Differential Modulation of Innate Antiviral Profiles in the Intestinal Lamina Propria Cells of Chickens Infected with Infectious Bursal Disease Viruses of Different Virulence.

Infectious bursal disease virus (IBDV) is one of the most important infectious diseases of poultry around the world. Gut-associated lymphoid tissues (GALT) are the first line of defense of the host against the infection. The purpose of this study was to investigate the role of innate immune antiviral signaling triggered by Toll-like receptor 3 (TLR3), as well as macrophage activation and cytokine response in the intestinal lamina propria (ILP) cells after the oral challenge of IBDV in relation to IBDV virulence and disease pathogenesis. The results showed that the expression levels of TLR3, IRF7, IFN-α/ß and the corresponding downstream antiviral factors OAS, PKR and Mx were all upregulated in the SPF chicken ILP cells at 8 h post-infection (hpi) and 12 hpi. Similarly, macrophages were activated, with the initial macrophage M1 activation observed at 8 hpi, but then it rapidly shifted to a non-protective M2-type. Both Th1 (IFN-γ, TNF-α, IL-12) and Th2 (IL-4 and IL-10) types of cytokines were differentially upregulated during the early stage of infection; however, the Th1 cytokines exhibited stronger activation before 8 hpi compared to those of the Th2 cytokines. Interestingly, differential regulations of gene expression induced by different IBDV strains with different virulence were detected. The HLJ0504-like very virulent (vv) IBDV strain NN1172 induced stronger activation of TLR3-IFN-α/ß pathway, macrophages and the Th1/2 cytokines' expression, compared to those induced by the attenuated strain B87 at 8 hpi and 12 hpi in the ILP cells. In conclusion, the innate antiviral response mediated by the TLR3-IRF7 pathway, macrophage activation and cytokine expression in the GALT cells at the early stage of IBDV infection was differentially modulated, and the HLJ0504-like vvIBDV strain triggered stronger activation than the attenuated vaccine strain, and that may play an important role in the progression of disease.

Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia.

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green-based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Characterization of antigenic variant infectious bursal disease virus strains identified in South Korea.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases of young chickens, causing considerable economic losses to the poultry industry. More than 30 years ago, an antigenic variant (av) pathotype of the IBD virus (IBDV) was reported to originate in, and subsequently spread among, poultry farms in the USA. Recently, a novel avIBDV lineage was identified in China and was shown to exhibit clear differences in its pathogenicity as well as molecular characteristics compared with the previously isolated variant strains. In this study, we conducted a passive surveillance of chicken carcasses submitted to our research division from June-December 2019, and detected the IBDV strains by reverse transcription PCR. Five avIBDV strains were isolated, and their pathogenicity was determined by necropsy and molecular analysis. Additionally, a coinfection field case involving an avIBDV strain and a very virulent IBDV (vvIBDV) strain was identified. Multiple sequence alignment and phylogenetic analysis of partial viral protein 1 (VP1) and hypervariable region (hv) VP2 genes revealed that those strains originated from two different avIBDV lineages. The co-occurrence of two sub-groups of avIBDVs in South Korea confirms for the first time the evolution of antigenic variant IBDV strains, and highlights the urgency for the development of new strategies for IBDV intervention in South Korea.RESEARCH HIGHLIGHTS Five avIBDV strains were identified in South Korea by passive surveillance test in 2019.A coinfection between two IBDV strains from different genogroups was reported in a field case.By phylogenetic analysis, Korean avIBDVs belonged to two distinct lineages of antigenic variant genogroup.

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus has a negative economic impact on broiler farms in Japan.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, immunosuppressive and causes a negative economic impact on poultry industry. IBDV-vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio) reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in the first and second flocks, respectively, using immunohistochemical staining and RT-PCR. IBDV isolates from both flocks were closely related to the China KM523643 strain. Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV infection was appeared to be the main influencing factor on BF atrophy and BB ratio reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary infection. The disease caused by IBDV had a negative economic impact in the farm. RESEARCH HIGHLIGHTS New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers. After vIBDV had infected broilers, at 21 days old they became immunosuppressed. High mortality at 30 days old in broilers was due to secondary infection. New vIBDV has a negative economic impact on broiler farms in Japan.

