蛋鸡感染 H9N2 AIV 后输卵管不同部位病毒的分布及病理组织学观察

为探索 H9N2 AIV感染蛋鸡致输卵管功能异常的机理,对蛋鸡感染 H9N2亚型 AIV后输卵管不同部位病毒载量与病理变化进行检测与观察。以 H9N2亚型 AIV 人工感染非免疫蛋鸡,在感染后3、5、7 d 分别采取鸡输卵管组织,real-time PCR 检测感染鸡输卵管组织中 AIV 病毒载量,同时 HE 染色后观察病理组织学变化。结果表明,real-time PCR 从鸡输卵管膨大部、峡部、子宫部和阴道部均检出了禽流感病毒,其中以膨大部和子宫部病毒载量最高,分别达5．27×104拷贝／μL±3．55×103拷贝／μL 和5．52×10 4拷贝／μL±3．14×103拷贝／μL。组织病理学观察发现蛋鸡感染 H9N2亚型 AIV 后输卵管膨大部、峡部和子宫部病变明显。观察到上皮细胞脱落坏死,炎性细胞浸润腺体,腺体间隙变大甚至溶解,腺体组织充血出血。本研究证实蛋鸡感染 H9N2亚型 AIV 后输卵管组织中 AIV 病毒载量与其病理变化存在明显的正相关。     

猪圆环病毒2型感染猪皮肤源树突状细胞炎性反应能力的变化

用PCV2 B1株经鼻腔接种40日龄SPF仔猪,于接种后3、7、14 d宰杀,收集皮肤源树突状细胞(DC).利用实时荧光定量PCR技术对感染仔猪皮肤源DC的IL-10、TNF-α、IFN-α、IL-8、趋化因子受体1(CCR1)、CCR5在mRNA转录水平的变化进行定量分析.结果表明,IFN-α在接种后3 d(3DPI)显著下调(P＜0.05),TNF-α、IL-10在7DPI时显著上调(P＜0.05);趋化因子IL-8在3、7、14 DPI时均下调,差异接近显著;MCP-1在感染后3、14DPI下调,7DPI均上调,但不显著;MIP-1β在3、7DPI明显上调,14DPI恢复正常;趋化因子受体CCR1、CCR5在3、7和14DPI均上调,且7DPI显著上调(P＜0.05).以上结果表明PCV2在感染早期可抑制DC炎性反应的能力,免疫应答失调,影响了动物机体的细胞和体液免疫功能的发挥.     

禽呼肠孤病毒感染鸡胚成纤维细胞后IL-17、IL-18和IFN-γ mRNA转录水平的动态变化

为了分析禽呼肠孤病毒(avian reovirus,ARV)S1133株感染鸡胚成纤维细胞(chicken embryo fibroblast,CEF)后,对其细胞因子IL-17、IL-18和IFN-γmRNA转录水平的影响,探讨禽呼肠孤病毒感染机制和宿主之间的作用关系。本试验将ARV-S1133感染CEF细胞后,运用荧光定量PCR技术,测定和分析ARV结构蛋白σC和CEF细胞的IL-17、IL-18及IFN-γ的mRNA动态转录水平。结果表明,ARV-S1133感染CEF细胞10h后病毒结构蛋白σC mRNA的相对表达量开始迅速上升,在48h达到最高峰(13 162.73倍);ARV-S1133感染后引起CEF细胞中的IL-17、IL-18和IFN-γmRNA表达量发生变化,IL-17和IFN-γmRNA转录水平在感染早期迅速上调,感染后6h达到第一个峰值,分别上调8.77倍和11.17倍,随后下降,在感染36、48、60h后大幅度上升,且在48h达到峰值,表达量分别为97.19倍和111.58倍。IL-18mRNA转录水平在整个感染过程中表达量较低,在感染6h后微量上调(1.39倍),在感染中后期,其表达量呈下调趋势,感染48h时,IL-18mRNA表达量最低,与对照组相比下调0.19倍;5个不同滴度的ARV病毒感染CEF细胞24h后,3个细胞因子mRNA的表达量与病毒滴度线性相关,IL-17和IFN-γmRNA转录水平与病毒滴度正相关,IL-18mRNA转录水平与病毒滴度负相关。结果表明,ARV感染后可诱导CEF分泌IL-17、IL-18、IFN-γ的mRNA转录水平上调,说明IL-17、IL-18、IFN-γ可能与禽呼肠孤病毒的复制和致病机制相关。     

