Transcriptional Profiling of Spleen Lymphocyte in Fowl Typhoid of Broilers
This study was carried out to investigate the differentially
expressed genome between S. gallinarum infected and uninfected
control in the spleen lymphocytes of Ross broiler chicks using microarray
analysis. GeneChip Chicken Genome Array containing 32,773 transcripts
corresponding to over 28,000 chicken genes for simultaneous expression
was used. The signal intensity of each gene was normalized and expressed
in fold change. A large numbers of genes were found with differential
expression majority of which are still unknown in chicken genome. Thirty
one known genes were found to have differential expression of which, 25
were up-regulated and 5 were down regulated. Majority of the up-regulated
genes belong to immune response system viz., IL8, IL1B, IL10, IL18,
IL17A, IL15, transferrin, IFNg, TLR2, TNFRSF1b, TNFRSF15 and the down
regulated genes were B-FIV, B-LA, SDF1, B-LBI, belonging to MHC-I and
II and CD1d. To validate the expression of these genes RT-PCR was done
using primers of 12 selected genes′ with total mRNA isolated from
spleen lymphocytes which has confirmed the similar pattern of expression
of all the genes as in microarray. The findings in this study have lead
to the identification of novel genes which may be useful in further studies
to understand the patho-physiology of fowl typhoid towards development
of diagnostics and therapeutics.
Fowl typhoid caused by Salmonella gallinarum (SG) is recognized worldwide
as a disease of social and economic significance. Salmonella enterica
serotype gallinarum is a non-motile, host adapted avian pathogens belonging
to Salmonella serogroup D (Shivaprasad, 1997).
The out break of fowl typhoid is characterized by increased mortality, anorexia,
greenish-yellow diarrhea and a drop in egg production (Brown
et al., 2007). Subacute outbreak leads to egg transmission to chicks
which increases dead or weak chicks. Though, it has largely been eradicated
from countries with intensive poultry industry for many years, fowl typhoid
caused by SG is still of considerable economic significance to the poultry industry
in many countries of Africa, the Middle East, Central and South America and
Asia (Shivaprasad, 2000). The quick detection of this
pathogen is therefore, extremely important.
Molecular understanding of the mechanism of the disease is important for developing
new diagnostic tools and in designing of disease specific therapies. Conventional
Kaufmann White scheme is still the only reliable method for serotyping of Salmonella,
however, it is time consuming and cumbersome (Ewing, 1986).
Use of classical methods based on biochemical tests /assays are tedious and
time consuming (Christensen et al., 1992; Shah
et al., 2001). Moreover, recent reports of intermediate strains with
variable biochemical pattern casts doubts on the validity of these biochemical
assays (Jia et al., 1993; Shah
et al., 2005).
The large scale analysis of gene function, is central to functional genomics.
Parallel quantitative display of genes is considered the most promising strategy
for biomarker discovery. Genomic analysis, which refers to large-scale study
of gene expression and function has gained great interest (Pandey
and Mann, 2000). Recently, genomic analysis has gained great interest for
the determination of biochemical processes involved in diseases (Fung
et al., 2000). The comparative characterization of gene expression
patterns in tissues has the potential to serve as the basis for new diagnostic
tools and in designing of disease specific therapies (Sinz
et al., 2002). Genomics is gaining popularity in the research on
animal and poultry diseases. Because of its biomedical utility, the poultry
will imminently join those vertebrates that have representative genomes sequenced
(Burt and Pourquie, 2003).
There are recent reports of gene expression due to Salmonella enteritidis
infection in chicken spleen (Zhou and Lamont, 2007) chicken
macrophage (Withanage et al., 2004) and chicken
intestine (Hemert et al., 2006). However, cellular
and molecular mechanisms of fowl typhoid is not yet well understood, because
of lack of large number of biomarkers for SG infection process in the poultry.
Gene expression in response to Salmonella in vivo and in vitro
in chicken mainly focused on cytokines and chemokines in various tissues (Kaiser
et al., 2000; Withanage et al., 2004;
Cheeseman et al., 2006). However, no detailed
study on genome analysis of fowl typhoid has been done yet.
Microarray technology is expected to revolutionize the biological research
field through the simultaneous analysis of gene expression patterns in the whole
genome scale. Hence, GeneChip Chicken Genome Array containing comprehensive
coverage of 32,773 transcripts corresponding to over 28,000 chicken genes was
used for the simultaneous investigation of gene expression changes in spleen
lymphocyte in chickens. The spleen plays a vital role in pathogenesis of Salmonella
infection of chickens (Hendersson et al., 1999).
Therefore, this study was carried out to investigate the differentially expressed
genome between S. gallinarum infected and uninfected control in the spleen
lymphocytes of chicken using microarray analysis.
