H9亚型HP株禽流感病毒繁殖规律的探索

为了研究H9亚型HP株禽流感病毒接种非免疫鸡胚后病毒的繁殖规律,试验用H9亚型HP株禽流感病毒接种非免鸡胚,记录不同时间段死亡的鸡胚数量,并分别收获各时间段死亡鸡胚的尿囊液,测定不同时间段尿囊液的病毒效价。结果表明,接种H9亚型HP株禽流感病毒后,非免鸡胚死亡高峰期出现在60～84 h,死亡数量占接种鸡胚数的80%以上,而该时间段死亡鸡胚尿囊液的病毒滴度也处于最高峰,最高到达10^9.63EID₅₀/mL,直到96 h病毒仍维持较高水平(10^9.50EID₅₀/m L),而96 h后病毒滴度开始衰减。     

Structural proteins of classic and variant strains of infectious bursal disease viruses.

Structural polypeptides of six tissue-culture-origin (BGM-70 continuous cell line) infectious bursal disease viruses representing classic and variant strains of serotype 1 and one serotype 2 strain were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, two of the variant strains were propagated in vivo in bursa of Fabricius and compared with those grown in cell culture. Differences among the structural proteins of serotype 1 viruses were minor and probably of no value in differentiating these viruses. However, distinct differences were observed between serotype 1 and 2 viruses. The bursa-derived viruses were different from those propagated in cell culture in molecular weights and in proportions of the proteins. The bursa-derived strains had protein migration patterns similar to those described for tissue-culture-incomplete virus particles.     

Comparison of the dot blot hybridization assay with antigen detection assays for the diagnosis of infectious bursal disease virus infections

The use of cDNA probes as diagnostic tools for detecting infectious bursal disease virus (IBDV) antigens was compared with the agar-gel precipitin (AGP) and immunofluorescence (IF) assays. Specific-pathogen-free chickens were inoculated with the STC or IN strains of IBDV in three separate experiments. Tissue samples were collected at various intervals postinoculation (PI) and examined for viral antigens using the IF assay, the AGP assay, and the hybridization assay. Viral antigen was detected by the AGP assay for a period of approximately 3 days beginning 2 days PI and by the IF assay for approximately 4 days beginning 2 days PI. Virus was detected by the hybridization assay through the length of all the experiments (longest experiment was 24 days) beginning 1 day PI. These results suggested that the hybridization assay is more sensitive than the IF assay and the AGP assay.     

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. II. Alterations of in vitro lymphoproliferation and in vivo immune responses.

To determine the functional impact of alterations in lymphocyte concentrations and ratios following infection with chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens, in vitro lymphoproliferation assays and in vivo responses to vaccination with several common viral agents were assessed at various time intervals post-inoculation (PI). Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation of splenic lymphocytes (SPL) collected from control birds could not be detected until 10-14 days PI. Infection with CAA was characterized by significantly higher PWM stimulation of SPL at 17 days PI and significantly lower PWM stimulation of peripheral blood lymphocytes (PBL) at 14 days PI, compared with uninfected controls. Concanavalin A and PWM stimulation of SPL was significantly increased in birds inoculated with IBDV alone. Lymphocytes harvested from birds inoculated simultaneously with CAA and IBDV had significantly lower responses. Effects on humoral and cell-mediated immunity following CAA and/or IBDV were determined by evaluating vaccination responses to Newcastle disease virus (NDV), fowl pox virus (FPV) and infectious laryngotracheitis virus (ILTV) during the acute phase of CAA infection (2 weeks PI). Vaccination of birds 2 weeks following CAA infection at 1 day of age resulted in decreased protection against NDV (85.7%) and ILTV (7.1%) challenge compared with protection rates in control birds (100% and 53.3% respectively). Infectious bursal disease virus infection was associated with decreased protection against NDV (60%) only. Concomitant infection at 1 day of age resulted in a greater reduction in NDV challenge protection (33.3%), slightly decreased FPV protection (87.5%), increased numbers of persistent FPV vaccination lesions and increased protection against ILTV challenge (71.4%). Vaccination of birds 2 weeks following CAA infection at 2 weeks of age resulted in slightly decreased NDV humoral antibody, development of persistent FPV vaccination lesions (17%) and increased immunity to ILTV challenge compared with control birds (83.3% vs. 66.7%). Chickens inoculated with IBDV alone displayed a more severe depression in NDV antibody titers and only a slight decrease in ILTV protection. Vaccination following concomitant infection at 2 weeks of age resulted in a higher percentage of FPV persistent vaccination lesions (39%) and greatly enhanced immunity to ILTV challenge (100%).     

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.     