H5N1亚型禽流感病毒和新城疫病毒感染鸡胚成纤维细胞IFN-β基因的动态表达

根据鸡β-干扰素(ChIFN-β)基因保守序列设计引物建立了检测鸡IFN-βmRNA表达水平的荧光定量RT-PCR(RRT-PCR)方法,并对H5N1亚型禽流感病毒(AIV)和新城疫病毒强毒株(vNDV)感染后3、6、12、24、30 h的鸡胚成纤维细胞(CEF)中IFN-βmRNA表达水平进行了检测。结果显示,建立的RRT-PCR特异性好,对鸡IFN-βmRNA的扩增效率为94.18%,线性范围为10-8~10-3,相关系数为0.992,最低能检出48拷贝/反应。AIV在感染CEF后6、12 h显著抑制IFN-βmRNA的表达,24 h开始诱导IFN-βmRNA表达,30 h时IFN-βmRNA水平显著升高;vNDV在感染CEF后的24 h内显著抑制IFN-βmRNA的表达,30 h时则显著诱导IFN-βmRNA表达。本试验结果为进一步研究H5N1和NDV与机体的相互作用提供了有价值的信息。     

猪繁殖与呼吸综合征病毒和猪圆环病毒2型共感染对外周血单个核细胞促炎细胞因子mRNA表达的影响

将猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）单感染和共感染6周龄健康仔猪,采用Real-ti me PCR技术对外周血单个核细胞（PBMC）中IL-1β、IL-6、IL-8和TNF-α等促炎细胞因子的mRNA表达进行定量分析。结果表明,病毒感染后,PRRSV感染组、PCV2感染组IL-1β、IL-6、IL-8和TNF-α的mRNA表达水平均上调,其中PRRSV感染组的IL-6、IL-8显著上调,PCV2感染组的IL-6、IL-8和TNF-α显著上调;PRRSV/PCV2共感染组仅有IL-8和TNF-α的mRNA表达水平上调且差异显著;并且,PRRSV/PCV2共感染组IL-1β、IL-6和IL-8的mRNA表达水平均低于单独感染组,仅TNF-α的mRNA表达水平显著高于单感染组。结果提示,TNF-α的过量表达可能在PRRSV和PCV2协同致病机制中扮演重要角色。     

鳗弧菌免疫诱导牙鲆IL-1β、IFN-γ和TNF-α基因表达变化

黏膜免疫是鱼类免疫系统的重要组成部分。多聚免疫球蛋白受体(p Ig R)能够介导黏膜抗体的转运和分泌,而细胞因子IL-1β、IFN-γ和TNF-α对p Ig R表达具有调节作用。本研究利用实时荧光定量PCR技术(q PCR)测定了牙鲆(Paralichthys olivaceus)组织中TNF-α、IL-1β和IFN-γ基因的表达动态。结果显示:浸泡和腹腔注射两种免疫方式皆可诱导3种基因发生显著上调表达;3种基因的上调表达在96 h内先上升,后降低到对照组水平,其中IL-1β和IFN-γ的上调幅度高于TNF-α,免疫后在牙鲆组织内的表达量分别比对照组高10~60倍和6~30倍,而TNF-α仅比对照组高2~4倍;不同免疫方式诱导的牙鲆各组织中3种基因的应答表达存在差异,浸泡免疫对黏膜免疫组织皮肤和鳃中的基因表达影响较大,显著上调达到峰值的时间短且峰值高,而注射免疫对系统免疫组织脾、头肾以及肝脏中的基因表达影响较大,其中在脾和头肾中注射免疫引起的IL-1β和IFN-γ的变化分别比对照组高50~60倍和30倍,浸泡组比对照组分别高15倍和10倍;前肠、中肠和后肠中,两种免疫方式诱导的3种细胞因子的上调表达量相近或浸泡组稍低。这些结果为进一步研究鱼类细胞因子IL-1β、IFN-γ和TNF-α对p Ig R的表达调节提供了资料。     

