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.     

Characterization of infectious bronchitis virus using monoclonal antibodies

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.     

A dot-immunobinding assay for infectious bronchitis virus

Common Whatman filter paper grade 1 and nitrocellulose membrane were compared for their sensitivity in a dot-immunobinding assay for detection of serum antibody titers to Arkansas avian infectious bronchitis virus (AIBV). For a blue to purple color detection, serum antibodies were bound to AIBV antigen adsorbed on the filter-paper discs or nitrocellulose membrane. Rabbit anti-chicken IgG horseradish-peroxidase (HRP) conjugate and hydrogen peroxide with 4-chloro-1-naphthol (HRP-color development reagent) were applied. The study indicates that very small amounts of antigen/antisera are needed for the dot-immunobinding assay. The test is sensitive, economical, and easy to run and can be completed within 6-8 hours.     

Detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibody

Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis.     

Isolation of avian infectious bronchitis virus from experimentally infected chickens.

Virus was recovered from the faeces of chickens infected at three or four weeks of age for more than 20 weeks after infection with commercial vaccines or with the T strain of avian infectious bronchitis virus (IBV). Virus was not recovered from the trachea, liver, spleen, bursa or kidneys of T strain infected birds longer than 29 days after infection at which point IBV was recovered from the bursa of a single infected bird. In a subsequent experiment IBV was recovered from the caecal lymph nodes and faeces of one of five birds 14 weeks after infection with a commercial vaccine but no virus was isolated from the trachea, kidneys, duodenum, bursa, ovaries or testes of any of the five birds at this time.     

感染IBV雏鸡气管肺及肾组织中P物质动态观察

鸡传染性支气管炎（IB）是由鸡传染性支气管炎病毒（IBV）引起的鸡的一种急性、高度接触性传染的病毒性疾病，主要侵害鸡的呼吸、泌尿生殖器官和消化等系统，可对不同日龄的鸡造成不同程度的危害。同时，IBV往往引起鸡只混合感染（如新城疫、慢性呼吸道病等），并可继发细菌性疾病，从而加重了对鸡群的危害。P物质是存在于呼吸道中并对呼吸道功能起着保护、调节作用的一种脑肠肽类物质，     

鸡传染性支气管炎病毒Real-time PCR方法的建立及其对感染鸡体内病毒的检测

本研究针对传染性支气管炎病毒（IBV）tl／CH／LDT3／03毒株的N基因（GenBank登录号为AY702975）设计并合成了一对引物,构建重组质粒作为阳性标准品,建立了检测IBV核酸的SYBRGreenⅠ荧光定量PCR方法。该方法可检测到初始模板中6．45×10copies／μL的病毒核酸。与常规PCR相比,敏感性高100倍。该检测方法特异性强,与其它禽源病毒如新城疫病毒（NDV）、传染性喉气管炎病毒（ILTV）、传染性法氏囊病毒（IBDV）、禽流感病毒（AIV）、马立克氏病毒（MDV）均不发生交叉反应;重复性试验的变异系数小于2．6％。结果表明,本试验建立的荧光定量PCR检测方法灵敏度高、特异性强、重复性好,可用于传染性支气管炎的临床诊断。同时应用本方法对人工感染IBV的SPF鸡的主要脏器盲肠扁桃体、肾脏、肺和气管进行了病毒RNA定量检测,结果表明,肾脏的病毒含量最高,盲肠扁桃体中病毒持续的时间最长,从而揭示了IBV在SPF鸡体内复制的动态变化,证实了感染鸡的临床表现与病毒滴度的时间相关性。     

Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.     

A monoclonal antibody-based immunoperoxidase monolayer (micro-isolation) assay for detection of type 1 and type 2 bovine viral diarrhea viruses.

A monoclonal antibody (mAb)-based immunoperoxidase monolayer assay (IPMA) for detection of bovine viral diarrhea virus (BVDV) was developed and compared with an existing bovine polyclonal antibody (pAb)-based IPMA. A pool of 5 mAbs, 4 mAbs produced to a type 1 BVDV and 1 mAb produced to a type 2 BVDV, was utilized in the mAb-IPMA. The mAbs were chosen for inclusion in the pool because of their broad cross-reactivities with type 1 and/or type 2 BVDV, their apparent avidities for antigen, their reactivity to different BVDV proteins, and their lack of competition for binding sites or their binding to unusual BVDV isolates. The mAb-IPMA outperformed the pAb-IPMA in staining, ease of reading test results, and relative sensitivity with a panel of known BVDV positive and negative sera. The relative sensitivities of the mAb-IPMA and pAb-IPMA were 100% and 93.5%, respectively, for 62 positive samples including several that were known to contain type 2 BVDV. With retesting, the pAb-IPMA gave a similar level of sensitivity as that of the mAb-IPMA. Both tests gave a specificity of 100% for 40 negative serum samples obtained from a BVDV-free herd.     

