Arch Virol (2015) 160:69–80 DOI 10.1007/s00705-014-2232-y
ORIGINAL ARTICLE
Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus Chih-Chun Lee • Bong-Suk Kim • Ching Ching Wu Tsang Long Lin
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Received: 16 April 2014 / Accepted: 9 September 2014 / Published online: 1 October 2014 Ó Springer-Verlag Wien 2014
Abstract Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-proteinexpressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNAvaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 9 102 times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p \ 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-a, IFN-b, IRF-1, C.-C. Lee B.-S. Kim C. C. Wu T. L. Lin (&) Department of Comparative Pathobiology, Purdue University, 406 S. University St, West Lafayette, IN 47907, USA e-mail: [email protected]
IRF-10, IL-1b, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p \ 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-c, IL-12(p40), IL-18, GM-CSF, GATA-3, p53, glucose transporter-2 and glucose transporter-3, were upregulated or significantly (p \ 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. Taken together, the results indicate that the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport, except for granzyme K and CD8, was not differentially expressed in DNA-vaccinated chickens protected from IBDV challenge.
Introduction Infectious bursal disease (IBD) is one of the most important viral diseases in the poultry industry worldwide and is characterized by its acute, highly contagious and immunosuppressive properties. The major target tissue of IBD is IgM-bearing B cells in the bursa of Fabricius. The infection in chickens between 3 and 6 weeks of age causes severe diarrhea, muscular hemorrhage, and bursal atrophy. IBD occurring in chickens less than 3 weeks of age induces few or no clinical signs but leads to an immunosuppressive status in recovered chickens, leading to subsequent vaccination failure and susceptibility to secondary infections [31]. Infectious bursal disease virus (IBDV) is a non-enveloped, double-stranded RNA virus with two genomic segments, A and B. A large open reading frame (ORF) in segment A encodes a polyprotein that is further processed
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into VP2 and VP3 to form the viral capsid, and VP4 to form a viral protease. The other ORF of segment A encodes a non-structural protein, VP5 [29], which is not essential for virus replication [30] but has been shown to induce apoptosis in chicken B cells and fibroblast cells to facilitate virus dissemination [47]. Segment B encodes VP1, an RNA-dependent RNA polymerase. Vaccination is the main strategy that is currently used to control IBD. Most commercially available vaccines consist of attenuated or killed IBDV. However, IBD outbreaks still occur in chicken farms worldwide due to the emergence of variant strains of IBDV and very virulent IBDV [26]. Variant IBDV strains have been prevalent in the United States since 1985 and the variant E (VE) strain is more common isolate than the other variants. Infection with the VE strain of IBDV results in rapid bursal atrophy with few clinical signs and gross lesions at 3 days post-challenge (dpc) [16]. Chicken embryos infected with the VE strain of IBDV display splenomegaly and liver necrosis, which are not observed in those infected with classical strains of IBDV [37]. In addition, vaccination with variant strains of IBDV induces better heterologous protection against classical strains of IBDV [17]. DNA vaccines encoding viral proteins induce protective responses against viruses and provide advantages over conventional vaccines [44]. DNA vaccines reduce the problem of revertant virulence and divergent mutants associated with live attenuated vaccines [8]. Previous studies have shown that DNA vaccines are effective in inducing humoral and/or cellular immunity and provide protection against virus infection [23]. A DNA vaccine encoding polyprotein VP243 in genomic segment A from the VE strain of IBDV has been developed and was shown previously to be efficacious [8, 9]. The antibody titer against IBDV measured by ELISA is low, but the virus-neutralization titer to IBDV increases in protected chickens receiving DNA vaccination [8, 9]. The virus antigen is not detected in the bursa and spleen in DNAvaccinated chickens after IBDV challenge by immunofluorescent assay (IFA) [10]. T helper 1 (Th1)-biased immune cell differentiation has been demonstrated in DNA-vaccinated chickens [10], and this could be due to the presence of unmethylated cytidine-phosphate-guanosine (CpG) motifs in the DNA vaccine. An unmethylated CpG motif has been shown to stimulate the innate immune response and proinflammatory cytokines [13, 14]. The aforementioned findings suggest that the effect of DNA vaccination on the bursal microenvironment during IBDV infection may affect the induction of cytokines and other cellular gene products involved in innate immunity, inflammation, immune cell regulation and apoptosis that are critical for anti-viral defense and protective immunity. Therefore, the protective effect of DNA vaccination against IBD may be related to differential expression of bursal
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RNA molecules (bursal transcriptome) upon IBDV infection. The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by kinetic analysis of the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport.
Materials and methods Chickens Specific-pathogen-free (SPF) embryonated chicken eggs obtained from Charles River Laboratories (North Franklin, CT, USA) were hatched out after incubation for 21 days. One-day-old SPF chickens were kept in Horsfall-Bauer isolators with food and water ad libitum. Protocols used for the animal study were approved by Purdue University Animal Care and Use Committee. Virus IBDV strain Variant E (VE), provided by Dr. John K. Rosenberger from University of Delaware, was used as the template to construct the plasmid for DNA vaccination and was also used to challenge chickens after DNA vaccination in the present study. DNA vaccination and infection challenge The DNA vaccine construct for expression of the IBDV large segment was described previously [9]. The animal experiment in the present study was described in a previous report [10]. Briefly, one hundred eighteen hatched SPF chickens were randomly divided into four groups as follows: negative control (CN); unvaccinated, challenged control (CC); DNA-vaccinated, unchallenged group (DN); and DNA-vaccinated, challenged group (DC). Chickens receiving DNA vaccination were injected intramuscularly three times with DNA vaccine containing 400 lg of pCR3.1-VP243 VE plasmid in phosphate-buffered saline (PBS) at 1, 7, and 14 days after hatching. Unvaccinated chickens were injected intramuscularly with PBS. In the groups challenged with IBDV, chickens were orally administered 8.2 9 102 times the embryonic infectious dose (EID)50 of IBDV strain VE 7 days after the last immunization. Bursae were collected at 0.5, 1, 3, 5, 7 and 10 dpc and stored in RNAlater (QIAGEN, Valencia, CA, USA) in a -20 °C freezer. Protection from IBDV challenge in a chicken was defined by a gross lesion score of 1 and bursal/body weight ratio not less than 2 standard deviations below the average ratio of the unchallenged control chickens [8].
Effect of IBDV DNA vaccination on bursal gene expression
RNA extraction and cDNA synthesis RNA was extracted using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s manual. In brief, 30 mg bursal tissue was homogenized twice for 30 s in 600 lL of RLT lysis buffer, using a MULTI-GEN 7 homogenizer (Pro Scientific Inc., Oxford, CT, USA). RNA was eluted with 40 lL of RNase-free water and stored in a -80 °C freezer. The quality and quantity of the RNA was evaluated spectrophotometrically using a GeneQuant 1300 (GE Healthcare Life Sciences, Piscataway, NJ, USA). Total RNA was treated with DNase to remove genomic DNA contamination. The reaction mixture contained 4 lg of RNA, 1x reaction buffer, and 1 unit of RQ1 RNase-free DNase (Promega, Madison, WI, USA) and was incubated at 37 °C for 1 h. The reaction was stopped by incubation at 70 °C for 15 min to deactivate DNase. cDNA synthesis was performed using DNase-treated RNA, Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and random primer (Sigma Genosys, The Woodlands, TX, USA). Briefly, DNase-treated RNA was added to 10 nmol of dNTP and 200 ng of random hexamer, heated to 65 °C for 5 min and immediately put on ice for 2 min. The reaction mixture containing 1x first-strand synthesis buffer, 5 mM DTT and 200 units of Superscript III reverse transcriptase in 20 lL was incubated at 50 °C for 60 min and then at 70 °C for 20 min to deactivate the reverse transcriptase. The resulting cDNA was stored in a -20 °C freezer for later use. Quantitative analysis of the transcriptome by real-time RT-PCR The gene expression levels of the cDNA were determined by real-time RT-PCR using a Rotor-Gene RG-3000 thermal cycler (QIAGEN). The quantification was carried out with either TaqMan-probe-based or SYBR Green–based chemistry. The design of primers and probes for IL-4, 28S and IFN-c was described previously [3], and primers for the other genes in the present study were designed using PrimerQuest (Integrated DNA Technologies Inc, Coralville, IA, USA). The sequences of primers and probes are summarized in Table 1. The probe-based PCR mixture contained 200 nM forward and reverse primers, 100 nM probe, and 1x Platinum Quantitative PCR SuperMix-UDG (Invitrogen). Thermal cycling was done at 50 °C for 2 min and 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 59 °C for 60 s. The fluorescent signal was acquired using the FAM/SYBR Green channel at 59 °C. The SYBR Green–based PCR mixture containing 200 nM forward and reverse primers and 1x EXPRESS SYBR GreenER qPCR universal qPCR Supermixes (Invitrogen) was subjected to 1 cycle of 50 °C for 2 min and 95 °C for
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2 min followed 40 cycles of 95 °C for 20 s and 60 °C for 60 s, and 1 cycle of final extension at 60 °C for 5 min. The fluorescent signal was acquired using the FAM/SYBR Green channel at the end of each extension stage. Melting curve analysis was performed between 65 °C and 95 °C, with a ramp speed at 1 °C/s. Melting curve analysis was used to evaluate the specificity of PCR amplification. Data analysis The expression of each gene was normalized to that of a housekeeping gene (GAPDH or 28S), so the normalized expression of a target gene to GAPDH or 28S was 2-DCt, where DCt = Cttarget gene - CtGAPDH (or Ct28S). For each gene, the normalized expression in groups DN, DC and CC relative to that in group CN was calculated using the formula 2-DDCt, where DDCttarget gene = each DCttarget gene in group DN, DC or CC – the average of DCttarget gene in group CN, and expressed as fold change. The fold changes were subjected to statistical analysis using a one-way ANOVA test with Duncan’s post hoc analysis (SPSS software, SPSS Inc, Chicago, IL, USA). A p-value less than 0.05 was considered statistically significant.