鸡传染性支气管炎病毒感染对雏鸡黏膜固有免疫相关受体和细胞因子转录水平的影响

为探讨鸡传染性支气管炎病毒(IBV)感染与宿主固有免疫系统的相互作用及其致病与免疫机制,本研究采用实时荧光定量RT-PCR方法检测IBV变异株GX-YL5感染鸡后不同时间点气管黏膜和哈德氏腺中固有免疫相关受体和细胞因子的mRNA水平以及IBV载量的动态变化。结果显示,气管黏膜和哈德氏腺中Toll样受体TLR2、TLR3、TLR6、TLR7、趋化因子MIP-1β和趋化因子受体CXCR4、CCR4在IBV感染后1~8天以及细胞因子IFN-α、IFN-β、IL-1β、IL-6、IL-10在IBV感染后1~5天,mRNA转录水平至少有一个时间点显著(P0.05)或极显著(P0.01)上调;感染后14~28天,一些受体和细胞因子的mRNA则出现下调;气管黏膜和哈德氏腺的IBV病毒载量在感染后立即上升,第3天达到峰值,随后下降。结果表明,IBV感染早期气管黏膜和哈德氏腺中固有免疫相关受体和细胞因子的mRNA转录水平显著上调,说明IBV感染对宿主固有免疫应答产生明显的影响。     

H5N1亚型禽流感病毒和新城疫病毒感染鸡胚成纤维细胞IFN-β基因的动态表达

根据鸡β-干扰素（ChIFN-β）基因保守序列设计引物建立了检测鸡IFN-β mRNA表达水平的荧光定量RT-PCR (RRT-PCR)方法,并对H5N1亚型禽流感病毒（AIV）和新城疫病毒强毒株（vNDV）感染后3、6、12、24、30 h的鸡胚成纤维细胞（CEF）中IFN-β mRNA表达水平进行了检测。结果显示,建立的RRT-PCR特异性好,对鸡IFN-β mRNA的扩增效率为94.18%,线性范围为10^-8～10^-3,相关系数为0.992,最低能检出48拷贝/反应。AIV在感染CEF后6、12 h显著抑制IFN-β mRNA的表达,24 h开始诱导IFN-β mRNA表达,30 h时IFN-β mRNA水平显著升高;vNDV在感染CEF后的24 h内显著抑制IFN-β mRNA的表达,30 h时则显著诱导 IFN-β mRNA表达。本试验结果为进一步研究H5N1和NDV与机体的相互作用提供了有价值的信息。      

鸡白痢沙门菌感染产蛋期蛋鸡的致病性分析

为了解鸡白痢沙门菌感染产蛋期蛋鸡致病特点,采集2022年2-11月感染产蛋期蛋鸡的肝脏等组织开展研究。对病鸡的肝脏、脾脏进行细菌分离,对分离菌株进行多重PCR鉴定和血清型检测;取病变的肝脏等组织制备病理切片以观察其病理变化;通过qRT-PCR分析肝脏、脾脏组织中鸡白痢沙门菌毒力基因和细胞因子mRNA的表达水平。结果表明,从送检的蛋鸡中分离出18株细菌,在麦康凯培养基上生长无色小菌落,多重PCR鉴定和血清型为鸡白痢沙门菌;病理切片可见心肌、肝脏及肺脏炎性细胞群浸润和细胞坏死;qRT-PCR检测显示感染鸡白痢沙门菌的病鸡诱导生物被膜相关基因(CsgB、RpoS、bcsA和PagC)、SPI-1基因(HilA、InvA、PrgH和SipC)和SPI-2基因(SteE、RcsC、SseL和SpiC)等毒力基因的表达水平上调;感染产蛋鸡的肝脏中IL-1β、IL-6、IL-4和IL-13的表达水平上调,IL-18和IFN-γ表达水平降低;脾脏中IL-6高度表达,IL-1β和IL-18表达水平显著降低。本研究中感染病鸡组织中鸡白痢沙门菌使生物被膜、SPI-1、SPI-2相关毒力基因和TH2分泌的细胞...     

