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。     

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。     

传染性法氏囊病免疫保护试验中3种评定指标的比较

100只SPF鸡均分为5组，A、B、c3组免疫后攻毒，接种3批次自制的IBD基因工程重组亚单位油乳剂疫苗；D组不免疫不攻毒，E组不免疫而攻毒。免疫后第22天，感染IBDV强毒株BC-6／85。攻毒后第4天，将所有存活的鸡只以颈脱臼致死，收集法氏囊，以3种方法和指标（法氏囊眼观病变；法氏囊显微病变；法氏囊中IBDV抗原检测）进行分析，以评定免疫保护率。结果显示，以这3种方法和指标评定，A组免疫保护率为90％，B、C组免疫保护率均为95％；试验鸡A3、A14、B7、C16、E1～E20，法氏囊眼观病变明显，法氏囊显微病理损伤评分为3～5分，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阳性；其他试验鸡，法氏囊眼观无明显异常，法氏囊显微病理损伤评分在3分以下，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阴性。结果表明，这3种方法和指标在IBD疫苗免疫保护试验评定中具有很好的一致性。     

Experimental cryptosporidiosis and infectious bursal disease virus infection of specific-pathogen-free chickens

Specific-pathogen-free chickens orally inoculated at 4 days of age with a moderately pathogenic vaccine strain of infectious bursal disease virus (IBDV) and/or at 5 days of age with Cryptosporidium baileyi oocysts remained free of overt clinical signs throughout a 16-day period postinoculation (PI). The prepatency period for C. baileyi oocyst shedding was shorter in chickens receiving higher numbers of oocysts, but once shedding was detected, there were no obvious differences in shedding patterns among groups receiving 10(3) through 10(6) oocysts. On days 8 and 16 PI, cryptosporidia were located primarily in the bursae of Fabricius. IBDV exposure was associated with bursal follicle atrophy, whereas C. baileyi infection resulted in bursal epithelial hypertrophy and hyperplasia, mild follicle atrophy, and heterophil infiltration of the bursal mucosa. Examination of experimental groups of 30 birds each indicated that concurrent infection with both agents resulted in more severe bursal lesions, more infected birds, and greater numbers of cryptosporidia in infected tissues. At the termination of the trial, 16 days PI, Cryptosporidium infection was associated with a 6% decrease in mean body weight compared with controls.     

The effects of age, route of exposure, and coinfection with infectious bursal disease virus on the pathogenicity and transmissibility of chicken anemia agent (CAA)

Specific-pathogen-free (SPF) chickens were inoculated with several different concentrations of chicken anemia agent (CAA) by the intra-abdominal, intratracheal, or oral routes. Based on lowered hematocrit values, the birds were most susceptible to CAA introduced by the intra-abdominal route. When SPF chickens were infected with infectious bursal disease virus (IBDV) at 1 day of age, they remained susceptible to CAA up to at least 21 days, whereas birds inoculated with CAA alone were susceptible only at 1 day of age. Infectious bursal disease virus introduced at 1 day of age also increased the susceptibility of birds to contact infection with CAA and resulted in increased mortality rates in CAA inoculates. The response of SPF birds to CAA infection varied following exposure at 1 day of age to two different strains of IBDV (STC and Variant-E). Chicken anemia agent contacts and inoculates infected with the Variant-E strain were affected 1 week earlier by CAA than by STC inoculates, as evidenced by depressed hematocrits. However, the total number of birds affected was similar for both the Variant-E and STC-inoculated chickens. Commercial broiler chickens inoculated at 1, 7, 10, and 14 days of age by non-parenteral routes with CAA or a combination of CAA and IBDV had mean hematocrits that were lower than controls. Several CAA-inoculated birds were considered anemic, with hematocrit values of 25 or less, while uninoculated birds remained within normal ranges.     

CAV与REV共感染SPF鸡对疫苗免疫反应的抑制作用

用1日龄SPF鸡人工感染鸡贫血病毒(CAV)和禽网状内皮增生病病毒(REV),探讨病毒感染对鸡体疫苗免疫反应的影响。结果表明,在用禽流感病毒(AIV,H5和H9)疫苗免疫后,CAV与REV单独感染均显著抑制了鸡体对H5和H9亚型禽流感病毒灭活疫苗的HI抗体反应,在CAV与REV共感染后,这种抑制作用更为明显。CAV单独感染后鸡体对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗的免疫反应受到抑制,但与对照组在统计学上的差异不显著,然而,CAV可以显著加重REV感染对鸡体在NDV和IBDV疫苗免疫后抗体反应的抑制作用。从而证实CAV与REV共感染在疫苗免疫抑制上有协同作用。     

Rocket Immunoelectrophoresis in the Diagnosis of Infectious Bursal Disease

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.     

