Gene expression responses to Riemerella anatipestifer infection in the liver of ducks

Abstract

Riemerella anatipestifer is one of the most economically important pathogens of farm ducks worldwide. The molecular mechanisms that underlie its pathogenesis, particularly the host response to R. anatipestifer infection, are poorly understood. The differentially expressed gene profile of duck livers at 24 h following R. anatipestifer infection was therefore investigated using suppression subtractive hybridizaton analysis. A total of 45 differentially expressed genes were identified, which primarily included genes for proteins involved in acute-phase response, inflammatory response, immune response, wound healing and iron homeostasis. For the expression level of 20 genes from those 45 analysed by quantitative reverse transcriptase-polymerase chain reaction at 8, 24 and 48 h post infection, significant differences were observed among the three time points of measurements. The result from this study revealed a gene expression profile of duck liver during R. anatipestifer infection, and those genes with a role in the immune response and wound healing deserving further investigation to elucidate their respective roles during infection.

Introduction

The duck is an important food bird, with more than 3 million metric tons of meat produced annually, primarily in Asia. Among the diseases of domestic ducks, R. anatipestifer infection is a major cause of considerable economic loss to duck production. R. anatipestifer is a Gram-negative bacterium that causes serious disease in ducks. The disease is characterized by fibrinous pericarditis, perihepatitis and meningitis (Sandhu & Rimler, 1997). Twenty-one serotypes of R. anatipestifer have been identified with no significant cross-protection reported (Loh et al., 1992; Pathanasophon et al., 1995, 2002). The infection is a continual problem in the intensive production of meat ducks, especially in China and other countries in Southeast Asia (Guo et al., 1982; Teo et al., 1992; Subramaniam et al., 2000; Cheng et al., 2003). Because a specific vaccine is currently unavailable, medication with antibiotics represents the only recourse. However, the subsequent development of antibiotic resistance is becoming another potential threat to food safety and human health.

Despite the devastating losses it causes to the duck industry, not much is known about the pathogenesis of R. anatipestifer infection and its virulence factors (Weng et al., 1999; Crasta et al., 2002; Zhou et al., 2009). The reported R. anatipestifer whole genome sequence should facilitate the understanding of disease mechanisms of this pathogen (Zhou et al., 2011). The pathogenesis of bacterial diseases and their development is a complex interactive process between the pathogen and the host. The R. anatipestifer infection usually occurs in an acute septicaemic form in ducklings, indicating that the early immune response of the host may play an important role in defence against infection. However, no information is known about the transcriptional response of a duck to R. anatipestifer infection. Previous studies indicated that the vertebrate liver is a major organ responsible for metabolism, and participates in host defence and tissue repair (Parker & Picut, 2005). Hence, the identification of differentially expressed genes in duck livers induced by R. anatipestifer infection may contribute to our understanding of the pathogenic process of R. anatipestifer infection.

Suppression subtractive hybridization (SSH) has been successfully used to identify host genes differentially expressed during infection or immune stimulation (Lin et al., 2007; Zhang et al., 2008; Tian et al., 2009). In the present study, the SSH analysis was employed to detect genes differentially expressed in duck livers during R. anatipestifer infection. After that, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the data obtained from SSH analysis.

Materials and Methods

Bacterium, experimental infection and tissue collection

R. anatipestifer RA-YM, a serovar 1 strain, was used as the challenge strain for this study. RA-YM is a field strain isolated from YunMeng county of Hubei province in China and is a highly virulent strain in ducks (Li et al., 2004). R. anatipestifer strain RA-YM was grown on Tryptic Soy agar (Difco, Detroit Michigan, USA) containing 5% newborn calf serum (Gibco, Carlsbad, California, USA) at 37°C for 24 h at 5% CO_2. A single colony was inoculated into 100 ml Tryptic Soy broth (Difco) and incubated at 37°C with shaking until an exponential growth phase was achieved. The final challenge inoculum concentrations were determined by plating 0.1 ml of a 10-fold serial dilution onto Tryptic Soy agar containing 5% newborn calf serum.

