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Ching-I Shen Ching-Ho Wang Jiunn-Wang Liao Tien-Wang Hsu Shu-Ming Kuo Hong-Lin Su 《Veterinary research》2010,41(1)
Here we introduce a culture system for the isolation, passaging and amplification of avian tracheal epithelial (ATE) cells. The ATE medium, which contains chicken embryo extract and fetal bovine serum, supports the growth of ciliated cells, goblet cells and basal cells from chicken tracheas on fibronectin- or matrigel-coated dishes. Non-epithelial cells make up less than 10% of the total population. We further show that ATE cells support the replication and spread of infectious bronchitis virus (IBV). Interestingly, immunocytostaining revealed that basal cells are resistant to IBV infection. We also demonstrate that glycosaminoglycan had no effect on infection of the cells by IBV. Taken together, these findings suggest that primary ATE cells provide a novel cell culture system for the amplification of IBV and the in vitro characterization of viral cytopathogenesis. 相似文献
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ZHAO Zeng-cheng LIN Shu-qian HUANG Zhong-li LI Gui-ming YANG Shi-fa FENG Min-yan SONG Min-xun FU Jian LI Ying SHANG Jing 《中国畜牧兽医》2017,44(1):282-288
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. 相似文献
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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。 相似文献
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In vitro studies with organ (oviduct and trachea) and chicken embryo kidney cell cultures were attempted to assess the pathogenicity of locally isolated infectious bronchitis virus (IBV-P:120) initially isolated from the oviduct of young chicks. In oviduct cultures infected with IBV, ciliary movements decreased as early as 24 hours postinoculation (PI), and on the 6th day ciliary movements ceased completely. Cytopathic changes were also noticed. Immunofluorescent antigen was detected from 1 to 6 days PI, the maximum being on the 3rd day. The characteristic microscopic changes in the oviduct explants were reduced by 24 hours PI and had completely ceased on the 5th day. Cytopathic effect and immunofluorescent antigen were present from 1 to 8 days PI, being maximum on the 5th day. Histological changes marked by loss of cilia, rounding of the epithelial cells, degeneration, and sloughing were detected from 2 to 8 days PI. Low-embryo-passaged (EP-7) IBV did not produce cytopathic effect on the chicken embryo kidney cell cultures. On the contrary, high-embryo-passaged (EP-14) virus produced cytopathic effect at the third tissue-culture-passage level. 相似文献
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本研究对分离自沈阳地区的一株传染性支气管炎病毒（ＳＹ毒株）进行了生物学特性的研究,同时成功地对其免疫原Ｓ１基因进行了ＲＴ－ＰＣＲ扩增、克隆与序列分析。 通过电镜观察、动物回归试验、血凝特性研究等试验验证分离自沈阳地区的ＳＹ毒株确实为一株传染性支气管炎病毒。气管环组织培养交叉中和试验结果表明,分离株ＳＹ株不同于参考毒株澳大利亚Ｔ、Ｈ５２、Ｍ４１,且不同于国内其它流行株ＨＤ、ＨＢ、ＸＢ、ＤＢ等,是一个新的变异株。 利用ＩＢＶ Ｓ１基因特异性寡聚核苷酸引物,经ＲＴ－ＰＣＲ扩增ＳＹ毒株的Ｓ１基因,得到预期的约１．７Ｋｂ片段;并将扩增所得ｃＤＮＡ插入克隆质粒ｐＵＣ１９的ＥｃｏＲⅠ／ＢａｍＨⅠ位点,在大肠杆菌ＤＨ５ａ中实现目的基因的克隆。经限制性核酸内切酶分析及ＰＣＲ鉴定,证实为阳性重组质粒,利用末端双脱氧链终止法对其测序,得到Ｓ１基因全长１６４０ｂｐ,包括整个开放阅读框。通过序列分析软件ＤＮＡＳＩＳ、ＰＲＯＳＩＳ、ＭＥＧＡ等软件对Ｓ１基因核苷酸序列及推导的氨基酸序列进行分析,结果表明：分离株ＳＹ与７株参考株和国内流行株ＨＤ株相比,无论是核苷酸序列同源百分率还是氨基酸序列同源百分离都较低,均未达到８０％,这就提示我们ＳＹ毒 相似文献
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鸡传染性支气管炎病理形态学及发病机理的研究 总被引：5，自引：1，他引：4
