鸡传染性支气管炎病毒LH2/01/10的分离鉴定

2001年在我国黑龙江省某鸡场疑似鸡传染性支气管炎的发病鸡群中，分离到一株病毒，将该病毒接种10日龄SPF鸡胚，取72h的尿囊液进行电镜观察并用1％胰酶处理，结果发现尿囊液中存在典型的冠状病毒，用胰酶处理过的尿囊液能凝集SPF鸡的红细胞，初步鉴定为鸡的传染性支气管炎病毒。将该病毒尿囊液再次接种10日龄SPF’鸡胚，通过病毒对鸡胚的致病作用、病毒超微形态特征以及病毒凝集鸡红细胞的特性等对该毒株进行研究，结果表明：经胰酶处理后的病毒尿囊液可凝集鸡红细胞。鸡胚的第二代病毒尿囊液(命名为LH2,／01／10)分别接种1日龄和15日龄的SPF鸡，发现对不同日龄的鸡表现不同的致病性，对1日龄接种鸡和同笼饲养的同居对照鸡致病力高，发病率为11／11，致死率分别为4／6和2／5；15日龄接种和同居感染鸡发病率为9／9，致死率分别为1／5和1／4。实验应用反转录一聚合酶链式反应技术对LH2／01／10的膜蛋白基因进行扩增、克隆和序列测定，结果表明该基因具有IBVM基因的共有分子特征．与IBV标准株M41的M基因核苷酸的同源性为90％，氨基酸的同源性为91％。这从分子水平进一步证实引起鸡群死亡的病毒为鸡传染性支气管炎病毒。     

Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens.

Outbreaks of an avian disease in infectious bronchitis-vaccinated chickens in China have led to the characterization of coronaviral isolates Q1, J2, and T3, which were isolated from proventricular tissues of the affected young layer flocks. Serologic analysis revealed that they could induce high titers of infectious bronchitis virus (IBV) antibodies in inoculated specific-pathogen-free (SPF) chickens in indirect enzyme-linked immunosorbent assay but were not neutralized by antisera specific to the IBV serotype M41 and the Australian T strain. In a pathogenicity experiment, the clinical signs and related gross lesions resembling those of field outbreaks were reproduced in SPF chickens, and viruses were reisolated from the damaged tissues, including trachea, proventriculus, duodenum, and cecal tonsil. Sequence data demonstrated the complete S1 amino acid sequences of these isolates were almost identical despite recovery from geographically different areas in China and had 47.3%-82.3% similarity in comparison with the 47 published S1 sequences. On the basis of genotyping and limited serology, the three isolates, which were responsible for field outbreaks of the disease, might be a new IBV variant.     

Infectious bronchitis virus nucleoprotein specific CTL response is generated prior to serum IgG

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.     

Influence of infectious bronchitis strains and vaccines on the incidence of Mycoplasma synoviae airsacculitis

A model system was used to study infectious bronchitis virus (IBV) and Mycoplasma synoviae (MS) interaction. The system involved exposure of chickens to IBV, followed by exposure to MS 2-5 days later. The chickens were subjected to a cold environment (10 +/- 2 C) for 3 weeks starting one day before MS exposure. Under these conditions, differences in the capacity of various strains of IBV to exacerbate MS airsacculitis was demonstrated. Exposure to IBV field isolates generally resulted in more air-sac lesions than did higher-egg-passaged laboratory strains and vaccine strains. Use of lower-egg-passaged vaccines resulted in a higher incidence of airsacculitis than did higher-egg-passaged vaccines. When chickens were IBV-vaccinated before being used in the model system, the incidence of airsacculitis was lowered, even though the chickens became infected by the challenge virus. Vaccination of MS-free chickens with IBV had no effect on airsacculitis incidence when MS exposure occurred after the vaccine reaction was past.     

Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus

Three virus isolates (ECV-1, -2, and -3) recovered from cloacae of chickens in flocks that experienced drops in egg production were identified as infectious bronchitis virus (IBV), based on characteristic embryo lesions, chloroform sensitivity, coronavirus morphology, and serology. Because these isolates were recovered from the cloacae of the hens, their tissue tropism was compared with the prototype strain of IBV, Massachusetts-41 (M-41), in experimentally inoculated chickens. During the 39-day period postinoculation (PI), virus isolation was attempted from digestive and respiratory tracts, kidney, and cloacal swabs. ECV-1, ECV-2, and M-41 were more frequently recovered from the cecal tonsils than from other tissues. ECV-1, ECV-3, and M-41 were also recovered from kidney for up to 39 days PI. ECV-2 and ECV-3 had a limited distribution in respiratory tissues, being isolated only sporadically from trachea, bronchus, and lung. Surprisingly, ECV-2 was isolated from esophagus at 2, 16, 30, and 39 days PI; otherwise, its distribution in other tissues was sporadic. Results confirmed that IBV, including M-41, can infect a variety of tissues and that some isolates may be recovered frequently from digestive tract tissues, particularly from the cecal tonsils.     

