Riemerella anatipestifer infection in ducks induces IL-17A production, but not IL-23p19

R. anatipestifer (RA) is one of the most harmful bacterial pathogens affecting the duck industry, and infection is associated with the production of proinflammatory cytokines, including IL-17A. Another proinflammatory cytokine, IL-23, is critical for the development of Th17 cells, which produce IL-17. However, IL-23 roles have not been studied in this infection. Here, we describe the identification and mRNA expression analysis of duck IL-23p19 (duIL-23p19) in splenic lymphocytes and macrophages stimulated with killed RA and in spleens of RA-infected ducks. Expression of duIL-23p19 transcript identified in this study was relatively high in livers of healthy ducks and was upregulated in mitogen-activated splenic lymphocytes as well as in splenic lymphocytes and macrophages stimulated with killed RA. In spleens of RA-infected ducks, expression levels of duIL-23p19 transcript were unchanged at all time points except on days 4 and 7 post-infection; however, duIL-17A and IL-17F expression levels were upregulated in both spleens of RA-infected ducks and splenic lymphocytes and macrophages stimulated with killed RA. In sera collected at 24 h after this infection, duIL-23p19 expression levels were unchanged, whereas IL-17A significantly upregulated. These results suggest that IL-23p19 does not play a critical role in the IL-17A response in early stages of RA-infected ducks.

Th17 cell differentiation and maturation is influenced by the cytokine environment, including transforming growth factor beta (TGF-β), IL-1β, IL-6, IL-21, and IL-23 24,25 . IL-23 is an important factor in the inflammatory response to infection and is the upstream cytokine that primarily promotes Th17 differentiation and proliferation 26 . IL-23 is a heterodimeric cytokine composed of the cytokine subunits IL-23p19 (IL-23) and IL-12p40, which is also shared with IL-12. IL-23 can act in an IL-17-dependent and IL-17-independent manner 27,28 . In ducks, duck IL-17A shared 84% amino acid sequence identity with chicken IL-17A, approximately 46-47% to mammalian homologues. The genomic structure of duck IL-17A consisted of three exons and two introns was quite similar to its chicken and mammalian counterparts 29 . Until now, only partial information is available on duck IL-12p40 of which gene sequence (XM_021268516) predicts to encode a putative 317 amino acid protein.
Our previous studies suggested that IL-17A is intimately associated with R. anatipestifer infection in ducks 9,11 . Thus, we were interested in elucidating any relationship between IL-23 and IL-17A during R. anatipestifer infection in ducks. Here, we provide the first description of a full-length duIL-23p19 cDNA and the expression profiles of duIL-23p19 transcript in various healthy tissues and mitogen-stimulated splenic lymphocytes using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also describe the comparative expression profiles of duIL-23p19 and related cytokines in duck splenic lymphocytes and macrophages stimulated with killed R. anatipestifer and in the spleens of R. anatipestifer-infected ducks.

Results
Molecular characterization of duIL-23p19. The full-length cDNA encoding a duck homologue of chicken IL-23p19 was first cloned from ConA-stimulated splenic lymphocytes. The cloned duIL-23p19 sequence contained a 564-bp open reading frame (ORF) predicted to encode a putative 187 amino acid protein with a calculated molecular weight of 20.5 kDa (non-glycosylated) and an isoelectric point of 8. 16. The duIL-23p19 amino acid sequence contains a signal sequence (amino acids , one potential N-linked glycosylation site (Asn-X-Ser/Thr) at position 94, and four cysteine residues involved in interchain disulphide bond formation (Fig. 1A,B). Analysis of the duIL-23p19 nucleotide sequence showed 37.8-66, 25.1-45.9, and 15.8% homology with that of poultry, mammals, and fish, respectively. Amino acid sequence comparison indicated that duIL-23p19 shares 40.4-62.1% amino acid sequence identity with avian counterparts, 31.5-36.1% with mammalian homologues, and 26.3% with the piscine equivalent ( Table 1). Comparison of duIL-23p19 with IL-23p19 proteins of other species using the MEGA6 programs revealed that this protein is more closely related to avian species than to the corresponding mammalian and piscine proteins. Phylogenetic analysis indicated a distinct distance from IL-12p35 and IL-12p40 proteins (Fig. 1C).