１５０只１８日龄雏鸡随机分为两组，试验组９０只接种传染性支气管炎病毒（ＩＢＶ－Ｍ４１）后３５天内分３０批依次于不同时间扑杀，作组织病理学、超微结构及病毒抗原位检查，对照组６０只作相同的检查。结果表明，ＩＢＶ攻击的靶器官是气管，气管的病变表现为粘膜上皮细胞的损伤和脱、残留的在皮细胞增殖形成复层上皮、粘膜固有层及粘膜下层大量淋巴细胞浸润及粘膜逐渐恢复的相互连续的病理过程。肺脏初级、次级支气管也有类似的 相似文献
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试验旨在观察鸡传染性支气管炎病毒（infectious bronchitis virus,IBV）和鸡新城疫病毒（newcastle disease virus,NDV）之间的增殖干扰现象,分析在免疫过程中两种疫苗存在的免疫干扰,为确定疫苗的免疫程序提供依据。采用完全随机试验设计,选取10日龄鸡胚尿囊腔接种不同浓度的IBV、NDV以及不同浓度混合的IBV和NDV,收集尿囊液测定其血凝效价（HA）;用不同浓度的IBV、NDV以及不同浓度混合的IBV和NDV分别免疫BALB/c小鼠及SPF雏鸡,收集血清,间接酶联免疫吸附法（ELISA）和血凝抑制（HI）试验测定IBV和NDV的抗体效价及抑制效价。结果表明,同胚培养时,无论先接种IBV后接种NDV还是先接种NDV后接种IBV或IBV和NDV同时接种,IBV对NDV均有干扰作用,而NDV对IBV没有干扰作用;接种IBV和NDV的小鼠,其产生IBV和NDV的抗体效价均低于单独免疫组,免疫的次序及免疫间隔时间对IBV和NDV的抗体效价均有影响,IBV对NDV的免疫效果具有较大的干扰作用。不同针混合免疫方式能提高IBV和NDV的抗体效价;接种IBV和NDV的雏鸡,免疫次序及免疫相隔时间对IBV和NDV的抗体效价均有影响,IBV对NDV的免疫效果具有较大的干扰作用;雏鸡和小鼠免疫血清HI试验数据表明,免疫次序及免疫间隔时间对IBV和NDV抗体的产生均有影响,但对NDV抗体的产生影响更明显,随着免疫间隔时间的增加,IBV和NDV的抗体水平呈增加趋势,雏鸡比小鼠更能敏感地反映出IBV对NDV的免疫干扰作用。混合注射时IBV和NDV的抗体水平均降低,IBV对NDV的免疫效果有干扰作用。因此,在同胚培养、小鼠及雏鸡的免疫中,IBV对NDV有干扰作用,而NDV对IBV无干扰作用。混合接种时IBV和NDV抗体效价均下降,IBV对NDV的免疫效果有干扰作用。免疫次序及免疫间隔时间对IBV和NDV抗体的产生均有影响,但随着免疫间隔的增加,IBV和NDV的抗体水平呈增加趋势。 相似文献
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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. 相似文献
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This experiment aimed to investigate the proliferation interference effects of the infectious bronchitis virus (IBV) and Newcastle disease virus (NDV),analyze the immune interference of two vaccines in the immune process,and provide a basis for determining vaccine immune program.This experiment was in a complete randomized design,10-day-old allantoic cavity of the chick embryo was inoculated with different concentrations of IBV and NDV and with a mixture of different concentrations of IBV and NDV,and then the allantoic fluid was collected for determination of the titer of hemagglutination (HA).Furthermore,BALB/c mice and SPF chicken were immunized with different concentrations of IBV and NDV and also with the mixed IBV and NDV with different concentrations,the blood of mice and chicken were collected,and antibody titer and inhibitory titer of IBV and NDV were determined by indirect enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.Experimental results showed that in the homeomorphic cultivation research,whether IBV and NDV were inoculated in a different order or inoculated at the same time,the inoculation of IBV behaved interference to the NDV,while the inoculation of NDV had no interference effects to IBV;Mice were inoculated with IBV and NDV,IBV and NDV antibody titers were lower than single immunized group,immune procedures and immune intervals often had effect on the titer of IBV and NDV antibody,IBV had interference on the immune effect of NDV,different needle mixed immunization can improve the antibody titer of IBV and NDV.In the group of SPF chicken inoculated with IBV and NDV,the order of the inoculation and the immune interval had effect on the titer of IBV and NDV antibody,IBV had a interference on the immune effect of NDV;In the immune serum inhibitory test,the data showed that the IBV and NDV antibody production were affected by the immune order of the inoculation and the interval,the effect on the NDV antibody was more obvious,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend,the chicken was more sensitive than