不同来源传染性支气管炎病毒诱导SPF鸡发病的免疫机制研究

试验旨在探讨不同来源的传染性支气管炎病毒(Infectious bronchitis virus,IBV)诱导SPF鸡发病的免疫机制。选用140只1日龄SPF白来航鸡,随机分为4组,3组攻毒组通过滴鼻点眼途径分别接种鸡源IBV强毒株、鸡源IBV弱毒株和野鸡源IBV毒株3个毒株,对照组以同种方式接种等量灭菌的磷酸盐缓冲液。在感染后12 h、36 h、72 h、7 d和14 d,每组随机选取5只进行剖检,并分别采集法氏囊、肾脏和气管组织,剩余鸡用于观察临床症状、发病及死亡情况。应用实时荧光定量PCR检测攻毒后不同时间点采集的各组织中IBV的病毒载量、Toll样受体(Toll-like receptors,TLRs)及部分细胞因子(白细胞介素(interleukin,IL)和干扰素(interferon,IFN))表达量的变化。结果显示,感染不同来源IBV毒株之后仅鸡源IBV强毒株感染组SPF鸡出现抑郁、翅膀下垂、甩头等典型的临床症状,且在感染后5~10 d共有7只死亡,死亡率为20%。病理剖检发现,感染鸡源IBV强毒株的鸡肾脏肿大、尿酸盐沉积和有花斑样病变,而感染野鸡源IBV毒株、鸡源IBV弱毒株和对照组的鸡无明显的眼观病变。实时荧光定量PCR结果显示,在鸡源IBV强毒株组的法氏囊、肾脏和气管3个组织中均检测到病毒。对照组和野鸡源IBV毒株组中均未检测到病毒,鸡源IBV弱毒株组只在部分组织中检测到病毒。在感染后72 h,鸡源IBV强毒株组与其他各组相比,TLR1、TLR2、TLR3、TLR5、TLR7和TLR15基因在法氏囊中的表达量均显著升高(P<0.05),IL-6和IFN-β参与更强烈的抗病毒免疫反应;在感染后7 d,鸡源IBV弱毒株组与其他各组相比,肾脏中TLR2、TLR3、TLR15、TLR21、IL-6和IL-18基因表达量均显著升高(P<0.05)。野鸡源IBV感染后36 h法氏囊组织中IFN-γ基因表达量显著上调(P<0.05)。综上所述,3个IBV毒株中仅鸡源IBV强毒感染引起SPF鸡典型临床发病症状与可视组织病变,且可提高SPF鸡组织中免疫相关因子的基因表达量。本研究结果揭示,不同来源的IBV对SPF鸡的不同致病性与其感染诱导的免疫反应不同有关。     

中药制剂对禽流感病毒H9N2、新城疫病毒、传染性支气管炎病毒和鸡毒支原体的攻毒保护试验

采用以野菊花、穿心莲、柴胡等10味中药提取的中药制剂,连续给22日龄SPF鸡口服5 d后,以禽流感病毒H9N2、新城疫病毒F4、传染性支气管炎病毒强毒株M41和支原体S6强毒分别攻击,观察SPF鸡临床症状、发病率、病理学变化和分离病毒。结果表明,此中药制剂75 mg/只口服组,新城疫病毒、禽流感病毒和传染性支气管炎病毒攻击后发病率分别为12.5%,0%,10%;37.5 mg/只剂量口服组发病率分别为50%,10%,10%,且低剂量组病毒分离阳性。攻击支原体后,以上两个剂量组SPF鸡病变指数分别3.5和5.5,研究证实本中药制剂可以控制以上4种病原所致的家禽呼吸道病的发生。     

Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.