Results Bursal transcriptome related to innate immunity The mRNA expression levels of Toll-like receptor 3 (TLR3) (Fig. 1A), melanoma differentiation-associated gene 5 (MDA5) (Fig. 1C), interferon-a (IFN-a) (Fig. 1D), IFN-b (Fig. 1E), IFN regulatory factor-1 (IRF-1) (Fig. 1F) and IRF-10 (Fig. 1G) in chickens in groups CN, DN and DC were similar to each other, but they were significantly (p \ 0.05) increased in chickens in group CC at 3, 5, 7 or 10 dpc, with the highest fold increases at 23.22-, 52.03-, 10.97-, 50.27-, 12.35- and 23.80-fold, respectively. The transcript levels of TLR7 (Fig. 1B) in the three groups were not significantly (p [ 0.05) different at each time point except for 0.5 dpc. No significant (p [ 0.05) difference in the expression level of interferon-stimulated gene 20 kDa (ISG-20)(Fig. 1H) was observed between group DN and group DC, while ISG-20 in chickens in group CC had higher or significantly (p \ 0.05) higher expressions at 1, 3, 5, 7 and 10 dpc. Bursal transcriptome related to inflammation The transcript levels of interleukin-1b (IL-1b) (Fig. 2A), IL-6 (Fig. 2B), IL-8 (Fig. 2C), inducible nitric oxide synthase (iNOS) (Fig. 2D) and IL-10 (Fig. 2G) increased significantly (p \ 0.05) in IBDV-challenged chickens in
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Table 1 Nucleotide sequences of primers and probes used in the present study Target gene
Forward sequence (50 ?30 )
Reverse sequence (50 ?30 )
GenBank accession no.
GAPDH
ATCAAGAGGGTAGTGAAGGCTGCT
TCAAAGGTGGAGGAATGGCTGTCA
NM_204305
IFN-a
CAATGCTTGGACAGCAGAGA
GTCTTGGAGGAAGGTGTGGA
XM_001231512
IFN-b
ACCAGGATGCCAACTTCTCTTGGA
ATGGCTGCTTGCTTCTTGTCCTTG
NM_001024836
IFN-c
GTGAAGAAGGTGAAAGATATCATGGA
GCTTTGCGCTGGATTCTCA
NM_205149.1
IFN-c probe
FAM-TGGCCAAGCTCCCGATGAACGA-TAMRA
TLR3
GCAAGGACAGTGCTCCATTT
CCCGGTAGTCTGTCAAGCTC
DQ780341
TLR7
TTGACAACCTTTCCCAGAGC
GTTGGTGGGCCATGTAAAAT
NM_001011668
MDA5
AATTCCTGCTGGCGCTGAAGAAAG
AAAGATGCCCAGCTCCAGACACTT
XM_422031.2
ISG-20
TACTTCCACCCCAAGGAGAG
CCACCAGGATCTTCTTCTGC
XM_413869.2
IRF-1
AAAGGCTTCTTCACCAACGA
GAACTCCAACTCTGCCGAAG
NM_205415.1
IRF-10 IL-1b
ACCAAATCCACCTGTGCTTC TCATCTTCTACCGCCTGGAC
GGAAGAGCTCTCGTGCAAAT GTAGGTGGCGATGTTGACCT
NM_204558.1 NM_204524
IL-2
CTGCAGTGTTACCTGGGAGA
CTTGCATTCACTTCCGGTGT
NM_204153.1
IL-4
AACATGCGTCAGCTCCTGAAT
TCTGCT AGGAACTTCTCCATTGAA
NM_001007079
IL-4 probe
FAM-AGCAGCACCTCCCTCAAGGCACC-TAMRA
IL-6
CTCCTCGCCAATCTGAAGTC
CCCTCACGGTCTTCTCCATA
NM_204628
IL-8
GCTCTGTCGCAAGGTAGGAC
GCGTCAGCTTCACATCTTGA
NM_205498
IL-10
CAGTCATCAGCAGAGCATGG
TTGATGGCTTTGCTCCTCTT
NM_001004414
IL-12 (p40)
CTGTGGCTCGCACTGATAAA
GAACATCTCAGTCGGCTGGT
NM_213578
IL-17a
GGCCGTAACGATTAGCAAGA
TCTTCTCATGGAGCACGTTG
NM_204460
IL-18
GGGAAGGAGAAGTTCCCAAA
TCAAAGGCCAAGAACATTCC
NM_204608
CD4
GGAGGAAGCTCATGTTTGGA
CTGCCACCTCATACCAGTGA
NM_204649.1
CD8
AGCTCAGAGCCAGGAACAAG
GTCTTTTGGCAGAGCACGAT
NM_205235.1
TGF-b4
GGACATGCAGAGCATTGCCAAGAA
GATCCTTGCGGAAGTCGATGTAGA
NM_001011691
GM-CSF
ACCCATGGACATCAGGGATA
GTTCGAGAAGAGGCGTTCAC
NM_001007078
GATA-3
TGCAAAAAGGTCCATGACAA
TAGTCAGCATGTGGCTGGAG
NM001008444
iNOS Granzyme A
TGGGTGGAAGCCGAAATA ACTCATGTCGAGGGGATTCA
GTACCAGCCGTTGAAAGGAC TGTAGACACCAGGACCACCA
U46504 NM_204457.1
Granzyme K
CGGGAAGCAACTGTTGAAAT
GAGTCTCCCTTGCAAGCATC
XM_423832
Caspase-3
GCAGCTGAAGGCTCCTGGTTTATT
TCTGCCACTCTGCGATTTACACGA
NM_204725
p53
CTCCAAGCAACAACAAACGCTGGA
TCCCAGTAAGATCCACCAACACAA
XM_419987
BCL-2
ACAAGGAGATGCGGGTACTG
CAGCGTTGTTCCCATACAGA
BX93598
GLUT-2
GTGGGGATGTGCTTCCAGTA
TCGTTTCGGGTACTTTGAGG
NM_207178.1
GLUT-3
ATGCTCTTCCCCTATGCTGA
AAAAGTCCTGCCCTTGGTCT
NM_205511.1
28S
GGCGAAGCCAGAGGAAACT
GACGACCGATTTGCACGTC
DQ018756
28S probe
FAM-AGGACCGCTACGGACCTCCACCA-TAMRA
group CC at 3, 5, 7 or 10 dpc, with the highest expression levels at 40.22-, 46.59-, 43.86-, 17.07- and 25.22-fold, respectively. The fold changes of these genes in chickens were similar between group DN and group DC at most time points. The expression levels of granzyme K (Fig. 2F) in chickens receiving DNA vaccination (groups DN and DC) were significantly (p \ 0.05) upregulated, ranging from 3.72- to 6.12-fold relative to the expression levels in chickens in group CN. The transcript levels of granzyme A (Fig. 2E) in chickens in group DN were similar to those in
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chickens in group CN. The expression levels of granzyme A and granzyme K in chickens in group CC were significantly (p \ 0.05) upregulated at 3, 5, 7, and 10 dpc, with the highest fold increases at 48.88 and 50.82 at 5 dpc, respectively. The expression level of transforming growth factor b4 (TGF-b4) (Fig. 2H) in chickens in group DC and group DN was not significantly different (p [ 0.05) at any time point except at 7 dpc, while that in chickens in group CC was significantly (p \ 0.05) lower than that in group DC at 1, 7 and 10 dpc.
