Comparative Pathological Changes Induced by Very Virulent Infectious Bursal Disease Virus Infection in Inoculated, Sentinel Pigeons and Chickens

The pathogenesis of highly virulent infectious bursal disease (IBD) virus (IBDV) infection was studied using 6-week-old intact and 5-week-old bursectomized chickens inoculated with highly virulent strain 90-11 or reference strain I. Chickens inoculated with 10(0.7) EID50 of strain 90-11 showed neither clinical signs nor lesions during the 4-day observation period. In contrast, birds inoculated with 10(2.7) or 10(4.7) EID50 developed severe clinical IBD, as well as gross and histologic lesions, typical of IBD, and produced IBDV antigen demonstrable by immunostaining in the bursa of Fabricius (BF), thymus, spleen and bone marrow from day 2 post-inoculation (PI) onwards. The antigen was also detected by the agar-gel precipitation and latex microsphere agglutination tests in a bursal suspension of these birds from day 2 or day 3 PI on. Birds inoculated with 10(6.1) EID50 of strain I developed only slight clinical signs at day 4 PI. Their lesion- and antigen-scores in the BF were almost the same as those in virulent strain-infected chickens, but lesion- and antigen-scores in the other organs were negligible. Bursectomized chickens inoculated with strain 90-11 did not develop clinical IBD despite the presence of infection that was evidenced by histologic lesions in the thymus and spleen as well as IBDV antigen demonstrable by immunostaining in these organs.

In this study, changes in cloacal temperature and clinical manifestations due to very virulent infectious bursal disease virus (vvIBDV) infection in pigeons (Columba livia domestica) and transmission to chickens were demonstrated. Thirty pigeons (3-6weeks old) and thirty chickens (3weeks old) divided into 4 groups (I-IV) were used for this study. Group I comprised of 10 uninoculated pigeons only; II comprised of 10 inoculated pigeons and 10 sentinel chickens; III comprised of 10 sentinel pigeons and 10 inoculated chickens, while IV comprised of 10 uninoculated chickens only. Pigeons in group II and chickens in group III were each inoculated with 0.20mL (titre of 109.76CID50/mL) of vvIBDV (Nigerian strain). Cloacal temperature was monitored and clinical manifestations scored post-inoculation (pi). Results indicated significant (P < 0.05) pyrexia at 2days pi (dpi), mild clinical signs and no mortality in inoculated pigeons. Significant (P < 0.05) pyrexia at 2-4 dpi, severe clinical signs and mortality (50%; 60%) were observed in inoculated and sentinel chickens. IBDV antigen and antibody were detected in pigeons and chickens. Pigeons showed response to vvIBDV infection thus suggesting susceptibility of pigeons to IBD. Sentinel chickens presented clinical manifestations of IBD and this suggests transmission from pigeons to chickens. This study therefore documents pyrexia and clinical manifestations due to vvIBDV infection in pigeons and successful transmission of the virus between pigeons and chickens.

This study examined the sequential pathological changes in the lymphoid organs (bursa of Fabricius, thymus, spleen and caecal tonsils) of 7-week-old Harco pullet chicks that showed severe clinical disease and lesions during a natural infection with a virulent infectious bursal disease virus. Clinical signs were sleepiness, droopy appearance, greenish-whitish diarrhoea, anorexia and prostration followed by death. Mortality rate was 78% within 3 days of the infection followed by recovery. Gross lesions were marked haemorrhages in the pectoral and thigh muscles, mucosa of the proventriculus and gizzard junction, and caecal tonsils. Bursa of Fabricius, thymus, spleen and kidneys were initially enlarged; however, bursa of Fabricius and thymus were later atrophic. Histologic lesions showed marked oedema, infiltration of heterophils, hyperaemia, and lymphoid depletion and hyperplastic corticomedullary layer in the bursa of Fabricius, lymphoid necrosis in thymus, spleen, and caecal tonsils. Lymphocytic depletion was marked in the bursa of Fabricius as early as day 1 of the infection, and in the spleen, thymus and caecal tonsils on day 2 of the infection. However, there were fibroplasias in the bursa of Fabricius and thymus but repopulation of lymphocytes in the spleen and caecal tonsils of birds sacrificed on day 6 of the infection. Confirmation of IBD was carried out using agar gel immunodiffusion test. The above observations showed that marked depletion of lymphocytes in the lymphoid organs correlated with marked clinical IBD while repopulation of lymphocytes in the spleen and caecal tonsils correlated with the recovery phase in pullet chicks. The description of the pathological changes in lymphoid organs caused by the IBDV currently circulating in Nigeria will be useful in assessing the time and recognition of early diagnostic features of the disease.Keywords: Infectious bursal disease, Lymphoid organs, Pathology, Pullets

Susceptibility of chicken Kupffer cells to Chinese virulent infectious bursal disease virus

