterophils, macrophages, and lymphocyte subpopulations (% of pulp
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cells). Heterophil concentrations in the blood decreased substantially (P
< 0.001) from 4.94 ± 0.34 to 0.81 ± 0.12 × 103/µL (K/µL) at 2 h postLPS injection, returned to normal at 4 h, increased above control levels
at 8 h (22.29 ± 2.1), and began returning to normal at 24 h. Monocyte
and lymphocyte concentrations in the blood also decreased at 2 h (P <
0.01), but returned to normal levels at 8 h for monocytes and 24 h for
lymphocytes. In the tissue, heterophil proportions were not different (P
> 0.05) from PBS-injected feathers at 0 or 2 h, but increased 20-fold at 4
h post-LPS injection to 23.2 ± 2.21% of pulp cells, continued increasing
at 8 h to 33.7 ± 4.88%, and began decreasing at 24 h to 26.5 ± 1.66%.
Macrophage proportions in the feather tissue were not different (P >
0.05) from controls at 0, 2, or 4 h, but increased 6-fold at 8 h and began
returning to normal at 24 h. We conclude that intradermal LPS injection
into growing feathers results in significant leukocyte recruitment into
the blood with corresponding recruitment levels reflected in the tissue
a few hours later.
Key Words: LPS, intradermal injection, inflammation, leukocytes
23 Cloning and functional characterization of the turkey macrophage migration inhibitory factor. M. Park*, S. Kim, and R. A.
Dalloul, Virginia Tech, Blacksburg, VA.
this study was to clone the turkey MIF (TkMIF) gene, express the active
protein, and characterize its basic function. The full-length TkMIF gene
was amplified from total RNA extracted from turkey spleen, followed
by cloning into a prokaryotic (pET28a) and a eukaryotic (pcDNA3.1)
expression vectors. Sequence analysis showed that TkMIF consists of
115 amino acids with 12.6 kDa molecular weight. Multiple sequence
alignment revealed 100% and 65% identity with chicken and duck
MIF, respectively, while TkMIF shared 93 and 67% identity with zebra
finch and falcon MIF, respectively. Recombinant TkMIF (rTkMIF) was
expressed in Escherichia coli and purified using Ni+-resin and endotoxin removal. SDS-PAGE analysis revealed an approximately 12 kDa
rTkMIF monomer in soluble form, similar to chicken MIF. Because there
is 100% identity between turkey and chicken MIF, Western blot analysis
with anti-chicken MIF antibody detected monomer (12 kDa) and dimer
(24 kDa) forms of TkMIF. Further, a chemotaxis assay performed using
a modified 48-well Boyden chamber and Diff-Quick staining resulted in
reduced migration of macrophages by rTkMIF (P < 0.05). In conclusion,
TkMIF shares sequence and functional similarities with chicken MIF
and further research of its role in turkey immune responses is warranted.
Key Words: macrophage migration inhibitory factor, turkey, chemotaxis, immunity
Macrophage migration inhibitory factor (MIF) is a soluble protein factor
that inhibits the random migration of macrophages and plays a pivotal
immunoregulatory function in innate and adaptive immunity. The aim of
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Poult. Sci. 93(E-Suppl. 1)

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