Very virulent infectious bursal disease virus infection caused changes in cloacal temperature and clinical manifestations in pigeons (Columba livia domestica) and is transmitted to sentinel chickens.

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 (3-6 weeks old) and thirty chickens (3 weeks 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 0.20 mL (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 2 days 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.

Antox® and Bactofort® mitigated the haematological alterations induced by a very virulent infectious bursal disease virus in chicks.

The study investigated the mitigating effects of two probiotics on blood parameters of ISA Brown chicks inoculated with a very virulent infectious bursal disease virus (vvIBDV). Two hundred chicks were assigned into four groups of 50 birds each. Groups A and B were administered Antox® in water and Bactofort® in feed daily from 1 to 42 days of age and inoculated with a vvIBDV at 28 days and C and D served as positive and negative controls, respectively. Blood samples were examined for changes in packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC), total white blood cell (TWBC), heterophil and lymphocyte counts seven days post inoculation. The PCV between groups A and C differed (P < 0.05) and in group B it was higher (P < 0.05) than that of group C. The Hb concentration between groups A, B and C differed (P < 0.05). There was a difference (P < 0.05) in RBC counts between groups A, B, C. Differences in TWBC between group A and C were significant (P < 0.05) and TWBC in group B was higher (P < 0.05) than that of group C. There was a significant difference in heterophil (P < 0.05) and lymphocyte (P < 0.05) count between group A and C, and B and C. Heterophil/lymphocyte ratio was significantly higher in positive control compared to groups A, B, C. Antox® and Bactofort® mitigated the deleterious effects of vvIBDV on blood parameters and can assist in cases of IBD outbreak.

Infection of bursal disease virus abrogates the extracellular glycoprotein in the follicular medulla.

In the medulla of bursal follicle, only the secretory dendritic cell (BSDC) is furnished with secretory machinery. The granular discharge of BSDC appears in membrane-bound and solubilized forms. Movat pentachrome staining proves that the solubilized form is a glycoprotein, which fills up the extracellular space of follicular medulla. The glycoprotein contributes to bursal microenvironment and may be attached to the surface of medullary lymphocytes. The secretory granules of BSDC may be fused, resulting in large, irregular dense bodies, which are the first sign of BSDC transformation to macrophage-like cells (Mal). To determine the effect of infectious bursal disease virus (IBDV) infection on the extracellular glycoprotein and BSDC, SPF chickens were experimentally infected with IBDV. On the surface of BSDC, the secretory substance is in high concentration, which may contribute to primary binding of IBDV to BSDC. The early distribution of IBDV infected cells is in consent with that BSDC. The IBDV infected BSDC rapidly transforms to Mal in which the glycoprotein staining appears. In the dense bodies, the packed virus particles inhibit the virus particles preventing the granular discharge, which may represent the first, early phase of virus replication cycle. The absence of extracellular glycoprotein results in alteration in the medullary microenvironment and subsequently B cell apoptosis. On the surface of medullary B cells, the solubilized secretory substance can be in much lower concentration, which results in secondary binding of IBDV to B cells. In secondary, late phase of virus replication cycle, the virus particles are not packed in electron dense substance which results in cytolytic lymphocytes and presence of virus in extracellular space. The Mal emigrates into the cortex, where induces inflammation, recruiting heterophil granulocyte and monocyte.

Ghrelin attenuates infectious bursal disease virus-induced early inflammatory response and bursal injury in chicken.

Studies demonstrated that chicken ghrelin mRNA was expressed in immune organs of chicken. However, it was not known for its functions in chicken immune system. This study aimed to investigate the effects of ghrelin on infectious bursal disease virus (IBDV)-induced acute inflammatory and bursal injury. Chickens were divided into 4 groups. One group was used as control ("C"). The other three groups incubated with IBDV on the 19th d, of which 2 were injected intraperitoneally with 0.5 nmol ("LG") or 1.0 nmol ("HG") ghrelin/100g body weight from 18th to 22nd d, respectively, and one was injected intraperitoneally with PBS ("I"). Results showed that cytokines including interleukin (IL)-6, IL-1ß, and IL-8 mRNA expression in I group were upregulated significantly after chickens infected with IBDV from 1 d post-infection (dpi) to 3 dpi (P < 0.05). However, the expression level of IL-6, IL-1ß, and IL-8 mRNA in LG and HG groups was 7.3, ∼43.3% as much as that of the I group at 2 dpi and 3 dpi (P < 0.05). Moreover, ghrelin administration attenuated significantly the bursal injury from 1 dpi to 7 dpi and prevents the reduction of bird weight gain at 5 dpi and 7 dpi, which were induced by IBDV (P < 0.05). The results indicated that ghrelin could play an important role in the immune system of chicken.

