Detection of vaccine-like infectious bursal disease (IBD) virus in IBD vaccine-free chickens in Japan

The prevalence of infectious bursal disease virus (IBDV) was studied in chickens, which had not been vaccinated against IBD. Fifty sera and forty-six bursae of Fabricius from chickens showing impaired growth, collected from 7 IBD vaccination-free farms in Japan were used for virus neutralization (VN) tests and RT-PCR for detection of IBDV genome corresponding to the VP2 hypervariable region. Of the fifty sera, 39 sera (78%) from 6 farms were VN antibodies positive. Of the forty-six bursae, 37 bursae (80.4%) from 6 farms were positive in the RT-PCR assay. The sequences of all the RT-PCR products detected in this study were closely related or identical to those of the vaccine strains. These results show that vaccine-like IBDV is prevalent even in IBD vaccine-free chicken farms in Japan.     

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.     

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.     

反转录─套式PCR方法检测鸡传染性法氏囊病病毒

采用蛋白酶 Ｋ 消化,酚、氯仿抽提的方法提取法氏囊匀浆和细胞培养液中的鸡传染性法氏囊病病毒（ Ｉ Ｂ Ｄ Ｖ）基因组 Ｒ Ｎ Ａ,用特异的寡核苷酸引物对其 Ｖ Ｐ２ 高变区进行反转录套式 Ｐ Ｃ Ｒ 扩增。应用该方法从一个法氏囊匀浆中即可特异地检出 Ｉ Ｂ Ｄ Ｖ Ｒ Ｎ Ａ,得到的扩增产物可用于分子流行病学的进一步分析,而从感染材料的处理到扩增结果的电泳检测,在两个工作日之内可轻松完成。本实验为法氏囊病病毒的诊断和分子流行病学的分析提供了一种快速、简便、可靠的手段。     

The effect of infectious bursal disease virus on the immune system of turkeys

In 1979 it was reported that an infectious bursal disease virus (IBDV) isolated from a case of respiratory disease of turkeys differed antigenically from the chicken isolates of this virus. We injected turkey poults with the turkey-originating TY89 and chicken-originating BD/6 isolates of IBDV and studied their effects on antibody production to the virus, serum immunoglobulin G (IgG), antibody response to sheep erythrocytes, in vitro response of peripheral blood lymphocytes to mitogens, and microscopic structure of the bursa of Fabricius. The chicken isolate BD/6 caused a significant decrease in the response to sheep erythrocytes, lower serum IgG, transient decrease in the response of lymphocytes to PHA, and mild microscopic lesions in the bursa of Fabricius. The turkey isolate TY89, however, caused no obvious damage to the immune system of the infected poults. We suggest that a partial and transient functional disorder of the immune system of poults can occur after infection with IBDV originating from chickens, even if the poults exhibited no clinical signs.     

ChMDA5通路参与传染性法氏囊病病毒致雏鸡法氏囊损伤的研究

传染性法氏囊病病毒（IBDV）为dsRNA病毒,可造成雏鸡法氏囊损伤进而发生免疫抑制;MDA5是能够特异性识别dsRNA病毒的模式识别受体。为研究鸡MDA5（chMDA5）信号通路在IBDV致雏鸡法氏囊病理损伤中的作用,试验选取50只14日龄SPF雏鸡随机分为IBDV感染组和空白对照组,每组25只,IBDV感染组雏鸡通过点眼、滴鼻感染IBDV JIC7株病毒液,0.6 mL·只^-1,空白对照组雏鸡经相同途径给予相同剂量无菌PBS,感染IBDV后第1、4、7、21及35天采集雏鸡法氏囊。采用qRT-PCR方法检测法氏囊中IBDV载量,chMDA5及chMDA5信号通路衔接蛋白（chIPS-1）、转录因子（chIRF3和chNF-κB）、下游产物细胞因子（chIFN-β、chTNF-α、chIL-1β、chIL-6） mRNA水平变化;间接免疫荧光法检测chMDA5蛋白表达变化,传统病理学方法检查法氏囊病理组织学变化。结果发现,雏鸡感染IBDV后,其法氏囊中chMDA5、chIPS-1、chIRF3、chNF-κB、chIFN-β、chTNF-α、chIL-1β、chIL-6的表达量均显著高于对照组,且法氏囊组织发生形态损伤,上述变化趋势与IBDV载量变化基本一致。结果表明,雏鸡法氏囊chMDA5及其信号转导通路可被IBDV激活,参与到IBDV感染雏鸡法氏囊损伤与抗损伤过程中。     

Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.     

