vvIBDV Infection Research Articles

Bursal transcriptome profiling of different inbred chicken lines reveals key differentially expressed genes at 3 days post-infection with very virulent infectious bursal disease virus.

Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.

Transcriptional profiles in bursal B-lymphoid DT40 cells infected with very virulent infectious bursal disease virus

BackgroundInfectious bursal disease virus (IBDV) causes a highly contagious, immunosuppressive disease in chickens. The virus mainly infects immature B lymphocytes in the bursa of Fabricius (BF). Chicken B cell line DT40, an avian leukosis virus-induced B cell line, supports very virulent IBDV (vvIBDV) infection in vitro and thereby serves as a good model for investigating the infection and pathogenesis of this virus. However, a transcriptome-wide understanding of the interaction between vvIBDV and B cells has not yet been achieved. This study aimed to employ time-course DNA microarrays to investigate gene expression patterns in DT40 cells after infection with vvIBDV strain LX.ResultsDT40 cells infected with vvIBDV exhibited alterations in the expression of many important host genes involved in signal transduction pathways, including MAPK signaling, PI3K/mTOR signaling, cell death and survival, BCR signaling, and antigen presentation. The changes in cellular mRNA levels identified by microarray analysis were confirmed for 8 selected genes using real-time reverse transcription-PCR. The upregulation of inflammatory cytokines and Toll-like receptors (TLRs) in the bursa of vvIBDV-infected chickens might involve excessive activation of the innate immune and inflammatory responses and contribute to tissue damage.ConclusionsThe present study is the first to provide a comprehensive differential transcriptional profile of cultured DT40 cells in response to vvIBDV infection and further extends our understanding of the molecular mechanisms underlying vvIBDV infection and pathogenesis.

Characterization of Chicken Splenic-Derived Dendritic Cells Following Vaccine and Very Virulent Strains of Infectious Bursal Disease Virus Infection.

Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain.

Prophylactic potential of resiquimod against very virulent infectious bursal disease virus (vvIBDV) challenge in the chicken

The study evaluated the prophylactic potential of resiquimod (R-848), a synthetic TLR7 agonist, against very virulent infectious bursal disease virus (vvIBDV) infection in chicken. Specific pathogen free White Leghorn chicks of three week age were treated with R-848 (50μg/bird, intramuscular) or PBS (n=26/group). Twenty four hour later, half of the birds from each group were challenged with 105 ELD50 of vvIBDV and observed for 10days. To understand the effect of R-848, immune response genes such as interferon (IFN)-β, IFN-γ, IL-1β, IL-4, iNOS and TLR7 were analyzed at 24 and 48h post-challenge in PBMCs ex vivo by real-time PCR (n=6/group). On day 4 post-challenge, representative birds (n=3/group) were sacrificed to study the bursal damage and IBDV antigen clearance. Immunosuppression was assessed by antibody response against live Newcastle disease virus (NDV) vaccine, which was administered on day 10 post-challenge. R-848 pre-treatment significantly upregulated the transcripts of each immune response gene studied (P<0.05). There was 50% mortality on vvIBDV challenge in control birds, while it was only 20% with R-848 group. R-848 pre-treatment reduced the bursal damage as indicated by lower bursal lesion score in histopathology, reduced IBDV antigen signal in immunohistochemistry and improved antigen clearance in agar gel immunodiffusion test. Further, it protected significantly against vvIBDV induced immunosuppression as indicated by HI antibody titre. It is concluded that pre-treatment of R-848 conferred partial protection from mortality and bursal damage while complete protection against immunosuppression in chicken when challenged with vvIBDV, which could be due to the upregulation of immune response genes.

Very Virulent Infectious Bursal Disease Virus Produces More-Severe Disease and Lesions in Specific-Pathogen-Free (SPF) Leghorns Than in SPF Broiler Chickens.