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Effect of IBDV DNA vaccination on bursal gene expression
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Fig. 1 Bursal transcriptome involved in innate immunity. Chickens receiving DNA vaccination were challenged with IBDV strain VE (group DC) or mock challenged (group DN), and the other chickens that did not receive DNA vaccination were challenged with IBDV (group CC) or mock challenged (group CN). Bursae were collected from chickens at 0.5, 1, 3, 5, 7 and 10 days post-challenge (dpc) to quantify gene transcripts of TLR3 (A), TLR7 (B), MDA5 (C), IFN-a
(D), IFN-b (E), IRF-1 (F), IRF-10 (G), and ISG-20 (H). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
Bursal transcriptome involved in immune cell regulation
group DC at any of the six time points. The expression level of CD8 in chickens in DN and group DC was significantly (p \ 0.05) higher than that in chickens in group CC at 0.5 dpc. No significant (p [ 0.05) difference was observed in the expression levels of IFN-c (Fig. 3D), IL-12(p40) (Fig. 3E) and IL-18 (Fig. 3F) between group DN and group DC. The
CD4 (Fig. 3A), CD8 (Fig. 3B) and IL-2 (Fig. 3C) exhibited significant (p \ 0.05) upregulation in chickens in group CC at 3, 5, 7 and 10 dpc, whereas there was no significant (p [ 0.05) difference between group DN and
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Fig. 2 Bursal transcriptome involved in inflammation. Bursae were collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc to quantify gene transcripts of IL-1b (A), IL-6 (B), IL-8 (C), iNOS (D), granzyme A (E), granzyme K (F), IL-10 (G), and TGF-b4 (H). The relative expression levels of these genes were normalized to GAPDH content
in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
transcripts of IFN-c, IL-12(p40) and IL-18 were upregulated or significantly (p \ 0.05) upregulated in chickens in group CC compared to those in the other two groups at 3, 5, 7 or 10 dpc, with the highest fold increases at 20.64-, 42.47-, and 6.77-fold, respectively. The transcript levels of IL-4 (Fig. 3H) in chickens from group DC were higher or significantly (p \ 0.05) higher than those in group DN and group CC at 5, 7 and 10 dpc,
Fig. 3 Bursal transcriptome involved in immune cell regulation. c Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc to quantify gene transcripts of CD4 (A), CD8 (B), IL-2 (C), IFN-c (D), IL-12(p40) (E), IL-18 (F), IL-4 (G), GM-CSF (H), GATA-3 (I), and IL-17a (J). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
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with the highest fold change at 6.59-fold. The expression levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Fig. 3G) and GATA-3 (Fig. 3I) were higher or significantly (p \ 0.05) higher in chickens in group CC at 3, 5, 7 and 10 dpc, while those between group DC and group DN did not differ significantly (p [ 0.05). The transcript levels of IL-17a (Fig. 3J) were upregulated or significantly (p \ 0.05) upregulated in chickens in group CC at 1, 3, 5, 7 or 10 dpc, with the highest fold changes at 10.90-fold, whereas those in group DN and group DC remained similar at all six time points.
Bursal transcriptome related to glucose transport
Bursal transcriptome related to apoptosis
The animal experiment in the present study was described previously [10]. The bursae collected were used to analyze the bursal transcriptome in the present study. The results from the animal experiment indicated that DNA-vaccinated chickens were protected from IBDV challenge, while severe bursal atrophy was observed in IBDV-infected chickens in group CC [10]. IBDV VP2 antigen was not present in the bursae of DNA-vaccinated chickens upon IBDV challenge, as determined by IFA, and a much lower virus load was detected by real-time RT-PCR in the bursae of DNA-vaccinated chickens when compared with that in IBDV-infected unvaccinated chickens [10], indicating that IBDV reached the bursae, spleen and cecal tonsils and replicated in these lymphoid tissues in DNA-vaccinated chickens, but the replication of IBDV was limited in
B-cell lymphoma-2 (Bcl-2) (Fig. 4A) was downregulated significantly (p \ 0.05) in chickens in group CC at 3, 5, 7 and 10 dpc. The transcript levels of Bcl-2 in group DN and group DC were similar throughout the study. The expression levels of p53 (Fig. 4B) in chickens in group CC were upregulated or significantly (p \ 0.05) upregulated at 3, 5, 7 and 10 dpc, while those in chickens in group DN and group DC were similar at all six time points. The transcript levels of caspase-3 (Fig. 4C) in chickens in group DN and group DC were not significantly (p [ 0.05) different at any of the time points, but those in group CC decreased significantly (p \ 0.05) at 3, 5, 7 and 10 dpc.
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Fig. 4 Bursal transcriptome involved in apoptosis. Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc were used to quantify gene transcripts of Bcl-2 (A), p53 (B) and caspase-3 (C). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression
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The expression of glucose transporter-2 (GLUT-2) (Fig. 5A) in IBDV-infected chickens in group CC was significantly (p \ 0.05) upregulated at 1 and 5 dpc and significantly (p \ 0.05) downregulated at 7 dpc. The expression of GLUT-3 (Fig. 5B) in chickens in group CC was higher or significantly (p \ 0.05) higher than that in group DN and group DC at 3, 5, 7, and 10 dpc.
levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
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Fig. 5 Bursal transcriptome involved in glucose transport. Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc were used to quantify gene transcripts of GLUT-2 (A) and GLUT-3 (B). The relative expression levels of these genes were normalized to GAPDH
content in each sample and are shown as fold changes over those in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
DNA-vaccinated chickens. This suggests that certain changes mediated by DNA-vaccination may help limit the growth of IBDV. In the present study, the expression levels of most selected genes in DNA-vaccinated chickens in group DN were similar to those in chickens in group CN, with the exception of granzyme K and CD8, which were upregulated in DNA-vaccinated chickens at 0.5 or 1 dpc, suggesting that the activated cellular immunity was mediated by DNA vaccination in the bursae. The expression levels of selected genes related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not elicited in DNA-vaccinated chickens upon IBDV challenge, whereas those in IBDV-challenged chickens in group CC showed differential expression.
than the emergence of clinical signs, as determined by bursal lesions and bursal/body weight ratio [10]. IRF-1 and IRF-10 are transcription factors regulating the antiviral immune response [32]. Consistent with the increase in type I IFNs, the significantly (p \ 0.05) enhanced expression of IRF-1 and IRF-10 in chickens in group CC after 3 dpc indicated the activation of the innate immune response. The upregulated expression of IRF-1 or IRF-10 was observed previously in IBDV-infected chickens [24, 35, 45].