A stable recombinant fowl poxvirus (rFPV) expressing the C-terminal region (119 amino acids) of the nucleocapsid (N) protein of an infectious bronchitis virus (IBV) strain Ch3 was constructed by inserting the coding sequence within the thymidine kinase gene of fowl poxvirus (FPV) by homologous recombination. The N protein was expressed under control of the vaccinia virus promoter P7.5 in chicken embryo fibroblast cell cultures as seen in immunofluorescence assay and in rFPV-inoculated specific-pathogen-free (SPF) chickens by detecting antibodies with enzyme-linked immunosorbent assay (ELISA). A homologous IBV strain (Ch3) and two heterologous IBV strains (Ch5 and H4) were used to inoculate SPF chickens in a challenge to examine the protective efficacy of the rFPV. When the chickens were challenged with IBV Ch3 or Ch5, the control birds had respiratory signs of infections bronchitis, whereas all the vaccinated birds were clinically normal although low levels of the IBV infection were detected by a differential ELISA. In contrast, in the chickens challenged with IBV H4, all control birds and vaccinated birds suffered from the highly lethal IBV H4 infection. Our results suggest that the C-terminal 119 amino acid of the nucleocapsid expressed by FPV is a host-protective antigen and may induce cross-protective immunity against illness among some IBV strains.     

Evaluation of the hemagglutination-inhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination

An infectious bronchitis virus (IBV) hemagglutination-inhibition (HI) test was used to assay serum-antibody titers after IBV vaccination of IBV-susceptible specific-pathogen-free broilers and commercial layers. Three-week-old broilers were vaccinated via eye-drop with IBV strains that represent the antigenic spectrum of commercial vaccines--Holland, Massachusetts 41 (41 Ms), Connecticut 46, Florida 18288, or JMK strain--and revaccinated 3 weeks later with either the same or a heterologous strain. Weekly serum samples were tested by IBV HI with homologous and heterologous antigens. Vaccinates, except for those vaccinated with the Holland strain, were HI-positive with homologous but not heterologous antigens by 1 to 2 weeks postvaccination. Sixteen-week-old IBV-vaccinated commercial layers were revaccinated with IBV Holland 52 (H 52) strain and subsequently infected with Arkansas 99 (Ark 99) and SE 17 strains. In contrast to the limited HI cross-reactivity of serum from IBV-vaccinated broilers, there were extensive cross-reactions in HI tests with 41 Ms, H 52, Ark 99, and SE 17 antigens of revaccinated layers. These results demonstrate that the IBV HI test is more strain-specific than previous reports indicate, especially when the test samples are from early postvaccination.     

Isolation of avian reovirus as a possible etiologic agent of osteoporosis ("brittle bone disease"; "femoral head necrosis") in broiler chickens

Avian reovirus was isolated from intestines of 3-to-7-day-old broiler chickens with enteritis from broiler houses where osteoporosis was a problem. The virus was purified in a cesium chloride gradient (buoyant density 1.37 gm/ml) and identified as a reovirus by electron microscopy. Specific-pathogen-free (SPF) chickens and commercial broiler chickens with anti-reovirus maternal antibodies inoculated at 1 day of age with the reovirus isolate developed lesions of femoral head fractures and/or osteoporosis; reovirus could be reisolated from the bone marrow and intestinal tracts of experimentally infected SPF birds. The reovirus isolate, although isolated from intestines, induced development fo tenosynovitis lesions in SPF and commercial broiler chickens.     

Serological monitoring on layer farms with specific pathogen-free chickens

To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected.     

白虎定喘口服液药效研究

为验证新兽药白虎定喘口服液的药效作用,进行了鸡传染性支气管炎病毒（infectious bronchitis virus,IBV）鸡胚增殖抑制试验、气管纤毛运动试验及SPF雏鸡人工感染IBV疗效试验。将试验药液与等量IBV液混合作用后,接种11日龄SPF鸡胚,37℃孵育168 h,取存活鸡胚肾脏进行IBV的RT-PCR检测,结果表明,药物组168 h鸡胚存活率为80%,阳性对照组为20%,两组间差异显著（P<0.05）;试验药物组RT-PCR检测无目的条带出现,而阳性对照组有目的条带出现。40日龄SPF鸡连续用药3 d后,气管内注入0.02 mL墨汁,测量1 min内墨汁移动的距离,结果表明药物组墨汁移动距离极显著大于生理盐水对照组（P<0.01）。17日龄SPF鸡用IBV-M41滴鼻,0.3 mL/只,24 h后开始灌服药液,1 mL/kg,连用5 d,结果表明,试验药物组与攻毒对照组在临床症状计分、痊愈率上均存在显著差异（P<0.05）;试验组气管黏膜组织切片未见明显病变,而阳性对照组有明显病理变化。以上试验结果表明,白虎定喘口服液对IBV具有较好的体外抑制作用,可显著促进气管纤毛的运动,对鸡传染性支气管炎（IB）具有较好的临床防制效果。     

