Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents.

We used in ovo technology to protect chickens against multiple diseases by inoculating vaccines containing mixtures of live viral agents. A single in ovo injection of a vaccine containing serotypes 1, 2, and 3 of Marek's disease virus (MDV), a vaccine strain of serotype 1 infectious bursal disease virus (IBDV), and recombinant fowl pox vaccine with HN and F genes of Newcastle disease virus (rFP-NDV) induced protection against virulent MDV, IBDV, Newcastle disease virus, and fowl poxvirus. The multiple-agent vaccine induced specific antibodies against the viral agents present in the mixture and did not adversely affect the survival of hatched chickens. Inoculation of a vaccine containing serotypes 1, 2, and 3 of MDV and IBDV did not affect hatchability of eggs, although the addition of rFP-NDV to the mixture reduced hatchability by 23%-26%. In ovo vaccination with a vaccine containing MDV and IBDV vaccine viruses did not exacerbate the inhibitory effect of individual viral agents on humoral and cellular immune competence.     

Protective immunity against infectious bursal disease virus in chickens in the absence of virus-specific antibodies

The role of cell-mediated immunity (CMI) in pathogenesis of infectious bursal disease virus (IBDV) was investigated. One-day-old specific pathogen-free chickens were treated with 3mg of cyclophosphamide (Cy) per chicken for 4 consecutive days and, 3 weeks later, infected with the IBDV-IM strain. Chickens were examined for: (a) mitogenic response of splenocytes to ConA, as an indicator of T-cell functions in vitro, (b) antibody against IBDV by ELISA, (c) IBDV genome in various tissues by RT-PCR and (d) immunological memory. At the time of IBDV infection, Cy-treated chickens had depleted bursal tissue (an avian primary B-cell lymphoid organ), severely compromised antibody-producing ability, but normal T-cell response to ConA. In primary infection, no detectable antibody against IBDV antigen in Cy-treated, IBDV-infected chickens was observed up to 28 days post-infection (PI), while IBDV genome was detected by RT-PCR in spleen, thymus, liver and blood until 10 days PI. Like intact control chickens infected with IBDV, Cy-treated, IBDV-infected chickens suppressed splenocytes responses to ConA from 5 to 10 days PI, suggesting that intact control as well as Cy-treated chickens responded similarly to IBDV infection in the early phase. Following re-infection with IBDV, no detectable secondary antibody response to IBDV as well as IBDV genome in tissues were observed in Cy-treated chickens, while intact control chickens developed vigorous secondary antibody response. Similar to intact control chickens infected with IBDV, Cy-treated chickens after second infection with IBDV did not suppress splenocyte response to ConA. These results suggested that in the absence of detectable anti-IBDV antibodies, protection of Cy-treated chickens from IBDV infection may occur via immunological memory mediated by CMI. We concluded that under normal conditions, IBDV induces a protective antibody response, however, in the absence of antibody, CMI alone is adequate in protecting birds against virulent IBDV.     

Embryo vaccination with infectious bursal disease virus alone or in combination with Marek's disease vaccine

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.     

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.     

Immunosuppressive effect of a highly virulent infectious bursal disease virus isolated in Japan.

The immunosuppressive effect of infectious bursal disease virus (IBDV) on vaccination against Newcastle disease (ND) was compared among 2-, 3-, and 4-week-old chickens inoculated with the highly virulent IBDV field isolate 90-11 and the reference serotype 1 strain GBF-1. In all age groups, isolate 90-11 severely suppressed antibody response to ND vaccination and protective vaccinal immunity against ND. In contrast, chickens inoculated with strain GBF-1 and vaccinated with ND vaccine were well protected from the ND virus challenge. The mitogenic response to phytohemagglutinin of splenic lymphocytes from chickens inoculated with isolate 90-11 or strain GBF-1 was significantly lower than that of uninoculated controls. There was no difference between the two inoculated groups in responsiveness, although lymphocyte depletion in the thymus was more severe in chickens inoculated with isolate 90-11 than in chickens inoculated with strain GBF-1.     

Pathology of avian adenovirus serotypes in the presence of Escherichia coli in infectious-bursal-disease-virus-infected specific-pathogen-free chickens

Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.5 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally. Those birds had been inoculated by eye drop at 1 day of age with a virulent infectious bursal disease virus (IBDV). Controls consisted of groups of chickens inoculated with: (a) IBDV and E. coli, (b) IBDV only, (c) E. coli only, and (d) no virus or E. coli. Gross pulmonary alterations at 5 days postinoculation (PI) included congestion and consolidation of one or both lungs of a chick inoculated with adenovirus serotype Ind-C and another inoculated with A-2. Histopathologic alterations in the lungs were those of multifocal interstitial and occasionally diffuse pneumonia. All 10 adenovirus serotypes elicited multifocal or diffuse pneumonia and bronchiolitis in one or more of the five birds per group necropsied at 5 days PI. T-8 and A-2 serotypes induced marked to serve diffuse pneumonia within 5 days; Ind-C, Stein, Tipton, 75-1A, B-3, and X-11 incited a mild diffuse pneumonia. In all groups, the pneumonic lesions were more severe 5 days PI than 12 days PI. Tracheitis was incited by Ind-C, Stein, T-8, and A-2 at 5 days PI; the lesions were minimal to marked in severity. None of the four control groups exhibited gross or histopathologic alterations except the two IBDV-infected groups, which exhibited bursal change.     