Pro-apoptosis effects of protocatechuic acid in the early stage of infectious bursal disease virus infection.

Infectious bursal disease virus (IBDV) is a very important small RNA virus in the family of Birnaviridae, which can cause severe immunosuppressive effects and pathological damages in young chickens. It can replicate in bursal lymphocytes and impede the growth and development of B cells, finally causing bursal lymphocytes apoptosis. Previous results have shown that protocatechuic acid (PCA) as an important phenolic compound could effectively improve the survival rate of chickens infected with IBDV. The current study aimed to explore how PCA influenced the pathogenesis of IBDV, especially lymphocyte apoptosis in the process of IBDV infection. The results showed that PCA could effectively alleviate bursal pathological changes at the early stage of IBDV invasion. Moreover, bursal lymphocyte apoptosis for tissue section samples was largely elevated by PCA by using the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method while the bursal lymphocyte apoptosis ratio was also increased by PCA by flow cytometry in the early stage of IBDV infection in vivo. Meanwhile, PCA could promote non-lymphocyte apoptosis in vitro. Further study displayed that the potential mechanisms mainly relied on regulation of the expressions of pro-apoptotic protein Bax and anti-apoptotic Bcl-2, thus speeding up the process of IBDV-infected cell apoptosis and preventing virus infection. Meanwhile, the results displayed that the PI3K/Akt and NF kappa B signal pathways might play an important role in promoting cell apoptosis after IBDV infection. Overall, PCA as a potent antiviral drug precursor is expected to be applied in the poultry industry as a substitute for clinical antiviral application.

Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells.

The understanding of virus-host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection.

Survival, growth and sexual maturation in Atlantic salmon exposed to infectious pancreatic necrosis: a multi-variate mixture model approach.

BACKGROUND: Outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon can result in reduced growth rates in a fraction of the surviving fish (runts). Genetic and environmental variation also affects growth rates within different categories of healthy animals and runts, which complicates identification of runts. Mixture models are commonly used to identify the underlying structures in such data, and the aim of this study was to develop Bayesian mixture models for the genetic analysis of health status (runt/healthy) of surviving fish from an IPN outbreak. METHODS: Five statistical models were tested on data consisting of 10 972 fish that died and 3959 survivors with recorded growth data. The most complex models (4 and 5) were multivariate normal-binary mixture models including growth, sexual maturity and field survival traits. Growth rate and liability of sexual maturation were treated as two-component normal mixtures, assuming phenotypes originated from two potentially overlapping distributions, (runt/normal). Runt status was an unobserved binary trait. These models were compared to mixture models with fewer traits (Models 2 and 3) and a classical linear animal model for growth (Model 1). RESULTS: Assuming growth as a mixture trait improved the predictive ability of the statistical model considerably (Model 2 vs. 1). The final models (4 and 5) yielded the following results: estimated (underlying) heritabilities were moderate for growth in healthy fish (0.32 ± 0.04 and 0.35 ± 0.05), runt status (0.39 ± 0.07 and 0.36 ± 0.08) and sexual maturation (0.33 ± 0.05), and high for field survival (0.47 ± 0.03 and 0.48 ± 0.03). Growth in healthy animals, runt status and survival showed consistent favourable genetic associations. Sexual maturation showed an unfavourable non-significant genetic correlation with runt status, but favourable genetic correlations with other traits. The estimated fraction of healthy fish was 81-85%. The estimated breeding values for runt status and (normal) growth were consistent for the most complex models (4 and 5), but showed imperfect correlations with estimated breeding values from the simpler models. CONCLUSIONS: Modelling growth in IPN survivors as a mixture trait improved the predictive ability of the model compared with a classical linear model. The results indicated considerable genetic variation in health status among survivors. Mixture modelling may be useful for the genetic analysis of diseases detected mainly through indicator traits.

Cell culture-adapted IBDV uses endocytosis for entry in DF-1 chicken embryonic fibroblasts.