MATERIALS AND METHODS
Twenty, one-day-old Ross broiler chicks were procured from Yanggi
Hatchery, Pyeongtaek, S. Korea. After six weeks of normal feeding the
chicks were randomly divided into two groups. One group was kept away
as uninfected control the other group was infected by intramuscular injection
with 1 mL of S. gallinarum (2.9x107 mL-1).
Randomly, 3 chickens were selected from each group after one week of infection
and spleens were collected after appropriate anaesthesia. Chicken spleens
were pooled together and lymphocytes were separated (Histopaque H-1070,
Isolation of Total RNA from Spleen Lymphocyte
Total RNA was isolated from chicken spleen lymphocyte samples collected
from control and infected chicken using a Trizol Reagent (Invitrogen,
USA). One milliliter of Trizol reagent was added to a 10 cm diameter dish
with cells and the cells were passed through a pipette several times.
The homogenized sample was incubated for 5 min at room temperature to
permit the complete dissociation of nucleoprotein complexes. The sample
was mixed with 0.2 mL of chloroform per 1 mL of Trizol reagent by shaking
vigorously by hand for 15 sec and incubated for 2 to 3 min. Then, the
samples were centrifuged at 12000 rpm for 15 min at 4°C. Following
centrifugation, the mixture was separated into a lower red phenol-chloroform
phase, an interphase and a colorless upper aqueous phase. The aqueous
phase was transferred to a fresh tube and precipitated the RNA from the
aqueous phase by mixing with isopropyl alcohol. Then 0.5 mL isopropyl
alcohol was added per 1 mL Trizol reagent used for the initial homogenization
and samples were incubated at room temperature for 10 min and centrifuged
at 12000 rpm for 10 min at 4°C. RNA pellet was washed by with 1 mL
of 75% ethanol per 1 mL of Trizol reagent. The RNA pellet was briefly
dried and 20 μL of RNAase-free water was added and incubated for
10 min at 56°C. Finally, the RNA concentration was measured in the
Hybridising and Analysis of Microarray
GeneChip Chicken Genome Array which contains comprehensive coverage
of 32,773 transcripts corresponding to over 28,000 chicken genes was used
for the simultaneous investigation of gene expression changes in spleen
lymphocyte in chickens.
The generation of GeneChip data from RNA isolated from spleen lymphocytes
of normal and infected chicken was performed by Seoulin Bioscience Corporation
(Seoul, Korea). About 5 μg total RNA from the normal and infected
chicken cells were used for labeling. Probe synthesis from total RNA samples,
hybridization, detection and scanning were performed according to standard
protocols from Affymetrix (California, USA). Briefly, cDNA was synthesized
using the One-Cycle cDNA Synthesis Kit (Affymetrix). Single-stranded (ss)
cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo
(dT) primers at 42°C for 1 h. Double-stranded (ds) cDNA was obtained
using DNA ligase, DNA polymerase I and RNase H at 16°C for 2 h, followed
by T4DNA polymerase at 16°C for 5 min. After cleanup using a Sample
Cleanup Module (Affymetrix, CA), ds cDNA was used for in vitro
transcription (IVT). cDNA was transcribed using the GeneChip IVT Labeling
Kit (Affymetrix) in the presence of biotin-labeled CTP and UTP. Then the
biotin-labeled IVT-RNA was fragmented and hybridized to the porcine genome
GeneChip array at 45°C for 16 h, according to the manufacturer′s
instructions. After hybridization, the arrays were washed in a GeneChip
Fluidics Station 450 with a non-stringent wash buffer at 25°C, followed
by a stringent wash buffer at 50°C. After washing, the arrays were
stained with a streptavidin-phycoerythrin complex. After staining, intensities
were determined with a GeneChip scanner, controlled by GeneChip Operating
Software (GCOS; Affymetrix).
To validate the differential expression of genes in infected chicken
spleen lymphocytes semi-quantitative RT-PCR analysis was performed using
specific primers for 12 selected genes and GAPDH as control (Table
1). cDNA was synthesized from RNA isolated from lymphocytes using
a SuperScriptTM II (Invitrogen, USA). The thermo cycler profile
was 5 min 94°C (initial denaturation) and then 22-30 cycles of 40
sec at 94°C (denaturation), 30 sec at 60-65°C (annealing), 40
sec at 72°C (extension) and followed by a 10 min final extension at
72°C. The PCR products were analyzed by electrophoresis (2.0% agarose
gels) in 40 mM Tris-acetate containing 1 mM EDTA (1x TAE) (in ethidium
bromide fluoresce). Quantitative analysis of PCR product was done using
a Sigma Gel software (Jandel Scientific) comparing with GAPDH standard.
||Primers used for validation of the expression of selected
genes in mRNA from spleen lymphocytes of fowl typhoid infected and
uninfected control chicken in RT-PCR analysis
||Image of gene chip hybridized with mRNA from spleen
lymphocyte of control (A) and Salmonella gallinarum infected
chicken (B) gene chip
This experiment was conducted to study the gene expression profile in
fowl typhoid by applying microarray analysis. Six week old chicks were
experimentally infected with S. gallinarum and lymphocytes from
spleen of control and Salmonella gallinarum infected chicken were
isolated. The gene expression profiles of spleen lymphocytes using GeneChip®
Chicken Genome Array were analyzed and results are explained (Fig.
