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PubMed2004

Comparative analysis of viral RNA and apoptotic cells in bursae following infection with infectious bursal disease virus.

Specific-pathogen-free (SPF) chickens infected with very virulent (vv) infectious bursal disease virus (IBDV) UPM94/273 developed lower pathogenicity compared to UPM97/61. Sequence analysis indicated that UPM94/273 is an exceptional vvIBDV. In this study, a SYBR Green I based real-time reverse transcriptase reaction assay was developed to measure viral RNA in the bursae of SPF chickens infected with IBDV. Specificity of the amplified products was confirmed by melting temperature analysis. A linear relationship was observed between the amount of input viral RNA and the threshold values for IBDV-specific product over five log10 dilutions. The viral RNA level following infection with UPM94/273 was significantly higher at day 1 and 2 post-inoculation (p.i.) compared to UPM97/61 infected chickens. However, chickens infected with UPM97/61 had significantly higher numbers of bursal cells undergoing apoptosis compared to UPM94/273 infected chickens. In both groups, the number of apoptotic cells and viral RNA levels peak at day 3 p.i. This study indicates that UPM97/61 and UPM94/273 have different efficiency of replication and percentage of apoptotic cells in bursae during the acute phase of IBDV infection.

Animals↗

PubMed2006

Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus.

The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.

Animals↗

PubMed2006

L-Arginine stimulates intestinal intraepithelial lymphocyte functions and immune response in chickens orally immunized with live intermediate plus strain of infectious bursal disease vaccine.

Infectious bursal disease (IBD) continues to pose potential threat to poultry industry all over the world. The disease can spell disaster not only through its infection but also by break of immunity in chickens vaccinated for other diseases. l-Arginine, a ubiquitous, semi-essential amino acid has emerged as an imunostimulant from variety of human and animal studies. In the present study, we demonstrate the stimulatory effects of l-arginine on intestinal intraepithelial lymphocyte (iIELs) functions as well as on systemic immune response in chickens orally vaccinated with live intermediate plus (IP) strain of IBD vaccine. Challenge studies with virulent IBDV revealed complete (100%) protection in IP+l-arginine group compared with 80% protection recorded in IP strain vaccinated chickens. Functional activities of iIELs evaluated by cytotoxicity assay demonstrated significantly high percentage cytotoxicity in IP+l-arginine groups compared with IP group (P<0.05). Proliferative response of iIELs against IBDV antigen and Con-A was also significantly higher in IP+l-arginine group. Similar results were obtained with peripheral blood mononuclear cell blastogenic response to IBDV and Con-A analyzed as an indicator of systemic cell-mediated immune response. Orally administered IP strain vaccine elicited good antibody titres in both the groups, IP and IP+l-arginine, however, the antibody titres were significantly higher in IP+l-arginine group compared with IP vaccinated group (P<0.05). These results clearly demonstrate that l-arginine stimulates intestinal and systemic immune response against IBDV.

Administration, Oral↗

PubMed2005

Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection.

Animals↗

PubMed2005

Genetic variations in maternal transfer and immune responsiveness to infectious bursal disease virus.

The immune responsiveness to infectious bursal disease virus (IBDV) in four native and crossbred chicken lines was compared. ELISA IBDV antibody titers in hen serum samples, yolk from matched eggs and sera from matched 1-day-old chicks from each chicken line with an identical vaccination program were measured, and plotted. There was considerable variation between lines in the measured IBDV specific antibodies, in vaccinated parent hens and in the amounts of inherited maternally derived antibodies in both yolk and progeny chicks. Differences in ratios of the inherited antibody level from hen to 1-day-old chicks were also found among different chicken lines. Breed differences in regressions of IBDV antibody levels in yolk to that of hen or progeny chicks' sera were also found, so prediction of serum titer of hen and/or progeny chicks from yolk are varied among chicken lines.

Animals↗

PubMed2004

VP1 protein of infectious bursal disease virus modulates the virulence in vivo.

