Tissue-print hybridization using a non-radioactive probe for the detection of infectious bursal disease virus.

Tissue-print hybridization was evaluated as a simplified means for detection of infectious bursal disease virus (IBDV) in the bursa of Fabricius from infected chickens. The assay employed a biotin-labeled synthetic oligonucleotide as a probe. The bound probe was detected using a color assay consisting of streptavidin conjugated to alkaline phosphatase. Bursae were imprinted onto nitrocellulose and then hybridized with the biotinylated probe. Bursal prints from IBDV-infected chickens were readily distinguished from control prints by color development and differences in signal intensity.     

Heteroduplex mobility assay for genotyping infectious bursal disease virus

A heteroduplex mobility assay (HMA) was developed to genotype infectious bursal disease virus (IBDV). This method analyzed 390-base pair (bp) polymerase chain reaction (PCR) products, encompassing the hypervariable region of the VP2 gene. IBDV strains from the United States and other countries were analyzed. The HMA was able to differentiate standard, antigenic variants and very virulent strains of IBDV. Minor differences between different strains from the same subtype were also detected. Close relationships between field IBDV with vaccines prepared with Delaware E strain were determined by HMA. The results obtained by HMA were confirmed by restriction fragment length polymorphism (RFLP) and phylogenetic analysis of nucleotide sequences. The HMA proved to be a useful technique to rapidly genotype different field strains of IBDV and should prove to be a useful tool in epidemiologic studies.     

鸡传染性法氏囊病病毒的快速检测

随着 IBDV变异株的出现 ,发病鸡常不产生法氏囊的肉眼病变 ,由于传染性和非传染性因素亦可引起淋巴细胞减少和法氏囊坏死 ,所以 ,采取组织病理学方法诊断 IBDV感染并不完全可靠。由于主动性抗体应答需要一定的时间及大多数雏鸡都带有母源抗体 ,因此血清学早期诊断也不完全可靠。该试验采用本所研究的 IBD快速试纸与经典的琼扩试验 ,在对 IBDV的检测效果上进行了详细的比较 ,介绍如下。1　材料与方法1 .1　 IBD快速检测试纸　由河南省农业科学院生物技术研究所制备提供。1 .2　试验鸡　 1日龄试验鸡 30 0只由河南农业大学试验鸡场提…     

传染性法氏囊病病毒检测系统的建立

为了建立传染性法氏囊病病毒(IBDV)检测系统,本试验在同一个试验技术平台上对9种IBDV检测方法进行了比较分析.IBDV野毒株盲传4～5代可适应于鸡胚和鸡胚成纤维细胞,死亡胚体水肿、出血,病变细胞圆缩、脱落.4个血清Ⅰ型1BDV毒株,用交叉中和试验可进一步分为3个血清亚型.用RT-PCR/SSCP技术分析4个毒株,扩增产物的电泳迁移率有差异.AGP、间接ELISA、夹心抑制ELISA以及中和试验等4种方法同时检测不同来源血清样品中的IBDV抗体,其他3种方法比AGP具有更高的敏感性,敏感度相差104倍.用AGP检测法氏囊组织样品,抗原效价可达到1:10,而细胞培养物和病鸡粪便则没有出现沉淀线;3种样品用夹心ELISA、RT-PCR、cDNA探针斑点杂交、RT-PCR/SSCP检测以及病毒分离等5种方法检测,均为阳性.     

Double-antibody sandwich ELISA for detection of infectious bursal disease virus.

A double-antibody sandwich ELISA was employed for detection of IBD virus in bursal suspension. Fifty IBD-free white leghorn chickens aged 5 weeks were experimentally infected with IBD virus. Bursae were collected 4, 8, 12, 24, 36 and 48 hours and 3, 4 and 5 days post-infection. An equal number of chickens acted as appropriate controls. The colour difference between a positive and a negative reaction was clearly distinguished with the naked eye. The cut-off level between ELISA negative and ELISA positive absorbance values was estimated at mean absorbance of negative controls plus three times the standard deviation.     

Improved preparation of ELISA antigen of infectious bursal disease virus.