Forty 10-day-old healthy Cherry Valley ducks (Anas platyrhynchos), not previously exposed to R. anatipestifer, were purchased from a commercial farm (Wuhan, China) and divided randomly into two groups: non-infected (n=15) and infected (n=25). A group of 25 ducks were inoculated at 12 days old via their foot pad with 0.5 ml suspension containing 1.0×10⁷ colony-forming units of R. anatipestifer RA-YM. The other group was left non-infected as controls. Ducks were inspected visually twice a day for clinical signs of R. anatipestifer infection (i.e. depression, anorexia, diarrhoea, ataxia, and lameness). At 8 h, 24 h and 48 h post infection, infected ducks were sacrificed and necropsied (n=4, n=11 and n=4, respectively). Livers from infected ducks and control ducks were collected and frozen in liquid nitrogen immediately before RNA extraction. Liver samples of infected ducks were confirmed to be culture positive, with homogenized liver samples yielding dense growth on Tryptic Soy agar plates. Seven liver samples of infected ducks and control ducks at 24 h post infection were used for SSH analysis. The experimental protocols described herein were approved by the Institutional Animal Experimental Committee of the Veterinary Faculty of Huazhong Agricultural University (Permit Number: 10–0818). All surgery was performed under chloral hydrate anaesthesia, and all efforts were made to minimize suffering.

RNA extraction and mRNA isolation

Total RNA was isolated from liver with TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instruction. The total RNAs from livers of infected or control ducks (n=7 per group) were each pooled for further experiments. All of the total RNA samples were subjected to mRNA purification using the Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). Their concentrations and quality were tested by measurement of absorbance at 260 nm and 280 nm and by agarose gel electrophoresis.

Construction of cDNA libraries by SSH

Two subtractive cDNA libraries were constructed by SSH using the PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, California, USA), according to the manufacturer's instructions. The forward library was screened for the up-regulated genes while the reverse library was examined for down-regulated genes. Briefly, the pooled liver mRNA (2 µg) from infected or control ducks (n=7 per group) was reverse transcribed into cDNA. For the forward subtraction, cDNA from control group of ducks was used as the driver, and cDNA from infected group was used as the tester. For the reverse subtraction, the cDNAs from the control and infected groups were employed as the tester and the driver, respectively. After digestion with RsaI, the tester cDNA was divided into two subpopulations, each of which was ligated with a unique adaptor at 16°C for 8 h. The adaptor 1-ligated or adaptor 2R-ligated tester cDNA was hybridized separately at 68°C for 8 h with an excess of driver cDNA after denaturalization at 98°C for 90 sec. The two hybridized samples were then mixed without denaturalization and were hybridized at 68°C for 12 h with an excess of denatured driver cDNA to further enrich for differentially expressed sequences. The resulting mixture was diluted and amplified by two suppressed PCRs. The primary PCR was performed using PCR primer from the kit. After incubating the reaction mixture at 75°C for 5 min to extend the adaptors, a 25 µl PCR reaction was subjected to 30 cycles at 94°C for 30 sec, 68°C for 30 sec, and 72°C for 90 sec. The amplified products were further diluted 10-fold. A 1 µl aliquot of diluted primary PCR products was used as template for the secondary nested PCR with 15 cycles of 94°C for 30 sec, 68°C for 30 sec, and 72°C for 90 sec using the primers, Nested 1 and Nested 2R, provided in the kit (Nested1, 5′-TCGAGCGGCCGCCCGGGCAGGT-3′; Nested2R, 5′-AGCGTGGTCGCGGCCGAGGT-3′). The PCR products obtained from the forward and reverse subtractions were separately cloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA), and transformed into Escherichia coli JM109 (Promega) to establish different cDNA libraries. Each recombinant clone was grown in 96-well plates containing 200 µl Luria broth medium supplemented with ampicillin (100 µg/ml). Stocks were made by addition of 15% glycerol followed by storage at −70°C.