Although membrane perforation was suggested as the means of penetration mediated by IBDV, the cellular mechanism being hijacked to facilitate its entry is largely unknown. In this study, the entry pathway of cell culture adapted IBDV (caIBDV) was characterized in DF-1 chicken embryonic fibroblasts. We observed that the entry of caIBDV was inhibited by bafilomycin A1 and CaEGTA which interfere with the function of vacuolar H(+)-ATPase (V-ATPase) and retain endosomal Ca(2+). This result suggests that the intact caIBDV particle was transported to the V-ATPase positive vesicles for uncoating and implicates an essential role of endocytosis during the viral entry. The IBDV-mediated endocytosis was demonstrated to be clathrin-independent. Instead, the entry of caIBDV in DF-1 was reduced under the inhibitions or depletions of lipid raft, c-Src tyrosine kinase, dynamin and actin polymerization. In summary, this study confirmed the role of endocytosis in caIBDV entry and characterized the route of its endocytosis.

Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells.

Aquatic birnavirus induces post-apoptotic necrotic cell death via a newly synthesized protein-dependent pathway. However, the involvement of viral genome-encoded protein(s) in this death process remains unknown. In the present study, we demonstrated that the submajor capsid protein, VP3, up-regulates the pro-apoptotic protein, Bad, in fish and mouse cells. Western blot analysis revealed that VP3 was expressed in CHSE-214 cells at 4 h post-infection (pi), indicating an early role during viral replication. We cloned the VP3 gene and tested its function in fish and mouse cells; VP3 overexpression induced apoptotic cell death by TUNEL assay. In addition, it up-regulated Bad gene expression in zebrafish ZLE cells by threefold at 12 h post-transfection (pt) and in mouse NIH3T3 cells by tenfold at 24 h pt. VP3 up-regulation of Bad expression altered mitochondria function, inducing mitochondrial membrane potential (MMP) loss and activating initiator caspase-9 and effector caspase-3. Furthermore, reduced Bad expression (65% reduction), MMP loss (up to 40%), and enhanced cell viability (up to 60%) upon expression of VP3 antisense RNA in CHSE-214 cells at 24 h post-IPNV infection was observed. Finally, overexpression of the anti-apoptotic gene, zfBcl-xL, reduced VP3-induced apoptotic cell death and caspase-3 activation at 24 h in fish cells. Taken together, these results suggest that aquatic birnavirus VP3 induces apoptosis via up-regulation of Bad expression and mitochondrial disruption, which activates a downstream caspase-3-mediated death pathway that is blocked by zfBcl-xL.

Acute pancreatitis.

Acute pancreatitis is an inflammatory disease of the pancreas. Acute abdominal pain is the most common symptom, and increased concentrations of serum amylase and lipase confirm the diagnosis. Pancreatic injury is mild in 80% of patients, who recover without complications. The remaining patients have a severe disease with local and systemic complications. Gallstone migration into the common bile duct and alcohol abuse are the most frequent causes of pancreatitis in adults. About 15-25% of pancreatitis episodes are of unknown origin. Treatment of mild disease is supportive, but severe episodes need management by a multidisciplinary team including gastroenterologists, interventional radiologists, intensivists, and surgeons. Improved understanding of pathophysiology and better assessments of disease severity should ameliorate the management and outcome of this complex disease.

Expression of Mx mRNA following infection with IPNV is greater in IPN-susceptible Atlantic salmon post-smolts than in IPN-resistant Atlantic salmon parr.

The Mx response was compared in parr and post-smolt Atlantic salmon following intra-peritoneal injection of the same dose of Infectious Pancreatic Necrosis Virus (IPNV) per g of fish. Mx gene expression, measured by quantitative RT-PCR in liver, showed a maximum level 3days after injection in parr with undetectable levels on day 7. In post-smolts, similar levels as in parr were attained on day 3, but levels then continued to rise on day 5 and 7 to about 10 times higher than the peak level in parr. Poly I:C injected parr showed Mx levels similar to IPNV injected post-smolts. Mortality from IPN in post-smolts occurred on days 6 and 7. Levels of IPN VP2 transcripts in parr were very low and did not increase with time, suggesting viral replication was low. Individual variation in levels of Mx and IPN VP2 gene transcripts was very high in post-smolts and although data is limited there was an inverse relationship between the levels of Mx and VP2, suggesting that individuals with high Mx levels on day 5 may be able to prevent viral replication. This contrasts with the response in parr, where IPN-resistance was not associated with a high Mx response.

Intracellular interference of infectious bursal disease virus.