T cell subset profile and inflammatory cytokine properties in the gut-associated lymphoid tissues of chickens during infectious bursal disease virus (IBDV) infection.

While infectious bursal disease virus (IBDV) mainly targets immature B cells and causes T cell infiltration in the bursa of Fabricius (BF) of chickens, the effect of IBDV infection on the properties of T cells and relevant cytokine production in avian gut-associated lymphoid tissues (GALTs) remains unknown. Here, we show that while the CD8+ T cell subset is not affected, IBDV infection decreases the percentage of CD4+ T cells in the cecal tonsil (CT), but not in esophagus tonsil, pylorus tonsil, and Meckel's diverticulum of GALTs, in contrast to BF and spleen, in which the proportion of CD4+ cells increases upon IBDV infection. Further, IBDV infection upregulates IFN-γ, IL-10, and the T cell checkpoint receptor LAG-3 mRNA expression in BF. In contrast, in CTs, IBDV infection significantly increases the production of IFN-ß and CTLA-4 mRNA, while no significant effect is seen in the case of IFN-γ, IL-10 and LAG-3. Together, our data reveal differential modulation of T cell subsets and proinflammatory cytokine production in different lymphoid tissues during the course of IBDV infection.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes.

Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family, causing severe immunosuppressive disease in chickens. The major capsid protein VP2 is responsible for the binding of IBDV to the host cell and its cellular tropism. In order to find proteins that potentially interact with IBDV VP2, a liquid chromatography-mass spectrometry (LC-MS) assay was conducted, and the host chicken CD74 protein was identified. Here, we investigate the role of chicken CD74 in IBDV attachment. Coimmunoprecipitation assays indicated that the extracellular domain of CD74 interacted with the VP2 proteins of multiple IBDV strains. Knockdown and overexpression experiments showed that CD74 promotes viral infectivity. Confocal assays showed that CD74 overexpression allows the attachment of IBDV and subvirus-like particles (SVPs) to the cell surface of nonpermissive cells, and quantitative PCR (qPCR) analysis further confirmed the attachment function of CD74. Anti-CD74 antibody, soluble CD74, depletion of CD74 by small interfering RNA (siRNA), and CD74 knockdown in the IBDV-susceptible DT40 cell line significantly inhibited IBDV binding, suggesting a pivotal role of this protein in virus attachment. These findings demonstrate that CD74 is a novel important receptor for IBDV attachment to the chicken B lymphocyte cell line DT40.IMPORTANCE CD74 plays a pivotal role in the correct folding and functional stability of major histocompatibility complex class II (MHC-II) molecules and in the presentation of antigenic peptides, acting as a regulatory factor in the antigen presentation process. In our study, we demonstrate a novel role of CD74 during IBDV infection, showing that chicken CD74 plays a significant role in IBDV binding to target B cells by interacting with the viral VP2 protein. This is the first report demonstrating that CD74 is involved as a novel attachment receptor in the IBDV life cycle in target B cells, thus contributing new insight into host-pathogen interactions.

New introduction of a very virulent infectious bursal disease virus in New York, USA.

Bursa tissue samples from a pullet flock in New York State that was experiencing immune suppression related disease were sent to our laboratory in 2018. A very virulent infectious bursal disease virus (vvIBDV) was identified in those samples through molecular and pathogenicity studies and designated 1/chicken/USA/1054NY/18. Phylogenetic analyses of the hypervariable VP2 nucleotide sequence region indicated that this strain belonged to genogroup 3 which comprises the vvIBDV. Partial sequence data of the VP1 gene indicated this virus also had a VP1 typical of vvIBDV. While vvIBDV have previously been identified in the United States in California and Washington State, the 1054NY vvIBDV was most closely related to isolates from Ethiopia, suggesting it is a new introduction into the U.S. The 1054NY vvIBDV was used to challenge four-week old specific-pathogen-free (SPF) layer chicks where it caused 100% morbidity and 68.7% mortality within 4 days. Upon necropsy, gross pathological findings in infected SPF birds included small yellowish coloured bursas, some with haemorrhages on the serosal and mucosal surfaces. Microscopic lesions included inflammation, severe lymphocyte necrosis, atrophy of the follicles and follicular depletion of lymphocytes. RESEARCH HIGHLIGHTS A very virulent infectious bursal disease virus (vvIBDV) was detected in a pullet flock in New York state, USA. Nucleotide sequence analysis of the vvIBDV VP2 gene indicates it is not related to previous US vvIBDV isolates and appears to be a new introduction into the US. The New York vvIBDV caused 100% morbidity and 68.7% mortality in four-week-old specific-pathogen-free chicks.