Screening for differential expression by dot blotting

Subtracted clones were subjected to screening for the differential expression of the unique genes as described previously (Tian et al., 2009). Briefly, the cDNA inserts were amplified by PCR with the nested primers Nested1 and Nested2R using the following conditions: 95°C for 1 min followed by 30 cycles of 95°C for 30 sec and 68°C for 1.5 min. The PCR products were analysed on 1.5% agarose gels to identify clones containing inserts. Individual PCR products were first denatured with 0.3 N NaOH, and spotted onto nylon membranes (Millipore, Billerica, Massachusetts, USA) in duplicates. The PCR products were then fixed by baking in an oven at 80°C for 2 h. DIG-labelled cDNA probes from either the forward or reverse subtractions were prepared, using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocols. To prepare probes, the DNA templates were denatured and treated with the DIG-High Primes for 12 h at 37°C, followed by heating. The duplicate membranes were hybridized at 65°C for 16 h with two probes generated from the forward and reverse subtractions, respectively. The clones that were hybridized to the tester probe alone or had five-fold greater signal intensity over the control as determined by densitometry were considered positive, and were then selected for further analysis. The images were quantified by densitometry scanning, followed by analysis with GeneSnap (Syngene, Frederick, Maryland, USA).

Database and sequence analysis

Plasmids from positive clones were extracted and their inserts were sequenced using the primer M13–47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) in an ABI 3700 automatic sequencer (Invitrogen Biotechnology Ltd, Shanghai, China). All cDNA sequences generated were analysed by comparing them with the GenBank database using the BLASTX and BLASTN.

Table 1.  Primers used in quantitative RT-PCR analysis for 20-gene transcripts relative to β-actin.

Gene	Primer	Size (base pairs)	Annealing temperature (°C)
FGC	F: 5′-TGGCTGGACAGTGTTACAGAGGAG-3′	172	58
	R: 5′-CAGGGTGTATGGCAGAGTGGATTG-3′
ALB	F: 5′-TATCCCAAGGCTTCATTCGCAG-3′	104	60
	R: 5′-CTGTCATCCATGCACTCCACCA-3′
TTR	F: 5′-GTGCTGGATGCAGTCAGAGGA-3′	101	58
	R: 5′-GTTTTCCCAGTGGCAAAGTCC-3′
HBAA	F: 5′-CAACGTCAAGGGTGTCTTCTCC-3′	129	58
	R: 5′ -CCGTGGGACAGGTCAAAGTG-3′
ApoA-I	F: 5′-TGGCTGAAGGACACCGAGAG-3′	128	58
	R: 5′-GTCCACTTGGCGGAGAACTG-3′
Ex-FABP	F: 5′-TTGCTCTGGCCTCCAACTCTG-3′	149	58
	R: 5′ -GATTGTCTCCCGTCTCCTACACC-3′
C7	F: 5′-CGAGGCATGAAACATCAGACAGTGG-3′	143	60
	R: 5′-AGTGAGAAAGTCTGGGCGGTTG-3′
MASP-2	F: 5′-ATTCCATCCGCATCTACTTCAC-3′	137	58
	R: 5′-AGCCTCCTCGGTGTCTGT-3′
PIT54	F: 5′-ATTACCATCTCCACCTCAGTCCG-3′	114	58
	R: 5′-TAAATCCCAGCCGTCGTCAC-3′
OTF	F: 5′-AGGACAAGTAACAGGATGGGAAGG-3′	126	58
	R: 5′-CAGAAGAAGCAGTGAAGGCAGAG-3′
FTH1	F: 5′-CGCATCTTCTTGCAGGACATC-3′	119	60
	R: 5′-TGCAGCTCCAACAGCGACT-3′
IL-6	F: 5′-TTCGACGAGGAGAAATGCTT-3′	150	60
	R: 5′-CCTTATCGTCGTTGCCAGAT-3′
OIH	F: 5′-AACAGGGAGCACAGGACAAACG-3′	123	60
	R: 5′-GCAGGCTACCAGAACACCACCAC-3′
AICL-2	F: 5′-ACCGCTCGCTGCATTTGG-3′	102	60
	R: 5′-GCAGGGTCTCGCACTTGGA-3′
DEFB	F: 5′-CCTTTACCTGCTCTTTGCTGTTCTC-3′	149	58
	R: 5′-ATCCCGAGGCAGCGAATGT-3′
CCL19	F: 5′-CAGGAAGGTCCCAAATAAAGGCA-3′	131	60
	R: 5′-TGTAGTAGGAGGTGGAAGCAAGTCG-3′
VASH1	F: 5′-CCTGGCTCCACAGACTTCTTGCT-3′	88	58
	R: 5′-GTTCCATTCCTGCTCCCCTATC-3′
VTN	F: 5′-TGGAGCTGGCTGCATAACGA-3′	107	58
	R: 5′-TTGTCCCCGACGAAGAAGTAGA-3′
BRP44L	F: 5′-CACAACTTATTCAAGGAGGACGAC-3′	109	58
	R: 5′-CTTGGTTGTGATTCGGGATGAG-3′
SCF25	F: 5′-TGTAGATGGGGCTGGAAACTGAC-3′	128	58
	R: 5′-CTGAACCAGTGACGCTGATTTCTC-3′
ACTB	F: 5′-TGGGTATGGAGTCCTGTGGCA-3′	147	58
	R: 5′-AGGGCTGTGATCTCCTTCTG-3′