Innate immunity The expression levels of innate immune genes in IBDVchallenged DNA-vaccinated chickens in group DC were generally similar to those in DNA-vaccinated chickens in group DN and did not change much compared to unvaccinated chickens in group CN, suggesting that the selected innate immune genes were not activated after DNA vaccination. The unchanged expression of the innate immune genes between chickens in group DC and group DN suggested that most of the entering viruses may have been eliminated and thus replicated poorly. The result was in line with the rapid clearance of IBDV in DNA-vaccinated chickens in the same animal experiment described previously [10]. The upregulated expression of TLR3 and MDA5 in IBDV-infected chickens in group CC suggested that TLR3 and MDA5 may be involved in sensing IBDV infection. The upregulation of TLR3 is observed in chicken embryo fibroblasts infected with IBDV [45]. The expression levels of IFN-a and IFN-b (type I IFN) in chickens in group CC were not upregulated until 3 dpc, indicating that the innate immune response had been activated since 3 dpc, earlier
Inflammation IL-1b and IL-6 induce an inflammatory response during IBDV infection [21]. IL-6 was shown in previous studies to be upregulated upon IBDV infection [20, 35]. The upregulated IL-1b and IL-6 in IBDV-challenged chickens in group CC in the present study indicated the occurrence of inflammation. Chicken IL-8 has been shown to attract neutrophils and naı¨ve T lymphocytes [46]. The increased expression of IL8 in IBDV-challenged chickens in group CC suggested the recruitment of heterophils and naı¨ve T lymphocytes to the bursa [39]. The upregulation of IL-8 by IBDV infection has been reported previously [24, 35]. Nitric oxide has multiple roles in host immune responses against pathogens, including direct antiviral activity against some enteroviruses, effects on T helper cell polarization, and oxidative stress through the formation of NO radicals [1]. In the present study, the increased expression of iNOS in IBDV-challenged chickens in group CC after 3 dpc suggested the activation of macrophages. Cytotoxic T lymphocytes mediate cytolytic activity through the secretion of granules containing perforin and granzymes [28]. Both granzyme A and granzyme K have trypsin-like activity and are able to cleave and activate caspase-3, which in turn activates caspase-activated DNase. The DNase damages the chromosomal DNA and
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leads to apoptosis. Human and mouse granzyme A induce the secretion of proinflammatory cytokines such as IL-1b, TNF-a and IL-6, thus playing a role in induction of inflammation [27]. Granzyme K appears to be a functionally redundant backup of granzyme A with some unique substrate specificity [6], and it provides protection against influenza virus challenge in mice lacking the expression of granzymes A and B [19]. Not much is known about chicken granzymes, but their upregulation during IBDV infection has been observed [35]. The clearance of IBDV has been suggested to be mediated by cytolytic molecules such as perforin and granzymes in cytotoxic T lymphocytes [33]. In the present study, the increased expression of granzyme K in chickens in group DN at 0.5 and 1 dpc suggested increased cytotoxicity induced by DNA vaccination. Enhanced cytotoxicity following DNA vaccination, as determined by increased granzyme B activity, has been demonstrated in mice and monkeys [2, 18]. Thus, DNA vaccination in the present study may have led to enhanced cytotoxicity, thereby contributing to the rapid clearance of IBDV. The upregulation of granzymes A and K in chickens in group CC at and after 3 dpc indicated the activation of cytotoxic T lymphocytes, as was shown previously [22, 33]. T cells infiltrated in the bursa are important for virus clearance during IBDV infection, and many of these cells are CD8? T cells [7, 22, 33], suggesting that cytotoxic T cell enzymes, such as granzymes A and K, may participate in virus clearance in IBDV-infected chickens.
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immunity, as was shown previously [11, 22, 36], while relatively unchanged expression of these cytokines in chickens in groups DN and DC indicated that IBDV challenge did not elicit Th1 immune response in DNAvaccinated chickens, most likely due to the rapid clearance of IBDV [10]. IL-4 promotes B cell activation and IgE production [3], and GM-CSF stimulates the differentiation and maturation of neutrophils, eosinophils and macrophages [40]. GATA-3 is a transcription factor found in Th2 lineage-committed T helper cells that stimulate a Th2 response [49]. The similar expression levels of IL-4, GM-CSF and GATA-3 between chickens in group DC and group DN suggested that IBDV challenge did not affect the expression of these genes in DNA-vaccinated chickens, most likely due to the rapid clearance of invading IBDV [10]. The enhanced expression of IL-4 (0.5 and 1 dpc), GM-CSF and GATA3 in IBDVinfected chickens suggested the activation of a Th2 immune response in IBDV-infected bursae. The expression of chicken IL-17a is induced in the gut after Eimeria tenella infection [15], and it also contributes to the immunopathology in the gut [48]. Upregulated or significantly (p \ 0.05) upregulated IL-17a in chickens in group CC suggested the activation of Th17 cells; however, this was not the case for chickens in groups DN and DC. Immunopathology caused by IBDV was suggested previously, since virus-induced pathology lasts several days after 5 dpc [43]. Therefore, chicken IL-17a may play a role during IBDV infection.
Immune cell regulation Apoptosis The relatively unchanged levels of CD4, CD8 and IL-2 in chickens in group DN suggested that DNA vaccination did not affect the expression levels of these genes. The significantly (p \ 0.05) increased expression of IL-2, CD4 and CD8 in IBDV-challenged chickens in group CC indicated the activation of helper and cytotoxic T cells in the bursa upon IBDV infection and corresponded to the quantification results of CD4- and CD8-positive T cells by immunohistochemistry staining in the same animal experiment [10]. The upregulated expression levels of CD4 and CD8 indicated bursal infiltration of T cells, which has been suggested to enhance virus clearance [36]. IFN-c mediates cellular immunity and thereby clears intracellular infection [42]. In addition, IFN-c induces Th1 cell differentiation and activates macrophages and cytotoxic T cells [5]. IL-18 induces the production of IFN-c and promotes the maturation of T and NK cells [12]. IL-12 activates cytotoxic T cells and NK cells and stimulates the production of IFN- c. Upregulation or significant (p \ 0.05) upregulation of IL-12, IL-18 and IFN- c in chickens of group CC suggested the activation of cellular
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IBDV-induced apoptosis at the later stage of infection is mediated by caspase and requires NF-kB activity [25]. Accumulating evidence suggest that IBDV-induced apoptosis occurs through the release of cytochrome c from mitochondria. Cytochrome c in the cytoplasm induces the activation of caspase-9 and the subsequent caspase-3 pathway, leading to the occurrence of apoptosis [41]. In the present study, the expression levels of Bcl-2, p53 and caspase-3 did not change much in chickens receiving DNA vaccination. The increased expression of p53 and the decreased expression of Bcl-2 in IBDV-infected chickens indicated the occurrence of apoptosis in the bursa during IBDV infection. The expression pattern of p53 and Bcl-2 was consistent with the progress of IBD, as shown previously [25]. However, a reduced level of caspase-3 expression was observed in the present study and in a previous study as well [35]. Since caspase-3 is synthesized as a zymogen and activated by proteolytic cleavage [4, 34], the unexpected downregulation of caspase-3 in IBDVinfected chickens in group CC may suggest a negative feedback mechanism during apoptosis.
Effect of IBDV DNA vaccination on bursal gene expression
Glucose transport It has been suggested that glucose transporter expression levels are increased during virus replication to meet higher metabolic demands [38]. In the present study, the expression of glucose transporters in DNA-vaccinated chickens remained relatively unchanged, suggesting that DNA vaccination did not change the expression of GLUT-2 and GLUT-3 in chickens. However, increased expression levels of GLUT-2 and GLUT-3 in IBDV-infected chickens in group CC were observed in the later stage of the infection, suggesting a higher demand for glucose during IBDV replication. In summary, among the genes examined in the present study, the significantly (p \ 0.05) upregulated granzyme K and CD8 in DNA-vaccinated chickens compared to those in unvaccinated chickens at 0.5 or 1 dpc suggested a possible link between the activation of cytolytic T lymphocytes and DNA vaccination. The expression levels of most genes related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport in DNAvaccinated chickens were not differentially expressed upon IBDV challenge, indicating an effect of DNA vaccination on rapid clearance of infecting IBDV, while those in IBDV-infected unvaccinated chickens were differentially expressed, indicating an effect of IBDV infection on the induction of the bursal transcriptome.