Infectious bursal disease virus (IBDV) is an important pathogen of chickens causing negative economic impacts in poultry industries worldwide. IBDV has a variable range of virulence, with very virulent (vvIBDV) strains being responsible for the greatest losses from mortality and decreased performance. Previous vvIBDV studies using conventional broilers reported resistance to lethal effects and decreased performance as compared to specific-pathogen-free (SPF) layers, but the potential contribution of the conventional vs. SPF status to resistance has not been examined. In this study we compared differences in the acute pathologic effects of infection by the California rA strain of vvIBDV for SPF white leghorn egg-laying chickens and SPF white Plymouth Rock broiler chickens over a 7-day experimental period. Based on the clinical signs and mortality observed, as well as on themore-severe pathologic changes in lymphoid tissues and kidneys, white leghorns were shown to be more susceptible to thedeleterious effects of vvIBDV infection than were white Plymouth Rocks. This study provides important information on theimpact of chicken breed on susceptibility to vvIBDV and the absence of impact from conventional vs. SPF status on the outcome.

Histamine levels in embryonic chicken livers infected with very virulent infectious bursal disease virus

Histamine is an endogenous nitrogenous compound with extensive effects on immunologic cells and involved in many physiological functions. The current aim was to determine histamine levels in embryonic liver and its association with the pathogenicity of a very virulent infectious bursal disease virus (vvIBDV) isolate serially passaged in chicken embryos. A vvIBDV isolate and the passaged viruses were inoculated into SPF embryonated chicken eggs (0.2ml per egg) via the chorioallantoic membrane. Embryonic livers were collected at 24, 48, 72, 96, and 120h post-inoculation and histamine contents were quantified by fluorescence spectrophotometry analyses. Results showed that the histamine content in embryonic livers infected with the original vvIBDV isolate and the early passaged viruses significantly increased 48h post-inoculation, as compared with the adapted IBDV isolate (p<0.01) and controls (p<0.01), with the concentration peaking from 72h to 96h. Most of the infected chicken embryos died from 48h to 96h post-inoculation. Moreover, the histamine content in dead embryos was markedly increased compared with live embryos (p<0.05), peaking at 72h post-inoculation (p<0.01). There was an association between histamine content in embryonic livers and an elevation in histidine decarboxylase activity. Taken together, our results suggest that an excess of histamine correlates with inflammatory responses during vvIBDV infection. This study provides an incremental step in the understanding of the pathogenesis of vvIBDV.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus.

BackgroundVery virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius.ResultsThe viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels.ConclusionThis study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.

Apoptotic response of chicken embryonic fibroblast cells to infectious bursal disease virus infections reflects viral pathogenicity.

Infectious bursal disease virus (IBDV) induces immunodeficiency in young chickens and apoptosis in chicken embryos. To understand the relation between the viral pathogenesis and the induction of cell death, chicken embryonic fibroblast (CEF) cells were infected with IBDV intermediate (im) and very virulent (vv) strains at different MOIs. The cell viability and DNA fragmentation were evaluated in infected cells. The cellular apoptotic pathway involve was investigated by determining the activities of caspase cascade. The imIBDV strain was replicated well in CEF cells and shown higher viral titers than vvIBDV. Apoptosis changes were observed only in vvIBDV-infected CEF cells at higher MOI 48 h post infection. Efflux of cytochrome c suggests that the intrinsic pathway of the apoptotic process induced by vvIBDV infection independently of virus replication. Prediction of caspase substrates cleavage sites revealed that different IBDV strains have conserved cleavage motif pattern for VP2 and VP5 viral proteins. These findings suggest the pathogenicity of IBDV strains might be involved in the induction of apoptosis in host cells.

Protection Effect of Vaxxitek HVT + IBD Vaccine Against Infectious Bursal Disease in Broiler Chickens

The very virulent infectious bursal disease virus (vvIBDV) infection cause constant and frequent outbreaks of IBD. In order to prevent the disease caused by vvIBDV, moderate-virulent live vaccines are usually used in broilers, but these vaccine influence the immune system of chickens and cause the immunosuppression. VAXXITEK® HVT+IBD bivalent vaccine, a novel vaccine produced by Merial Company, is the recombinant vaccine generated by inserting IBDV VP2 gene into the HVT genome. The recombinant vaccine could prevent Marek’s disease and IBD of chickens at the same time after vaccinated by the subcutaneous route at 1-day-old. The objective of this study was to evaluate protection effect of this new vaccine against IBD in broilers. The results of the lab and field immunization trial showed that the vaccination of VAXXITEK® HVT+IBD vaccine did not damage the bursa of broilers; the level of IBD antibody of the VAXXITEK® HVT+IBD vaccination groups was higher than IBD-BLEN® vaccination groups at 21-day-old; the protection effect of the VAXXITEK® HVT + IBD vaccination groups was better than IBD-BLEN® vaccination groups, especially at 21-28 day. The results indicated that VAXXITEK® HVT + IBD vaccine could provide better protection to broiler chickens against IBD.DOI: http://dx.doi.org/10.3329/pa.v24i1-2.19102 Progress. Agric. 24(1&amp;2): 69 - 78, 2013

