Detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa

In situ hybridization and immunohistochemistry were utilized to identify tissues infected in ovo with infectious bronchitis virus (IBV). Chicken embryos were inoculated in ovo (chorioallantoic sac) with the Arkansas (Ark) serotype of IBV at 18 days of age. At 24, 48, 72, and 120 hr postinfection (HPI), bursa, lung, spleen, heart, and thymus were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. The digoxigenin-labeled antisense S1 riboprobe detected viral mRNA in the cytoplasm of respiratory epithelial cells in the primary bronchus at 24, 48, and 72 HPI. Viral mRNA was detected in bursa samples collected at 48 hr. Immunohistochemistry detected viral antigens in epithelial cells of the parabronchi and bursal tissues at 24 and 48 hr, respectively. No viral mRNA or antigen was detected by in situ hybridization or immunohistochemistry, respectively, in heart, thymus, or spleen at any time after inoculation. On the basis of these data, IBV apparently initially infects lung tissue, then migrates to and infects cells of the bursa. These results indicate that in situ hybridization can be useful in detection of IBV-infected chickens and in understanding the pathogenesis and virulence of IBV infection.     

Detection of infectious bursal disease vaccine viruses in lymphoid tissues after in ovo vaccination of specific-pathogen-free embryos.

Control of infectious bursal disease virus (IBDV) by vaccination is important for poultry production worldwide. Two vaccines, an IBDV immune complex (ICX) vaccine and an IBDV-2512 vaccine, were administered at 100 mean embryo infectious dose to specific-pathogen-free 18-day-old broiler embryos in ovo. At 3, 6, 9, 15, and 21 days post in ovo vaccination (PIOV), bursa, spleen, and thymus tissues were collected and analyzed for virus protein by antigen capture chemiluminescent enzyme-linked immunosorbent assay (ELISA). Chicks were bled and antibody titers were determined by the antibody ELISA. At 21 days PIOV, chickens were challenged with a 1:500 dilution of an antigenic standard IBDV strain. At 28 days PIOV, birds were euthanatized and bursa weight:body weight ratios were determined. Embryos vaccinated with either vaccine exhibited 92% hatchability; however, within 1 wk of hatch, birds vaccinated with IBDV-2512 showed 56% mortality, whereas those given IBDV-ICX had only 3.2% mortality. Both IBDV-ICX and IBDV-2512 vaccines were detected in bursa, spleen, and thymus at day 3 PIOV. A 5-day delay in virus replication was observed with IBDV-ICX vaccine. By day 15 PIOV, the IBDV-ICX was no longer detectable in the bursa and spleen but persisted in the thymus. The IBDV-2512 vaccine persisted in the spleen and thymus on day 15 PIOV. By day 21 PIOV, neither vaccine virus was detected in any lymphoid organ. This assay can be useful in the early detection of vaccine virus in the tissues of chickens vaccinated via the in ovo route. Both vaccines caused bursal atrophy at all times PIOV. The IBDV-2512 caused splenomegaly at day 6 PIOV, whereas splenomegaly was not seen in IBDV-ICX-vaccinated birds until day 9 PIOV. Thymus atrophy was observed in IBDV-2512-vaccinated chicks from day 3 PIOV, whereas this occurred on day 15 PIOV in IBDV-ICX-vaccinated birds. Bursa weight: body weight ratios in IBDV-ICX-vaccinated unchallenged and vaccinated challenged birds were not different (P < 0.05).     

鸡传染性法氏囊病的病理学研究

人工接种２８日龄非免疫鸡传染性法氏囊病病毒（ＩＢＤＶ）后,对感染鸡的法氏囊、胸腺、脾、盲肠扁桃体、哈德氏腺、肝、肾进行病理组织学检查。感染后４８ｈ,法氏囊淋巴组织最早出现坏死且长久存在。其他淋巴器官的病变出现较迟,程度轻微且恢复较快。ＩＢＤＶ单抗免疫荧光检测,法氏囊及其他淋巴器官中均检测到病毒,接种后１２ｈ法氏囊中即检出病毒,持续时间也最长（攻毒后１２ｄ）,其次是盲肠扁桃体（攻毒后８ｄ）。攻毒１３ｄ以后,上述器官均未检测到病毒。法氏囊粘膜上皮的扫描电镜观察,攻毒后２ｄ,上皮细胞肿胀,微绒毛减少或消失。攻毒后３ｄ,局部上皮细胞坏死、脱落,并向整个粘膜层扩展,攻毒后１０ｄ,上皮层基本修复。     