Following transfection of COS-7 cells with a construct expressing duIL-23p19-MYC, the molecular weight of duIL-23p19 was determined in PNGase F-treated lysates and supernatants. An 18.8-kDa protein (asterisk in Fig. 2A; calculated molecular weight of 20.5 kDa) is likely the backbone of duIL-23p19, and the protein of approximately 20.9 kDa (arrows) represents an N-linked glycosylated form in cell lysates and supernatants ( Fig. 2A). The expression of the duIL-23p19 mRNA in various healthy tissues and mitogen-activated splenic lymphocytes was monitored by qRT-PCR. The duIL-23p19 mRNA was detected in all tested tissues. Compared to lung duIL-23p19 mRNA expression, the highest levels of this mRNA were detected in the liver, while moderate levels were found in the small intestine, skin, thymus, and brain. Moreover, very low levels of duIL-23p19 transcript were found in the fat, large intestine, and spleen (Fig. 2B). The expression level of duIL-23p19 transcript was generally upregulated in ConA-, LPS (E. coli)-, and poly I:C-stimulated splenic lymphocytes compared to unstimulated cultured splenic lymphocytes (Fig. 2C). mRNA expression profiles of IL-23 and Th17 cytokines in splenic lymphocytes and macrophages stimulated with killed R. anatipestifer. Our previous work indicated that IL-17A and IL-17F were significantly increased in ducks infected with R. anatipestifer and in duck splenic lymphocytes activated with killed R. anatipestifer 9,11 . IL-23p19 has been reported to play an important role in IL-17 production 27,28 ; thus, the mRNA expression profiles of IL-23p19, IL-12p40, IL-17A, and IL-17F were investigated in splenic lymphocytes (Fig. 3) and macrophages (Fig. 4) stimulated with killed R. anatipestifer. All four cytokines were significantly higher at all time points in splenic lymphocytes and macrophages stimulated with killed R. anatipestifer compared with levels in unstimulated cultured controls. IL-23p19 expression showed 9-36.7-fold change in lymphocytes (Fig. 3A) and 9.4-2091.5-fold change in macrophages (Fig. 4A), while IL-12p40 expression showed a 61.5-106.6-fold change in lymphocytes (Fig. 3B) and a 4.8-116-fold change in macrophages (Fig. 4B). Furthermore, the expression levels of IL-17A and IL-17F transcripts were markedly upregulated in splenic lymphocytes (Fig. 3C,D) and macrophages (Fig. 4C,D) activated with killed R. anatipestifer compared to unstimulated cultured controls. These results suggested that both duIL-23p19 and IL-17A cytokines are significantly higher in splenic lymphocytes and macrophages treated with killed R. anatipestifer. mRNA expression profiles of IL-23 and Th17 cytokines in R. anatipestifer-infected ducks. The mRNA expression profiles of IL-23p19, IL-12p40, IL-17A, and IL-17F were investigated on days 1, 4, and 7 in the spleens of R. anatipestifer-infected ducks (Fig. 5). When compared to healthy controls, expression levels of duIL-23p19 mRNA were not different at day 1 after infection but were higher by 1.7-fold at day 4 and by 2.4-fold at day 7 post-infection (Fig. 5A). The mRNA expression levels of IL-12p40, the other subunit of IL-23, were lower at days 1 and 4, and unchanged at day 7 after infection in the spleens of R. anatipestifer-infected ducks (Fig. 5B). In addition, IL-17A and IL-17F expression levels were generally higher in R. anatipestifer-infected ducks (Fig. 5C,D).
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Expression of duIL-23p19 was not elevated at early stage of R. anatipestifer infection in ducks.
To examine the in vivo expression levels of duIL-23p19 during the early time points post-infection, the transcript expression profiles of IL-23p19, IL-12p40, IL-17A, and IL-17F in the spleen were investigated at 4, 8, and 12 h post-infection with R. anatipestifer (Fig. 6). When compared to healthy controls, IL-23p19 mRNA expression levels were unchanged at all time points in the spleen of R. anatipestifer-infected ducks (Fig. 6A). IL-12p40 mRNA expression levels showed a 0.6-2.1-fold change in the spleen of R. anatipestifer-infected ducks (Fig. 6B). However, IL-17A and IL-17F expression levels were higher at all time points post-infection in duck spleens (Fig. 6C,D).