大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织细胞因子表达及损伤程度的影响

本试验旨在研究体外感染金黄色葡萄球菌与大肠杆菌对奶牛子宫内膜组织中细胞因子白介素-6(IL-6)、IL-1β和IL-8的表达及损伤程度的影响。以体外培养的奶牛子宫内膜组织作为研究对象,采用1×10~5～1×10~9 CFU/mL大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织进行体外感染,通过实时荧光定量PCR和ELISA方法检测两种细菌刺激后奶牛子宫内膜组织中IL-6、IL-1β及IL-8 mRNA与蛋白的表达量,并用HE染色法观察两种细菌感染后奶牛子宫内膜组织病理学切片。结果显示,1×10~5～1×10~9 CFU/mL大肠杆菌体外感染后,奶牛子宫内膜组织中IL-6、IL-1β和IL-8 mRNA表达量均极显著高于空白对照组(P0.01);金黄色葡萄球菌感染浓度为1×10~5、1×10~6 CFU/mL时,IL-6、IL-1βmRNA表达量极显著高于空白对照组(P0.01),感染浓度为1×10~7 CFU/mL时,IL-6、IL-1βmRNA表达量显著高于空白对照组(P0.05),感染浓度为1×10~6 CFU/mL时,IL-8 mRNA表达量显著高于空白对照组(P0.05),其他感染浓度均与空白对照组无显著差异(P0.05)。奶牛子宫内膜组织感染1×10~5～1×10~9 CFU/mL金黄色葡萄球菌和大肠杆菌时,IL-6、IL-1β及IL-8蛋白表达量均极显著高于空白对照组(P0.01)。相同浓度的大肠杆菌感染奶牛子宫内膜组织后,IL-6、IL-1β及IL-8 mRNA与蛋白表达量均极显著高于金黄色葡萄球菌感染组(P0.01)。HE切片染色结果显示,大肠杆菌感染后仍有部分上皮细胞保留,而金黄色葡萄球菌感染后上皮细胞全部脱落。本试验结果表明,大肠杆菌与金黄色葡萄球菌感染奶牛子宫内膜组织后,引起的炎症反应不同。大肠杆菌感染后,促炎性细胞因子被显著上调,而金黄色葡萄球菌感染后破坏子宫内膜上皮细胞程度更加严重。     

Alteration in lymphocyte responses,cytokine and chemokine profiles in chickens infected with genotype VII and VIII velogenic Newcastle disease virus

Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus–host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM⁺ B cells and KUL01⁺ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P < 0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P < 0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4 dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3 dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4 dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4 dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.     

两株不同J亚群禽白血病毒株诱发组织增生病变和细胞因子表达的比较

旨在比较ALV-J-SD1005毒株和ALV-J-NX0101毒株感染鸡只致病性、诱发先天性免疫因子和致肿瘤因子表达的差异,用感染剂量为10³TCID50的两株病毒分别颈部皮下接种75只1日龄海兰褐鸡,感染后7、14、21 d检测体重、肿瘤病变、死亡率、血液和皮下纤维组织中病毒含量以及肝中鸡TRIM25、MDA5、IRF7、IFN-α/β、14-3-3σ、P53、STAT1等免疫因子或肿瘤因子的mRNA表达量。结果显示,雏鸡感染ALV-J-SD1005毒株后最早于第10天出现纤维组织增生,14 d致纤维组织增生率为100%(18/18),死亡率为5.2%(1/19);随着感染日龄的增加,增生组织指数和死亡率不断升高,21 d时分别达34.4%(74.5/209.5)和58.3%(7/12)。血液和皮下纤维组织的病毒载量显著升高;同时,显著上调鸡肝中MDA5、IRF7、P53等基因的表达量,下调IFN-α/β和14-3-3σ基因的表达量;而鸡TRIM25基因呈现感染早期(7 d)表达显著下调,后期(14~21 d)表达显著上调。ALV-J-NX0101毒株感染后21 d未检测到肿瘤发生,也没有鸡只死亡,但见鸡血液等组织中病毒载量显著增多,鸡TRIM25、MDA5、IRF7、IFN-β、IFN-α基因表达显著下降,STAT1基因表达显著上调。上述结果可以看出,ALV-J-SD1005毒株与ALV-J-NX0101毒株在感染鸡体内诱发不同的抗病毒反应和抗肿瘤反应,导致产生明显不同的病毒增殖和致病特点。本研究为深入理解两株ALV-J病毒致肿瘤机制、探索新诊断标识提供重要科学依据。     