Replication of infectious bursal disease virus in continuous cell lines

Three mammalian continuous cell lines--MA-104, Vero, and BGM-70--were tested for their ability to support replication of infectious bursal disease virus (IBDV). Selected tissue-culture-adapted vaccine strains and tissue-culture-adapted field isolates of IBDV replicated in the MA-104, Vero, and BGM-70 cells; cytopathic effects were most pronounced in the BGM-70 cells. The cytopathic effects of the viruses in BGM-70 cells and chicken embryo fibroblast (CEF) cultures were similar. Virus-neutralization titers of selected serum samples determined in BGM-70 cultures compared well with those obtained from CEF cultures.     

Effect of bursal cell number on the pathogenesis of infectious bursal disease in chickens

The pathogenesis of infectious bursal disease (IBD) in chickens neonatally chemically bursectomized (CB) by cyclophosphamide and subsequently inoculated with various numbers of bursal cells was examined. CB chickens inoculated with at least 62.5 X 10(6) bursal cells were as susceptible to IBD clinical manifestations (as determined by gross and microscopic evaluation of bursal tissues, virus recovery from spleen, and antibody titer) as intact chickens following inoculation with virus at 5 weeks of age. In contrast, CB chickens inoculated with 2.5 X 10(6) or fewer bursal cells were refractory to the IBD clinical manifestations compared with intact chickens or CB chickens inoculated with 62.5 X 10(6) or more bursal cells. Results from this study suggest that the availability of a large number of bursal cells is an essential factor in the development of IBD.     

不同方法对鸡胚和法氏囊中囊病病毒抗原含量的检测比较

用单抗介导的抗原捕获－酶联免疫吸附试验（AC－ELISA），琼脂扩散试验（ACP）快速诊断试纸条（ISK）对感染同株强毒或弱毒囊病病毒（IBDV）的鸡胚和鸡法氏囊中的病毒抗原效价进行了检测比较，结果表明，上述3种方法测得法氏囊中温毒IBDV抗原效价分别为10^6.0(AC-ELISA),2^-3.0(AGP)t 10^-3.0，测得鸡胚中强毒IBDV抗原效价依次为10^-1.0,0和0。强毒株接种鸡胚后，只有第1代鸡胚组织可被AC－ELISA测到低效价的IBDV抗原，而第2代以后则未能测到病毒抗原，弱毒疫苗株IBDV感染鸡后，法氏囊中可测到低效价抗原，但感染鸡胚后则无可测性病毒抗原。     

Pathology of avian adenovirus serotypes in the presence of Escherichia coli in infectious-bursal-disease-virus-infected specific-pathogen-free chickens

Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.5 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally. Those birds had been inoculated by eye drop at 1 day of age with a virulent infectious bursal disease virus (IBDV). Controls consisted of groups of chickens inoculated with: (a) IBDV and E. coli, (b) IBDV only, (c) E. coli only, and (d) no virus or E. coli. Gross pulmonary alterations at 5 days postinoculation (PI) included congestion and consolidation of one or both lungs of a chick inoculated with adenovirus serotype Ind-C and another inoculated with A-2. Histopathologic alterations in the lungs were those of multifocal interstitial and occasionally diffuse pneumonia. All 10 adenovirus serotypes elicited multifocal or diffuse pneumonia and bronchiolitis in one or more of the five birds per group necropsied at 5 days PI. T-8 and A-2 serotypes induced marked to serve diffuse pneumonia within 5 days; Ind-C, Stein, Tipton, 75-1A, B-3, and X-11 incited a mild diffuse pneumonia. In all groups, the pneumonic lesions were more severe 5 days PI than 12 days PI. Tracheitis was incited by Ind-C, Stein, T-8, and A-2 at 5 days PI; the lesions were minimal to marked in severity. None of the four control groups exhibited gross or histopathologic alterations except the two IBDV-infected groups, which exhibited bursal change.     

Isolation of infectious bursal disease virus from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec.

Infectious bursal disease viruses (IBDVs) were isolated from turkeys showing symptoms of arthritis and respiratory disease in commercial poultry farms in the province of Quebec, Canada. Synovial fluids collected from hock joints of arthritic birds and peripheral blood leukocytes obtained from the birds with respiratory problems were used for virus isolation in embryonated chicken eggs, and Vero and BGM-70 cell cultures. The infected cells were evaluated for the presence of IBDV by indirect immunofluorescence assay using monoclonal antibodies. The viruses were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral genome and by electron microscopy. Although one of these turkey isolates tested was neutralized by serotype 1-specific commercial chicken antisera, preliminary results indicated that there are antigenic differences between the Quebec isolate, IBDV QT-1, and the existing strains of IBDV belonging to serotype 1.     

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。     

Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.     