传染性法氏囊病病毒（IBDV）人工感染SPF鸡的病理学观察

取３２０只３０～４０Ｈ龄ＳＰＦ鸡，经滴鼻和点眼接种传染性法氏囊病病毒（ＩＢＤＶ），接种后２４～７２ｈ出现死亡，病死率５１．２５％，死亡鸡剖检变化主要为法氏囊肿胀、出血，甚至呈紫葡萄样，脾脏肿大、出血，骨骼肌出血，腺胃肌胃交界处出血。病理组织学变化，呈现以法氏囊淋巴滤泡内髓质淋巴细胞坏死为主的特征性病变，并可在巨噬细胞浆内发现病毒包涵体。电镜观察，在法氏囊内淋巴细胞、异染性细胞、巨噬细胞浆内，见大量晶格状排列的病毒粒子和包涵体，表明ＩＢＤＶ首先损害法氏囊淋巴滤泡髓质内未成熟的Ｂ淋巴细胞，病毒在淋巴细胞内以包涵体方式增殖。     

A study of the epidemiology of infectious bursal disease in poultry in Queensland, 1976–1979

The epidemiology of infectious bursal disease (IBD) was studied by serology and sometimes by visual examination of the bursa of Fabricius in poultry flocks in Queensland during 1976–1979.
Ten flocks, each of approximately 30,000 meat breeding chickens, were surveyed. All chickens had maternally-derived antibody against IBD virus (IBDV) at hatching and active antibody was not detected while the chickens were brooded on rearing farms. When distributed to breeding farms, 7 of the flocks developed antibody when 11 to 25 weeks of age. The remaining 3 flocks were vaccinated by infection of 10% of the birds and within 4 weeks more than 80% of the chickens had developed precipitating antibody to IBDV.
Blood samples of 20 to 30 broiler chickens were collected at slaughter (7 to 9 weeks of age) from each of 312 broiler flocks raised on 37 contract farms. While the samples from 21 flocks were without detectable antibody to IBDV, all serum samples for 263 flocks contained antibody. The ratio of bursal weight to bodyweight was significantly lower in birds from 144 flocks having antibody to IBDV than in birds from 10 flocks that were without detectable antibody. In sequential studies, IBDV antibody became demonstrable in 27 of 30 flocks when the chickens were one to 6 weeks of age and was accompanied by bursal atrophy.
Serological investigation of 4 flocks of layer breeding chickens on a multi-age farm at approximately monthly intervals resulted in antibody to IBDV being detected at every examination.
Serological tests and bursal examinations were carried out weekly in 2 flocks each of 4000 layer chickens between one and 20 weeks of age. Serum antibody developed in one flock at 4 weeks of age and in the other at 17 weeks of age. In both flocks, bursal atrophy occurred concurrently with the development of antibody.     

Organ culture of chicken bursa as a model to study the pathogenicity of infectious bursal disease virus isolates.

In vitro study with chicken bursal organ culture was attempted to assess the pathogenicity of locally isolated infectious bursal disease virus (IBDV) initially isolated from the bursa of naturally infected birds. In bursal organ culture, lymphoblastic transformation was noticed as early as 24 hr postinoculation and reached maximum at 72 hr postinoculation. The other microscopic changes were increased number of macrophages and formation of plasma cells. The IBDV antigen was detected 24 hr onward by coagglutination test with antibody coated Staphylococcus aureus strain Cowan I. On the basis of lesion score, the three isolates of IBDV (A, B, and C) were graded as virulent (B isolate) and moderately virulent (A and C isolates). A similar pattern of pathogenicity was also observed in the in vivo pathogenicity studies in chicken based on bursa: body weight ratio and histopathologic lesion score. The bursal organ culture thus provides a useful experimental model to differentiate the IBDV isolates on the basis of their virulence.     