Study on Efficacy of Baihu Dingchuan Oral Liquid

In order to verify the effect of Baihu Dingchuan oral liquid of new veterinary drugs,the effects of infectious bronchitis virus (IBV) on the proliferation inhibition test, tracheal cilia movement test and SPF chickens infected with IBV were tested.The test solution was mixed with an equal amount of IBV solution,inoculation of 11-day-old SPF chicken embryo,incubation at 37℃ for 168 h.RT-PCR was performed to detect IBV in kidney of live chick embryo.The results showed that the survival rate of 168 h chicken embryo was 80% in the drug group,but the positive control group was 20%, and there was significant difference between two groups (P<0.05). In the test drug group, no band was detected by RT-PCR, but the target band appeared in the positive control group.After 40 days old SPF chickens were administered continuously for 3 days, 0.02 mL of ink was injected into the trachea. The distance of ink movement in 1 min was measured. The results showed that the migration distance of ink was extremely significantly higher than that of saline control group (P<0.01).In the 17-day-old SPF chickens, IBV-M41 was infused into the nasal mucosa with 0.3 mL. After 24 h, 1 mL/kg was administered for 5 days. The results showed that there were significant differences in clinical symptom score and cure rate between the test drug group and the control group (P<0.05). There was no significant pathological changes in the tracheal mucosa of the experimental group, but the pathological changes in the positive control group were significant. The above results showed that Baihu Dingchuan oral liquid had good inhibitory effect on IBV in vitro, which could promote the movement of tracheal cilia, and had good clinical control effect on infectious bronchitis.     

Isolation and characterization of new infectious bronchitis virus variants in Hungary

Two agents not agglutinating chicken erythrocytes were isolated, one in each of two flocks, from organ samples and tracheal swabs taken from 4- to 7-week-old chicks of 8 broiler flocks experiencing respiratory signs. Virus isolation was done in embryonated SPF hen's eggs. Morphological changes of the embryos, appearing as dwarfing or curling into a spherical form, usually occurred in the 3rd or 4th passage on postinoculation (PI) days 5-9. Some embryos had swollen kidneys covered with urate. Electron microscopy of ultrathin sections of these kidneys revealed the presence of virions reminiscent of coronaviruses. Similar viral particles were seen in resuspended pellets of isolates concentrated by ultracentrifugation. Based on embryo changes, cross-neutralization tests with type-specific antisera, physicochemical tests, results obtained in cell cultures, and electron microscopic findings the two isolates were identified as infectious bronchitis virus (IBV). By cross-neutralization tests the isolates differed from IBV reference strains M41 and H52 and can be considered distinct variants. Elucidation of their epizootiological role requires further investigations.     

鸡传染性支气管炎病毒CQ/01/2004株的分离与鉴定

2004年从重庆某肉用鸡场疑似鸡传染性支气管炎的病鸡中采集病料，按常规处理后接种9～10日龄鸡胚，通过鸡胚连续传代培养3代，并对该分离毒株的鸡胚致病性、血凝性和NDV的干扰特性进行检测．同时进行了动物回归试验。结果表明，该分离株具有IBV感染特征，可使鸡胚胚体出血、蜷缩、矮化；该分离毒株无直接血凝性，对NDV有明显的干扰作用；动物回归试验中有75％的感染鸡在10d内发病或死亡。剖检病死鸡可见肾苍白、肿胀，肾小管内充塞大量尿酸盐，支气管有出血点、有大量粘液。采用反转录．聚合酶链式反应（RT-PCR）技术对CQ／01／2004的纤突蛋白S1基因进行扩增、克隆和序列测定，结果表明该基因具有IBVSl基因的共有分子特征，将测序结果提交GenBank进行同源性检索，发现分离株CQ／01／2004和J株S1的同源性最高，核苷酸同源性为94％，氨基酸同源性为89．4％，与M41的核苷酸同源性为80．6％，氨基酸同源性为78．0％。试验结果表明，分离的病毒株CQ／01／2004为鸡传染性支气管炎病毒。     

Experimental reproduction of transmissible viral proventriculitis by infection of chickens with a novel adenovirus-like virus (isolate R11/3)