Nephropathogenesis of chickens experimentally infected with various strains of infectious bronchitis virus

Four-day-old specific-pathogen-free chickens were inoculated by eyedrop with four different strains (Gray, JMK, CV56b, and Wolgemuth) of infectious bronchitis virus (IBV). Birds were monitored clinically and euthanatized at 1, 4, 7, and 14 days postinfection and tissues were collected for virus isolation, histopathologic examination, in situ hybridization (ISH), and immunohistochemistry (IHC). Clinical disease was severe in chickens infected with Wolgemuth, but no overt disease was observed with the other strains. Virus was isolated from the kidneys of chickens infected with the Gray-, CV56b-, and Wolgemuth-strains of IBV. Histologically, interstitial nephritis was evident in chickens infected with these same 3 strains. However, viral nucleic acid and antigen were detected only with Wolgemuth-infected kidneys by ISH and IHC. These results indicate that the pathological changes in kidneys from chickens infected with Gray and CV56b may not have resulted from the cytolytic action of the virus.     

Kinetics of lymphocytic subsets in chicken tracheal lesions infected with infectious bronchitis virus

The kinetics of T-cells (CD3 positive (+), CD4+ and CD8+ cells) and B-cells (IgG+, IgM+ and IgA+ cells) in chicken trachea were studied immunohistochemically and histopathologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 16 hr to 6 days post-inoculation (p.i.) with a peak on 4 days p.i. A few IgG+, IgM+ and IgA+ cells were detected in the submucosa from 8 hr p.i. Thereafter IgG+ and IgM+ cells were gradually increased in number, and dramatically increased from 3 days p.i., peaked on 4 days p.i., and gradually decreased after 5 days p.i. IgA+ cells were detected in a small number than IgG+ and IgM+ cells during the all experimental period. These B cells mainly existed in the lamina propria, and some cells were recognized in the interepithelial space. After 14 days p.i., small number of IgG+ and IgM+ cells were detected in the germinal center of lymph follicles in the lamina propria. From 24 to 60 hr p.i., a few number of CD3+, CD4+ and CD8+ cells were detected at the perivascular area in the lamina propria. After 3 or 4 days p.i., each positive T-cells increased rapidly in number, and reached on the peak at 5 days p.i. CD3+, CD4+ and CD8+ cells tend to distribute diffusely, perivascular area, and surrounding area of CD4+ cells, respectively. CD4+ cells were dramatically decreased from 7 days p.i., and CD3+ and CD8+ cells were decreased from 14 days p.i. No T-cells were detected in the lymph follicles in the lamina propria.     

鸡传染性支气管炎病毒沈阳分离株组织嗜性的研究

鸡传染性支气管炎病毒(IBV)不同毒株对两个主要的靶器官气管和肾脏的侵嗜倾向和损伤程度有很大差异,这种差异是决定病毒致病性的原因之一.目前,由于国内分离的IBV主要是以致病性、临床症状和病理变化作为IBV病型和鉴定指标,其准确性与可靠性存在很大的差异.因此,在实验室条件下将病毒分离株回归动物试验,通过不同的试验方法进行鉴定是非常必要的.本试验为探讨IBV SY分离株的组织嗜性,应用H.E.染色法和IFA技术对IBV感染后的SPF鸡不同脏器和病理变化及抗原定位进行了研究.     

The rapid detection of Aujeszky's disease virus in pigs by direct immunoperoxidase labelling

Direct immunoperoxidase labelling on impression smears of brain and pharynx was compared with virus isolation and direct immunofluorescence for the detection of Aujeszky's disease virus in experimentally-infected pigs. Immunoperoxidase labelling was as sensitive as immunofluorescence and more sensitive than virus isolation for tissue that had been stored at room temperature (approximately 20 degrees C) for up to 144 h.     

核酸探针检测鸡传染性支气管炎病毒方法的建立及应用

以地高辛(DIG)标记鸡传染性支气管炎病毒(IBV)pol基因的保守片段制成核酸探针,与IBV参考株、新城疫病毒、传染性法氏囊病病毒、禽流感病毒、正常鸡胚尿囊液及正常鸡肾组织等的RT-PCR产物进行斑点杂交,以检测探针的特异性,结果该探针仅与IBV毒株的RT-PCR产物杂交呈阳性,与对照病毒和组织的RT-PCR产物杂交呈阴性.敏感性试验显示,探针最低能检出约3.4pg的IBV RT-PCR产物.用该方法检测了38份疑似IBV临床病料,31份阳性;而用RT-PCR法扩增IBV S2基因确诊为阳性的只有29份.对人工接种IBV H52弱毒苗鸡咽喉和肛门拭子32份进行检测,检出15份阳性.结果表明,利用DIG探针检测IBV的RT-PCR产物,特异性和敏感性强,可重复,能克服RT-PCR非特异性反应和探针Northern杂交的不稳定性.