鸡传染性法氏囊病超强毒Gx与疫苗毒Gt在鸡体内的复制研究

本试验利用鸡传染性法氏囊病超强毒Gx及其致弱疫苗毒Gt为对象,研究超强毒与弱毒株在鸡体主要免疫器官内的复制情况,以探讨两类毒株表现不同生物特性的原因。分别利用鸡胚半数致死量和鸡胚成纤维细胞半数感染量对超强毒Gx和疫苗株Gt进行病毒滴度的测定;再利用荧光定量RT-PCR对两类毒株进行病毒量的校准。以相同量的病毒对2周龄SPF鸡进行攻毒。攻毒试验表明超强毒Gx能造成47.5%的死亡,法氏囊、脾脏、胸腺等免疫器官均严重损伤;而疫苗毒Gt无致死,且未能造成任何病理可见的损伤。病毒的体内复制情况表明超强毒相对于疫苗毒复制更为迅速,病毒载量更高。     

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.     

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. I. Kinetic alterations of avian lymphocyte subpopulations.

The potential effect of chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens was determined by measuring alterations in hematocrit values, lymphoid organ-to-body weight ratios and lymphoid cell concentrations at 4, 7, 10, 14, 17, 21, 28 and 42 days post-inoculation (PI). Lymphocyte subpopulations were identified and counted by flow cytometry using cell suspensions stained with monoclonal antibodies (Mabs) for panlymphocytes (K55), cytotoxic T-cells (CTLA3), T-helper cells (CT3), Ia-expressing cells (P2M11) and macrophages (P7). Chicken anemia agent induced a substantial but transient decrease in hematocrit value, thymus-to-body weight ratio and bursa-to-body weight ratio between 7 and 21 days PI corresponding to a generalized lymphocytopenia in the thymus, bursa and spleen. However, cytotoxic T-cell, T-helper cell and Ia-expressing cell concentrations increased in the bone marrow of birds inoculated with CAA alone or in combination with IBDV during the same time period. T-helper-to-cytotoxic T-cell ratios increased in the thymus and spleen during severe lymphocytopenia, indicating a selective decrease in cytotoxic T-cells. T-helper-to-cytotoxic T-cells ratios increased in the bone marrow, indicating a selective increase in T-helper cell concentrations. The increase in Ia-expressing cells in the bone marrow may be a reflection of increased number of activated T-cells which express Ia antigen. Infectious bursal disease virus alone induced a persistent depression of Ia-expressing cells in the bursa and the spleen and no measurable change in the bone marrow lymphocyte subpopulations. Chickens inoculated simultaneously with CAA and IBDV experienced clinical signs observed in chickens inoculated with each virus separately with a prolonged acute phase prior to recovery or mortality.     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens

Specific-pathogen-free chickens inoculated with isolate VA (variant A) or isolate IM of infectious bursal disease virus (IBDV) were examined for mitogenic response to T-cell mitogens, primary and secondary antibody response to sheep erythrocytes and Brucella abortus, and gross and histologic lesions in thymus and bursa. Both isolates induced comparable depression in the mitogenic and antibody response, and both caused extensive gross and histologic lesions in the bursa of Fabricius. However, bursal necrosis induced by the IM isolate was accompanied by an inflammatory response, whereas the inflammatory component was lacking in the lesion induced by the VA isolate. Furthermore, the IM isolate induced extensive lesions in the thymus, but the VA isolate did not.     

Quantitative measures of disease in broiler breeder chicks of different major histocompatibility complex genotypes after challenge with infectious bursal disease virus

Criteria for evaluating genetic differences in resistance and susceptibility to infectious bursal disease (IBD) within a commercial broiler breeder line of chickens were compared. Line A broiler breeder chickens were challenged with graded doses of Animal and Plant Health Inspection Service (APHIS) strain IBD virus (IBDV) and evaluated at 2 time points, 3 days postinoculation (PI) and 10 days PI. Measures obtained at both time points included bursa to body weight, bursa histology, bursa lymphocyte count, and percentage of T cells in the bursa. Furthermore, viral load in the bursa was determined 3 days PI and anti-IBDV antibody titers, 10 days PI. A dose of 50 50% embryo infective dose caused IBD in about half the line A birds at the 10-day time point, and this dose was chosen for further studies. The data were analyzed for correlation among the various measures. Comparison of the 3-day- and 10-day-PI bursa lymphocyte counts indicated that birds challenged with low doses of virus suffered lymphocyte depletion at the 3-day time point, but many or all (depending on the dose) recovered by the 10-day time point. With a viral dose that caused bursal atrophy in about half the birds by 10 days PI, families segregating for 2 major histocompatibility complex (MHC) haplotypes were compared in terms of resistance to IBD. Results indicated that there was no difference among the 3 MHC genotypes in incidence of IBD by any of the disease measures.     

