Detection of infectious bursal disease viruses using in situ hybridization and non-radioactive probes.

The in situ hybridization assay was developed for the detection of infectious bursal disease virus (IBDV) infections in chickens. Bursal tissue samples were harvested 4 days following infection with the ST-C, MD, E, IN, or SAL IBDV strain. The cDNA clones STC-243, located on genome segment A, and STC-119, located on genome segment B, were used to prepare non-radioactive probes. Probes were labeled with digoxigenin and detected the homologous ST-C virus and also heterologous viruses in bursal tissue sections. No positive cells were observed in tissue sections from uninfected control chickens.     

传染性法氏囊病病毒Vero细胞培养条件优化的研究

鸡传染性法氏囊病病毒（ＩＢＤＶ）在Ｖｅｒｏ细胞上增殖条件的优化研究结果表明，ＩＢＤＶ在Ｖｅｒｏ细胞上敏感性有所提高；降低轿清含量，中性ｐＨ环境，适当高的细胞密度有利于ＩＢＤＶ的增殖。     

原位PCR检测鸡传染性法氏囊病病毒

采取临床病例鸡的法氏囊制成石蜡切片,经蛋白酶K处理、原位RT-PCR后,用地高辛精标记探针进行原位杂交检测传染性氏囊病病毒（IBDV）。结果10个病例有8个呈阳性,2个阴性。本试验尝试利用具有极高灵敏度的原位PCR方法,从组织中检测低拷贝甚至单拷贝的目标序列,以鉴别隐性感染或混合感染,为IBDV的诊断和分子流行病学的分析提供了一种新的手段。     

Evaluation of serological, histological and immunocytochemical methods for the detection of infectious bursal disease virus infection in broiler flocks in New Zealand

AIMS: TO study and compare three diagnostic methods for the detection of infectious bursal disease virus (IBDV) infection. METHODS: Samples of sera and bursae were collected from two flocks from each of two broiler farms (Farms A and B) in which IBD had occurred or was suspected to have occurred. Sera were tested in ELISA and agar gel precipitation tests for the presence of IBD antibodies. Bursae were examined histologically for evidence of IBD lesions. An immunocytochemical test was developed to detect IBDV antigens in sections of bursa. RESULTS: Bursae from serologically negative, 45-day-old birds from Farm A, Flock 1 and from serologically positive 49-day-old birds from Farm B, Flock 1 had histological and immunocytochemical evidence of IBDV infection. Birds from Farm A, Flock 2, sampled 12 months after the sampling of Flock 1, and specific-pathogen-free birds, showed no evidence of IBDV infection by any of the three diagnostic methods. Birds from Farm B, Flock 2, sampled on four occasions, were positive for IBD at 20 days of age by histology and immunocytochemistry, but did not seroconvert until 42 days of age. CONCLUSIONS: Serological testing is not a reliable method for the detection of IBDV infection in New Zealand broiler flocks because antibodies may not have developed to detectable levels by the time of slaughter. Histological examination of bursae allowed the demonstration of IBD-like lesions, but these need to be differentiated from those caused by other agents. The immunocytochemistry test was able to detect early IBDV infection. It provided a rapid, definitive diagnosis and may be useful in control programmes. The results from Farm A demonstrate that strict biosecurity measures can be successful in the eradication of IBDV.     

Isolation and propagation of infectious bursal disease virus using the ovine kidney continuous cell line.

Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.     

Lysis of myelocytes in chickens infected with infectious bursal disease virus

In specific-pathogen-free chickens infected with the highly virulent HPS-2 strain or virulent reference GBF-1 strain of infectious bursal disease virus (IBDV), pathologic changes of the bone marrow were investigated. On histologic examination, bone marrow lesions were prominent in the HPS-2 group but only mild in the GBF-1 group. The bone marrow of the HPS-2 group showed severe lysis and depletion of heterophil myelocytes with pyknotic nuclear alteration 2-3 days after inoculation. On examination with an electron microscope, heterophil myelocytes were characterized by shrinkage of the cytoplasm and peripheral condensation of nuclear chromatin. IBDV particles were not detected in altered myelocytes. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method demonstrated a positive reaction in only heterophil myelocytes. In contrast, nucleosomal DNA fragmentation in HPS-2-infected bone marrow cells was indiscernible by agarose gel electrophoresis. These findings indicate that lysis of bone marrow cells is selectively induced in heterophil myelocytes at an early stage after IBDV infection and independent of virus replication.     