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ORIGINAL ARTICLE
Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus Chih-Chun Lee • Bong-Suk Kim • Ching Ching Wu Tsang Long Lin
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Received: 16 April 2014 / Accepted: 9 September 2014 / Published online: 1 October 2014 Ó Springer-Verlag Wien 2014
Abstract Infectious bursal disease virus (IBDV) infection destroys the bursa of Fabricius, causing immunosuppression and rendering chickens susceptible to secondary bacterial or viral infections. IBDV large-segment-proteinexpressing DNA has been shown to confer complete protection of chickens from infectious bursal disease (IBD). The purpose of the present study was to compare DNAvaccinated chickens and unvaccinated chickens upon IBDV challenge by transcriptomic analysis of bursa regarding innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. One-day-old specific-pathogen-free chickens were vaccinated intramuscularly three times at weekly intervals with IBDV large-segment-protein-expressing DNA. Chickens were challenged orally with 8.2 9 102 times the egg infective dose (EID)50 of IBDV strain variant E (VE) one week after the last vaccination. Bursae collected at 0.5, 1, 3, 5, 7, and 10 days post-challenge (dpc) were subjected to real-time RT-PCR quantification of bursal transcripts related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport. The expression levels of granzyme K and CD8 in DNA-vaccinated chickens were significantly (p \ 0.05) higher than those in unvaccinated chickens upon IBDV challenge at 0.5 or 1 dpc. The expression levels of other genes involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not upregulated or downregulated in DNA-vaccinated chickens during IBDV challenge. Bursal transcripts related to innate immunity and inflammation, including TLR3, MDA5, IFN-a, IFN-b, IRF-1, C.-C. Lee B.-S. Kim C. C. Wu T. L. Lin (&) Department of Comparative Pathobiology, Purdue University, 406 S. University St, West Lafayette, IN 47907, USA e-mail: [email protected]
IRF-10, IL-1b, IL-6, IL-8, iNOS, granzyme A, granzyme K and IL-10, were upregulated or significantly (p \ 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. The expression levels of genes related to immune cell regulation, apoptosis and glucose transport, including CD4, CD8, IL-2, IFN-c, IL-12(p40), IL-18, GM-CSF, GATA-3, p53, glucose transporter-2 and glucose transporter-3, were upregulated or significantly (p \ 0.05) upregulated at 3 dpc and later in unvaccinated chickens challenged with IBDV. Taken together, the results indicate that the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport, except for granzyme K and CD8, was not differentially expressed in DNA-vaccinated chickens protected from IBDV challenge.
Introduction Infectious bursal disease (IBD) is one of the most important viral diseases in the poultry industry worldwide and is characterized by its acute, highly contagious and immunosuppressive properties. The major target tissue of IBD is IgM-bearing B cells in the bursa of Fabricius. The infection in chickens between 3 and 6 weeks of age causes severe diarrhea, muscular hemorrhage, and bursal atrophy. IBD occurring in chickens less than 3 weeks of age induces few or no clinical signs but leads to an immunosuppressive status in recovered chickens, leading to subsequent vaccination failure and susceptibility to secondary infections [31]. Infectious bursal disease virus (IBDV) is a non-enveloped, double-stranded RNA virus with two genomic segments, A and B. A large open reading frame (ORF) in segment A encodes a polyprotein that is further processed
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into VP2 and VP3 to form the viral capsid, and VP4 to form a viral protease. The other ORF of segment A encodes a non-structural protein, VP5 [29], which is not essential for virus replication [30] but has been shown to induce apoptosis in chicken B cells and fibroblast cells to facilitate virus dissemination [47]. Segment B encodes VP1, an RNA-dependent RNA polymerase. Vaccination is the main strategy that is currently used to control IBD. Most commercially available vaccines consist of attenuated or killed IBDV. However, IBD outbreaks still occur in chicken farms worldwide due to the emergence of variant strains of IBDV and very virulent IBDV [26]. Variant IBDV strains have been prevalent in the United States since 1985 and the variant E (VE) strain is more common isolate than the other variants. Infection with the VE strain of IBDV results in rapid bursal atrophy with few clinical signs and gross lesions at 3 days post-challenge (dpc) [16]. Chicken embryos infected with the VE strain of IBDV display splenomegaly and liver necrosis, which are not observed in those infected with classical strains of IBDV [37]. In addition, vaccination with variant strains of IBDV induces better heterologous protection against classical strains of IBDV [17]. DNA vaccines encoding viral proteins induce protective responses against viruses and provide advantages over conventional vaccines [44]. DNA vaccines reduce the problem of revertant virulence and divergent mutants associated with live attenuated vaccines [8]. Previous studies have shown that DNA vaccines are effective in inducing humoral and/or cellular immunity and provide protection against virus infection [23]. A DNA vaccine encoding polyprotein VP243 in genomic segment A from the VE strain of IBDV has been developed and was shown previously to be efficacious [8, 9]. The antibody titer against IBDV measured by ELISA is low, but the virus-neutralization titer to IBDV increases in protected chickens receiving DNA vaccination [8, 9]. The virus antigen is not detected in the bursa and spleen in DNAvaccinated chickens after IBDV challenge by immunofluorescent assay (IFA) [10]. T helper 1 (Th1)-biased immune cell differentiation has been demonstrated in DNA-vaccinated chickens [10], and this could be due to the presence of unmethylated cytidine-phosphate-guanosine (CpG) motifs in the DNA vaccine. An unmethylated CpG motif has been shown to stimulate the innate immune response and proinflammatory cytokines [13, 14]. The aforementioned findings suggest that the effect of DNA vaccination on the bursal microenvironment during IBDV infection may affect the induction of cytokines and other cellular gene products involved in innate immunity, inflammation, immune cell regulation and apoptosis that are critical for anti-viral defense and protective immunity. Therefore, the protective effect of DNA vaccination against IBD may be related to differential expression of bursal
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RNA molecules (bursal transcriptome) upon IBDV infection. The purpose of the present study was to compare DNA-vaccinated chickens and unvaccinated chickens upon IBDV challenge by kinetic analysis of the bursal transcriptome involved in innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport.
Materials and methods Chickens Specific-pathogen-free (SPF) embryonated chicken eggs obtained from Charles River Laboratories (North Franklin, CT, USA) were hatched out after incubation for 21 days. One-day-old SPF chickens were kept in Horsfall-Bauer isolators with food and water ad libitum. Protocols used for the animal study were approved by Purdue University Animal Care and Use Committee. Virus IBDV strain Variant E (VE), provided by Dr. John K. Rosenberger from University of Delaware, was used as the template to construct the plasmid for DNA vaccination and was also used to challenge chickens after DNA vaccination in the present study. DNA vaccination and infection challenge The DNA vaccine construct for expression of the IBDV large segment was described previously [9]. The animal experiment in the present study was described in a previous report [10]. Briefly, one hundred eighteen hatched SPF chickens were randomly divided into four groups as follows: negative control (CN); unvaccinated, challenged control (CC); DNA-vaccinated, unchallenged group (DN); and DNA-vaccinated, challenged group (DC). Chickens receiving DNA vaccination were injected intramuscularly three times with DNA vaccine containing 400 lg of pCR3.1-VP243 VE plasmid in phosphate-buffered saline (PBS) at 1, 7, and 14 days after hatching. Unvaccinated chickens were injected intramuscularly with PBS. In the groups challenged with IBDV, chickens were orally administered 8.2 9 102 times the embryonic infectious dose (EID)50 of IBDV strain VE 7 days after the last immunization. Bursae were collected at 0.5, 1, 3, 5, 7 and 10 dpc and stored in RNAlater (QIAGEN, Valencia, CA, USA) in a -20 °C freezer. Protection from IBDV challenge in a chicken was defined by a gross lesion score of 1 and bursal/body weight ratio not less than 2 standard deviations below the average ratio of the unchallenged control chickens [8].