Infectious bursal disease viruses (IBDVs), belonging to the Birnaviridae family, cause severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius (BF). Different pathotypes of IBDVs, including cell culture-adapted viruses, differ markedly in virulence, which is characterized by mortality and bursal damage. To study the molecular determinants of virulence in IBDV, the genomic segments A and B of GLS bursa-derived (GLSBD) and tissue culture-adapted (GLSTC) viruses were cloned and sequenced. Comparison of the deduced amino acid sequences of segments A and B revealed only two amino acid substitutions at positions 87 (Q --> R) and 261 (P --> L) in segment B, and at positions 253 (Q --> H) and 284 (A --> T) in segment A; the latter of which has been shown to be involved in tissue culture adaptation and attenuation of the virus. To study the function of VP1 protein encoded by segment B, reassortant viruses between tissue culture-adapted strains, GLSTC and D78, and GLSBD were recovered using the reverse genetics system. The recombinant virus rGLSBDB containing segment B of GLSBD was able to replicate in Vero and chicken embryo fibroblast (CEF) cells but exhibited delayed replication kinetics. To evaluate the characteristics of these viruses in vivo, 3-week-old chickens were given equal doses of parental viruses or reassortant viruses by ocular inoculation. The pathological lesions and viral antigen distribution in BF were analyzed at 1, 2, or 3 days postinfection. Parental GLSBD and the recovered rGLSBDB viruses propagate most efficiently in the BF and cause severe bursal lesions, whereas the tissue culture-adapted GLSTC virus replicates less efficiently and induces mild bursal lesions at 3 days postinfection. Taken together, our results demonstrate that the VP1 protein of IBDV is involved in the efficiency of viral replication and modulates the virulence in vivo.

Animals↗

PubMed1995

Apoptosis in chicken embryos induced by the infectious bursal disease virus.

Fifteen-day-old fertile eggs (specific pathogen-free) were inoculated with the infectious bursal disease virus (IBDV) by the allantoic route and were opened and examined 2, 4 or 6 days later. The bursas of Fabricius (BFs) were collected and processed for DNA extraction, flow cytometry, and light and electron microscopy. Cellular DNA was subjected to electrophoresis on 1.5% agarose gel and stained with ethidium bromide. Intense internucleosomal DNA fragmentation was detected in IBDV-infected bursas. Cytograms from cell suspensions derived from infected BFs displayed an increased population of cells with either high density and small size (apoptotic cells) or small size and high uptake of ethidium bromide (necrotic cells). Light and electron microscopical examination of the IBDV-infected BFs revealed death of lymphoid cells without surrounding inflammatory reaction, but with condensation of nuclear chromatin, crescent formation, and nuclear and cellular fragmentation. These data indicated that infection of chicken embryos with IBDV induced apoptosis in bursal lymphoid cells.

Animals↗

PubMed2000

Effect of ascorbic acid supplementation on the immune response of chickens vaccinated and challenged with infectious bursal disease virus.

One-day-old chickens were divided into two groups and reared under similar conditions. One group was fed a diet supplemented with 1000ppm ascorbic acid and the other group was fed an identical diet, but not supplemented with ascorbic acid. Both groups were vaccinated against infectious bursal disease (IBD) at 7 days of age and challenged orally with 4x10(5) of 50% embryo-lethal-dose IBDV 14 days later. The number of anti-IBDV antibody secreting cells, production of interleukin-2 (IL-2) by splenocytes, number of CD4(+), CD8(+) and IgM(+) cells in spleen and IgM(+) cells in bursa of Fabricius were compared between the two groups at 7 days (prior to vaccination), 21 days (14 days post-vaccination and prior to challenge) and 31 days (10 days post-challenge) of age. The number of CD8(+) in spleen at 7 days of age and IgM(+) cells in bursa at 7, 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Production of IL-2 by splenocytes was higher as indicated by higher stimulation indices in ascorbic acid supplemented group. The number of anti-IBDV IgG antibody secreting cells in spleen at 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Dietary supplementation of ascorbic acid may ameliorate the immunosuppression caused by IBDV vaccination and improve humoral and cellular immune responses.

Animals↗

PubMed1998

Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens.

We examined the effects of infectious bursal disease virus (IBDV) on splenic T cells and macrophages. In acute IBDV infection, splenocytes responded poorly to Con A stimulation. However, when T cells were isolated from whole spleen cells, purified T cells responded normally to Con A. This result indicated that functional T cells were present in the spleen but mitogen-induced proliferation of T cells was being suppressed by other cells. Previous studies indicated that soluble factors from suppressor cells may mediate this inhibition of T cell mitogenesis. We thus examined the effects of IBDV on spleen adherent cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantitate the expression of several cytokine genes in splenic macrophages. In acute IBDV infection, splenic macrophages exhibited enhanced gene expression of type I interferon (IFN), chicken myelomonocytic growth factor (cMGF), an avian homolog of mammalian IL-6, and 9E3/CEF4, an avian homolog of mammalian IL-8. Mitogen-stimulated spleen cell cultures also produced elevated levels of nitric oxide. The elevation of cytokine gene expression by macrophages occurred transiently during the acute phase of viral infection and coincided with in vitro inhibition of T cell mitogenic response of spleen cells.