the mice to reflect the immune interference of IBV on NDV.IBVand NDV antibodies titer was also reduced in the groups that was inoculated with the mixed IBV and NDV,IBV had a interference on the immune effect of NDV.In the homeomorphic cultivation research and animal research,IBV behaved interference to the NDV,while NDV had no interference effects to IBV.IBV and NDV antibody titers decreased when mixed immunization,IBV had a interference on the immune effect of NDV.The order of the inoculation and the immune interval had effect on the antibody titer of IBV and NDV,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend. 相似文献
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The ability of nine strains of avian infectious bronchitis virus (IBV) to induce chicken interferon has been investigated using Semliki Forest virus for the tests. The Beaudette, H120 and Connecticut 46 strains induced interferon in the allantoic fluids of embryonated hens' eggs, the highest titre (1 : 30) being associated with Beaudette; but these as well as the Massachusetts M-41 and H52 strains failed to yield interferon in primary monolayer cultures of chick kidney cells as did all nine strains in organ cultures of chick embryo trachea. None of six strains of IBV investigated was susceptible to the inhibitory effects of chicken interferon. 相似文献
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鸡胚盲肠上皮细胞原代培养与鉴定 总被引：2，自引：1，他引：1
为研究鸡E.tenella的宿主细胞--盲肠上皮细胞体外培养技术,为E.tenella损伤机制及抗球虫药的研究提供体外模型,分别用胰蛋白酶法、胶原酶Ⅰ法、嗜热菌蛋白酶法、嗜热菌蛋白酶+胶原酶Ⅰ法和中性蛋白酶Ⅰ+胶原酶Ⅺ法分离纯化原代鸡胚盲肠上皮细胞,通过测定细胞活力、细胞团块比例、总细胞产量和细胞团块产量,筛选出鸡胚盲肠上皮细胞最佳分离方法,并进行了纯化和培养,对培养细胞进行了鉴定.结果表明:嗜热菌蛋白酶消化、低速离心去除单细胞、差速贴壁除去成纤维细胞,为最佳分离和纯化盲肠上皮细胞的方法;分离纯化的细胞接种后分别于第3-5天、第10-11天进入对数生长期,可存活14 d以上;经形态学观察、碱性磷酸酶染色、扫描电镜鉴定为盲肠上皮细胞,所分离的细胞培养至第4、7、11天时上皮细胞比例分别为81.67%、84.33%和72.00%.用该法可获得纯度较高的原代鸡胚盲肠上皮细胞. 相似文献
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Kotani T Shiraishi Y Tsukamoto Y Kuwamura M Yamate J Sakuma S Gohda M 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2000,62(2):129-134
All stages of degeneration and regeneration in chicken tracheal epithelium were studied morphologically following an intratracheal inoculation of infectious bronchitis virus (IBV). Viral antigen was detected in the cytoplasm of tracheal epithelium from 1 to 7 days post-inoculation (d.p.i.) with a peak on 3 d.p.i. At 1 d.p.i., almost all epithelial cells were involved in the degeneration. At this time, labelling index of bromodeoxyuridine (BrdU) in the basal cells showed significantly high value compared with control. At 2 and 3 d.p.i., a great number of basal cells were recognized, but the BrdU labelling index tended to decrease. At 4 and 5 d.p.i., the BrdU labelling index of basal cells significantly decreased than 1 d.p.i., and a few number of regenerated immature ciliated epithelia appeared. At 6 to 11 d.p.i., the ciliated columnar epithelia increased rapidly in number, and returned to the normal appearance except for non-ciliated patch by 13 d.p.i. These results suggested that the tracheal epithelial cells infected with IBV degenerated within 24 hours and proliferating activity of basal cells functioned immediately, and 3 to 4 days later, these basal cells were differentiated to the ciliated epithelia. 相似文献
