Morphologic observations of new type gosling viral enteritis virus (NGVEV) virulent isolate in infected duck embryo fibroblasts

The morphogenesis of the new type gosling viral enteritis virus (NGVEV) and the characteristic ultrastructural changes in the duck embryo fibroblasts (DEFs) were investigated by ultrathin sectioning and transmission electron microscopy after monolayer DEFs were experimentally infected with a virulent NGVEV strain. The investigation demonstrated that typical NGVEV particles were round, with a diameter ranging from 75 nm to 90 nm and that they were present in both the nucleus and cytoplasm of the infected DEFs. The mature virions contained nucleocapsids and nucleic acids. The virion penetrated the DEF, replicated, and matured in the nucleus, and they were finally released into the extracellular space via budding and disruption of the cytoplasmic membrane. With the appearance of progeny NGVEV, certain virus-related structures that were densely electron stained, which were circular, U-shaped, or irregular in appearance, could be observed in the cytoplasm of the infected DEFs. In this research, we first detected three types of intracytoplasmic inclusion bodies during the NGVEV infection, which always contained a number of NGVEV particles. Furthermore, we detected that NGVEV could induce apoptosis in DEFs, which had not been reported previously. The morphologic changes of apoptosis included shrinking of the apoptotic cells, chromatin condensation and margination, appearance of vacuoles on the cytoplasmic membrane, and the formation of apoptotic bodies. The mitochondria were ultracondensed and aggregated into compact clusters during apoptosis.     

Immunohistochemical detection and localization of new type gosling viral enteritis virus in paraformaldehyde-fixed paraffin-embedded tissue

To determine the distribution and localization of new type gosling viral enteritis virus (NGVEV) in paraformaldehyde-fixed paraffin-embedded tissues of experimentally infected goslings, for the first time, an immunohistochemical (IHC) staining method was reported. Anti-NGVEV polyclonal serum was obtained from the rabbits immunized with purified NGVEV antigen, which was extracted by caprylic-ammonium sulphate method and purified through High-Q columns anion exchange chromatography. Three-day-old NGVEV-free goslings were orally inoculated with NGVEV-CN strain suspension as infection group and phosphate buffered saline solution (PBS) as control group, respectively. The tissues were collected at sequential time points between 0.5 and 720 h post inoculation (PI), and prepared for IHC staining and ultra-structural observation. The positive immunoreactivity could be readily detected in the lymphoid and gastrointestinal organs of infected goslings as early as 48 h PI, in the liver, kidney, pancreas and myocardium from 72 h, and in the cerebrum and cerebellum from 96 h, while it was hardly detected in the respiratory organs at any time. The positive staining reaction could be detected in NGVEV-infected goslings until 600 h PI, and no positive staining cell could be observed in the controls. The highest levels of viral antigen were found in the bursa of Fabricius (BF), thymus, proventriculus, gizzard and intestine tract, moreover, the liver, kidney, spleen, myocardium and pancreas were intensively and widely stained. The target cells had a ubiquitous distribution, especially included the epithelial cells, endothelial cells, superficial and crypt mucosal cells, glandular cells, fibrocytes, macrophages and lymphocytes, which served as the principal sites for antigen localization. The ultra-structural observation by transmission electron microscope (TEM) further indicated that NGVEV particles could be widely detected in the lymphoid and digestive organs of infected goslings from 72 h PI onwards. This work may be useful not only for offering a possibility of routine diagnosis of NGVE, but also for better understanding of the pathogenesis of the disease.     

雏鹅新型病毒性肠炎病毒强毒在感染鸭胚体内形态发生学和宿主细胞超微病理学研究

将雏鹅新型病毒性肠炎病毒（NGVEV）强毒CH株经尿囊腔途径人工感染10日龄鸭胚,应用透射电镜和超薄切片技术研究病毒在宿主细胞内的形态发生及各组织器官的超微结构变化。结果表明：感染后不同时间剖杀及死亡鸭胚的尿囊膜、肠、心、肝、脑和肌胃组织中,均观察到60～70nm的病毒粒子。病毒粒子主要通过与细胞膜融合而进入细胞质内,然后在细胞核内进行复制和装配。最后病毒粒子通过核膜和细胞膜破裂的方式被释放。病毒侵害的主要靶细胞包括鸭胚尿囊膜上皮细胞、肠上皮细胞、肠道平滑肌细胞、成纤维细胞、肝细胞、肌胃黏膜上皮细胞和心肌细胞等,表现为细胞核内外膜间隙严重扩张,细胞质整体结构严重空化。病毒侵害的主要靶细胞器包括粗面内质网和线粒体,表现为粗面内质网扩张呈囊状;尿囊膜上皮细胞的线粒体出现固缩和异常聚集变化,而其他组织细胞的线粒体均表现为肿胀和嵴断裂、消失。本试验还发现NGVEV可诱导宿主细胞发生严重的细胞凋亡现象,表现为细胞皱缩,胞核内染色质密度增高,核固缩成一个或数个团块凝聚在核膜周边,胞质浓缩深染并形成凋亡小体。     