Adjuvant effects of interleukin-18 in DNA vaccination against infectious bursal disease virus in chickens

Interleukin-18 (IL-18) is an important cytokine with multiple functions in innate and acquired immunity. In this study, chicken IL-18 was evaluated for its adjuvant effects on the protective immunity of a DNA vaccine carrying the VP243 gene of IBDV. Groups of 14-day-old SPF chickens were given twice at 2-week intervals with 100μg of the plasmid DNA vaccine pCAGVP243, pCAGVP243-IL-18 and the blank vector pCAGGS, respectively, and challenged with vvIBDV (HLJ0504 strain) 2 weeks later. Chickens immunized with plasmid pCAGVP243-IL-18 carrying both VP243 and IL-18 genes induced significantly higher levels of antibodies, lymphocyte proliferation responses and of the cytokines IL-4 and IFN-γ than those injected with pCAGVP243 encoding the VP243 gene alone. Furthermore, pCAGVP243-IL-18 provided higher protection (93%) against vvIBDV challenge in chickens than pCAGVP243 (60%), as evidenced by the absence of clinical signs, mortality, and bursal atrophy. These results indicated that the cytokine IL-18 could enhance the immune responses and protection efficacy of DNA vaccine against IBDV infection in chickens, highlighting the potential value of chicken IL-18 as an adjuvant in the prevention of vvIBDV infection.

DNA prime–protein boost vaccination enhances protective immunity against infectious bursal disease virus in chickens

Infectious bursal disease virus causes an acute contagious immunosuppressive disease in chickens. Using VP2 protein from IBDV (Gx strain) as the immunogen, the goal of the current study was to evaluate the immune responses and protective efficacy elicited by different prime-boost vaccination regimens (DNA only, protein only, and DNA plus protein) in chickens. The results indicated that both pCAGoptiVP2 plasmid and rVP2 protein induced humoral and cellular immune responses. Chickens in the DNA prime-protein boost group developed significantly higher levels of ELISA and neutralizing antibodies to IBDV compared with those immunized with either the DNA vaccine or the protein vaccine alone (P<0.05). Furthermore, the highest levels of lymphocyte proliferation response, IL-4 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rVP2 protein. Additionally, chickens inoculated with the DNA prime-protein boost vaccine had 100% protection against challenge with vvIBDV, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. In contrast, chickens receiving the DNA vaccine and the rVP2 protein vaccine had 67% and 80% protection, respectively. These findings demonstrated that the DNA prime-protein boost immunization strategy was effective in eliciting both humoral and cellular immune responses in chickens, highlighting the potential value of such an approach in the prevention of vvIBDV infection.

Acute hypersensitive-like injury in specific-pathogen-free chickens after infection with very virulent infectious bursal disease virus

Very virulent infectious bursal disease virus (vvIBDV) can cause systemic inflammatory syndromes and acute death in specific-pathogen-free chickens within 72 h. However, the subtle mechanism of these severe inflammatory responses has been unsatisfactorily resolved until now. This study determined the kinetics of mast cells, tryptase, eosinophilic major basic protein, and eotaxin expression in specific-pathogen-free chickens after vvIBDV infection. Results showed that mast cell population, tryptase activity, major basic protein, and eotaxin expression were increased markedly in the vvIBDV-infected animals compared with the controls, with a significant difference in the bursa. Acute inflammatory lesions and high mortality were observed in vvIBDV-infected chickens. These observations implicate activated mast cells and eosinophils as important participants in vvIBDV-induced acute inflammatory lesions through mediators released in a short timeframe.