Association of increased rate of condemnation of broiler carcasses due to hepatic abnormalities with immunosuppressive diseases in the broiler chicken industry in Saskatchewan

The objective of this study was to identify the causative agents of hepatitis observed in broiler chickens at processing. Livers of chickens from 16 broiler farms in Saskatchewan with gross lesions of hepatitis were collected at processing. In addition to routine bacterial isolation and histopathological examination, serologic studies for infectious bursal disease virus (IBDV) and Chicken anaemia virus (CAV), calculation of the ratio of the weight of the bursa of Fabricius (BF) to body weight (BBW), and histopathological examination of the BF were done. Of the 264 livers with gross lesions, 83% had multifocal to coalescing necrotizing hepatitis, 16% had perihepatitis, and 1% had hemorrhages. No definitive causative microorganisms were isolated from the hepatic lesions; however, no significant bacterial isolations were made. Bursal atrophy, low BBW ratio, and high titer of antibody against IBDV each correlated with the rate of total condemnations (P = 0.0188, P = 0.0001, and P = 0.0073, respectively). Nucleotide sequencing of IBDV isolated from the BF identified the variant strains Delaware-E and 586. Condemnation because of hepatic lesions was correlated with titer of antibody against IBDV and BBW (P = 0.016 and P = 0.027). The results of this study demonstrate that hepatic lesions in Saskatchewan chickens are not currently caused by a primary bacterial pathogen but are associated with indicators of immunosuppression that is likely due to variant 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.     

Viral genotyping of infectious bursal disease viruses isolated from the 2002 acute outbreak in Spain and comparison with previous isolates

An infectious bursal disease (IBD) outbreak occurred in the east region of Spain in the spring of 2002 and rapidly spread thorough the whole country, although proper vaccination programs were applied. In this report, 33 infectious bursal disease viruses (IBDVs) isolated from this outbreak were characterized by nucleotide sequencing of the VP2 gene hypervariable region and were compared with reference IBD strains and the 1990s Spanish IBDVs in order to determine possible emergence of IBDV isolates with modified antigenic or virulent properties. Moreover, histopathologic and immunohistochemical studies of those cases where bursal tissues were available were carried out. Of the 33 isolates, 23 were identified as very virulent IBDVs (vvIBDVs), whereas the other 10 isolates were classified as attenuated or intermediate virulence classical strains and could possibly be IBDV live vaccine strains used in the immunization of these chickens. Results of this study indicate that wIBDV isolates from the 2002 Spanish outbreak are closely related with those from the 1990s outbreak. However, acute IBD cases have not been reported in Spain during these 10 yr. Genetic, management, and environmental factors likely related with IBD reemergence in Spain are discussed. Moreover, our results indicate that good correlation exists between the IBDV subtype present in the field and the degree of lesions in bursa tissue, as well as the immunohistochemistry staining.     

Digoxigenin-labeled nucleic acid probe for the detection of infectious bursal disease virus in infected cells.

A cDNA probe was synthesized from the VP-4 region of a virulent field isolate of infectious bursal disease virus (IBDV). The probe was labeled during synthesis with a non-radioactive steroid hapten, digoxigenin. The probe was used to develop a hybridization assay to detect the presence of IBDV in infected cell-culture and tissue suspensions from the bursa of Fabricius of infected chickens. The test was rapid, reproducible, and sensitive, and it could detect four serologic subtypes of IBDV, including the GLS-5 isolate.     

传染性法氏囊病病毒NN1107广西株的分离鉴定及遗传进化分析

为研究从广西南宁市某鸡场疑似患传染性法氏囊病的鸡群中分离鉴定出的一株传染性法氏囊病病毒NN1107株的分子特征,通过反转录-聚合酶链反应特异扩增后进行克隆、序列分析。NN1107株VP2基因高变区(vVP2)的克隆测序和序列比较结果显示,序列符合超强毒传染性法氏囊病病毒(vvIBDV)的分子特征;其与广西vvIBDV毒株的核苷酸同源性在96%～99.6%之间。根据vVP2核苷酸序列同源性绘制的遗传进化树结果显示,NN1107株属于vvIBDV毒株群,与2004年、2005年、2007年和2010年流行毒株BH09、NNTZ(3)、NN07122、HP1001的亲缘关系最近,与疫苗株B87(in)、Bursine-2、FW2512株的亲缘关系较远。     

安徽地区IBDV超强毒株的分离鉴定和VP2基因序列分析

经鸡胚绒毛尿囊膜(CAM)接种、易感鸡接种试验、电镜观察,从安徽地区疑似病鸡的法氏囊组织分离到3株传染性法氏囊病毒。分离株人工感染4周龄鸡,致死率分别为92%、83%、67%。接种9～10SPF鸡胚测得的鸡胚半数致死量(ELD50)分别为10-6.8/0.2mL、10-5.4/0.2mL、10-4.6/0.2mL。应用Nested-PCR分别对3株分离株VP2基因高变区进行克隆测序和序列分析,结果表明:3个分离株与国内外参考超强毒株的核苷酸同源性为97.2%～99.5%,氨基酸同源性为99.3%～100%,VP2高变区核苷酸和推导的氨基酸符合传染性法氏囊病病毒超强毒株特征。