小鹅瘟和雏鹅新型病毒性肠炎二联PCR检测试剂盒组装和初步应用

本试验旨在研制小鹅瘟和雏鹅新型病毒性肠炎二联PCR检测试剂盒,观察临床应用效果。根据小鹅瘟和鹅病毒性肠炎基因序列,设计合成一对特异性引物,对PCR反应条件进行优化,并分别对试剂盒的特异性、敏感性、重复性和保存期进行研究,之后进行临床扩大试验。该二联PCR诊断试剂盒能扩增出预期的小鹅瘟375 bp核酸片段和鹅病毒性肠炎223 bp核酸片段,与鸭瘟病毒、鹅副黏病毒无交叉反应;最低能检测到小鹅瘟病毒10⁴ ELD₅₀/mL,雏鹅新型病毒性肠炎病毒2.5×10^-1ELD₅₀;不同批次的试剂盒对同一样品检测结果一致;常温下至少可保存5个月;临床应用效果显著。该二联PCR检测试剂盒能够对小鹅瘟和雏鹅病毒性肠炎早期诊断、流行病学调查和卫生检疫提供了检测手段。     

痛风雏鹅肠道菌群通过TLR4/MyD88/NF-κB通路促进肾损伤的研究

旨在研究痛风雏鹅的肠道菌群对肾损伤的影响及作用机制。本研究选取具有典型痛风特征的雏鹅15只,相同日龄及生长环境下的健康雏鹅15只,利用16S rDNA测序技术分析盲肠内容物中菌群的差异;用改良苦味酸法测定血清肌酐（Cr）,用酶比色法测定血清尿酸（UA）,用脲酶-谷氨酸脱氢酶法测定血清尿素氮（BUN）;用RT-qPCR和Western blot法测定肾组织Toll样受体4（TLR4）、髓样分化因子（MyD88）、核转录因子-κB （NF-κB）、白细胞介素1β（IL-1β）、肿瘤坏死因子ɑ（TNF-ɑ）及白细胞介素6（IL-6）的mRNA和蛋白表达量。并将肠道菌群移植给无菌小鼠进行验证。结果显示：1）与健康雏鹅比较,痛风雏鹅肠道菌群16S rDNA测序所得操作分类单元（OTUs）数量、Chao 1指数及Shannon指数均显著降低（P<0.01）,变形菌门、埃希菌属、变形菌属及肠球菌属等细菌丰度显著增加,痛风雏鹅肠道菌群在免疫系统、细菌感染、膜传输及核苷酸代谢等代谢途径参与度较高;2）与健康雏鹅比较,痛风雏鹅肾组织中TLR4、MyD88、NF-κB、IL-1β、TNF-ɑ及IL-6的mRNA表达及蛋白表达均显著升高（P<0.05或P<0.01）,且这些分子表达量与Cr、BUN及UA呈正相关,与埃希菌属、变形菌属及肠球菌属等菌属丰度呈强正相关关系;3）移植痛风雏鹅肠道菌群小鼠的肾小管发生变性,肾组织TLR4、MyD88、NF-κB的mRNA表达量及血中Cr、BUN、肾损伤因子-1（KIM-1）等水平显著高于移植健康雏鹅菌群的小鼠;给予移植痛风雏鹅菌群小鼠TAK-242后,可回调这些变化。综上表明,痛风雏鹅肠道菌群发生了变化,并可激活LTR4/MyD88/NF-κB通路,诱发炎症反应,促进肾损伤的发生。     