鸭瘟病毒强毒株在感染鸭实质器官内的增殖与分布

鸭瘟病毒(DPV)CHv强毒株经皮下注射、滴鼻和口服3种途径分别感染20日龄天府肉鸭,于攻毒后10、30、60、90min以及4、12、48、72h和9、15d每组分别剖杀2只鸭,采集心、肝、脾、肺、肾、脑、胸腺、法氏囊、哈德氏腺等实质器官,应用TaqMan-MGB探针实时荧光定量PCR对DPV在这些器官的分布和增殖进行检测。结果表明,DPV分布到具体器官的速度与感染的途径、鸭的解剖结构密切相关,其中皮下注射是DPV分布到各实质器官速度最快的途径。30min于皮下感染鸭的肝、脾、胸腺、法氏囊、哈德氏腺、肺、脑、肾,口服感染鸭的肺和法氏囊,滴鼻感染鸭的心脏和哈德氏腺均检测到DPV-DNA;90min所有受检样品中检测到DPV-DNA。鸭抗DPV感染的免疫器官的重要性依次是脾、胸腺、法氏囊和哈德氏腺,30min内DPV-DNA分布到脾、胸腺、法氏囊的速度和数量决定了DPV感染的潜伏期和疾病的严重程度。不同途经感染鸭的相同器官在同一时间内的DPV-DNA拷贝数大多以皮下感染鸭为最高。DPV致死鸭的法氏囊和肾是DPV-DNA含量最高的实质器官。     

Characterization of infectious bursal disease viruses isolated in 2007 from Delmarva commercial broiler chickens

A study was performed in 2007 to isolate and characterize infectious bursal disease viruses (IBDVs) in commercial broilers grown in the Delmarva (DMV) Peninsula region of the United States. Bursae of Fabricius were collected weekly from 1 to 4 wk of age from broilers on 10 farms with a history of poor performance. Microscopic pathology was used to determine the infectious bursal disease (IBD) status of the broilers. Bursae from 1- and 2-wk-old broilers did not show IBD microscopic lesions. Moreover, broilers on 1 of the 10 farms were IBD lesion free at 3 and 4 wk of age. However, 3 of 9 and 9 of 9 farms yielded broilers with IBD-affected bursae from 3- and 4-wk-old commercial broilers, respectively. Ten IBDV isolates were recovered from 3 of 3 lesion-positive bursal pools at 3 wk of age and 7 of 9 lesion-positive bursal pools at 4 wk of age. Analysis of the viral protein (VP) 2 genes identified all isolates as serotype 1 Delaware (Del) variant viruses. Five field isolates, each representing different molecular clades of the Delaware variant viruses, were selected for further study. Experimental infection of specific-pathogen-free white leghorn chickens with isolates DMV/4813/07, DMV/4947/07, DMV/4955/07, DMV/5038/07, and DMV/5041/07 produced gross and microscopic pathology of the bursa consistent with Delaware variant infection. Monoclonal antibody testing showed DMV/4813/07, DMV/4947/07, DMV/ 4955/07, and DMV/5041/07 to be similar to previous recognized variant viruses. However, DMV/5038/07 was found to be unreactive with the monoclonal antibodies that typically recognize reference strains STC, Del E, GLS, RS593, and AL2. In a challenge of immunity study, 10-day-old progeny from breeders immunized with a commercially available inactivated IBDV vaccine containing the Del E and classic strains were protected to a lesser degree against isolate DMV/5038/07 compared to Del E challenge based on microscopic lesion scores (P < 0.01) of the bursa. This result suggests the virus is antigenically different from the Del E strain contained in the vaccine. Collectively, the monoclonal antibody and progeny challenge of immunity findings suggest DMV/5038/07 is antigenically different from the Del E strain contained in the vaccine.