Effect of IBDV DNA vaccination on bursal gene expression
RNA extraction and cDNA synthesis RNA was extracted using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s manual. In brief, 30 mg bursal tissue was homogenized twice for 30 s in 600 lL of RLT lysis buffer, using a MULTI-GEN 7 homogenizer (Pro Scientific Inc., Oxford, CT, USA). RNA was eluted with 40 lL of RNase-free water and stored in a -80 °C freezer. The quality and quantity of the RNA was evaluated spectrophotometrically using a GeneQuant 1300 (GE Healthcare Life Sciences, Piscataway, NJ, USA). Total RNA was treated with DNase to remove genomic DNA contamination. The reaction mixture contained 4 lg of RNA, 1x reaction buffer, and 1 unit of RQ1 RNase-free DNase (Promega, Madison, WI, USA) and was incubated at 37 °C for 1 h. The reaction was stopped by incubation at 70 °C for 15 min to deactivate DNase. cDNA synthesis was performed using DNase-treated RNA, Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and random primer (Sigma Genosys, The Woodlands, TX, USA). Briefly, DNase-treated RNA was added to 10 nmol of dNTP and 200 ng of random hexamer, heated to 65 °C for 5 min and immediately put on ice for 2 min. The reaction mixture containing 1x first-strand synthesis buffer, 5 mM DTT and 200 units of Superscript III reverse transcriptase in 20 lL was incubated at 50 °C for 60 min and then at 70 °C for 20 min to deactivate the reverse transcriptase. The resulting cDNA was stored in a -20 °C freezer for later use. Quantitative analysis of the transcriptome by real-time RT-PCR The gene expression levels of the cDNA were determined by real-time RT-PCR using a Rotor-Gene RG-3000 thermal cycler (QIAGEN). The quantification was carried out with either TaqMan-probe-based or SYBR Green–based chemistry. The design of primers and probes for IL-4, 28S and IFN-c was described previously [3], and primers for the other genes in the present study were designed using PrimerQuest (Integrated DNA Technologies Inc, Coralville, IA, USA). The sequences of primers and probes are summarized in Table 1. The probe-based PCR mixture contained 200 nM forward and reverse primers, 100 nM probe, and 1x Platinum Quantitative PCR SuperMix-UDG (Invitrogen). Thermal cycling was done at 50 °C for 2 min and 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 59 °C for 60 s. The fluorescent signal was acquired using the FAM/SYBR Green channel at 59 °C. The SYBR Green–based PCR mixture containing 200 nM forward and reverse primers and 1x EXPRESS SYBR GreenER qPCR universal qPCR Supermixes (Invitrogen) was subjected to 1 cycle of 50 °C for 2 min and 95 °C for
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2 min followed 40 cycles of 95 °C for 20 s and 60 °C for 60 s, and 1 cycle of final extension at 60 °C for 5 min. The fluorescent signal was acquired using the FAM/SYBR Green channel at the end of each extension stage. Melting curve analysis was performed between 65 °C and 95 °C, with a ramp speed at 1 °C/s. Melting curve analysis was used to evaluate the specificity of PCR amplification. Data analysis The expression of each gene was normalized to that of a housekeeping gene (GAPDH or 28S), so the normalized expression of a target gene to GAPDH or 28S was 2-DCt, where DCt = Cttarget gene - CtGAPDH (or Ct28S). For each gene, the normalized expression in groups DN, DC and CC relative to that in group CN was calculated using the formula 2-DDCt, where DDCttarget gene = each DCttarget gene in group DN, DC or CC – the average of DCttarget gene in group CN, and expressed as fold change. The fold changes were subjected to statistical analysis using a one-way ANOVA test with Duncan’s post hoc analysis (SPSS software, SPSS Inc, Chicago, IL, USA). A p-value less than 0.05 was considered statistically significant.
Results Bursal transcriptome related to innate immunity The mRNA expression levels of Toll-like receptor 3 (TLR3) (Fig. 1A), melanoma differentiation-associated gene 5 (MDA5) (Fig. 1C), interferon-a (IFN-a) (Fig. 1D), IFN-b (Fig. 1E), IFN regulatory factor-1 (IRF-1) (Fig. 1F) and IRF-10 (Fig. 1G) in chickens in groups CN, DN and DC were similar to each other, but they were significantly (p \ 0.05) increased in chickens in group CC at 3, 5, 7 or 10 dpc, with the highest fold increases at 23.22-, 52.03-, 10.97-, 50.27-, 12.35- and 23.80-fold, respectively. The transcript levels of TLR7 (Fig. 1B) in the three groups were not significantly (p [ 0.05) different at each time point except for 0.5 dpc. No significant (p [ 0.05) difference in the expression level of interferon-stimulated gene 20 kDa (ISG-20)(Fig. 1H) was observed between group DN and group DC, while ISG-20 in chickens in group CC had higher or significantly (p \ 0.05) higher expressions at 1, 3, 5, 7 and 10 dpc. Bursal transcriptome related to inflammation The transcript levels of interleukin-1b (IL-1b) (Fig. 2A), IL-6 (Fig. 2B), IL-8 (Fig. 2C), inducible nitric oxide synthase (iNOS) (Fig. 2D) and IL-10 (Fig. 2G) increased significantly (p \ 0.05) in IBDV-challenged chickens in
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Table 1 Nucleotide sequences of primers and probes used in the present study Target gene
Forward sequence (50 ?30 )
Reverse sequence (50 ?30 )
GenBank accession no.
GAPDH
ATCAAGAGGGTAGTGAAGGCTGCT
TCAAAGGTGGAGGAATGGCTGTCA
NM_204305
IFN-a
CAATGCTTGGACAGCAGAGA
GTCTTGGAGGAAGGTGTGGA
XM_001231512
IFN-b
ACCAGGATGCCAACTTCTCTTGGA
ATGGCTGCTTGCTTCTTGTCCTTG
NM_001024836
IFN-c
GTGAAGAAGGTGAAAGATATCATGGA
GCTTTGCGCTGGATTCTCA
NM_205149.1
IFN-c probe
FAM-TGGCCAAGCTCCCGATGAACGA-TAMRA
TLR3
GCAAGGACAGTGCTCCATTT
CCCGGTAGTCTGTCAAGCTC
DQ780341
TLR7
TTGACAACCTTTCCCAGAGC
GTTGGTGGGCCATGTAAAAT
NM_001011668
MDA5
AATTCCTGCTGGCGCTGAAGAAAG
AAAGATGCCCAGCTCCAGACACTT
XM_422031.2
ISG-20
TACTTCCACCCCAAGGAGAG
CCACCAGGATCTTCTTCTGC
XM_413869.2
IRF-1
AAAGGCTTCTTCACCAACGA
GAACTCCAACTCTGCCGAAG
NM_205415.1
IRF-10 IL-1b
ACCAAATCCACCTGTGCTTC TCATCTTCTACCGCCTGGAC
GGAAGAGCTCTCGTGCAAAT GTAGGTGGCGATGTTGACCT
NM_204558.1 NM_204524
IL-2
CTGCAGTGTTACCTGGGAGA
CTTGCATTCACTTCCGGTGT
NM_204153.1
IL-4
AACATGCGTCAGCTCCTGAAT
TCTGCT AGGAACTTCTCCATTGAA
NM_001007079
IL-4 probe
FAM-AGCAGCACCTCCCTCAAGGCACC-TAMRA
IL-6
CTCCTCGCCAATCTGAAGTC
CCCTCACGGTCTTCTCCATA
NM_204628
IL-8
GCTCTGTCGCAAGGTAGGAC
GCGTCAGCTTCACATCTTGA
NM_205498
IL-10
CAGTCATCAGCAGAGCATGG
TTGATGGCTTTGCTCCTCTT
NM_001004414
IL-12 (p40)
CTGTGGCTCGCACTGATAAA
GAACATCTCAGTCGGCTGGT
NM_213578
IL-17a
GGCCGTAACGATTAGCAAGA
TCTTCTCATGGAGCACGTTG
NM_204460
IL-18
GGGAAGGAGAAGTTCCCAAA
TCAAAGGCCAAGAACATTCC
NM_204608
CD4
GGAGGAAGCTCATGTTTGGA
CTGCCACCTCATACCAGTGA
NM_204649.1
CD8
AGCTCAGAGCCAGGAACAAG
GTCTTTTGGCAGAGCACGAT
NM_205235.1
TGF-b4
GGACATGCAGAGCATTGCCAAGAA
GATCCTTGCGGAAGTCGATGTAGA
NM_001011691
GM-CSF
ACCCATGGACATCAGGGATA
GTTCGAGAAGAGGCGTTCAC
NM_001007078
GATA-3
TGCAAAAAGGTCCATGACAA
TAGTCAGCATGTGGCTGGAG
NM001008444
iNOS Granzyme A
TGGGTGGAAGCCGAAATA ACTCATGTCGAGGGGATTCA
GTACCAGCCGTTGAAAGGAC TGTAGACACCAGGACCACCA
U46504 NM_204457.1
Granzyme K
CGGGAAGCAACTGTTGAAAT
GAGTCTCCCTTGCAAGCATC
XM_423832
Caspase-3
GCAGCTGAAGGCTCCTGGTTTATT
TCTGCCACTCTGCGATTTACACGA
NM_204725
p53
CTCCAAGCAACAACAAACGCTGGA
TCCCAGTAAGATCCACCAACACAA
XM_419987
BCL-2
ACAAGGAGATGCGGGTACTG
CAGCGTTGTTCCCATACAGA
BX93598
GLUT-2
GTGGGGATGTGCTTCCAGTA
TCGTTTCGGGTACTTTGAGG
NM_207178.1
GLUT-3
ATGCTCTTCCCCTATGCTGA
AAAAGTCCTGCCCTTGGTCT
NM_205511.1
28S
GGCGAAGCCAGAGGAAACT
GACGACCGATTTGCACGTC
DQ018756
28S probe
FAM-AGGACCGCTACGGACCTCCACCA-TAMRA
group CC at 3, 5, 7 or 10 dpc, with the highest expression levels at 40.22-, 46.59-, 43.86-, 17.07- and 25.22-fold, respectively. The fold changes of these genes in chickens were similar between group DN and group DC at most time points. The expression levels of granzyme K (Fig. 2F) in chickens receiving DNA vaccination (groups DN and DC) were significantly (p \ 0.05) upregulated, ranging from 3.72- to 6.12-fold relative to the expression levels in chickens in group CN. The transcript levels of granzyme A (Fig. 2E) in chickens in group DN were similar to those in
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chickens in group CN. The expression levels of granzyme A and granzyme K in chickens in group CC were significantly (p \ 0.05) upregulated at 3, 5, 7, and 10 dpc, with the highest fold increases at 48.88 and 50.82 at 5 dpc, respectively. The expression level of transforming growth factor b4 (TGF-b4) (Fig. 2H) in chickens in group DC and group DN was not significantly different (p [ 0.05) at any time point except at 7 dpc, while that in chickens in group CC was significantly (p \ 0.05) lower than that in group DC at 1, 7 and 10 dpc.