Transmissible viral proventriculitis (TVP) was experimentally reproduced in 2-wk-old specific-pathogen-free chickens and commercial broiler chickens by eyedrop inoculation of adenovirus-like virus (AdLV), isolate R1 1/3. No clinical signs and no weight gain depression were observed in chickens inoculated with AdLV (R11/3); however, gross and microscopic lesions characteristic of TVP were present in proventriculi of inoculated chickens. Proventriculi of AdLV (R11/3)-inoculated chickens were markedly enlarged, compared with sham-inoculated controls, by day 7 postinoculation (PI). Microscopic lesions in proventriculi of inoculated chickens were detected beginning on day 3 PI and consisted of degeneration and necrosis of glandular epithelium, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration; no microscopic lesions were observed in other tissues. AdLV (R11/3) antigens were detected in proventriculi by immunohistochemistry on days 3-10 PI in inoculated SPF chickens and days 3-21 PI in inoculated commercial broiler chickens; no viral antigens were detected in other tissues. AdLV (R11/3) was reisolated from proventriculi of inoculated SPF and commercial broiler chickens on days 5 and 7 PI. No virus, viral antigens, or lesions were detected in proventriculi collected from sham-inoculated chickens. These findings indicate an etiologic role for AdLV (R11/3) in TVP.     

Effect of serial embryo passage of an Arkansas-type avian infectious bronchitis virus isolate on clinical response, virus recovery, and immunity

Infectious bronchitis virus (IBV) Arkansas-type DPI strain (Ark DPI) was attenuated by serial passage in chicken embryos. Virus of passage 50 was less pathogenic for day-old maternally immune broiler-type chickens than virus of passage 10 or 25 as determined by clinical response to vaccination, virus isolation in respiratory (trachea) and nonrespiratory (kidney and cloaca) tissues, and weight-gain studies. Chickens vaccinated with virus of passage 10, 25, or 50 were at least 80% resistant to homologous virus challenge of the upper respiratory tract 4 and 6 weeks postvaccination. Serum antibody production by passage-50-vaccinated chickens was comparatively low at 4 weeks but at 6 weeks was similar to that in chickens vaccinated with virus of passage 10 or 25.     

Comparison of a tissue-culture virus-neutralization test and the enzyme-linked immunosorbent assay for measurement of antibodies t infectious bronchitis

Two serological tests, the virus-neutralization (VN) test in tissue culture using a tissue-cell-adapted virus and the enzyme-linked immunosorbent assay (ELISA), were compared to detect antibodies against Massachusetts 41 and Connecticut 46 strains of Infectious Bronchitis Virus (IBV). The VN test was conducted in wells of microplates by the usual procedure. The two strains of IBV were adapted after 20 serial passages to induce CPE in 24 hours in chickens embryos kidney cells (CEKC). The ELISA test was carried out using partially virus following ultracentrifugation of each stain of IBV as antigen. The ELISA test detected higher geometric mean antibody titers (GMT) against both strains of IBV than did the VN test. One hundred four serum samples taken at 1, 3, 5, 9, 22, 24, and 26 weeks of age from a flock of chickens vaccinated with the Mass strain three times and the Conn strain of IBV two times during the growing period showed higher antibody titer responses to the Conn 46 than to the Mass 41 strain. Maternal antibodies in chicks one week of age were readily detected by the ELISA test, whereas low or insignificant titers were found by the VN test. Sera of vaccinated chickens collected following challenge with Mass 41 or Conn 46 strain of IBV showed that the ELISA was more sensitive and showed higher titers than did the VN test. Although the VN test showed no rise in GMT in the same sera tested with the heterologous virus, the ELISA showed a slight increase or cross-reaction. The serum samples from the unchallenged control group showed no change in GMT with either test or IBV strain.     

鸡传染性支气管炎病毒血凝抑制试验抗原的研制

为应用血凝抑制(HI)试验检验鸡传染性支气管炎疫苗免疫效力(血清、卵黄抗体水平),建立了鸡传染性支气管炎病毒HI试验抗原的制备方法。该方法是通过选取抗原谱最广的鸡传染性支气管炎病毒(IBV)M41株,经SPF鸡胚增殖培养36h后无菌收取鸡胚尿囊液,4℃、12 000r/min离心10min,上清用聚乙二醇(PEG)20000浓缩100倍;兔源A型产气荚膜梭菌中国标准株(C57-1株)37℃增殖培养18h,4℃、12 000r/min离心10min取上清,经PEG 20000透析袋浓缩5倍后通过0.20μm滤膜过滤除菌,然后将IBV液和A型产气荚膜梭菌菌液(含α毒素)二者按一定比例混合后经37℃恒温振荡感作2h,4℃经48h后制成。经大量试验表明,制备的IBV HI试验抗原效价高、稳定性好,可替代进口抗原应用于鸡群IBV疫苗免疫后血清抗体及卵黄抗体的HI效价检测。