Viral Competition and Maternal Immunity Influence the Clinical Disease Caused by Very Virulent Infectious Bursal Disease Virus

The very virulent form of infectious bursal disease virus (vvIBDV) causes an immunosuppressive disease that is further characterized by the rapid onset of morbidity and high mortality in susceptible chickens. In 2009, vvIBDV was first reported in California, U. S. A., and since that time only a few cases of acute infectious bursal disease attributed to vvIBDV have been recognized in California. In other countries where vvIBDV has become established, it rapidly spreads to most poultry-producing regions. Two factors that may be involved in limiting the spread or reducing the severity of the clinical disease caused by vvIBDV in the U. S. A. are maternal immunity and competition with endemic variant strains of the virus. In this study, the ability of vvIBDV to infect and cause disease in maternally immune layer chickens was examined at weekly intervals over a 5-wk period during which their neutralizing maternal antibodies waned. Birds inoculated with vvIBDV at 2, 3, and 4 wk of age seemed healthy throughout the duration of the experiment, but macroscopic and microscopic lesions were observed in their bursa tissues. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay also confirmed the presence of vvIBDV RNA in their bursa tissues, indicating this virus was infecting the birds even at 2 wk of age when neutralizing maternal antibodies to infectious bursal disease virus were still relatively high (> 2000 geometric mean antibody titer). No mortality was observed in any birds when inoculated at 2, 3, or 4 wk of age; however, inoculation at 5 and 6 wk of age resulted in 10% and 20% mortality, respectively. Three experiments on the competition between vvIBDV and the two variant viruses T1 and FF6 were conducted. In all three experiments, specific-pathogen-free (SPF) birds that were inoculated with only the vvIBDV became acutely moribund, and except for Experiment 1 (62% mortality) all succumbed to the infection within 4 days of being exposed. When the variant viruses were inoculated into SPF chickens at 4 wk of age and the vvIBDV was administered 10 days later, a real-time reverse transcriptase (RT)-PCR assay was positive for the variant T1 and FF6 viral RNA but negative for vvIBDV RNA, indicating the vvIBDV isolate could not establish an infection in the bursa tissues of these birds. No clinical disease or mortality was observed in the birds, but macroscopic and microscopic lesions typical of a variant virus infection were observed. When the interval was shortened to 2 days, both variant viruses and vvIBDV were detected by RT-PCR, but the vvIBDV viral load in the bursa tissue was significantly lower compared with that of birds inoculated with vvIBDV alone. No clinical disease or mortality was observed, and macroscopic and microscopic lesions in the bursa of these birds were not typical of a vvIBDV infection but instead resembled a pathogenic classic virus infection. When the variant viruses and vvIBDV were inoculated simultaneously into 4-wk-old SPF chickens only the vvIBDV RNA was detected in the bursa tissues of the birds, and no reduction in viral load as measured by real-time RT-PCR was detected. Mortality (30%-40%) was reduced in these duel-infected birds compared with the 100% mortality observed in birds that received vvIBDV alone. The data suggest that maternal immunity and competition with variant viruses are acting to reduce the clinical signs and anticipated mortality after an vvIBDV infection, which may increase the chances that vvIBDV is not being recognized in commercial poultry operations.

Impaired intestinal mucosal immunity in specific-pathogen-free chickens after infection with very virulent infectious bursal disease virus

Diarrhea is one of the most important clinical side effects that occurs in chickens after infection with bursal disease virus. However, the kinetics of the intestinal mucosal immune parameters in infectious bursal disease virus (IBDV)-infected birds remains unclear. Forty-five specific-pathogen-free chickens were randomly divided into 2 groups [30 chickens in the very virulent IBDV (vvIBDV)-infected group and 15 chickens in the control group] to determine the effect of vvIBDV infection on intestinal mucosal immunity. All chickens were raised in negative-pressure isolators and fed the same diet. Results indicate that severe histologic lesions were observed in the intestinal tract in the vvIBDV-infected group, and the villus heights of the duodenum, jejunum, and ileum were decreased after vvIBDV infection (P < 0.01 or P < 0.05). The number of intestinal intraepithelial lymphocytes, mast cells, and goblet cells decreased significantly in the vvIBDV-infected group compared with the control (P < 0.01 or P < 0.05) on d 1, 2, and 3 postchallenge with vvIBDV. Very virulent IBDV infection reduced the expression of each fragment of intestinal secretory IgA at all 3 time points. Intestinal alkaline phosphatase decreased dramatically on d 2 and 3 postchallenge with vvIBDV. Taken together, these results indicate that vvIBDV infection contributes to considerable damage to the normal structure of the intestine and intestinal mucosal immune parameters of specific-pathogen-free chickens. Our research helps to increase an understanding of the pathogenesis of diarrhea induced by vvIBDV infection. These findings provide new insights into the pathogenesis and pathophysiology of vvIBDV infection.