Infectious bursal disease virus tissue tropism and pathogenesis of the infection in chickens by application of in situ PCR, immunoperoxase and HE staining.

Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.

Effect of IBDV infection on the interfollicular epithelium of chicken bursa of Fabricius.

In the chicken bursa of Fabricius (BF), the interfollicular epithelium (IFE) consists of cylindrical- and cuboidal-shaped cells. Among the cylindrical-shaped epithelial cells, mucus-producing and caveolin-1 (Cav-1)-expressing cells can be distinguished. Occasionally, the cuboidal-shaped cells also express Cav-1, which suggests that they are precursors of both mucus-producing and Cav-1-expressing cells. Very virulent infectious bursal disease virus (IBDV) impedes the differentiation of Cav-1-expressing cells and shifts the differentiation of cuboidal cells towards mucus-producing cells. In control birds exclusively, the IFE surface shows a mucous membrane, but after IBDV infection, the surfaces of both IFE and FAE are also covered by a mucous membrane. After IBDV infection, the cells of FAE also produce mucus, providing evidence for cell transformation. In late postinfection (pi; 28 d pi), the Cav-1 expression returned in the IFE cells, whereas the follicle (the primary lymphoid organ) underwent atrophy. The appearance of the renewed Cav-1-positive cells is similar to that of the normal basal cell, but they randomly locate in different levels of IFE, suggesting the loss of epithelial polarity. Between days 2 and 7 pi, the Cav-1 expression in the endothelial cells of the cortico-medullary capillary web is variable, which may explain the hemorrhage in several infected birds. The IBDV infection stops the Cav-1 expression and subsequently the cholesterol efflux into the bursal lumen. In the infected birds, the high cholesterol level may further worsen the clinical syndrome of IBDV.

Bursal immunopathology responses of specific-pathogen-free chickens and red jungle fowl infected with very virulent infectious bursal disease virus.

Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection.

Exacerbated Apoptosis of Cells Infected with Infectious Bursal Disease Virus upon Exposure to Interferon Alpha.

Infectious bursal disease virus (IBDV) belongs to the Birnaviridae family and is the etiological agent of a highly contagious and immunosuppressive disease (IBD) that affects domestic chickens (Gallus gallus). IBD or Gumboro disease leads to high rates of morbidity and mortality of infected animals and is responsible for major economic losses to the poultry industry worldwide. IBD is characterized by a massive loss of IgM-bearing B lymphocytes and the destruction of the bursa of Fabricius. The molecular bases of IBDV pathogenicity are still poorly understood; nonetheless, an exacerbated cytokine immune response and B cell depletion due to apoptosis are considered main factors that contribute to the severity of the disease. Here we have studied the role of type I interferon (IFN) in IBDV infection. While IFN pretreatment confers protection against subsequent IBDV infection, the addition of IFN to infected cell cultures early after infection drives massive apoptotic cell death. Downregulation of double-stranded RNA (dsRNA)-dependent protein kinase (PKR), tumor necrosis factor alpha (TNF-α), or nuclear factor κB (NF-κB) expression drastically reduces the extent of apoptosis, indicating that they are critical proteins in the apoptotic response induced by IBDV upon treatment with IFN-α. Our results indicate that IBDV genomic dsRNA is a major viral factor that contributes to the triggering of apoptosis. These findings provide novel insights into the potential mechanisms of IBDV-induced immunosuppression and pathogenesis in chickens.IMPORTANCE IBDV infection represents an important threat to the poultry industry worldwide. IBDV-infected chickens develop severe immunosuppression, which renders them highly susceptible to secondary infections and unresponsive to vaccination against other pathogens. The early dysregulation of the innate immune response led by IBDV infection and the exacerbated apoptosis of B cells have been proposed as the main factors that contribute to virus-induced immunopathogenesis. Our work contributes for the first time to elucidating a potential mechanism driving the apoptotic death of IBDV-infected cells upon exposure to type I IFN. We provide solid evidence about the critical importance of PKR, TNF-α, and NF-κB in this phenomenon. The described mechanism could facilitate the early clearance of infected cells, thereby aiding in the amelioration of IBDV-induced pathogenesis, but it could also contribute to B cell depletion and immunosuppression. The balance between these two opposing effects might be dramatically affected by the genetic backgrounds of both the host and the infecting virus strain.