The hematological and serum biochemical alterations following very virulent infectious bursal disease virus (IBDV) infection in pigeons and chickens were evaluated in this study. Sixty IBDV seronegative birds comprising 30 (3–6 weeks old) pigeons and 30 (3 weeks old) chickens were randomly divided as follows: 10 uninoculated pigeons only, 10 inoculated pigeons + 10 sentinel chickens, 10 inoculated chickens + 10 sentinel pigeons, and 10 uninoculated chickens. Inoculated birds were administered 0.20 ml of inoculum containing very virulent IBDV (vvIBDV). Blood was collected postinoculation/exposure (pi/pe) and processed for hematology and biochemistry. The results revealed significantly (P < 0.05) increased packed cell volume, decreased mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC) in inoculated and sentinel chickens. Total leukocyte count (TLC), heterophil, and heterophil/lymphocyte (H/L) ratio were increased from 3 dpi/dpe in inoculated pigeons and from 3 to 14 dpi/dpe in inoculated and sentinel chickens. At 10 and 14 dpi/dpe, there was significantly (P < 0.05) increased serum total protein and globulin concentrations and decreased albumin/globulin ratio in pigeons and chickens. Serum urea concentration showed significant (P < 0.05) increase in inoculated and sentinel chickens at 3 and 4 dpi/dpe. To the authors' knowledge, this is the first report on hematological and serum biochemical alterations due to vvIBDV infection in pigeons.

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

Infectious bursal disease virus (IBDV) infection causes highly contagious and immunosuppressive disease in poultry. The thymus, serving as the primary organ for T cell maturation and differentiation, plays an important role in the pathogenicity of IBDV in the infected chickens. However, there are no reports on the molecular pathogenesis of IBDV in the thymus currently. The aim of the study was to elucidate the molecular mechanisms underlying the pathogenicity of a field very virulent (vv) IBDV strain NN1172 in the thymus of SPF chickens using integrative transcriptomic and proteomic analyses. Our results showed that a total of 4,972 Differentially expressed genes (DEGs) in the thymus of NN1172-infected chickens by transcriptomic analysis, with 2,796 up-regulated and 2,176 down-regulated. Meanwhile, the proteomic analysis identified 726 differentially expressed proteins (DEPs) in the infected thymus, with 289 up-regulated and 437 down-regulated. Overall, a total of 359 genes exhibited differentially expression at both mRNA and protein levels, with 134 consistently up-regulated and 198 genes consistently down-regulated, as confirmed through a comparison of the RNA-seq and the proteomic datasets. The gene ontology (GO) analysis unveiled the involvement of both DEGs and DEPs in diverse categories encompassing cellular components, biological processes, and molecular functions in the pathological changes in IBDV-infected thymus. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the host mainly displayed severely disruption of cell survival/repair, proliferation and metabolism pathway, meanwhile, the infection triggers antiviral immune activation with a potential emphasis on the MDA5 pathway. Network inference analysis identified seven core hub genes, which include CDK1, TYMS, MCM5, KIF11, CCNB2, MAD2L1, and MCM4. These genes are all associated with cell-cycle regulating pathway and are likely key mediators in the pathogenesis induced by NN1172 infection in the thymus. This study discovered dominant pathways and genes which enhanced our understanding of the molecular mechanisms underlying IBDV pathogenesis in the thymus.

Two experiments were carried out to examine the effects of feeding an uncontaminated control diet (CON) or a Fusarium toxin-contaminated diet (FUS; 10.7 mg deoxynivalenol [DON]/kg diet) to growing broilers, which were either uninfected or infected with infectious bursal disease virus (IBDV) beginning at 1 day post hatch. Broilers had been infected at three weeks post hatch with either a classical virulent infectious bursal disease virus (IBDV-IM, Exp. 1) or a very virulent IBDV (vvIBDV, Exp. 2) strain. The effects of the DON-contaminated diet in combination with the virus-infection on the bursa of Fabricius and spleen were determined at 3 and 6–7 days post infection. The transient development of the bursa oedema and the bursa atrophy was not significantly affected by the diet after infection with the different IBDV-strains. The histopathological lesions were more severe in IBDV-IM-infected birds at 6 days post infection when additionally exposed to the FUS diet as compared to the FUS-free feed. Most parameters of the bursa of Fabricius and spleen protein turnover (e.g. fractional protein synthesis rate, protein, DNA and RNA content and derived indices) were significantly and interactively influenced by infection and stage of infection. The vvIBDV-infected birds responded with a more pronounced depressing effect on the fractional protein synthesis rate after feeding the DON-containing FUS diet when compared to their IBDV-IM-infected counterparts, where the opposite effect was observed. It can be concluded that feeding a FUS diet to IBDV-infected broilers might modulate the virulence-dependent pathogenesis of an IBDV infection.

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.