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs ( p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased ( p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.

Mast cell mediated inflammatory response in chickens after infection with very virulent infectious bursal disease virus

The potential role of the mast cells in the invasion of very virulent infectious bursal disease virus (vvIBDV) is unknown. We evaluated mast cell activity and tryptase production after vvIBDV infection in special pathogen-free (SPF) chickens using cytochemistry and immunohistochemistry analyses. The results were as follows: (1) severe histologic lesions were observed in the thymus, spleen, cloacal bursa, liver, kidney and other tissues. vvIBDV viral antigens were detected and presented extensively in the parenchymatous organs, in particular, the cloacal bursa, liver, kidney, thymus, spleen and pancreas. (2) In the vvIBDV-infected group, the mast cell population increased markedly in the liver, kidney, thymus, glandular stomach, spleen and cloacal bursa on days 1, 2 and 3 after vvIBDV infection ( p < 0.05). However, very few mast cells were observed in those same tissues in the controls, especially in the bursa of Fabricius. (3) Tryptase, a marker for activated mast cells, has a positive correlation with mast cell distribution. The mast cells identified in the tissues were likely to be activated since they were associated with cell degranulation and the presence of tryptase. Furthermore, the co-localization of mast cells, and presence of vvIBDV antigens suggests that the mast cells were activated by vvIBDV infection. Our results also suggest that tryptase may contribute to the inflammation of acute IBD induced by vvIBDV infection. Our research contributes to the further understanding of inflammatory response mechanisms and the contribution of mast cell activity to this process.

DNA Immunization Against Very Virulent Infectious Bursal Disease Virus with VP2‐4‐3 Gene and Chicken IL‐6 Gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2-4-3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin-6 (ChIL-6) genes were constructed and designated as pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 respectively. Several DNA vaccination experiments were performed: 1-week-old chickens were intramuscularly injected with only plasmid pcDNA3-VP2, pALTER-MAX-VP2-4-3 or mixture with pALTER-MAX-ChIL-6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 three times conferred protection for 90% of chickens. Enzyme-linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER-MAX-ChIL-6 were higher than those immunized simply with plasmid pcDNA3-VP2 or pALTER-MAX-VP2-4-3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT-PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2-4-3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL-6 plasmid significantly increased the protection after challenge with the very virulent strain.

Protection against very virulent infectious bursal disease virus in chickens immunized with DNA vaccines

Plasmid DNA vaccines pcDNA-VP2 expressing only VP2 protein and pcDNA-VP243 expressing VP2, VP4 and VP3 proteins of very virulent infectious bursal disease virus (vvIBDV) Korean SH/92 strain were constructed. The expression of viral proteins from constructed DNA vaccines was confirmed by an in vitro transcription/translation system and transfection in COS-7 cells. To investigate the protective efficacy of these DNA vaccines, 2-week-old chickens were injected intramuscularly and intraperitoneally with pcDNA-VP2 and pcDNA-VP243 twice at 2-week intervals. On week 2 after the second immunization, chickens were orally challenged with the vvIBDV SH/92 strain and observed for 10 days. Antibodies specific to IBDV were not detected by enzyme-linked immunosorbent assay in DNA vaccination groups before challenge but were induced after challenge. The immunized groups exhibited a higher survival rate and lower bursal atrophy as compared with the non-immunized groups after challenge. The survival rates of pcDNA-VP243 and pcDNA-VP2 groups were 70 and 50%, respectively, but the survival rate of challenge control group was only 10%. In the ConA-induced lymphocyte proliferation assay of peripheral blood and splenic lymphocytes, the immunized groups showed significantly higher proliferation responses ( P<0.05) than non-immunized groups. The maintenance of T cell proliferation activity in DNA vaccination groups may be closely related to the protection against vvIBDV. These results suggest that our plasmid DNA vaccines induced high protective immunity against vvIBDV, in which cell mediated immune response rather than humoral immune response seemed to contribute to the protection of chickens against vvIBDV infection.