鸡传染性法氏囊病病毒的PCR检测法

用聚合酶链反应（ＰＣＲ）技术直接检测鸡传染性法氏囊病病毒，可快速、特异及敏感地检出实验毒株和病鸡法氏囊内存在的微量病毒核酸。对病鸡、进口种鸡的实测结果表明，本法对诊断鸡传染性法氏囊病病毒具有重要的实用意义     

In vitro infection of fractionated chicken lymphocytes by infectious bursal disease virus

Lymphocytes from bursa of Fabricius and thymus of chickens were purified and separated into the three cell subsets--T, B, and null cells--by the techniques of nylon fiber columns and cytotoxicity tests. The in vitro susceptibility of the fractionated lymphocytes to a virulent strain of infectious bursal disease virus (IBDV) was studied by using immunofluorescence as the infection criterion. B cells were highly susceptible. By contrast, T cells and null cells were insusceptible to infection by IBDV. The relationship between the target cells for virus infection and those B cells that possessed surface immunoglobulin (SIg) was tested. B cells were further divided into SIg(M)- and SIg(G)-bearing cells by immunoadsorbent columns employing anti-immunoglobulin M(IgM) (mu-specific) or anti-IgG (gamma-specific) sera coated with Sephadex. The SIg(M)-bearing cells were highly susceptible. These results suggest strongly that SIg(M)-bearing B cells were the target cells for infection by IBDV.     

Single and combined infections of specific-pathogen-free chickens with infectious bursal disease virus and an intestinal isolate of reovirus

The susceptibility of 1-day-old and 7-day-old specific-pathogen-free chickens to infection with a virulent strain of infectious bursal disease virus (IBDV) or an intestinal isolate of avian reovirus, or a combination of the two, was investigated. Chickens infected with IBDV and reovirus had more severe pathological lesions than chickens infected with either virus alone, and prior infection with IBDV enhanced the pathogenicity of enteric reovirus. Virus recovery was attempted from bursa, spleen, thymus, liver, intestine, pancreas, cecal tonsils, heart, and tarso-metatarsal tendons. Viruses were recovered from all tissues sampled for either IBDV or reovirus isolation, and indications were that infection with IBDV before infection with reovirus led to longer persistence of reovirus in some tissues. Antibodies to IBDV or reovirus were measured by the virus neutralization test and enzyme-linked immunosorbent assay. Chickens infected with IBDV had lower (P less than 0.05) antibody responses to reovirus than chickens infected with reovirus alone.     

The Characterization of Infectious Bursal Disease Virus Strains/Isolates from Field Outbreaks in India

Pahar, B. and Rai, A., 1997. The characterization of infectious bursal disease virus strains/isolates from field outbreaks in India. Veterinary Research Communications, 21 (4), 289-301Three infectious bursal disease virus (IBDV) isolates were adapted to culture in chick embryo fibroblast cells in which they produced a cytopathic effect. The isolates were identified as IBDV by virus neutralization tests using a standard hyperimmune serum against infectious bursal disease, physicochemical properties and their pathogenicity in chick embryos and chicks. The IBDV S394 strain was antigenically different from IBDV S194/IBDV S494 as well as from the IBDV Intermediate Georgia strain, one of the vaccine strains in use in India.     

CAV基因T程亚单位苗与IBDV二联灭活疫苗的研究

IBDV为自行分离的VVIBDV COB－C1组织毒,毒价为CELD5010^5.0/0.2mL,CAV为VP1和VP2基因克隆进家蚕杆状病毒转基因载体质粒中,后经重组,筛选后获得的重组VP1和VP2基因产物,IBDV经甲醛灭活后与CAV按适当比例混合。1：4与白油佐剂研制成油包水型乳化剂二联疫苗,经安全试验、免疫保护试验证明该二联灭活疫苗安全有效,免疫种鸡群后可使其后代产生IBDV和CAV抗体,雏鸡得到较好的保护。     

An immunoperoxidase monoclonal antibody stain for rapid diagnosis of infectious bursal disease

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.