Genome-wide microarray analysis showed differential expression of 802
genes, 430 genes were upregulated of which 25 are known and 372 genes
were down regulated of which 6 are known in chicken cells. However, a
large number of differentially expressed genes have not yet been described
in chicken genome database. Gene Chip Operating Software (GCOS) and randomized
microarray (RMA) statistical analysis showed reproducibly differentially
expressed genes. The differentially expressed genes were functionally
annotated with reports from literature. Visualization of these differentially
expressed genes by hierarchical clustering demonstrated that expression
of spleen lymphocytes from normal and infected chicken were similar to
each other. Gene Ontology (http://www.geneontology.org;
GO) annotations were determined for each gene product represented. GO
terms are consistent descriptions of gene products on terms of the biological
processes they are involved in, the cellular components in which they
exist and molecular functions they perform.
||Up regulated genes as expressed in spleen lymphocyte
between fowl typhoid infected and uninfected control chicken in microarray
||Down regulated genes as expressed in spleen lymphocyte
between fowl typhoid infected and uninfected control chicken in microarray
A total of 31 known genes were found to have differential (2 fold change)
expression of which 25 genes were up-regulated and 6 genes were down regulated.
Among the up-regulated genes majority belong to immune response system
(Table 2) and the down regulated genes mostly belong
to MHC-I and MHC-II (Table 3). The important up-regulated
expression of genes observed in spleen lymphocytes are IL1B, IL8, IL10,
IL15, IL17A, IL18,IL 21/22 transferrin, IFNg, TLR2, TNFRSF1B, TNFRSF15
and the down regulated genes were B-FIV, B-LA, SDF1, B-LBI, belonging
to MHC-I and II and CD1d. To validate the expression of genes RT-PCR was
done with mRNA isolated from spleen lymphocytes which has confirmed the
similar expression of all the 12 genes (Fig. 2). A comparison
of gene expression in microarray and RT-PCR analysis is presented in Table
||Comparison of expressions of selected genes in mRNA
from spleen lymphocyte of fowl typhoid infected and uninfected control
chicken in RT-PCR analysis
||Comparision of the level of differential expressions
of selected genes in spleen lymphocyte between fowl typhoid infected
and uninfected control chicken in microarray and RT-PCR analysis
Study of the interaction between host and pathogen at molecular level has become
a major research area in functional genomics. In this study a search was made
for biomarkers in fowl typhoid, though, no report is available about the functional
genomic search on fowl typhoid. Differential expression of cytokines and chemokines
such as interleukin-6 (IL-6), IL-1b, IL-10, IL-12a, IL-12b, IL-18, CXCR1, CXCR4
with infection of Salmonella enteritidis or Salmonella typhimurium
in chickens and chicken lymphocytes have been reported in many studies (Kaiser
et al., 2000; Kogut et al., 2003; Beal
et al., 2005; Withanage et al., 2004,
2005; Wigley et al., 2005,
2006; Cheeseman et al., 2006).
Microarray technology has the advantage of simultaneously investigating the
expression of thousands of genes, to enable the study of gene interactions and
signal pathways. The current study utilized a chicken microarray to investigate
differential gene expression between Salmonella gallinarum inoculated
and non-inoculated birds.
This is the first study that has determined the genome-wide molecular
expression pattern of spleen lymphocyte from experimental fowl typhoid
and thus provided comprehensive insight into avian salmonellosis. In the
present study experimental fowl typhoid was successfully induced by intramuscular
inoculation in 6 weeks old chickens and the same has been confirmed by
pathogen isolation, gross necropsy finding and clinical symptoms. Large
number of important genes were found with differential expression using
microarray analysis many of them are related to immune response system
which were upregulated (IL1B, IL8, IL10, IL15, IL17A, IL18, transferrin,
IFNg, TLR2). A number of genes related to MHC class I and class II (B-FIV,
B-LA, SDF1, B-LBI) chemokine CXC and CD1d were down regulated.
Chemokines, cytokines and their receptors play very important roles in Salmonella
infection in chickens (Withanage et al., 2004).
Many chemokines, cytokines and their receptors were differentially expressed
with SG infection in the current study and IL 1b is an important among them.
In poultry IL1B recognized as pro-inflammatory and stimulates immune systems.