Analysis of mRNA transcripts by quantitative RT-PCR

The differential expression of 20 gene mRNA transcripts was verified using quantitative one-step RT-PCR. Total RNA extraction from four individual liver samples from the control or infected group of ducks at 8 h, 24 h, and 48 h post infection served as a template for amplification using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Carlsbad, California, USA). RT-PCR transcripts from each duck specimen were amplified and detected using the QuantiTect SYBR Green RT-PCR kit (Qiagen). The specific primers listed in Table 1 were synthesized by Invitrogen Biotechnology Ltd and were designed based on submitted sequences of the clones. Thermal cycling parameters were as follows: 50°C for 30 min, 95°C for 1 min, 40 cycles at 95°C for 15 sec, 58°C or 60°C for 15 sec (the annealing temperature varied depending on the gene specific primers), and 72°C for 20 sec. At the extension step, fluorescent data acquisition was performed. Following thermal cycling, disassociation curve analysis was performed at 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec. Data acquisition was performed at the final 60 to 95°C ramp and the final 95°C step. Analysis of the disassociation curves confirmed that fluorescent signal was generated only from specific cDNA transcripts. The housekeeping gene β-actin was used to normalize the level of target gene expression due to its uniform expression levels over a variety of tissue and treatment conditions (Skovgaard et al., 2009; Tian et al., 2009). Each individual sample was run in triplicate. The average critical threshold cycle (Ct) value was used for calculating relative quantification. The fold-change in expression of the target gene is presented as 2^−ΔΔCt, where:

Statistical analysis

Data from the cDNA dot blotting hybridization and quantitative RT-PCR (fold-change) were analysed by the Student's t test using SPSS12.0. (SPSS Inc., Chicago, IL, USA) P < 0.05 was considered to be significant difference and P < 0.01 was considered to be highly significant difference.

Results

Screening of the SSH cDNA libraries

Two cDNA libraries, duck liver forward (DLF) and duck liver reverse (DLR), were established by SSH analysis. While the DLF library (subtraction of control from R. anatipestifer-infected liver) contained R. anatipestifer-infection up-regulated genes, the DLR library (subtraction of R. anatipestifer-infected liver from the control) contained genes down-regulated by R. anatipestifer infection. The inserts of both libraries were analysed by PCR and were found to have size ranging from 200 to 1100 base pairs. These clones were further screened by two rounds of dot blot. A total of 181 positive clones were produced and sequenced. Forty-five sequences, 36 from the DLF library and nine from the DLR library, were finally obtained (Table 2). We therefore detected 45 genes (36 up-regulated genes and nine down-regulated genes) that were differentially expressed in the duck livers after R. anatipestifer infection over the control uninfected ducks.

Table 2.  Differentially expressed genes in the livers of ducks (A. platyrhynchos) following R. anatipestifer infection.