5株H9N2亚型禽流感病毒血凝素蛋白分子特征分析和豚鼠间传播能力的评估

为研究H9N2亚型禽流感病毒(AIV)在哺乳动物间的传播能力,本研究以豚鼠为模型评价了5株H9N2亚型AIV在豚鼠体内的复制能力和水平传播能力,并分析了5株病毒血凝素(HA)蛋白的分子特征。结果表明,5株病毒均属于CK/Beijing谱系,其中2株病毒的HA具有人样受体特征(Lys226),2株病毒具有禽样受体特征(Gln226),而A/Chicken/JN/Li-2/2010(H9N2)株在该位点的氨基酸残基为苯丙氨酸(Phe226)。裂解位点分析表明,5株病毒均具有低致病性AIV特征。个别病毒的潜在糖基化位点存在增加或缺失现象。感染试验表明,5株病毒均能够在豚鼠呼吸道复制。并且在鼻甲骨处复制稳定,平均病毒滴度为2.01 Log EID50/mL～4.5 Log EID50/mL。传播试验表明,所有病毒株的人工接种豚鼠的鼻洗液中均能够检测到病毒,最长排毒期为接毒后第8 d,而接触组豚鼠鼻洗液中未检测到病毒。本研究表明,5株H9N2亚型AIV均属于CK/Beijing谱系,部分病毒株的HA蛋白已具备人样受体结合特征,并且关键氨基酸位点(226位)处出现新的突变。5株病毒均能够在豚鼠呼吸道复制并通过上呼吸道排毒,但不能在豚鼠间同群传播。     

Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens

Chickens were intranasally inoculated with Chilean H7N3 avian influenza (AI) viruses of low pathogenicity (LP) (H7N3/LP), high pathogenicity (HP) (H7N3/HP), and a laboratory derivative (02-AI-15-#9) (H7N3/14D) from the LPAI virus to determine pathobiologic effects. All chickens inoculated with H7N3/HP AI virus became infected and abruptly died 2 or 3 days postinoculation, but a few showed moderate depression before death. The H7N3/HP AI virus produced focal hemorrhages of the comb, petechial hemorrhage at the esophageal-proventricular junction and proventricular mucosa, edema and congestion of the lung, petechiation of the spleen, and generalized decrease in body fat. Histologically, severe necrosis, hemorrhage, and inflammation were primarily identified in lungs and the lymphoid tissues. All tissues sampled from the H7N3/HP AI group were positive for the AI viral antigen, predominantly in endothelium of blood vessels throughout most tissues and less frequently in histiocytes and cellular debris of lymphoid tissues. Even less consistently, cardiac myocytes, hepatocytes, Kupffer cells, glandular epithelial cells, microglial cells, and neurons became infected. These studies suggest the Chilean H7N3/LP AI virus was poorly infectious for chickens and may have been recently introduced from a nongalliform host. By contrast, the H7N3/HP AI virus was highly infectious and lethal for chickens. The H7N3/HP AI virus had a strong tropism for the cardiovascular system, principally vascular endothelium, which is similar to the viral tropism demonstrated previously with other H5 and H7 HPAI viruses. Interestingly, the H7N3/LP AI virus on intravenous inoculation replicated in cardiac myocytes, a feature of HPAI and not LPAI viruses, which further supports the theory that the H7N3/LP AI virus was in transition from LP to HP.     

Pathogenesis of marble spleen disease in bursectomized and non-bursectomized ring-necked pheasants following oral inoculation with cell-culture-propagated virus.

Seventy-two 13-week-old ring-necked pheasants were inoculated orally with 5.0 x 10(2) tissue-culture infective dose (TCID) of cell-culture-propagated marble spleen disease virus. Inoculated birds exhibited neither mortality nor clinical disease. Gross and histologic lesions were typical of marble spleen disease. The mean splenic weight was significantly (P less than 0.02) higher in inoculated birds than in controls between 6 and 10 days postinoculation (PI). The histologic splenic lesions, which consisted of reticuloendothelial cell hyperplasia, intranuclear inclusions within reticuloendothelial cells, and lymphoid depletion, were most prominent between 6 and 10 days PI. In a second experiment, 1-day-old pheasants were chemically bursectomized by dosing birds with 1.2 mg cyclophosphamide on 3 consecutive days. At 7 weeks of age, 54 bursectomized birds were inoculated orally with 5.0 x 10(2) TCID of marble spleen disease virus. Gross and histologic lesions were detected in one of the inoculated pheasants, but the mean splenic weight was not significantly different from control birds at any time PI. These results are evidence of the role of the bursa of Fabricius in the pathogenesis of marble spleen disease.     

朗德鹅禽流感病毒的分离与鉴定

用禽流感病毒ELISA试剂盒对某朗德鹅养殖场的病鹅气管粘液进行了检测，发现5份粘液样本均呈禽流感阳性；随后取相应气管组织材料接种于9～11日龄鸡胚分离病毒．发现尿囊液能使鸡红细胞发生凝集，用禽流感病毒H5、H7、H9标准阳性血清和新城疫病毒、传染性支气管炎病毒、传染性喉气管炎病毒、传染性法氏囊炎病毒抗血清作HI试验，结果禽流感病毒H5亚型抗血清的血凝抑制滴度达到2^7，而禽流感病毒H7、H9亚型及其他病毒抗血清无血凝抑制滴度，说明从朗德鹅分离到的病毒为H5亚型禽流感病毒。     

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.