A search for dominant-negative mutant polypeptides hampering infectious bursal disease virus (IBDV) replication has been undertaken. We have found that expression of a mutant version of the VP3 structural polypeptide known as VP3/M3, partially lacking the domain responsible for the interaction with the virus-encoded RNA polymerase, efficiently interferes with the IBDV replication cycle. Transformed cells stably expressing VP3/M3 show a significant reduction (up to 96%) in their ability to support IBDV growth. Our findings provide a new tool for the characterization of the IBDV replication cycle and might facilitate the generation of genetically modified chicken lines with a reduced susceptibility to IBDV infection.

Interference between mild and pathogenic strains of infectious bursal disease virus in chickens.

Infectious bursal disease virus is a contagious, immunosuppressive disease of young chickens that is controlled by vaccination. Cross-protection occurs between different strains of the virus as a result of shared neutralizing epitopes. However, interactions between two antigenically similar strains (a mild and a pathogenic) coinfecting the same host have not been investigated. Groups of specific-pathogen-free chickens were inoculated with a mild strain followed by a pathogenic strain at 0, 16, 24, or 48 hr postinoculation (PI) with a mild strain. Virus persistence and the predominant strain of the virus were determined by reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis, respectively, in bursas at 2, 4, 8, 14, and 21 days PI with the pathogenic strain. Severity of infection was assessed by the bursa/body weight ratios and histopathologic lesion scores. The mild virus interfered with replication of the pathogenic virus. The greatest interference was observed when the pathogenic strain was inoculated 24 hr PI with the mild strain. The interference phenomenon observed might be due to competition for host receptor sites or production of cytokine(s). This interference phenomenon could have practical implications for vaccine usage and protection.

Antigenic and molecular characterization of recent infectious bursal disease virus isolates in China.

Eleven infectious bursal disease virus (IBDV) strains isolated recently from China were compared with the early classical virulent strain CJ801, the chicken embryo fibroblast-adapted (CEF) variant strain GZ902, and the attenuated vaccine strains BJ836, BK912, and LM to discern the evolutionary characteristics of IBDV in China at both antigenic and genetic levels. Virus neutralization (VN) assay showed that all ten very virulent (vv) IBDV strains belong to the same subtype as attenuated strains, whereas the other variant isolate strain BX could be attributed to other subtype of the variant strain GZ902. Antigen-capture ELISA (AC-ELISA) determined by a panel of monoclonal antibodies (Mabs) against classical and variant strains showed further that among these vv strains, nine strains except for strain NC had no reaction with neutralizing Mab B69. The vv strains SC and YV had no reaction with non-neutralizing Mabs 2B8 and 2C4, respectively, whose epitopes were located in classical IBDV strains. On the other hand, there is no alteration in antigenic epitopes located in the variant strain BX as that of the variant GZ902. Sequence comparison of the highly variable region (HVR) of the VP2 proteins showed that these vv strains had 98.6-100.0% identities to European and Asian vv strains at amino acid level. For the vv strains NC, SC, and YV, all had one amino acid substitution at the major hydrophilic domains, indicating that new vv strains are evolving. In addition, the vv strains DMS and NC had amino acid residue 279N as well, showing that the substitution of amino acid at this position might not be related to the virulence of IBDV. The variant strain BX had one amino acid substitution in the two major hydrophilic domains and two unique amino acids 249K and 254S as the other early variant strains, and shared 97.3% of amino acid identity to the variant strain VarE. Phylogenetic analysis suggests that the recent Chinese vvIBDVs and the previous European and Asian vv strains still belong to a genetic group and the variant strain BX to the other genetic group, which is more closely related to the European classical virulent strain F52/70 and the American classical virulent strain STC than to the early Chinese classical virulent strain CJ801, showing that the recent vv and variant strains that spread widely in the country might be derived from Europe and America than from early Chinese classical virulent strains.

Role of intrabursal T cells in infectious bursal disease virus (IBDV) infection: T cells promote viral clearance but delay follicular recovery.

Infectious bursal disease virus (IBDV) induces an acute, highly contagious immunosuppressive disease in young chickens. We examined the role of T cells in IBDV-induced immunopathogenesis and tissue recovery. T cell-intact chickens and birds compromised in their T cell function by a combination of surgical thymectomy and Cyclosporin A treatment (Tx-CsA) were infected with an intermediate vaccine strain of IBDV (Bursine 2, Fort Dodge). Our data revealed that functional T cells were needed to control the IBDV-antigen load in the acute phase of infection at 5 days post infection. The target organ of IBDV, the bursa of Fabricius, of Tx-CsA-birds had a significantly higher antigen load than the one of T cell-intact birds (P < 0.05). Tx-CsA-treatment abrogated the IBDV-induced inflammatory response and significantly (P < 0.05) reduced the incidence of apoptotic bursa cells and the expression of cytokines such as interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in comparison to T cell-intact birds. T cell-released IL-2 and IFN-gamma may have mediated the induction of inflammation and cell death in T cell-intact birds. The IBDV-induced upregulation of tumor necrosis like-factor (TNF) expression was comparable between T cell-intact and Tx-CsA-birds. Tx-CsA-birds showed a significantly faster resolution of IBDV-induced bursa lesions than T cell-intact birds (P < 0.05). This study suggests that T cells modulate IBDV pathogenesis in two ways: a) they limit viral replication in the bursa in the early phase of the disease at 5 days post infection, and b) intrabursal T cells promote bursal tissue damage and delay tissue recovery possibly through the release of cytokines and cytotoxic effects.