Functional classification	Gene symbol	Homologous gene	Accession number	Clone	Library
Acute-phase response	FGA	Fibrinogen alpha chain	JK342147	I41C6	DLF
	FGB	Fibrinogen beta chain	JK342148	I7F7	DLF
	FGC	Fibrinogen gamma chain	JK342149	I21C6	DLF
	AT	Antithrombin	JK342150	I7B10	DLF
	ALB	Albumin	JK342151	N4G10	DLR
	TTR	Transthyretin	DQ334337	N12C11	DLR
	HBAA	Haemoglobin, alpha 1	JK342152	N2F11	DLR
	ApoA-I	Apolipoprotein A-I	JK342153	N1G7	DLR
	Ex-FABP	Extracellular fatty acid binding protein	JK342154	I8B4	DLF
Complement cascade	C7	Complement component 7	DR763842	I61A5	DLF
	CFH	Complement regulator factor H	JK342155	I41H9	DLF
	MASP-2	Mannan-binding lectin serine peptidase 2	JK342156	I21G4	DLF
Metal ion binding/transport	PIT 54	PIT 54 protein	JK342157	I7E12	DLF
	OTF	Ovotransferrin	JK342158	I5G6	DLF
	FTH1	Ferritin, heavy polypeptide 1	JK342159	I5F4	DLF
	HPX	Hemopexin	JK342160	I3E11	DLF
	Alpha-2-M	Alpha-2-macroglobulin precursor	JK342161	I7D7	DLF
	Steap3	protein (LOC424275)	JK342162	I2F11	DLF
	CP	Ceruloplasmin precursor (Ferroxidase)	JK342163	I22F4	DLF
	SOD1	Superoxide dismutase 1, soluble	JK342164	I61A12	DLF
Inflammation response	IL-6	Interleukin-6	AB191038	I2D9	DLF
	PGES2	Prostaglandin E synthase 2	JK342165	I43E2	DLF
	NFRB	Nuclear factor related to kappaB binding protein	JK342166	I1H8	DLF
Immune/defence response	OIH	Ovoinhibitor	JK342167	I7A4	DLF
	Nedd4	Nedd4 family interacting protein 1-like	JK342168	I8C2	DLF
	AICL-2	Activation-induced C-type lectin 2	JK342169	I21E1	DLF
	DEFB	Beta-defensin	JK342170	I2D2	DLF
	DAD1	Defender against cell death 1	JK342171	I7A6	DLF
	CCL19	Chemokine C-C motif ligand 19	DR766004	I41B5	DLF
Wound healing	VASH1	Vasohibin	JK342172	N2H9	DLR
	VTN	Vitronectin	JK342173	N2F1	DLR
	BRP44L	Brain protein 44-like	JK342174	N4G7	DLR
	MAEA	Macrophage erythroblast attacher	JK342175	I1E5	DLF
Other function	COX1	Mitochondrion cytochrome c oxidase subunit I	EU009397	I6G2	DLF
	COX2	Mitochondrion cytochrome c oxidase subunit II	EU009397	I6C12	DLF
	COX3	Mitochondrion cytochrome c oxidase subunit III	EU009397	I7C5	DLF
	OCIAD1	OCIA domain containing 1	JK342176	I7A5	DLF
	RAB18	RAB18, member RAS oncogene family	JK342177	I8B12	DLF
	GHITM	Growth hormone inducible transmembrane protein	JK342178	I42A11	DLF
	AdK	Adenosine kinase	JK342179	I61C3	DLF
	GNB2L1	guanine nucleotide binding protein (G protein) Beta polypeptide 2-like 1	JK342180	I21G7	DLF
	GUSB	Glucuronidase, beta	JK342181	I43A12	DLF
	EEF1A1	Eukaryotic translation elongation factor 1 alpha 1	JK342182	I2G10	DLF
	Scf25	Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6	JK342183	N4H5	DLR
	STT3	STT3, subunit of the oligosaccharyltransferase complex, homologue A	JK342184	N5A9	DLR

The proteins encoded by the 45 identified differentially expressed genes were involved in many functions including those in acute-phase response (APR), inflammation response, immune/host defence response, wound healing, iron homeostasis and other functions. A conserved APR was evident in the significantly up-regulated or down-regulated transcripts during R. anatipestifer infection, which include coagulation factors, transport proteins and complement components. Several highly abundant acute-phase proteins (APPs) in the DLF library were the fibrinogen system (FGA, FGB, FGG), transport proteins (FTH1, PIT54), COX and DEFB. These genes were detected at least five-fold higher over the control, even reaching a 33-fold change for FGG. A number of additional genes believed to play important roles in the inflammation response, immune/host defence response, and cellular responses to infection were also up-regulated during infection. These included IL-6, PEGS2, NFRB, CCL19, MASP-2, Nedd4 and AICL-2.