In vitro rapid clearance of infectious bursal disease virus in peripheral blood mononuclear cells of chicken lines divergent for antibody response might be related to the enhanced expression of proinflammatory cytokines

Infectious bursal disease (IBD) is an acute and highly contagious viral disease of young chickens caused by infectious bursal disease virus (IBDV). An effective way to control IBDV would be to breed chickens with a reduced susceptibility to IBDV infection. In the present work, we used chickens selected for high and low specific responses to sheep red blood cells (SRBC) (H and L, respectively) to assess the susceptibility of differential immune competent animals to IBDV infection. The peripheral blood mononuclear cells (PBMCs) of high SRBC line (HL) and low SRBC line (LL) were infected with IBDV and viral RNA loads were determined at different time post-IBDV infection. Chicken orthologues of the T helper 1 (Th1) cytokines, interferon-γ (IFN-γ) and interleukin-2 (IL-2); a Th2 cytokine, IL-10; a pro inflammatory cytokine, IL-6; the CCL chemokines, chCCLi2, chCCLi4 and chCCLi7; colony stimulating factor, GM-CSF; and a anti-inflammatory cytokine, transforming growth factor β-2 (TGFβ-2) were quantified. The expression of chCCLi2, chCCLi4 and chCCLi7 was significantly higher in L line as compared to H line. However, in H line the viral RNA loads were significantly lower than in L line. Therefore, the upregulated chemokines might be associated with the susceptibility to IBDV. The expression of IFN-γ, IL-2 and IL-6 was significantly higher in H line as compared to L line. We assume that the higher proinflammatory cytokines expression in H line might be related to the rapid clearance of virus from PBMCs. Significantly higher levels of IL-10 and TGFβ-2 mRNAs in L line might be related to the pathogenesis of IBDV. In conclusion, selection for antibody responses appears to influence the expression profiles of chemokines and cytokines against IBDV. Further, the selection for high SRBC response might improve the immuno-competence of chickens against IBDV.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

禽网状内皮组织增生病病毒感染对SPF雏鸡法氏囊细胞Bcl-2和C-myc基因表达及凋亡的影响

【目的】 探究禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)感染禽类后导致免疫器官发生细胞凋亡的机理。【方法】 以1日龄SPF雏鸡为试验对象,将100只SPF雏鸡随机均分为REV感染组和未感染病毒的对照组,REV感染组雏鸡经腹腔感染500 μL REV稀释液,对照组雏鸡经相同途径注射等量灭菌生理盐水,于病毒感染后第1、7、14、21、28和42天,2组雏鸡随机各抽取5只,心脏采血处死雏鸡后快速摘取法氏囊。分别应用HE染色和病理切片成像系统测定分析法氏囊细胞核浆比,TUNEL细胞凋亡原位检测试剂盒测定凋亡细胞数,免疫组化法测定Bcl-2和C-myc阳性细胞数量,实时荧光定量PCR和ELISA法分别检测法氏囊Bcl-2和C-myc 基因mRNA表达和蛋白含量。【结果】 ①REV感染1日龄SPF雏鸡后21~42 d,其法氏囊淋巴细胞凋亡百分比显著或极显著高于对照组雏鸡(P<0.05;P<0.01);②SPF雏鸡感染REV后21和28 d,其法氏囊细胞核浆比显著低于对照组雏鸡(P<0.05);③REV感染SPF雏鸡法氏囊中Bcl-2和C-myc阳性细胞数在病毒感染后21和28 d显著高于对照组雏鸡(P<0.05);④REV感染SPF雏鸡后21 d,其法氏囊Bcl-2和C-myc 基因mRNA表达极显著高于对照组雏鸡(P<0.01)。⑤SPF雏鸡感染REV后,其法氏囊中Bcl-2蛋白含量较对照组雏鸡有不同程度的增加,其中21和28 d分别差异极显著(P<0.01)和显著(P<0.05),病毒感染组雏鸡的C-myc蛋白含量也始终高于对照组雏鸡,且21和28 d极显著增高(P<0.01)。【结论】 REV感染所致SPF雏鸡法氏囊细胞Bcl-2和C-myc的mRNA表达以及蛋白含量异常均与病毒感染导致的法氏囊细胞凋亡密切相关,而法氏囊细胞凋亡数量增加与REV感染引发的机体免疫机能抑制密切相关。     

Molecular and phenotypic characterization of infectious bursal disease virus isolates

Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.     