Protective effects of rabbit sacculus-derived antimicrobial peptides on SPF chicken against infection with very virulent infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. Although the PDE and PFG domains of the capsid protein VP2 contribute significantly to virulence and fitness, the detailed molecular basis for the pathogenicity of IBDV is still not fully understood. Because residues 253 and 284 of VP2 are not the sole determinants of virulence, we hypothesized that other residues involved in virulence and fitness might exist in the PDE and PFG domains of VP2. To test this, five amino acid changes selected by sequence comparison of the PDE and PFG domains of VP2 were introduced individually using a reverse genetics system into the virulent strain (rGx-F9VP2). Then reverse mutations of the selected residues 249 and 256 were introduced individually into the attenuated strain (rGt). Seven modified viruses were generated and evaluated in vitro (CEF cells) and in vivo (SPF chicken). For residue 249, Q249R could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while R249Q could reduce in vitro and elevate in vivo the replication of rGt; meanwhile Q249R reduced the virulence of rGx-F9VP2 while R249Q increased the virulence of rGt, which indicated that residue 249 significantly contributed to the replication and virulence of IBDV. For residue 256, I256V could elevate in vitro and reduce in vivo the replication of rGx-F9VP2 while V256I could reduce in vitro but didn’t change in vivo the replication of rGt; although V256I didn’t increase the virulence of rGt, I256V obviously reduced the virulence of virulent IBDV. The present results demonstrate for the first time, to different extent, residues 249 and 256 of VP2 are involved in the replication efficiency and virulence of IBDV; this is not only beneficial to further understanding of pathogenic mechanism but also to the design of newly tailored vaccines against IBDV.

Since 1990, highly virulent infectious bursal disease virus (IBDV), which induces high mortality, has been infecting even vaccinated flocks in Japan. We report the efficacy of three live vaccines that are available in Japan. Two mildly attenuated strains (A and B) and one intermediate strain (C) were each tested both in specific-pathogen-free (SPF) chickens and in commercial chickens that have maternal antibodies against IBDV. Chickens were vaccinated at 20 days old and challenged with highly virulent IBDV 10 days post-vaccination. Protection was determined 7 days after challenge by measuring bursa/body weight ratios, histopathological lesions, and antibody responses to IBDV. All three lie vaccines conferred protection to SPF chickens. However, only vaccine C protected 100% of vaccinated commercial chickens against highly virulent IBDV; Vaccines A and B respectively protected three-fourths and none of vaccinated commercial chickens from severe bursal lesions. Vaccines A, B, and C and highly virulent IBDV induced bursal lesions in 3%, 0%, 23%, and 61% of inoculated commercial chickens, respectively. These results suggest that serological determination of the optimum vaccination time for each flock is required to effectively control highly virulent IBDV in the field. The optimum vaccination timing could be approximated by titrating the maternal IBDV antibodies of 1-day-old chicks by an enzyme-linked immunosorbent assay or by an agar gel precipitin test.

Infectious bursal disease (IBD) or known as Gumboro, is a disease that attacks chicken older than 3 weeks, caused by famili Birnaviridae virus. Gumboro in Indonesia was firstly reported in 1983 and until now is commonly found. Very virulent IBD virus causes high morbidity and mortality that can even reach 100%. Clinical symptoms are exhibited as sluggish chicken, dropped wings and cloacal pasting. At gross examination, the bursa was found swollen, with yellowish fluid or hemorrhagic 3 days after infection. The bursa will get atrophy from 7 days post-infection. Meanwhile, the non virulent IBD virus causes subclinical symptoms. Chicken that survived, became stunted or dwarfed. On gross and histopathological findings, the bursa Fabricius has mild lesion and will recover at 14 days post-infection. Diagnosis of IBD can be determined based on pathological observation, supported by immunohistochemical examination and laboratory confirmation of disease by agar gel immunodiffusion, polymerase chain reaction techniques, antigen capture enzyme linked immunosorbent assay and isolation. Detection of antibodies can be made by serum neutralization technique or enzyme linked immunosorbent assay. Prevention can be done by routine vaccination in the field when the maternal antibodies have declined. The review describes the etiology, epidemiology, pathogenesis clinical symptoms, pathological discription and control of the disease to improve the knowledge of poultry farmer or people who are interested in poultry health. Key words: Gumboro, etiology, epidemiology, pathology, diagnosis

Studies with specific-pathogen-free chickens revealed that chicks hatching from eggs inoculated at the 18th day of embryonation with infectious bursal disease (IBD) vaccine viruses of low virulence (isolates TC-IBDV and BVM-IBDV) developed antibody against IBD virus (IBDV) and resisted challenge with virulent IBDV at 3 weeks of age or older. Embryo vaccination did not adversely affect hatchability of chicks or survival of hatched chicks. Chicks embryonally vaccinated with TC-IBDV had transient histologic lesions in the bursa of Fabricius at hatch. Similar but milder lesions were also noted in chickens that received TC-IBDV at hatch. The level of protection following embryo vaccination with TC-IBDV and BVM-IBDV was similar to that following vaccination with the same vaccines at hatch. Vaccine viruses of moderate virulence (isolates BV-IBDV and 2512-IBDV) were not suitable as vaccines in embryos lacking maternal antibody to IBDV, because the vaccinated chicks developed acute IBD after hatch. Isolate 2512-IBDV was not pathogenic for embryos bearing maternal antibody to IBDV. Maternal antibody against IBDV interfered with efficacy of embryo vaccination with BVM-IBDV but not with 2512-IBDV. Embryo vaccination with a mixture of vaccines against IBD and Marek's disease resulted in protection of hatched chicks against challenge with virulent IBDV and Marek's disease virus.

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