Down-regulated genes in the DLR library included several APPs (ALB, HBAA, Apo-A1, TTR) and others. The observed decrease in mRNA for the albumin-related protein implies that the duck resembles mammals in this regard. Interestingly, two transcripts with important functions were identified. These included VASH1, which is believed to be involved in regulation of angiogenic processes and was the first identified to be down-regulated during bacterial infection, and BRP44L, which functions in the apoptotic process.

Confirmation of differentially expressed genes by quantitative RT-PCR

Quantitative RT-PCR analysis was performed to analyse the expression profiles of the 20 selected SSH-identified genes at three different time points of infection with R. anatipestifer. The relative levels of 20 genes were normalized to β-actin transcript in R. anatipestifer infection or control groups (Table 3). Compared with the non-infected control ducks, a significant change in duck liver gene expression was observed at more than one time point for all 20 genes during R. anatipestifer infection, which is consistent with the experimental strategy.

Table 3.  Results of gene expression analyses of duck livers at different points following R. anatipestifer infection.

	8 h	24 h	48 h
Gene	2^−ΔΔCt±SD	2^−ΔΔCt±SD	2^−ΔΔCt±SD
FGC	3.52±0.12*	2.594±0.41*	3.17±0.06
ALB	0.356±0.02**	0.538±0.08*	0.19±0.03*
TTR	0.123±0.04**	0.235±0.08**	0.053±0.03**
HBAA	0.156±0.08*	0.148±0.072*	0.393±0.01**
ApoA-I	0.31±0.11	0.24±0.06**	1.988±0.02**
Ex-FABP	25.4±0.58**	21.90±0.69**	137.85±0.39*
C7	1.733±0.05*	1.988±0.12*	2.252±0.10*
MASP-2	3.02±0.12*	5.57±0.21*	15.88±0.21*
PIT54	2.07±0.31	6.32±0.26**	6.42±0.22*
OTF	1.566±0.08*	1.83±0.07*	2.415±0.01**
FTH1	2.05±0.02**	2.291±0.11**	2.12±0.05*
IL-6	3.86±0.04**	2.71±0.14*	2.57±0.04*
OIH	2.963±0.04*	1.968±0.17*	12.252±0.16*
AICL-2	2.944±0.30*	1.877±0.04*	4.438±0.06**
DEFB	1.43±0.02*	4.199±0.04**	1.77±0.01**
CCL19	2.26±0.01**	2.16±0.21*	3.09±0.14*
VASH1	0.655±0.05*	0.397±0.04*	0.35±0.06*
VTN	2.202±0.11*	2.405±0.15*	2.745±0.07**
BRP44L	0.319±0.09	0.29±0.14**	0.139±0.05**
SCF25	0.231±0.09**	0.61±0.09**	0.689±0.03*
The levels of mRNA transcripts for 20 genes were determined by quantitative RT-PCR. Relative quantification of gene amplification was performed using the Ct value; fold-change in expression of the target gene is presented as 2^−ΔΔCt. Statistical differences were analysed by Student's t test. *P< 0.05, significant; **P< 0.01, highly significant.

Three immune-related genes (AICL-2, CCL19 and DEFB) were identified as significantly up-regulated at the three time points in the SSH study and were confirmed by quantitative RT-PCR. The four genes (PIT54, Ex-FABP, OIH and MASP-2) exhibited significantly higher levels of gene expression in R. anatipestifer-infected ducks at 48 h post infection compared with the control ducks. The decreased expression of ApoA-I has been described in mammals as inversely associated with the levels of circulating IL-6 and TNF-α. Surprisingly, the expression level of IL-6 was significantly up-regulated over the 48-h period of infection with R. anatipestifer. However, ApoA-I was down-regulated at 8 h and 24 h and significantly up-regulated at 48 h post infection.

Discussion

Although R. anatipestifer is a pathogen of major importance to duck farming industries worldwide, the molecular events of R. anatipestifer infection are largely uncharacterized. In this study, the ducks were first inoculated with R. anatipestifer and then 8 h, 24 h and 48 h later total RNA was isolated from the livers of infected and non-infected ducks. Subsequently, cDNA was synthesized from mRNA and subjected to SSH analysis for identification of differentially expressed genes. The data showed that 45 genes were differentially expressed in infected duck livers compared with the non-infected control duck livers. The expression level of 20 genes in the duck liver was also analysed by quantitative RT-PCR at 8 h, 24 h and 48 h post R. anatipestifer infection. To our knowledge, these data are the first report of gene expression profiles during R. anatipestifer infection that may provide insight into the molecular basis of R. anatipestifer infections.