Proinflammatory cytokine expression in the lung of pigs with porcine circovirus type 2-associated respiratory disease

Porcine circovirus type 2 (PCV2) causes a significant health problem for the swine industry worldwide. In this study, we investigated the cytokine expression profiles (IFN-γ, IL-1α, IL-8, and IL-10) in the lungs of pigs with PCV2-associated respiratory disease. The mRNA expressions of IL-1α and IL-8 were significantly up-regulated in pigs with PCV2-associated respiratory disease, while IL-10 expression was not detected. These results suggest that the increased expressions of proinflammatory cytokines in the lungs may play an important role in the immunopathologic response in pigs with PCV2-associated respiratory disease.     

Profiling pro-inflammatory cytokine and chemokine mRNA expression levels as a novel method for selection of increased innate immune responsiveness

Previous studies using F1 reciprocal crosses and two parental lines of broilers show the sire is instrumental in determining the in vitro leukocyte function and cytokine/chemokine profile. Since the innate immune response is the primary means young chickens have to protect themselves, we hypothesize utilizing a novel genomics approach to select sires based on an elevated pro-inflammatory cytokine and chemokine profile. By identifying sires with increased pro-inflammatory cytokine (interleukin [IL]-1beta and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels, we expect the progeny will also have elevated profiles. We characterized the pro-inflammatory cytokine and chemokine profile of 119 sires using quantitative real-time RT-PCR (qRT-PCR) and identified two populations with inherently high and low mRNA expression levels of IL-1beta, IL-6, CXCLi2, and CCLi2. Select high and low sires were then used to produce progeny for the second phase of the trial. Blood samples were collected from 214 progeny and the cytokine and chemokine mRNA expression levels determined. Progeny from high sires had significantly (P相似文献   

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.     

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01(+) PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1beta, IL-6, IL-8, IL-10, IL-18 and IFN-gamma mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.     

干扰素刺激基因对羊口疮病毒在OFTu细胞中增殖的抑制效应

羊口疮病毒（ORFV）是重要的人兽共患病病原,不仅严重危害养羊业,而且威胁人类健康。干扰素刺激基因（stimulator of interferon genes,STING）作为细胞的DNA感受器,在机体天然免疫中起重要作用。为探索STING在ORFV感染中的作用及其对病毒复制的影响,本研究构建了ORFV感染羊胚胎鼻甲细胞（OFTu）的模型,分析了ORFV感染细胞后对STING及其相关基因的动态表达,探索了STING基因在干扰表达和过表达状态下对ORFV在细胞上增殖的影响。结果表明,ORFV感染OFTu细胞后,STING、cGAS、TBK1、IRF3、IRF7、IL-6、IFN-β、IL-1β和TNF-α的转录明显升高。OFTu细胞过表达STING可导致RIG-1、DDX41、IFI16、IRF3、IRF7、IL-6、TNF-α、IFN-α和IFN-β等基因转录上调。OFTu细胞在STING过表达状态下感染ORFV可介导TBK-1、IRF3、IFN-β和TNF-α的转录升高,抑制ORFV的复制;在STING表达干扰的状态下,ORFV感染OFTu细胞降低了TBK-1、IRF3、IFN-β和TNF-α的转录,增加了ORFV的复制。这表明STING蛋白能够增强抗病毒细胞因子的表达,抑制ORFV在OFTu细胞中的增殖,研究结果为深入理解STING在羊口疮病毒感染和复制中的作用提供了科学的理论依据,也为深入探索ORFV感染和致病的分子机制提供了基础数据。