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Effect of IBDV DNA vaccination on bursal gene expression
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Fig. 1 Bursal transcriptome involved in innate immunity. Chickens receiving DNA vaccination were challenged with IBDV strain VE (group DC) or mock challenged (group DN), and the other chickens that did not receive DNA vaccination were challenged with IBDV (group CC) or mock challenged (group CN). Bursae were collected from chickens at 0.5, 1, 3, 5, 7 and 10 days post-challenge (dpc) to quantify gene transcripts of TLR3 (A), TLR7 (B), MDA5 (C), IFN-a
(D), IFN-b (E), IRF-1 (F), IRF-10 (G), and ISG-20 (H). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
Bursal transcriptome involved in immune cell regulation
group DC at any of the six time points. The expression level of CD8 in chickens in DN and group DC was significantly (p \ 0.05) higher than that in chickens in group CC at 0.5 dpc. No significant (p [ 0.05) difference was observed in the expression levels of IFN-c (Fig. 3D), IL-12(p40) (Fig. 3E) and IL-18 (Fig. 3F) between group DN and group DC. The
CD4 (Fig. 3A), CD8 (Fig. 3B) and IL-2 (Fig. 3C) exhibited significant (p \ 0.05) upregulation in chickens in group CC at 3, 5, 7 and 10 dpc, whereas there was no significant (p [ 0.05) difference between group DN and
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Fig. 2 Bursal transcriptome involved in inflammation. Bursae were collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc to quantify gene transcripts of IL-1b (A), IL-6 (B), IL-8 (C), iNOS (D), granzyme A (E), granzyme K (F), IL-10 (G), and TGF-b4 (H). The relative expression levels of these genes were normalized to GAPDH content
in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
transcripts of IFN-c, IL-12(p40) and IL-18 were upregulated or significantly (p \ 0.05) upregulated in chickens in group CC compared to those in the other two groups at 3, 5, 7 or 10 dpc, with the highest fold increases at 20.64-, 42.47-, and 6.77-fold, respectively. The transcript levels of IL-4 (Fig. 3H) in chickens from group DC were higher or significantly (p \ 0.05) higher than those in group DN and group CC at 5, 7 and 10 dpc,
Fig. 3 Bursal transcriptome involved in immune cell regulation. c Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc to quantify gene transcripts of CD4 (A), CD8 (B), IL-2 (C), IFN-c (D), IL-12(p40) (E), IL-18 (F), IL-4 (G), GM-CSF (H), GATA-3 (I), and IL-17a (J). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
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Effect of IBDV DNA vaccination on bursal gene expression
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with the highest fold change at 6.59-fold. The expression levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Fig. 3G) and GATA-3 (Fig. 3I) were higher or significantly (p \ 0.05) higher in chickens in group CC at 3, 5, 7 and 10 dpc, while those between group DC and group DN did not differ significantly (p [ 0.05). The transcript levels of IL-17a (Fig. 3J) were upregulated or significantly (p \ 0.05) upregulated in chickens in group CC at 1, 3, 5, 7 or 10 dpc, with the highest fold changes at 10.90-fold, whereas those in group DN and group DC remained similar at all six time points.
Bursal transcriptome related to glucose transport
Bursal transcriptome related to apoptosis
The animal experiment in the present study was described previously [10]. The bursae collected were used to analyze the bursal transcriptome in the present study. The results from the animal experiment indicated that DNA-vaccinated chickens were protected from IBDV challenge, while severe bursal atrophy was observed in IBDV-infected chickens in group CC [10]. IBDV VP2 antigen was not present in the bursae of DNA-vaccinated chickens upon IBDV challenge, as determined by IFA, and a much lower virus load was detected by real-time RT-PCR in the bursae of DNA-vaccinated chickens when compared with that in IBDV-infected unvaccinated chickens [10], indicating that IBDV reached the bursae, spleen and cecal tonsils and replicated in these lymphoid tissues in DNA-vaccinated chickens, but the replication of IBDV was limited in
B-cell lymphoma-2 (Bcl-2) (Fig. 4A) was downregulated significantly (p \ 0.05) in chickens in group CC at 3, 5, 7 and 10 dpc. The transcript levels of Bcl-2 in group DN and group DC were similar throughout the study. The expression levels of p53 (Fig. 4B) in chickens in group CC were upregulated or significantly (p \ 0.05) upregulated at 3, 5, 7 and 10 dpc, while those in chickens in group DN and group DC were similar at all six time points. The transcript levels of caspase-3 (Fig. 4C) in chickens in group DN and group DC were not significantly (p [ 0.05) different at any of the time points, but those in group CC decreased significantly (p \ 0.05) at 3, 5, 7 and 10 dpc.
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Fig. 4 Bursal transcriptome involved in apoptosis. Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc were used to quantify gene transcripts of Bcl-2 (A), p53 (B) and caspase-3 (C). The relative expression levels of these genes were normalized to GAPDH content in each sample and are presented as fold changes over expression
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Discussion
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The expression of glucose transporter-2 (GLUT-2) (Fig. 5A) in IBDV-infected chickens in group CC was significantly (p \ 0.05) upregulated at 1 and 5 dpc and significantly (p \ 0.05) downregulated at 7 dpc. The expression of GLUT-3 (Fig. 5B) in chickens in group CC was higher or significantly (p \ 0.05) higher than that in group DN and group DC at 3, 5, 7, and 10 dpc.
levels in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
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Fig. 5 Bursal transcriptome involved in glucose transport. Bursae collected from chickens at 0.5, 1, 3, 5, 7 and 10 dpc were used to quantify gene transcripts of GLUT-2 (A) and GLUT-3 (B). The relative expression levels of these genes were normalized to GAPDH
content in each sample and are shown as fold changes over those in chickens in group CN. Bars indicate mean ± standard deviation based on five or four chickens. The letters above the bars indicate p \ 0.05 among the three groups as determined by one-way ANOVA
DNA-vaccinated chickens. This suggests that certain changes mediated by DNA-vaccination may help limit the growth of IBDV. In the present study, the expression levels of most selected genes in DNA-vaccinated chickens in group DN were similar to those in chickens in group CN, with the exception of granzyme K and CD8, which were upregulated in DNA-vaccinated chickens at 0.5 or 1 dpc, suggesting that the activated cellular immunity was mediated by DNA vaccination in the bursae. The expression levels of selected genes related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport were not elicited in DNA-vaccinated chickens upon IBDV challenge, whereas those in IBDV-challenged chickens in group CC showed differential expression.
than the emergence of clinical signs, as determined by bursal lesions and bursal/body weight ratio [10]. IRF-1 and IRF-10 are transcription factors regulating the antiviral immune response [32]. Consistent with the increase in type I IFNs, the significantly (p \ 0.05) enhanced expression of IRF-1 and IRF-10 in chickens in group CC after 3 dpc indicated the activation of the innate immune response. The upregulated expression of IRF-1 or IRF-10 was observed previously in IBDV-infected chickens [24, 35, 45].