Among the differentially expressed genes, the most noticeable category was the APR genes. The APR is the systemic inflammatory component of innate immunity, which is believed to help restore homeostasis after infection or other events involving some degree of tissue destruction. These APR genes identified after R. anatipestifer infection included fibrinogen system genes (FGA, FGB, FGG), PIT54, OTF, FTH1, HPX, CP, STEAP3, Ex-FABP, A2M, AT, TTR, ApoA-1, ALB and HBAA (Table 2). Similar subsets of APR were reported to be differentially expressed in pig (Chen et al., 2009), dairy cows (Jiang et al., 2008) and teleost fish (Gerwick et al., 2007; Peatman et al., 2007; Peatman et al., 2008), demonstrating the conservation of function of the majority of APPs.

Regulation of iron homeostasis was a key aspect of the APR observed in this study. Several genes involved in iron binding, transport and storage were up-regulated following R. anatipestifer infection. These were PIT54, OTF, FTH1, HPX, CP and STEAP3. Among these genes, PIT54, OTF and FTH1 were up-regulated to a larger extent 48 h after R. anatipestifer infection by quantitative RT-PCR. PIT54, a soluble member of the family of scavenger receptor cysteine-rich proteins, functions as the major haemoglobin-binding protein that provides protection against the oxidative damage caused by released haemoglobin. Interestingly, the PIT54 gene exists only in birds (Wicher & Fries, 2006). Consistent with a protective role against free haemoglobin, PIT54 may be implicated in regulation of heterophil function by inhibiting the overproduction of the reactive oxygen species (Iwasaki et al., 2001). OTF, another up-regulated gene, has been known to have iron-binding properties. OTF has also been shown to have antimicrobial activities that are independent of iron status. It efficiently prevents the adhesion and invasion of Chlamydophila psittaci and avian Marek's disease virus to tissue cells and modulates the biological responses of macrophages and heterophils (Giansanti et al., 2005; Beeckman et al., 2007). Moreover, OTF and its derived peptides have been found to be capable of killing Gram-negative bacteria, including E. coli, Salmonella enteritidis and C. psittaci (Ibrahim et al., 2000). We speculate that OTF peptides probably kill R. anatipestifer since R. anatipestifer is also a Gram-negative bacterium and thus shares a common outer membrane with other aforementioned Gram-negative bacteria. Recent work has also demonstrated that blood OTF concentration is modulated under inflammation and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens (Xie et al., 2002; Rath et al., 2009). Ferritin is a major intracellular iron storage protein and functions as a cytoprotectant by sequestering iron to minimize the formation of reactive oxygen species. We observed that FTH1 was also up-regulated during R. anatipestifer infection compared with control individuals by quantitative RT-PCR. Ferritin up-regulation has also been described after immune challenge in starfish (Beck et al., 2002) and sea bass (Neves et al., 2009). In addition, the expression of the heavy chains of ferritin is under the control of the NF-κB signalling pathway in mammals. This pathway plays a major role in the immune response, inducing the expression of immune-related genes, and limiting reactive oxygen species-induced apoptosis (Bubici et al., 2006). These genes involved in iron homeostasis were differentially expressed, suggesting that physiological mechanisms within the host may attempt to limit free iron availability to inhibit bacterial growth and avoid iron-induced cellular damage.