Infectious bursal disease virus infection leads to changes in the gut associated-lymphoid tissue and the microbiota composition.

Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease. IBD virus (IBDV) is the causative agent, which may lead to high morbidity and mortality rates in susceptible birds. IBDV-pathogenesis studies have focused mainly on primary lymphoid organs. It is not known if IBDV infection may modify the development of the gut associated lymphoid tissues (GALT) as well as the microbiota composition. The aim of the present study was to investigate the effects of IBDV-infection on the bursa of Fabricius (BF), caecal tonsils (CT) and caecum, and to determine the effects on the gut microbiota composition in the caecum. Commercial broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 (Experiment 2) or 15 (Experiment 1) days post hatch (dph). Virus replication, lesion development, immune parameters including numbers of T and B lymphocytes, macrophages, as well as the gut microbiota composition were compared between groups. Rapid IBDV-replication was detected in the BF, CT and caecum. It was accompanied by histological lesions including an infiltration of heterophils. In addition a significant reduction in the total mucosal thickness of the caecum was observed in vvIBDV-infected birds compared to virus-free controls (P < 0.05). vvIBDV infection also led to an increase in T lymphocyte numbers and macrophages, as well as a decrease in the number of B lymphocytes in the lamina propria of the caecum, and in the caecal tonsils. Illumina sequencing analysis indicated that vvIBDV infection also induced changes in the abundance of Clostridium XIVa and Faecalibacterium over time. Overall, our results suggested that vvIBDV infection had a significant impact on the GALT and led to a modulation of gut microbiota composition, which may lead to a higher susceptibility of affected birds for pathogens invading through the gut.

Molecular characterization of very virulent infectious bursal disease virus strains circulating in Egypt from 2003 to 2014.

In the present study, four very virulent infectious bursal disease virus (vvIBDV) isolates from flocks of chickens with vaccination failure in Egypt in 2003, 2007, 2010 and 2014 were characterized. The four viruses, designated USC2003, USC2007, USC2010 and USC2014, were detected by reverse transcription PCR, subjected to sequencing of both genomic segments (A and B) and compared with geographically and phylogenetically diverse IBDV strains. Phylogenetic analysis of segment A (complete) and B (partial) revealed a close relationship between Egyptian and vvIBDV reference strains of European and Asian origin. The sequences of segments of A and B the current Egyptian isolates were 96.1-98.2% and 96.5-98.7% identical, respectively, to those of other known vvIBDV isolates. The deduced amino acid sequences of VP1, polyprotein (pVP2-VP4-VP3) and VP5 revealed the presence of putative virulence determinants of Egyptian isolates compared with vvIBDV and less virulent (classical and variant) strains. The Egyptian isolates also possess unique amino acids substitutions within the hypervariable region of VP2 that differ from those of other reference IBDV strains. Further studies may be necessary to determine the pathogenic significance of these amino acid substitutions to fully understand the molecular epidemiology and evolution of IBDV.

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine.

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×103 EID50) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8+ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

A novel "in-feed" delivery platform applied for oral DNA vaccination against IPNV enables high protection in Atlantic salmon (Salmon salar).

BACKGROUND: DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. METHODS: We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. RESULTS: The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mgpDNA/kgfish⋅d), and 66% for double dose (2mgpDNA/kgfish⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). CONCLUSION: The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide.

Circulating strains of variant infectious bursal disease virus may pose a challenge for antibiotic-free chicken farming in Canada.