Serotype 1 of infectious bursal disease virus (IBDV) adapted to chicken embryo fibroblasts (CEF) was used for the preparation of enzyme-linked immunosorbent assay (ELISA) antigen. After several passages of diluted viruses in CEF cultures, the titer of seed virus increased to 1.2 x 10(8) plaque-forming units/ml. Purified virus prepared from this seed virus had high titers of antigen and was less nonspecific than that from low titer of seed virus in an ELISA. The nonspecific reaction of purified virus decreased further after treatment with Triton X-100. When the specificity of this treated antigen was examined with specific-pathogen-free chicken sera before and during lay and with 14 antisera to some major avian viruses, this ELISA antigen had no nonspecific reaction and was specific to antibodies to serotypes 1 and 2 of IBDV.     

An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus.

The application of the indirect enzyme-linked immunosorbent assay (ELISA) for detection of infectious bursal disease virus antibodies in chicken serum was investigated. The test procedure involved the coating of concentrated infectious bursal disease virus antigen onto polystyrene tubes, followed by the addition of chicken anti-infectious bursal disease virus serum and horseradish peroxidase labeled rabbit anti-chicken globulin. As an indicator substrate, 5-aminosalicylic acid, with the oxidant H2O2 was added. The reaction was stopped by 3M NaOH and the colour intensity of the reaction mixtures read in a spectrophotometer at 449 nm. The ELISA test was found to be a precise, sensitive and reproducible means of measuring infectious bursal disease virus antibodies in chicken and turkey sera.     

Correlation of enzyme-linked immunosorbent assay titers with protection against infectious bursal disease virus.

We examined the utility of baculovirus-expressed infectious bursal disease virus (IBDV) proteins to act as antigens in the enzyme-linked immunosorbent assay (ELISA). The three IBDV protein antigens tested included 1) a truncated VP2, 2) whole VP2, and 3) the polyprotein products VP2, VP3, and VP4. Serum samples from 2-wk-old commercially reared broilers were collected and tested in the three ELISAs. Serum samples were obtained from 34 different commercial broiler flocks. An average of 14 serum samples (range = 11-17) were tested for each flock. The ELISA results were compared with the percentage of protection of these birds following challenge with IBDV. Fifty 2-wk-old chicks from each of the 34 broiler flocks were challenged with STC classic virus or Del-E variant virus. At 7 days postchallenge, the bursa from each of the birds was removed and bursa/body weights were recorded. Percentage of protection was determined by the number of birds in each challenge group that had normal relative bursal weights compared with unchallenged controls. No evidence was found of a relationship between ELISA data generated with the polyprotein antigen (VP2, VP3, VP4) and percentage of protection observed in the STC and Del-E challenged birds. A significant relationship was found between ELISA data and percentage of protection to STC and Del-E when the truncated VP2 or whole VP2 antigens were used in the ELISA. The results of this study indicate that predicting the percentage of protection against classic or variant IBDV strains in broilers from vaccinated breeder flocks can be improved when VP2 is used as the only antigen in the ELISA.     

用改良的RT-PCR技术快速检测传染性法氏囊病病毒

本研究将最新的病毒核酸纯化技术和单管ＲＴ－ＰＣＲ技术相结合,应用于ＩＢＤＶ的诊断研究,对４０余个病毒样品进行扩增反应,均取得令人满意的效果。该技术灵敏快速,从核酸纯化到电脉检测ＰＣＲ产物只需５～６ｈ,反应灵敏度比传统方法至少提高１００倍。所设计的引物对毒株的适用范围较广,对１２个ＩＢＤＶ毒株和１２个病变法氏囊样品均得到分子量一致并与设计相符的扩增产物     

马立克氏病病毒感染鸡对新城疫疫苗和法氏囊疫苗免疫反应的影响

研究了马立克氏病病毒(MDV)不同致病型毒株感染鸡后对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗抗体反应的影响。结果表明，2周龄接种MDV超强毒株RBIB和7日龄接种MDV强毒株GA的鸡HI效价均显著低于对照鸡，但在本试验条件下尚未发现MDV感染对法氏囊病病毒疫苗免疫效果有显著影响。     

对某鸡场传染性法氏囊病的诊治体会

传染性法氏囊病是养鸡场常发的一种急性传染病，其发病率高、病程短、死亡率高，雏鸡感染后还可导致免疫抑制，并可诱发多种疫病或使多种疫苗免疫失败。最常见的是导致鸡新城疫、鸡传染性支气管炎的免疫失败，给养鸡业带来巨大的经济损失。我县某养鸡场今年年初暴发该病，现将其诊断及治疗过程报告如下。     

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.