IL1B activates a range of cell including macrophages and T cell which lead to
produce other cytokine and chemokines (Wigley and Kaiser,
2003). The up-regulated expression of IL1B gene might lead to the high level
of this cytokines which as a consequence lead to fever (Wigley
and Kaiser, 2003). IL-1b mRNA level was elevated with infection of SG in
the present study. An increased IL-1b mRNA level was also observed in heterophils
(Ferro et al., 2004) and in spleen tissue (Zhau
and Lamont, 2007) chicken infected with SE. Withanage et
al. (2004) reported higher mRNA level of IL-1b in spleen, cecum, ileum
and liver at different time points post-infection with S. typhimurium in
Rhode Island Red chickens. A higher level of IL-1b mRNA was detected in spleen
and cecal tonsils at day 6 with challenge of S. typhimurium in birds
(Beal et al., 2005). Upregulation of pro-inflammatory
cytokine might be crucial to recruit lymphocyte cells to the site of infection,
to subsequently mount an efficient innate and adaptive immune response to infection.
IL8 is a chemokine having chemotactic activity for specific leukocytes (Wuyts
et al., 1998) which appears to specifically activate neutrophils
in response to infection in human. In chicken IL8 is equivalent to mammalian
IL8 though functionally it is designated as chicken chemotactic and angiogenic
factor (CAF) (Kaiser et al., 1999). It has been
reported that IL8 produced an influx of heterophils in chicken gut with S.
typhimurium infection (Henderson et al., 1999)
which suggested the induction of IL8. Therefore, it appears to be natural to
have induction of this chemokine at gene label in spleen lymphocytes in present
study as also reported in mammalian Salmonella infection induced IL8
in the gut epithelium (Wallis and Galyov, 2000).
IL 15 in mammals is a T cell growth factor stimulate the growth of T lymphocytes,
NK cells (Kennedy et al., 1998). IL15 induces proliferation,
cytokine production and migration of T cells and also stimulates proliferation
of number of other cells including B lymphocytes. Recently, it has been proposed
that IL-15 plays a major role in driving autoimmune thyroiditis in obese strains
of chickens (Kaiser et al., 2002). IL-18 in mammals
induces production of IFN-g (Okamura et al., 1995).
It is produced at high levels in the liver by macrophages and Kupffer cells
and plays important role in the initiation of cell mediated immune response
(Wigley and Kaiser, 2003). The chicken IL-18 gene is
a quarter in length and a bioassay has shown the similar response of chicken
IL-18 as its human counterpart (Kaiser et al., 2001).
IFN-g is type II interferon which plays vital role in macrophage activation
and modulation of the immune system (DeMayer and DeMayer,
1988). It is produced primarily by T lymphocytes and natural killer (NK)
cells leading to activation of macrophage antimicrobial activity. It has potent
macrophage activating factor activity that is heat and pH labile (Lowenthal
et al., 1995).
Besides, several other chemokines, their receptors and cytokines were also
differentially regulated in the current study as mentioned earlier. Upregulated
gene expression of MIP-1, IL-8, CXCR4 with infection of SE and/or S. typhimurium
has been found in other studies (Ferro et al.,
2004; Swaggerty et al., 2004, 2006;
Withanage et al., 2004; Wigley
et al., 2006). The upregulation of these genes suggests that these
cytokines are important in the initiation of the pro-inflammatory response to
defend the host against SE infection. Several genes related to MHC were down
regulated in this study. However, several genes of the T-cell surface glycoprotein
family were significantly upregulated in the broiler with SE infection (Zhou
and Lamont, 2007). This might be a different kind of response in case of
SG which needs further confirmation. The CD complex, is essential for signal
transduction of the T cell receptor and proper T-cell responses against infections
(Janeway et al., 1999). Upregulation of the CD3
complex in chickens with SE infection was reported by Zhau and Lamont (2007)
to be associated with activation of the T cell receptor, which would then modulate
T cell immune response to fight SE infection. However, in this study a down
regulation of CD1 was observed.
The search for differentially expressed genes is an important means to understand
pathological pathways to find markers of disease for diagnosis and for treatments.
There are recent efforts towards this direction for Salmonella enteritidis
in chicken spleen (Zhou and Lamont, 2007) and chicken
intestine (Hemert et al., 2006). As far as we
know it has not been tried before for understanding the fowl typhoid using spleen
lymphocytes of chicken with microarray analysis, hence it is difficult to compare
the observations. The current study presents the microarray analysis Salmonella
gallinarum infection in chickens and provides an new initiative to study
molecular and cellular mechanisms of gene expression in fowl typhoid disease.
These findings demonstrated the power of microarray technology to study molecular
mechanisms of Salmonella infection in chickens. This also has laid a
foundation to further study cellular and molecular mechanisms of Salmonella
gallinarum infection in poultry.
This research was fully supported by a grant from Biogreen21 program,
Rural Development Administration, Republic of Korea.
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