Antibiotic-free and safe animal products are most desirable among consumers. However, ensuring safe poultry products is a challenging task when the chicken immune system is compromised. Infectious bursal disease virus (IBDV) causes immunosuppression and predisposes chickens to secondary infections. Breeder vaccination against IBDV is routinely practiced for producing chicks with maternally-derived antibody (MAb) to prevent infection in newly hatched chicks. The majority of IBDV circulating in Canadian farms are variant strains (vIBDV). Whether circulating vIBDV strains are immunosuppressive in chicks or are amenable to current vaccine regimens has not previously been tested through challenge studies. In this study, one-day-old broiler chicks (n=240) carrying MAb were obtained from broiler breeders vaccinated with commercial IBDV vaccines. In the first set of experiments (n=40/group), at six days post-hatch, one group was challenged with a Canadian field isolate, vIBDV (strain-SK09) (3×10(3) EID50). The second and the third groups (controls) were inoculated with non-immunosuppressive IBDV D-78 (10×10(3) TCID50) and saline, respectively. Histopathological examination on days 14 and 30 post-challenge revealed that despite the high level of MAb, vIBDV (SK09) caused severe bursal damage in chicks. Another set of experiments with treatment groups as above, demonstrated that pre-exposure of chicks with vIBDV (SK09) caused immunosuppression resulting in significantly higher mortality and disease severity in chicks challenged with a virulent strain of Escherichia coli (E. coli). Our data provide evidence that IBDV strains circulating in Canada are immunosuppressive, not amenable to current anti-IBDV vaccination strategy, and a potential threat to antibiotic-free chicken farming.

Efficient self-assembly and protective efficacy of infectious bursal disease virus-like particles by a recombinant baculovirus co-expressing precursor polyprotein and VP4.

BACKGROUND: Virus-like particle (VLP) technology is considered one of the most promising approaches in animal vaccines, due to the intrinsic immunogenic properties as well as high safety profile of VLPs. In this study, we developed a VLP vaccine against infectious bursal disease virus (IBDV), which causes morbidity and mortality in chickens, by expressing a baculovirus in insect cells. METHODS: To improve the self-proteolytic processing of precursor polyprotein (PP), we constructed a recombinant baculovirus transfer vector that co-expresses PP and the VP4 protease gene of IBDV. RESULTS: Expression and VLP assembly of recombinant proteins and antigenicity of the VLP were examined by Western blotting, ELISA, and transmission electron microscopy. In animal experiments, vaccination with the recombinant VLP induced strong and uniform humoral immunity and provided complete protection against challenge with very virulent (vv) IBDV in SPF chickens (n = 12). As determined by the bursa of Fabricius (BF)/body weight (B/BW) ratio, the protection against post-challenge bursal atrophy was significantly higher (P < 0.001) in VLP-vaccinated birds than in non-vaccinated controls. CONCLUSIONS: Since the protective efficacy of the VLP vaccine was comparable to that of a commercially available inactivated vaccine, the recombinant VLP merits further investigation as an alternative means of protection against vvIBD.

Runting Stunting Syndrome Associated with Transmissible Viral Proventriculitis in Broiler Chickens.

This report describes an outbreak of transmissible viral proventriculitis (TVP) associated with runting stunting syndrome (RSS) in 25- and 28-day-old broiler chickens, in which chicken proventricular necrosis virus (CNPV) was detected. Clinical signs included poor uniformity, very small birds for their age, increased mortality, and culling of smaller birds. Almost all birds necropsied exhibited moderate to severely enlarged proventriculi with diffusely pale serosa and thickened walls. Microscopically the proventriculi had lesions of degeneration and necrosis of the epithelium of the proventricular glands, accompanied by lymphocytic inflammation and glandular hyperplasia, with occasional formation of lymphoid nodules within the glandular parenchyma. Immunohistochemistry staining for CPNV was positive. Positive staining was generally found in the cytoplasm of glandular epithelial cells in the form of finely granular brown pigment. CPNV RNA was detected in the proventriculi by reverse transcriptase-PCR (RT-PCR). Other findings included mild enteritis in a few birds and small bursa of Fabricius. Direct electron microscopy performed on the intestinal samples was negative for viral particles. RT-PCR analysis of bursae was positive for infectious bursal disease virus (IBDV). In conclusion, this report associates TVP with RSS by describing an outbreak in which TVP attributable to CPNV was the most commonly found lesionin chickens with a clinical history compatible with RSS. Therefore, TVP should be considered as a possible differential diagnosis in cases compatible with RSS.