Next, we investigated IL-17A and IL-23p19 expression in the sera of ducks at 24 h post-infection with R. anatipestifer (Fig. 7). When compared to healthy controls (53.5 ± 10.7), IL-17A expression levels were significantly higher in the sera of R. anatipestifer-infected ducks (63.8 ± 11.2) (Fig. 7A). However, IL-23p19 expression levels were unchanged post-infection in duck sera (Fig. 7B). Taken together, these observations suggested that expression levels of duIL-23p19 mRNA and protein, unlike IL-17A, are unchanged at early stage of R. anatipestifer-infected ducks.

Discussion
IL-17A can be produced via IL-23-independent and -dependent mechanisms 27,30 . Our previous report suggested that IL-17A was significantly upregulated in ducks infected with R. anatipestifer 9,11 , a harmful bacterial pathogen that affects the duck farming industry. The IL-23/IL-17A pathogenic pathway has been implicated in organ-specific autoimmune inflammation 31 , psoriasis 32 , joint autoimmune inflammation 33 , colitis [34][35][36] , and hepatitis 37 . Therefore, this study was designed to investigate whether IL-23, which is the primary cytokine controlling Th17 development, is a critical immune factor in R. anatipestifer infection in ducks. To accomplish this, we first identified duIL-23p19 from ConA-stimulated duck splenic lymphocytes and described its molecular weight and mRNA expression profiles in healthy duck tissues as well as in R. anatipestifer-stimulated lymphoid cells and spleens of R. anatipestifer-infected ducks.
IL-23, which belongs to the IL-12 cytokine family, occurs as a heterodimer cytokine composed of a distinct p19 subunit (known as IL-23p19, IL-23 alpha subunit, or IL-23A) and a subunit to IL-12 (known as IL-12p40) 26 . In this study, we cloned the p19 subunit of IL-23 (duIL-23p19) and found that it contains a 564-bp ORF, predicted to encode 187 amino acids, including a 26-amino acid signal peptide and a single N-glycosylation site. The presence of a signal peptide in a sequence suggests that the protein is secreted via the classical pathway. The presence of a single predicted glycosylation site in the duck protein is in agreement with the presence of one putative N-glycosylation site in the chicken homologue 38 and other avian species but is in contrast to the absence of an N-glycosylation site in the mammalian (human and mouse) counterparts 39 and the five N-glycosylation sites in the zebrafish counterpart 40 . Interestingly, PNGase F-treated lysates and supernatants of COS-7 cells transfected with the duIL-23p19 construct displayed an 18.8-kDa protein, whereas the untreated cell lysates and supernatants exhibited an approximately 20.5-kDa protein ( Fig. 2A), indicating the possibility of N-linked glycosylation in duIL-23p19.
Multiple sequence alignment of the deduced amino acid sequences of the IL-23p19 homologues from mammals, fish, and avians, including ducks, revealed conservation of amino acids in the cloned IL-23p19, particularly of the cysteine residues important for disulphide bond formation (C78/90 in human, C75/87 in zebrafish, and C86/98 in duck) 39 , the cysteines important for interacting with the IL-12p40 subunit (C82 in duck, C77 in swine, C74 in human, and C75 in mouse) 41 . In addition, the tryptophan residue is the signature hallmark of the binding site to the signaling receptors such as the gp130 cytokine family (W158 in human, W148 in zebrafish, and W153 in duck) 42 . Computer-assisted phylogenetic analysis showed that duIL-23p19 is more closely related to the identified IL-23p19 cytokines than IL-12p40 cytokines. Moreover, duIL-23p19 formed a branch in the same cluster with other avian IL-23p19 molecules. Amino acid comparison of duIL-23p19 also showed higher identities and similarities to avian species compared to its mammalian and piscine counterparts. www.nature.com/scientificreports www.nature.com/scientificreports/ Two isoforms (p19a and p19b) of IL-23p19 cDNA were identified in both rainbow trout and Atlantic salmon. The expression levels of the two isoforms were low in most healthy uninfected tissues of rainbow trout and Atlantic salmon 43 . Expression levels of the p19a form were significantly upregulated in the spleen and head kidney of rainbow trout infected with the bacterial pathogen Yersinia ruckeri and in the head kidney of rainbow trout infected with viral haemorrhagic septicaemia virus (VHSV). In general, however, p19b expression was not significantly different between infected animals and time-matched uninfected controls 43 . In rainbow trout head kidney macrophages stimulated with poly I:C and peptidoglycan, p19a expression was high compared to p19b expression, which was not responsive or exhibited only a relatively small increase 43 . In our study, expression levels of the duIL-23p19 transcript were significantly upregulated in ConA-, LPS-, and poly I:C-stimulated splenic lymphocytes at all time points examined (Fig. 2C). Thus, our data indicate that duIL-23p19 cDNA more closely resembles the IL-23p19a form than the IL-23p19b form from rainbow trout. Moreover, the upregulated expression of duIL-23p19 in R. anatipestifer-stimulated splenic cells is in accord with the expression of IL-23p19 in in vitro LPS-stimulated zebrafish leucocytes 40 , chicken macrophage HD11 cells and CU91 T cells 38 , mouse peritoneal macrophages 44 , and human periodontal ligament cells 45 .