There were several down-regulated acute-phase genes differentially expressed during R. anatipestifer infection, including ApoA-1, TTR, ALB and HBAA. ApoA-I, the major protein component of high-density lipoprotein, is a multifunctional apolipoprotein that, besides its contribution in cholesterol transport and metabolism, plays important defensive functions in mammals and lower vertebrates. Experiments have shown that the expression level of ApoA-I decreased in duck liver at 8 h and 24 h after R. anatipestifer infection but its expression was significantly up-regulated at 48 h, which is contrary to mammals, in which the hepatic ApoA-I expression is down-regulated under pro-inflammatory conditions, and with fish, in which no significant differences were found in gene expression between livers of healthy and diseased fish (Carpintero et al., 2005; Villarroel et al., 2007); these results suggest perhaps a small functional difference among these species. TTR, also known as thyroxin-binding prealbumin, is a serum protein that is a negative acute-phase reactant in humans (Ingenbleek & Young, 1994). Levels of TTR are routinely measured as an indicator of health status. Following Streptococcus suis type 2 infection, TTR showed a negative APR with serum concentrations dropping significantly at 2 days following infection (Campbell et al., 2005). Therefore, it is possible that the use of down-regulated APP genes, such as ApoA-1 or TTR, in combination with up-regulated APP genes in an acute-phase index could represent valuable parameters for monitoring the health of ducks. Furthermore, a complete analysis of APP responses would be useful when microbial or other inflammatory stimuli target different cytokine networks.

Infections with Gram-negative bacteria induce an inflammatory response. The up-regulated expression of the IL-6 gene was observed in duck livers during R. anatipestifer infection, reflecting an ongoing inflammatory response. A previous report has described that histologically the numerous cellular infiltrates consisted of mononuclear cells and heterophilic leukocytes during R. anatipestifer infection (Dougherty et al., 1955). Correspondingly, we observed an up-regulated expression of CCL19, a molecule involved in leukocyte migration. CCL19 is a member of the CC chemokines that is produced by neutrophils in response to microbial pathogens. Its receptor, CCR7, is expressed on mature dendritic cells and T-cell subsets. Up-regulation of CCL19-like genes after bacterial infection has also been reported in rainbow trout and Atlantic salmon (Boshra et al., 2006; Peatman et al., 2007). The ability of neutrophils to produce CCL19 is significant in orchestrating the recruitment of immune cells to the inflamed sites and therefore contributes to the regulation of the immune response. Furthermore, the neutrophil-derived antibiotic peptide β-defensin was selectively chemotactic for CCR7-expressing dendritic cells and T cells (Yang et al., 1999; Akahoshi et al., 2003). The up-regulated expression of CCL19 and DEFB genes was observed at 8 h, 24 h and 48 h post infection, which may indicate that increased production of CCL19 and β-defensin by heterophilic leukocytes initiates antigen-specific immune responses by recruiting dendritic cells and T cells to the site of R. anatipestifer invasion.

AICL-2 was shown to be up-regulated 48 h post infection. Further studies are being done to analyse the in vivo role of AICL-2 during infection. C-type lectins are a superfamily of proteins with a variety of functions in the immune response. AICL is an activation-induced C-type lectin encoded by the human natural killer (NK) gene complex, which has been implicated in the regulation of the function of NK cells and other lymphocytes. An up-regulated level of AICL was found during the early activation of PMA-stimulated peripheral blood lymphocytes (Eichler et al., 2001). AICL has been identified as a myeloid-specific activating receptor that is up-regulated by Toll-like receptor stimulation. AICL functions as a ligand of the human activating NK cell receptor NKp80. Cross-linking of both NKp80 and AICL stimulates secretion of pro-inflammatory cytokines. In co-cultures of NK cells and monocytes, cytokine release was partially dependent on NKp80 engagement (Welte et al., 2006). Chicken C-type lectin-like receptors B-NK and B-lec have clear homology to specific receptors encoded in mammalian NK complex (Rogers et al., 2005).

In summary, SSH analysis of differentially expressed genes in duck livers after infection with R. anatipestifer indicated that the APR, a component of the innate immune systems of the host, was rapidly induced. The majority of APPs were strongly differentially regulated. Several transcripts involved in iron homeostasis were also highly induced, suggesting that iron has an important function in physiological metabolism and defence mechanisms. The differential regulation of several immune and wound healing-associated genes indicated that the duck liver probably plays an important role in pathogen recognition and defence. These genes with roles in the immune response and wound healing deserve further investigation to elucidate their respective roles during R. anatipestifer infection.

Conflict of interest statement

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Acknowledgements

This work was funded by the China Postdoctoral Science Foundation (Grant No. 20100471192), the Wuhan Key Project of Science and Technology (201021037378–3) and the Chen Guang Project of Wuhan City (201150431114). The authors thank Dr Christina Lora M. Leyson (University of Georgia, USA) for her helpful discussions and manuscript revision.