Caracterización clínico-patológica de tres aislamientos del virus de la infección de la bolsa de Fabricio, obtenidos en granjas comerciales de pollo de engorda en México / Clinical and pathological characterization of three isolates of infectious bursal disease virus obtained from commercial broiler flocks in Mexico

Se aislaron tres virus de la infección de la bolsa de Fabricio (VIBF) a partir de muestras obtenidas de muestras en granjas avícolas en México, aquéllos se denominaron H, P y G. Se determinaron sus propiedades antigénicas mediante la técnica de ELISA por captura de antígenos, utilizando los anticuerpos monoclonales (Mab) B-29, R-63, BK-9, B-69 y 57. Se evaluó su virulencia en aves inoculadas mediante la observación de signos clínicos, mortalidad, proporción del peso bursal con el peso corporal, lesiones microscópicas, persistencia de los virus en tejidos linfoides, serología contra VIBF y medición de la respuesta inmune hacia Brucella abortus y eritrocitos de ovino. Los virus P y G presentaron los epítopes reconocidos por lo B-29, R-63 y B-69, que están presentes en las cepas clásicas de VIBF, el virus H no pudo ser reconocido por ningún Mab. Los tres aislamientos indujeron la presentación subclínica de la IBF en las aves inoculadas, se observó marcada atrofia bursal con deplesión linfoide severa, persistencia del VIBF en bolsa hasta los catorce días posinoculación y seroconversión de las aves a partir de la segunda semana después de la inoculación. A pesar de que no se detectó plenamente un estado de inmunodepresión mediante la respuesta a los dos antígenos utilizados, no se puede descartar un eventual efecto inmunodepresor por el aislamiento "H". Tampoco se detectaron diferencias en la virulencia de los tres VIBF aislados

Apoptosis in cell cultures induced by infectious bursal disease virus following in vitro infection.

Chicken embryo fibroblasts (CEFs) and Vero cells infected with infectious bursal disease virus (IBDV) exhibited the biochemical feature of apoptosis. Agarose gel electrophoresis of DNA extracted from IBDV-infected cells revealed the characteristic laddering pattern of DNA fragmentation, which was more intense in infected CEFs than in Vero cells. The appearance of apoptotic nucleosomal DNA fragments in IBDV-infected CEFs was independent of virus replication and occurred at an early stage following in vitro infection.

Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains.

In order to study replication efficiency of infectious bursal disease virus (IBDV) in lymphoid tissues, both the virus titers and the virus antigen titers in four lymphoid tissues were compared among chickens inoculated with Ehime/91 (about 50% mortality), J1 (no mortality), or K (attenuated) IBDV strains during 1-7 days postinoculation (PI). IBDV antigens in tissue homogenates were detected by an enzyme-linked immunosorbent assay. In the bursa of Fabricius, higher virus titers were maintained for 1-3 days PI in chickens inoculated with Ehime/91 or J1 strains, whereas the virus titers increased gradually and reached to the peak on 3 days PI in chickens inoculated with the K strain. There were no clear differences in both the virus and the virus antigen titers in bursae and thymus between chickens inoculated with Ehime/91 and J1 stains. However, the virus and/or the virus antigen titers in spleen and bone marrow of chickens inoculated with Ehime/91 strain were higher than those of the J1-inoculated chickens. Immunohistochemical analyses indicated that larger numbers of IBDV antigen-positive cells were detected in spleen and bone marrow of the Ehime/91 group than in those of the J1 group. There was almost no detectable virus and virus antigens in thymus, spleen, and bone marrow of the K-inoculated chickens throughout the experiment. These results suggest that efficient replication of IBDV not only in the bursa but also in the spleen and the bone marrow may be required for clinical infectious bursal disease.