Acute Disease↗

PubMed2001

Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus.

Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA.

Animals↗

PubMed1999

Susceptibility of local Nigerian and exotic chickens to infectious bursal disease by contact exposure.

One hundred 6-week-old susceptible cockerels were inoculated with a pathogenic strain of infectious bursal disease virus (IBDV) and kept in the same pen as 100 each of 6-week-old pullets, local chickens and broilers. The cockerels developed depression and diarrhoea on day 3 post inoculation (PI) and most of the pullets and some of the local chickens and broilers showed similar signs on day 4 PI. Loss in weight was severe and similar in the pullets and local chickens, being significantly greater than that in the broilers from days 3-11 PI. The total mortality was 85%, 66.7%, 30% and 20% for the pullets, cockerels, local chickens and broilers, respectively. The lesions were more severe in the pullets and local chickens than in the broilers. IBDV antigen and antibody were detected, respectively, in all the bursal and serum samples from the infected chickens tested. The contact exposure method used in this study simulates better what happens in nature than inoculation with IBDV. The reduced mortality observed among the local chickens, compared with that (61.5%) seen in earlier studies using intraocular inoculation of IBDV, may have been due to behavioural differences that tend to result in their ingesting a relatively low dose of the virus.

Animals↗

PubMed2000

Rocket immunoelectrophoresis in the diagnosis of infectious bursal disease.

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.

Animals↗

PubMed2001

Molecular characterisation of very virulent infectious bursal disease viruses in Taiwan.

The very virulent infectious bursal disease virus (vv IBDV) RNA in the bursa of Fabricius and spleen from experimentally infected chickens or field samples was detected by in situ hybridisation (ISH) with subsequent reverse transcription (RT)-polymerase chain reaction (PCR) and sequence analysis. The VP 2 gene of vv IBDV was detected by ISH in infected chicken tissues with a cloned digoxigenin (DIG)-labelled cDNA probe. To verify ISH, RT - PCR was used to amplify two 643- and 500-base pair fragments on the VP 2 gene of IBDV in the bursa of Fabricius. With all isolates, two c DNA fragments of 643 and 500 bp long, respectively, were generated as expected and further confirmed the specificity of ISH. Analysis of the hypervariable region (HVR) of the VP 2 gene revealed that a serine-rich heptapeptide SWSASGS located at amino acids 326-332 was conserved in recent Taiwanese strains, and two amino acid substitutions were found in the classical Taiwanese strains at positions 330M and 331W. Three amino acids were unique to the vv strains at positions 222A, 256I and 294I, compared with classical and variant strains. Sequence and phylogenetic analysis showed that the recent Taiwanese strains were closely related, very similar to vv IBDV s from Europe, China, Japan, and Africa, and distantly related to the Taiwanese classical strains.

Amino Acid Sequence↗

PubMed1996

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Animals↗

PubMed2004

Dogs as potential carriers of infectious bursal disease virus.

In this study, the possibility that dogs could eventually be carriers of infectious bursal disease virus (IBDV) after having eaten (voluntarily or accidentally) IBDV-infected chicks has been evaluated. A single Beagle dog was fed chicks infected by a very virulent IBDV strain (vvIBDV). Afterwards, the presence and viability of IBDV in the faeces was assessed. Viable vvIBDV was detected in the dog's faeces for 2 days after the initial ingestion, which indicates excretion of vvIBDV. Comparison by molecular techniques of the administered and excreted virus using reverse transcription-polymerase Chain reaction and enzymatic digestion confirmed that the initial virus maintained the same characteristics after being excreted. We believe that this study could be of great interest for a better understanding of the epidemiology of IBD disease on farms where dogs live close to avian facilities.

Animal Feed↗

PubMed2006

The anti-nuclease humoral immune response of broiler chickens exposed to Staphylococcus aureus, infectious bursal disease virus and chicken anaemia virus in an experimental model for bacterial chondronecrosis and osteomyelitis.