The proinflammatory cytokine IL-23 is essential for the differentiation and preservation of Th17 cells, which are the primary source of IL-17A 46 . Thus, to investigate the possibility of a connection between IL-23p19 and IL-17A, duck splenic lymphocytes and macrophages were treated with heat-killed R. anatipestifer. Expression levels of duIL-23p19 transcript, as well as IL-12p40, IL-17A, and IL-17F, were generally elevated in both immune cell types at all time points (Figs 3 and 4). Moreover, macrophages were more sensitive to this stimulation than lymphocytes. More specifically, duIL-123p19 expression at 4, 8 and 12 h following stimulation with R. anatipestifer was markedly upregulated by 9.0-, 36.7-, and 18.5-fold in the splenic lymphocytes and by 2091.5-, 1724.0-, and 9.4-fold in the splenic macrophages, respectively, indicating that macrophages are more sensitive than lymphocytes to R. anatipestifer stimulation. The primary sources of IL-23p19 and IL-17A are different. Lymphocytes, including CD4 + αβ-T cells (Th17) and γδ-T cells, are primary sources of IL-17A, while IL-23p19 is produced by activated dendritic cells and macrophages 13,16,26,47 . In addition, human dendritic cells stimulated with killed gram-negative bacteria, but not with killed gram-positive bacteria, exhibited elevated expression of IL-23p19 transcripts 48 .
Unlike our in vitro results, expression levels of duIL-23p19 mRNA were unchanged at the early time points (4,8,12, and 24 h) of R. anatipestifer infection in ducks, although the expression levels were higher slightly by 1.7and 2.4-fold at days 4 and 7 post-infection, whereas IL-17A mRNA expression levels were upregulated at all time points. Similar to mRNA expression patterns, expression levels of duIL-23p19 were unchanged in sera collected at 24 h after R. anatipestifer infection, whereas IL-17A expression levels were significantly upregulated (Fig. 7). Level of IL-23p19 in zebrafish infected with Mycobacterium marinum increased by 5-7-fold on day 1 and 4-fold on day www.nature.com/scientificreports www.nature.com/scientificreports/ 6 after infection 40 . After intraperitoneal injection of LPS in C57BL/6 J, plasma levels of IL-23p19 peaked as early as 3 h 44 . Expression levels of IL-23p19 in rainbow trout infected with Yersinia ruckeri were markedly upregulated 820-fold in spleens and 449-fold in head kidneys on day 1 after infection 43 . IL-23 has pathogenic roles in many autoimmune diseases 28 , while IL-23 is also important for protection against infections, such as Listeria monocytogenes 49 and Citrobacter rodentium 50 .
In conclusion, we cloned the duIL-23p19 gene to determine whether the upregulation of IL-17A expression in R. anatipestifer-infected ducks is related to IL-23. Unlike splenic lymphocytes and macrophages stimulated with R. anatipestifer, expression levels of duIL-23p19 were not significantly upregulated at early time points in R. anatipestifer-infected ducks. These results suggest that during R. anatipestifer infection, IL-17A can have biological functions independent of duIL-23p19, especially at early times post-infection.