Persistent infection with chicken anaemia virus and some effects of highly virulent infectious bursal disease virus infection on its persistency

Chicken anaemia virus (CAV) infectivity and the effect of highly virulent infectious bursal disease virus (hv IBDV) infection on CAV's infectivity were examined in chickens inoculated with CAV or inoculated dually with CAV and hv IBDV. Five chickens inoculated dually with hv IBDV at 35 days old and then with CAV at 40 days old exhibited no clinical signs of disease, but showed atrophic bursae of Fabricius when necropsied 4 weeks later. Upon examining the chickens at 7 days postinoculation (dpi) with CAV, it was found that hv IBDV infection had inhibited production of virus neutralising (VN) antibody to CAV, and that it was possible to recover CAV from plasma of these chickens. Although VN antibody to CAV appeared after 14 dpi, CAV was recovered from blood cells (BC s) at high titres ranging from 10(2.5)to 10(5.5)TCID(50)/0.1 ml, 7 to 28 dpi in IBDV -induced immunosuppressed chickens. In addition, CAV was sporadically recovered, using rectal swabs, from the dually inoculated chickens at low titers, ranging from 10(1.0)to 10(2. 0)TCID(50)/0.1 ml). In contrast, although CAV was recovered from BC s in most of the chickens inoculated with CAV alone, the titers were lower (10(1.0)to 10(2.5)TCID(50)/0.1 ml). No CAV was detected from the rectal swabs of these chickens. The results of virus recovery were confirmed by polymerase chain reaction. This study first examined the persistency of CAV in BC s and the effective enhancement of primary CAV infection as a result of immunosuppression caused by hv IBDV infection.     

Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens

The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.     

Reproduction of hydropericardium syndrome in three-week-old cyclophosphamide-treated specific-pathogen-free chickens by adenoviruses from inclusion body hepatitis

The pathogenicity of serotype 8 group I avian adenovirus (GIAAV) strains (TR630 and Saga97 strains) from inclusion body hepatitis (IBH) against cyclophosphamide (CY)-treated 3-wk-old specific-pathogen-free (SPF) chickens was examined. SPF chickens were inoculated intramuscularly with 10(7) plaque-forming units of viruses. Both strains from IBH could produce hydropericardium and mortality in CY-treated chickens as hydropericardium syndrome (HPS) that serotype 4 GIAAV strains cause, although they could not induce either hydropericardium or mortality in nontreated chickens. Histologically, hepatocytic necrosis with intranuclear inclusions, pancreatic acinar necrosis with intranuclear inclusions, and epicardial edema were seen in CY-treated chickens inoculated with GIAAV from IBH. Immunohistochemically, these inclusions were positive against GIAAV antigen. There were neither histologic lesions nor positive reactions against GIAAV antigen in nontreated chickens inoculated with GIAAV from IBH. From the present findings, pathogenic characteristics of IBH strains and HPS strains in the chickens were essentially the same.     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

Effect of viral structure and replication characteristics on the pathogenesis of infectious bursal disease]

Infectious bursal disease (IBD) is a highly contagious disease of young chickens, which occurs world-wide, and is responsible for severe losses in poultry industries. In birds surviving an acute infection, lymphoid cells in the bursa of Fabricius are destroyed, resulting in B-cell-dependent immunodeficiency. This causes increased susceptibility to diseases by otherwise harmless agents. The decisive role in the pathogenesis of IBD is played by the bursa, representing the target organ of the aetiological agent, infectious bursal disease virus (IBDV). By adaptation of IBDV to chicken embryo cells, we obtained several variants of a pathogenic wild type strain. These variants had altered abilities for replication in actively dividing B lymphocytes and, consequently, had altered pathogenic properties. An IBDV isolate from turkeys, non-pathogenic for chickens, was used to create reassortant virus strains. The virological and the biological characterization of these IBDV variants is reported.     

不同源传染性法氏囊病病毒的分离及其生物学特性分析

用传染性法氏囊病病毒 ( IBDV)单克隆抗体夹心抑制 EL ISA对麻雀、鸭、鹅进行了血清学调查 ,阳性率分别为 7.4% ( 4/ 54 )、95.5%( 3 6 3 / 3 80 )、9.4% ( 1 1 / 1 1 7)。从 IBD阳性麻雀、IBD病鸡以及同群饲养的鸭、鹅体内分离到了 4株不同源病毒 ,IBDV单克隆抗体夹心 EL ISA、Dig-标记 IBDV c DNA探针斑点杂交和交叉中和试验证明该病毒均为 IBDV血清 型。病毒可适应并致死鸡胚 ,适应于鸡胚成纤维细胞并产生细胞病变效应 ( CPE)。病毒代谢抑制试验证明其基因组为 RNA,病毒对乙醚不敏感 ,p H2 .0不能灭活病毒 ,p H 1 2可使病毒失去感染性 ,56℃作用 3 h病毒仍有活性 ,70℃ 1 h可灭活病毒。以上结果表明 ,从鸡、麻雀、鸭、鹅体内分离到的 IBDV生物学性质比较稳定且非常相似。本研究结果提示 IBDV已在我国广泛流行 ,麻雀、鸭、鹅可成为 IBDV携带者或传染源 ,在 IB-DV传播和生态变异中起重要作用