This study aimed to develop an enzyme-linked immunosorbent assay to detect antibody associated with Staphylococcus aureus that is produced during the chicken immune response to this organism. The protein nuclease was tested for suitability as an antigen to detect antibody in sera from broiler chickens that had been exposed to aerosolized S. aureus on day 1 post hatch during experiments to reproduce bacterial chondronecrosis and osteomyelitis. An enzyme-linked immunosorbent assay was developed to measure the levels of nuclease antibody in 509 chicken sera from various experiments, which also enabled the examination of the influence of factors such as the S. aureus infection status and co-infection with chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) on nuclease antibody levels. Positive levels of nuclease antibody were detected in 71% of serum samples from chickens inoculated with S. aureus, CAV and IBDV, while positive levels were detected in 35% of chickens inoculated with S. aureus only. The influence of CAV and IBDV on the number of chickens with positive levels was most prominent in chickens aged up to 42 days. The study showed that nuclease-specific antibodies form part of the humoral immune response in broiler chickens that have been exposed to S. aureus. Co-infection with CAV and IBDV appeared to promote development of antibody in birds younger than 42 days; however, the presence of antibody did not necessarily prevent systemic infection.

Aging↗

PubMed2005

Infectious bursal disease virus-induced immunosuppression in the chick is associated with the presence of undifferentiated follicles in the recovering bursa.

Infectious bursal disease virus (IBDV) causes an acute cytolytic infection in chicken B lymphocytes resulting in destruction of the B-cell population. Most severe depletion occurs in the bursa of Fabricius, where the immunoglobulin repertoire is developed by gene conversion. Chicks surviving IBDV infection are immunosuppressed despite repopulation of the bursa with B cells. Here we show that infection of neonatal chicks with a classical virulent IBDV strain (F52/70) causes severe bursal Bcell depletion with recovery after about one week. Two distinct types of bursal follicles developed: large reconstituted follicles and small poorly developed follicles lacking a discernible cortex and medulla. The presence of large numbers of undifferentiated follicles was associated with inability to mount antibody responses to IBDV itself and after immunization with Salmonella Enteritidis bacterin, indicating that B cells in these follicles are unable to produce peripheral B-cells with an effective immunoglobulin repertoire. Additionally a number of inflammatory foci were observed in the recovering bursa. These foci contained few B cells at the margins, but large numbers of CD4(+) and CD8(+) cells, scattered gammadelta(+) T-cells and macrophages, and small central aggregates of dendriticlike cells expressing the CD40 antigen.

Animals↗

PubMed2000

Ascorbic acid supplementation improved antibody response to infectious bursal disease vaccination in chickens.

The purpose of the present study was to determine if supplementation of ascorbic acid (AA) to the diet would have a beneficial effect on infectious bursal disease (IBD) vaccination of chickens for protection against infectious bursal disease virus (IBDV) infection. Two hundred forty specific pathogen-free (SPF) chickens were divided into eight experimental groups. A 2 x 2 x 2 factorial arrangement in a completely randomized design was used; AA supplementation at 1,000 ppm in the diet, vaccination, and challenge were the main effects. Prior to challenge and 10 d after challenge, serum AA concentration, serum corticosterone concentration, ELISA antibody titer to IBDV, body weight, bursa-to-body weight (B:B) ratio, and bursal histological score (BHS) were determined. Nonvaccinated chickens fed a diet supplemented with AA did not exhibit clinical signs or mortality following challenge, whereas AA-unsupplemented counterparts had 100% cumulative morbidity and 30% cumulative mortality. Serum AA levels of AA-supplemented and vaccinated chickens were significantly (P < 0.05) higher than AA-unsupplemented and vaccinated chickens. Fourteen days following vaccination, significantly (P < 0.05) higher ELISA titers to IBDV were observed in vaccinated chickens supplemented with AA as compared to AA-unsupplemented counterparts. Ascorbic acid-supplemented chickens, especially those also vaccinated, had higher body weight gains as compared to the AA-unsupplemented chickens. Ascorbic acid-supplemented chickens challenged with IBDV did not show any clinical signs or mortality. The results suggest that supplementation of AA at 1,000 ppm in the diet has beneficial effects on antibody response to IBD vaccination and body weight gain.

Animals↗