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A recombinant DNA probe with specificity for the 3' end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2,4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation. 相似文献
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腺胃病变型鸡传染性支气管炎病毒的分离鉴定 总被引：28，自引：8，他引：28
从１９９７年在辽宁省大连市某鸡场发生的一种以腺胃肿大为特征的鸡的传染病病例中收集腺胃组织（Ｄ９７１）接种ＳＰＦ鸡胚,传至１３代,具有规律性死亡和典型胚胎病变特征,ＥＩＤ５０为１０－６．４８／０．２ｍｌ。Ｄ９７１毒株感染ＳＰＦ鸡胚尿囊液经负染电镜观察,可见直径为８０～１２０ｎｍ,有囊膜和纤突的冠状病毒粒子。Ｄ９７１毒株经卵磷脂酶Ｃ处理后能凝集鸡红细胞,并能特异的被Ｄ９７１多抗所抑制。用反转录聚合酶链反应获得了Ｄ９７１毒株免原（Ｓ１）基因ｃＤＮＡ,经１％琼脂糖凝胶电泳可见１．７ｋｂ的特征性条带。用Ｄ９７１毒株接种ＳＰＦ鸡,能引起与自然病例相似的病理变化,并分离到病毒。用Ｄ９７１毒株可直接感染ＳＰＦ鸡胚成纤维细胞。结果证实从腺胃病变型ＩＢ鸡群中分离的Ｄ９７１毒株是冠状病毒科ＩＢＶ成员。 相似文献
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Isolation and identification of four infectious bronchitis virus strains in China and analyses of their S1 glycoprotein gene 总被引：4，自引：0，他引：4
Between 2003 and 2005, four strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in China. The results from chicken embryo cross-neutralization assays showed that all the four isolates were relative to strain A2 of IBV, which was isolated in 1996 in Beijing and related to strain 4/91. The S1 gene of the spike protein was amplified and sequenced. The nucleotide and amino acid sequence of the S1 gene had a similar degree of identity (88.98-99.28%) among the four Chinese IBV isolates. The identity of the S1 protein gene between the four Chinese IBV isolates and 14 strains of other IBVs varied from 70.06 to 81.59%. Phylogenetic analysis suggested that there are at least four groups of IBVs circulating in China and the disease outbreaks might have been caused by infection of multiple strains of IBV. 相似文献
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Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis. 相似文献
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Between 2006 and 2009, seven strains of infectious bronchitis (IB) virus (IBV) were isolated from vaccinated chicken flocks on different chicken farms in China. The pathogenic characters of seven IBV strains were assessed. Each of the seven strains was infective to the test chickens and could induce an immune response. The results from chicken embryo cross-neutralization assays showed that these strains were antigenically distinct from classic IBV strains of H120, M41, Conn, and Gray. Compared to H120 vaccine strain, point mutation, short insertion, and deletion occurred at many positions in the S1 protein of the seven strains. Five of the seven strains had the motif (HRRRR), which was identical to that of the epidemic IBV strains in China. Two new motifs (HRLRR and RRIRR) emerged in the isolated strains. The homology of the nucleotide and amino acid sequences of the S1 gene among the seven isolates was 81.7%-99.7% and 79.0%-99.4%, respectively. These seven strains were also genetically different from the vaccine strains and non-China IBV strains but closely related to large numbers of Chinese strains. The seven isolates and 36 reference IBV strains were clustered into six distinct groups (I-VI). The seven strains were categorized into groups I, II, and III, forming a big phylogenetic branch, which is closely related to Chinese IBVs, whereas the vaccine strains belonging to group VI are genetically distant from groups I, II, and III. The results from this study indicate that different IBV strains cocirculate in the chicken population in China. 相似文献