Innate immunity The expression levels of innate immune genes in IBDVchallenged DNA-vaccinated chickens in group DC were generally similar to those in DNA-vaccinated chickens in group DN and did not change much compared to unvaccinated chickens in group CN, suggesting that the selected innate immune genes were not activated after DNA vaccination. The unchanged expression of the innate immune genes between chickens in group DC and group DN suggested that most of the entering viruses may have been eliminated and thus replicated poorly. The result was in line with the rapid clearance of IBDV in DNA-vaccinated chickens in the same animal experiment described previously [10]. The upregulated expression of TLR3 and MDA5 in IBDV-infected chickens in group CC suggested that TLR3 and MDA5 may be involved in sensing IBDV infection. The upregulation of TLR3 is observed in chicken embryo fibroblasts infected with IBDV [45]. The expression levels of IFN-a and IFN-b (type I IFN) in chickens in group CC were not upregulated until 3 dpc, indicating that the innate immune response had been activated since 3 dpc, earlier
Inflammation IL-1b and IL-6 induce an inflammatory response during IBDV infection [21]. IL-6 was shown in previous studies to be upregulated upon IBDV infection [20, 35]. The upregulated IL-1b and IL-6 in IBDV-challenged chickens in group CC in the present study indicated the occurrence of inflammation. Chicken IL-8 has been shown to attract neutrophils and naı¨ve T lymphocytes [46]. The increased expression of IL8 in IBDV-challenged chickens in group CC suggested the recruitment of heterophils and naı¨ve T lymphocytes to the bursa [39]. The upregulation of IL-8 by IBDV infection has been reported previously [24, 35]. Nitric oxide has multiple roles in host immune responses against pathogens, including direct antiviral activity against some enteroviruses, effects on T helper cell polarization, and oxidative stress through the formation of NO radicals [1]. In the present study, the increased expression of iNOS in IBDV-challenged chickens in group CC after 3 dpc suggested the activation of macrophages. Cytotoxic T lymphocytes mediate cytolytic activity through the secretion of granules containing perforin and granzymes [28]. Both granzyme A and granzyme K have trypsin-like activity and are able to cleave and activate caspase-3, which in turn activates caspase-activated DNase. The DNase damages the chromosomal DNA and
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leads to apoptosis. Human and mouse granzyme A induce the secretion of proinflammatory cytokines such as IL-1b, TNF-a and IL-6, thus playing a role in induction of inflammation [27]. Granzyme K appears to be a functionally redundant backup of granzyme A with some unique substrate specificity [6], and it provides protection against influenza virus challenge in mice lacking the expression of granzymes A and B [19]. Not much is known about chicken granzymes, but their upregulation during IBDV infection has been observed [35]. The clearance of IBDV has been suggested to be mediated by cytolytic molecules such as perforin and granzymes in cytotoxic T lymphocytes [33]. In the present study, the increased expression of granzyme K in chickens in group DN at 0.5 and 1 dpc suggested increased cytotoxicity induced by DNA vaccination. Enhanced cytotoxicity following DNA vaccination, as determined by increased granzyme B activity, has been demonstrated in mice and monkeys [2, 18]. Thus, DNA vaccination in the present study may have led to enhanced cytotoxicity, thereby contributing to the rapid clearance of IBDV. The upregulation of granzymes A and K in chickens in group CC at and after 3 dpc indicated the activation of cytotoxic T lymphocytes, as was shown previously [22, 33]. T cells infiltrated in the bursa are important for virus clearance during IBDV infection, and many of these cells are CD8? T cells [7, 22, 33], suggesting that cytotoxic T cell enzymes, such as granzymes A and K, may participate in virus clearance in IBDV-infected chickens.
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immunity, as was shown previously [11, 22, 36], while relatively unchanged expression of these cytokines in chickens in groups DN and DC indicated that IBDV challenge did not elicit Th1 immune response in DNAvaccinated chickens, most likely due to the rapid clearance of IBDV [10]. IL-4 promotes B cell activation and IgE production [3], and GM-CSF stimulates the differentiation and maturation of neutrophils, eosinophils and macrophages [40]. GATA-3 is a transcription factor found in Th2 lineage-committed T helper cells that stimulate a Th2 response [49]. The similar expression levels of IL-4, GM-CSF and GATA-3 between chickens in group DC and group DN suggested that IBDV challenge did not affect the expression of these genes in DNA-vaccinated chickens, most likely due to the rapid clearance of invading IBDV [10]. The enhanced expression of IL-4 (0.5 and 1 dpc), GM-CSF and GATA3 in IBDVinfected chickens suggested the activation of a Th2 immune response in IBDV-infected bursae. The expression of chicken IL-17a is induced in the gut after Eimeria tenella infection [15], and it also contributes to the immunopathology in the gut [48]. Upregulated or significantly (p \ 0.05) upregulated IL-17a in chickens in group CC suggested the activation of Th17 cells; however, this was not the case for chickens in groups DN and DC. Immunopathology caused by IBDV was suggested previously, since virus-induced pathology lasts several days after 5 dpc [43]. Therefore, chicken IL-17a may play a role during IBDV infection.
Immune cell regulation Apoptosis The relatively unchanged levels of CD4, CD8 and IL-2 in chickens in group DN suggested that DNA vaccination did not affect the expression levels of these genes. The significantly (p \ 0.05) increased expression of IL-2, CD4 and CD8 in IBDV-challenged chickens in group CC indicated the activation of helper and cytotoxic T cells in the bursa upon IBDV infection and corresponded to the quantification results of CD4- and CD8-positive T cells by immunohistochemistry staining in the same animal experiment [10]. The upregulated expression levels of CD4 and CD8 indicated bursal infiltration of T cells, which has been suggested to enhance virus clearance [36]. IFN-c mediates cellular immunity and thereby clears intracellular infection [42]. In addition, IFN-c induces Th1 cell differentiation and activates macrophages and cytotoxic T cells [5]. IL-18 induces the production of IFN-c and promotes the maturation of T and NK cells [12]. IL-12 activates cytotoxic T cells and NK cells and stimulates the production of IFN- c. Upregulation or significant (p \ 0.05) upregulation of IL-12, IL-18 and IFN- c in chickens of group CC suggested the activation of cellular
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IBDV-induced apoptosis at the later stage of infection is mediated by caspase and requires NF-kB activity [25]. Accumulating evidence suggest that IBDV-induced apoptosis occurs through the release of cytochrome c from mitochondria. Cytochrome c in the cytoplasm induces the activation of caspase-9 and the subsequent caspase-3 pathway, leading to the occurrence of apoptosis [41]. In the present study, the expression levels of Bcl-2, p53 and caspase-3 did not change much in chickens receiving DNA vaccination. The increased expression of p53 and the decreased expression of Bcl-2 in IBDV-infected chickens indicated the occurrence of apoptosis in the bursa during IBDV infection. The expression pattern of p53 and Bcl-2 was consistent with the progress of IBD, as shown previously [25]. However, a reduced level of caspase-3 expression was observed in the present study and in a previous study as well [35]. Since caspase-3 is synthesized as a zymogen and activated by proteolytic cleavage [4, 34], the unexpected downregulation of caspase-3 in IBDVinfected chickens in group CC may suggest a negative feedback mechanism during apoptosis.
Effect of IBDV DNA vaccination on bursal gene expression
Glucose transport It has been suggested that glucose transporter expression levels are increased during virus replication to meet higher metabolic demands [38]. In the present study, the expression of glucose transporters in DNA-vaccinated chickens remained relatively unchanged, suggesting that DNA vaccination did not change the expression of GLUT-2 and GLUT-3 in chickens. However, increased expression levels of GLUT-2 and GLUT-3 in IBDV-infected chickens in group CC were observed in the later stage of the infection, suggesting a higher demand for glucose during IBDV replication. In summary, among the genes examined in the present study, the significantly (p \ 0.05) upregulated granzyme K and CD8 in DNA-vaccinated chickens compared to those in unvaccinated chickens at 0.5 or 1 dpc suggested a possible link between the activation of cytolytic T lymphocytes and DNA vaccination. The expression levels of most genes related to innate immunity, inflammation, immune cell regulation, apoptosis and glucose transport in DNAvaccinated chickens were not differentially expressed upon IBDV challenge, indicating an effect of DNA vaccination on rapid clearance of infecting IBDV, while those in IBDV-infected unvaccinated chickens were differentially expressed, indicating an effect of IBDV infection on the induction of the bursal transcriptome.
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