Materials and Methods
Animals and infection. Pekin ducklings (Joowon, ASTA Ducks, Korea) were housed in wire cages and raised in a temperature-controlled condition with constant light and had access to food and water ad libitum throughout the duration of the experiment. Sixty two-week-old ducks were challenged intramuscularly with 5 × 10 7 CFU of R. anatipestifer serotype 7 in 200 µl phosphate buffer saline (PBS). The control birds were administered 200 µl PBS intramuscularly. R. anatipestifer used in this study were isolated from a commercial duck farm in Changwon, Gyeongnam Province, Korea, and serotyped at Chonbuk National University, Korea. The isolates were grown on agar plates supplemented with 5% sheep blood (Asan Pharmaceutical, Korea) for 48 h in a 37 °C incubator under 5% CO 2 . A single colony was inoculated into tryptic soy broth (TSB; Difco, USA) and incubated at 37 °C in a shaking incubator until logarithmic growth phase was achieved. The final concentration of the inoculum was determined by 10   Based on the partial sequence information, 5′/3′ rapid amplification of cDNA ends (RACE) was performed to obtain the full-length duIL-23p19 cDNA using specific primers (Table 2), high-fidelity DNA polymerase (Bioneer, Korea), and the 5′/3′ RACE kit (2 nd Generation; Roche Applied Sciences, Germany) according to the manufacturer's protocol. RT-PCR products were cloned to the TA Vector (RBC, Taiwan) and were sequenced (Macrogen, Korea). RT-PCR was performed on a DNA engine thermocycler (Bio-Rad, Hercules, CA, USA) with the following parameters: 5 min at 95 °C; 35 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C; and a final 5-min extension at 72 °C.

Sequence analysis. Protein sequence and amino acid alignment were analysed using Expert Protein
Analysis System (ExPASy; www.expasy.org/tools/) and Clustal Omega multiple sequence alignment (www. ebi.ac.uk/Tools/msa/clustalo/), respectively. Percent identity and similarity of the duIL-23p19 sequence to its known and predicted vertebrate homologues were assessed using EMBOSS Needle pairwise sequence alignment (www.ebi.ac.uk/Tools/psa/emboss_needle). The signal peptide sequence was predicted using the SignalP 4.1 Server (www.cbs.dtu.dk/services/SignalP), and the potential N-linked glycosylation site was assessed using the NetNGlyc 1.0 Server (www.cbs.dtu.dk/services/NetNGlyc/). Theoretical pI value and predicted molecular weight were calculated using Compute pI/Mw (www.expasy.org/compute_pi). Evolutionary analysis was performed with a phylogenetic tree that was constructed using the neighbour-joining method in the MEGA6 program with a bootstrap value of 10,000 51 .
Cell culture. Duck splenic lymphocytes were obtained from a two-week-old healthy duck as previously described 52 . Briefly, aseptically collected spleens of ducks were gently separated using a cell strainer (SPL Life Science, Korea) to obtain a single cell suspension, and the splenic lymphocytes were collected using Ficoll-Paque PLUS (GE Health Life Sciences, UK) following the manufacturer's instructions. Cells were collected, washed twice with PBS, and then cultured in Dulbecco's modified eagle's medium (DMEM; Gibco Life Technologies, USA) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 unit/ml) in a 41 °C incubator under 5% CO 2 . Splenic lymphocytes were resuspended to a concentration of 5 × 10 6 cells/ml and stimulated with 10 µg/ml concanavalin A (ConA; Amersham Bioscience, Sweden), 25 µg/ml polyinosinic:polycytidylic acid (poly I:C; Sigma-Aldrich, Germany), 10 µg/ml lipopolysaccharide (LPS from Escherichia coli, 0111:b4; www.nature.com/scientificreports www.nature.com/scientificreports/ Sigma-Aldrich), or heat-killed R. anatipestifer (1 × 10 6 CFU/ml) for 4, 8, and 24 h. Duck primary macrophages were obtained from spleens of 2-week-old ducks as previously described 52,53 . Briefly, spleens were homogenized and sieved using a syringe plunger and passed through a cell strainer to obtain a single cell suspension. Cells were collected, washed twice with PBS and resuspended in a culture flask with DMEM supplemented with 10% FBS and penicillin-streptomycin (10,000 unit/ml). Cells were then incubated in a 41 °C incubator under 5% CO 2 for 2 days to allow adherence of cells. Non-adherent cells were removed by washing with DMEM, and adherent cells were cultured until reaching 80% confluency. Adherent cells were seeded (5 × 10 6 cells/ well) and were treated with heat-killed R. anatipestifer (1 × 10 6 CFU/ml) for 4, 8, and 24 h. Heat killed R. anatipestifer was prepared by placing in a water bath at 100 °C for 5 min 9,11 . COS-7 cells were grown as described above in a 37 °C incubator under 5% CO 2 .