免疫胶体金试纸膜和AGP检测vvIBDV GX株在SPF鸡体内的侵染性变化规律

对28日龄无特定病原(SPF)鸡通过点眼、滴鼻方式人工感染了传染性法氏囊病病毒(IBDV)超强毒株GX;自感染第2天至第9天,每天用免疫胶体金试纸膜和琼脂扩散试验(AGP)两种方法检测九种组织(法氏囊、脾脏、胸腺、肾脏、肝脏、盲肠、扁桃体、肺脏、心脏、直肠内容物)的病毒含量变化,初步探索出传染性法氏囊病病毒在鸡体内各组织的侵染性变化规律。     

Humoral and cell-mediated immune responses in chickens with infectious bursal disease

Primary and secondary immune responses to Newcastle disease virus (NDV) was evaluated in chickens infected with infectious bursal disease virus (IBDV) at one and 28 days of age. The geometric mean primary hemagglutination-inhibition antibody titers (GMT) of chickens infected with IBDV at one day of age was significantly lower (P less than or equal to 0.01) than those infected at 28 days of age. Infection with IBDV had no influence on secondary immune response to NDV. The effect of IBDV infection at one day of age on the cell-mediated immunity of chickens was evaluated by skin allograft acceptance or survival time. There was no significant difference between the percentage of grafts accepted in IBDV infected and noninfected control chickens. However, the mean graft survival time in the IBDV infected chickens was significantly longer (P less than or equal to 0.05) than those in the control group. This suggested a suppression of cell-mediated immunity due to IBDV infection.     

鸡传染性法氏囊病毒超强毒株的分离及生物学特性鉴定

用SPF鸡及鸡胚，从吉林某鸡场病死鸡的法氏囊组织到一株超强毒株（J20株）。此病毒不能凝集鸡的红细胞，可以被IBDV标准阳性血清检出IBDV抗原。通过对J2001分离毒株进行回归实验，测定病毒效价EID50达10^6/0.2ml，发病率为100％，死亡达70％；病毒理化特性试验，电镜观察等证实J20株为鸡传染性法氏囊病超强毒株。     

Sequential changes in the number of surface immunoglobulin-bearing B lymphocytes in infectious bursal disease virus-infected chickens

Variations of B lymphocytes bearing surface immunoglobulin (SIg) M [SIg(M)] and G [SIg(G)] were studied in the spleen and peripheral blood of chickens infected with infectious bursal disease virus (IBDV). The proportion of SIg-bearing B lymphocytes and SIg(M)- and SIg(G)-bearing B cells in chickens infected at one day of age decreased from 1 week postinfection (p.i.) onward and was significantly lower at 8 weeks p.i. In chickens infected at 4 weeks, the percentage of SIg-bearing B cells decreased severely during the first 2 weeks p.i. The decrease of SIg(M)-bearing B cells preceded that of SIg (G)-bearing B cells: the lowest percentage of SIg(M)-bearing B cells has observed 2 to 3 days p.i., and that of SIg(G)-bearing B cells was seen 4 days p.i. The results suggest that SIg(M)-bearing B cells are the major target for IBDV infection.     

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。     

INTERACTION BETWEEN INFECTIOUS BURSAL DISEASE VIRUS AND NEWCASTLE DISEASE VIRUS IN CHICKENS

The Australian strain of infectious bursal disease virus (IBDV), 002/73, affected the response of chickens to Newcastle disease virus (NDV). The titre of serum antibodies to NDV in chickens infected with IBDV was significantly lower than that of birds infected with NDV alone. It also appeared that IBDV affected NDV excretion from chickens as NDV was more frequently isolated from chickens infected with IBDV, IBDV infection did not alter the pathogenicity of NDV in chickens. This Australian strain of IBDV therefore appeared to be immunodepressive in one-day-old chickens.