Plasmid construction and cell transfection. The sequence of duIL-23p19 cDNA with a MYC-tag for expression (duIL-23p19-MYC) was amplified by PCR from single-stranded cDNA using the following specific primers: forward 5′-GATCAAGCTTATGGCCCCGCTCCGTCTCCT-3′ and reverse 5′-GATCGAATTCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGCGGGTGCTGGCGCCGTAGG-3′. The primers included HindIII and EcoRI restriction enzyme sites, indicated by a single solid underline, and a MYC-expressing sequence, indicated by a dashed underline. PCR products were purified using the www.nature.com/scientificreports www.nature.com/scientificreports/ England Biolabs, Ipswich, MA, USA) when necessary, and then heated at 95 °C for 5 min. Proteins were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene difluoride (PVDF; Bio-Rad) membranes. The membranes were blocked with 4% nonfat dry milk (Sigma-Aldrich, St. Louis, MO, USA) in PBS containing 0.05% Tween-20 (PBS-T) for 2 h at room temperature and then incubated with monoclonal anti-MYC mouse antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. The membranes were then washed five times with PBS-T and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Promega) in PBS-T with 4% nonfat dry milk for 1 h at room temperature. The membranes were washed with PBS-T five times, incubated with chemiluminescent reagent EZ West Lumi plus (Dogen Bio Co., LTD, Korea), and visualized using the ChemiDoc Imaging System (Bio-Rad).
qRT-PCR. Spleens, splenic lymphocytes, and macrophages from ducks infected with R. anatipestifer and time-matched unstimulated and uninfected control samples were subjected to qRT-PCR in duplicate. Samples were collected and homogenized using a grinder (Dalhan Sci., Korea) for spleens and a vortex for cells. Total RNA was isolated from the samples using RiboEx (GeneAll, Korea), purified with RNeasy mini kit (Qiagen), and stored at −70 °C. The total RNA concentration was measured using a nano-spectrophotometer (Optizen, Korea) and treated with DNase I to remove contaminating genomic DNA. A single-strand cDNA was synthesized from the total RNA using random hexamer primers and the Quantitect reverse transcription kit (Qiagen). Then, qRT-PCR analysis was performed in duplicate using CFX 96 real-time PCR system (Bio-Rad) with SYBR Green (Bioneer) and gene-specific primers ( Table 2). In order to verify the presence of a single amplification product without primer dimers, a melting curve was obtained at the end of each amplification program. Relative expression levels of individual transcripts from infected ducks and stimulated splenic lymphocytes and macrophages were compared with those of the control uninfected ducks and unstimulated lymphoid cells using Bio-Rad CFX software respectively and were quantified using the 2 −ΔΔ CT method with β-actin as a reference gene for normalization.
Enzyme linked immunosorbent assay (ELISA). Two-week-old ducks were challenged intramuscularly with 5 × 10 7 CFU of R. anatipestifer serotype 7 in 200 µl phosphate buffer saline (PBS). The control birds were administered 200 µl PBS intramuscularly. Whole blood was obtained after 24 h, and serum was separated after clotting by centrifugation. Aliquots were then taken and stored at −70 °C until test were performed. Serum Statistical analysis. Data were analysed using the Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test using InStat ® software (GraphPad, USA). Differences were considered statistically significant when *P < 0.05, **P < 0.01, or ***P < 0.001. The data were expressed as the mean values ± standard error (SE).  Figure 7. Serum IL-23p19 and IL-17A levels in R. anatipestifer-infected ducks. Two-week-old ducks were challenged intramuscularly with 5 × 10 7 CFU of R. anatipestifer serotype 7 in 200 µl phosphate buffer saline (PBS). The uninfected control birds (NC) were administered 200 µl PBS intramuscularly. IL-17A and IL-23p19 levels in serum obtained at 24 h after infection were determined using the duck IL-17A and chicken IL-23 ELISA kits, respectively. COS-7 cells were transiently transfected with duIL-23p19-MYC construct. Cell lysates were collected after 48 h and used as positive control for chicken IL-23 ELISA. The results from one representative experiment of two independent experiments are shown. Data are shown as the mean ± SE (n = 10). *P < 0.05. RA, sera from R. anatipestifer-infected ducks.

Target Genes Purpose
Orientation and sequence (5′-3′) References  Table 2. Primer sequences used for IL-23p19 cloning and quantitative RT-PCR of cytokine expression.