Morphological and biochemical aspects of apoptosis,oncosis and necrosis

Recent investigations have demonstrated the need for a precise differentiation of various forms of cell death such as apoptosis, oncosis, necrosis and programmed cell death. Apoptosis is marked by cellular shrinking, condensation and margination of the chromatin and ruffling of the plasma membrane with eventually breaking up of the cell in apoptotic bodies. Cell death marked by cellular swelling should be called oncosis, whereas the term necrosis refers to the morphological alterations appearing after cell death. Apoptosis and oncosis are therefore pre-mortal processes, while necrosis is a post-mortal condition. The term programmed cell death refers to the 'fixed' pathway followed by dying cells, whether or not with the characteristic morphology of apoptosis. Three mechanisms are actually known to be involved in the apoptotic process: a receptor-ligand mediated mechanism, a mitochondrial pathway and a mechanism in which the endoplasmic reticulum plays a central role. All three mechanisms activate caspases which are responsible for the characteristic morphological changes observed during apoptosis. A review of the different methods used for detecting apoptotic cells demonstrates that most of these techniques are not entirely specific.     

小鹅瘟病原学与检测方法的研究

从患病雏鹅的病料样本中分离鉴定出一株小鹅瘟野生强毒.采用鹅胚增殖病毒,以琼脂扩散试验从感染鹅胚尿囊液中检出小鹅瘟病毒抗原;以感染鹅胚尿囊液人工接种5日龄健康雏鹅,复制出与自然感染病例相类似的病变; 通过电镜观察到典型的小鹅瘟病毒粒子.该分离毒株核酸分子量为5 000 bp.制备的小鹅瘟沉淀抗原可以检测患病雏鹅血清中的抗体,监测治疗血清的滴度和疫苗接种的效果.标记的小鹅瘟荧光抗体能直接检测患病雏鹅组织中的病毒抗原.针对编码主要结构蛋白VP3的基因设计合成一对引物,扩增出与预期长度相符的特异性DNA片段.     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

Anatid herpesvirus 1 CH virulent strain induces syncytium and apoptosis in duck embryo fibroblast cultures

Anatid herpesvirus 1 (AHV-1) CH virulent strain was first isolated from an infected duck and it was found that this virus strain could induce cytopathic effect (CPE) in duck embryo fibroblast (DEF). Following AHV-1 infection, DEF showed morphological changes such as cell rounding, improved refractivity and detachment from the culture surface. However, its pathological characteristics were not adequately known. Related studies were performed and the results showed that syncytium formation could be observed as the other type of CPE in AHV-1 infection. Hematoxylin-eosin staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining of infected DEF were each used to visualize the shape and distribution of chromatin within nuclei and nuclear fragmentation was observed. Chromatin condensation and margination, as well as formation of apoptotic bodies were observed by transmission electron microscopy (TEM). DNA ladder formation was detected in AHV-1 infected cells and apoptosis of the infected DEF was also detected by flow cytometry analysis of Annexin V-FITC/PI staining method. Therefore, it was suggested that AHV-1 virulent strain can induce syncytium and apoptosis in DEF. Syncytium formation and apoptosis observed in this study may contribute to the elucidation of AHV-1 pathogenesis.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.     

铅引起细胞急性死亡的途径

以不同质量浓度的铅（１０、２０、４０、８０mg/Ｌ）作用于Ｗish细胞３、６、１２、２４h,探索了铅对细胞急性死亡的影响。结果表明,１０mg/Ｌ的铅使Ｗish细胞出现;了以 环死为特征的细胞死亡铅导致细胞死亡可通过２条不同途径：一个途径为一定剂量的铅通过启动细胞内的凋亡程序,出现以细胞体积缩小、胞质浓缩、ＤＮＡ琼脂糖电泳呈梯状图像、流式细胞仪检测出现明显凋亡峰为特征的细胞凋亡;另一个途径是在铅质量浓度过大时,导     

固始鸡中枢免疫器官细胞和自然凋亡细胞超微结构研究

应用透射电子显微镜技术,对固始鸡17日龄胚胎、4周龄和19周龄固始鸡正常发育的中枢免疫器官超微结构进行了研究。结果表明,固始鸡胚胎胸腺和法氏囊超微结构不同于4周龄和19周龄固始鸡;在中枢免疫器官内免疫细胞存在着自然凋亡现象,主要表现为细胞核的变化,其次是线粒体增生。核质凝集为新月形、C字形、圆环形、花瓣状等多种形态,凋亡细胞裂解为凋亡小体,最后被巨噬细胞吞噬。     

Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study

The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.     

流式细胞术在细胞凋亡研究中的应用

流式细胞术做为一种高新生物技术,在动物医学的各个领域,包括细胞生物学、病毒学、肿瘤学、免疫学和病理学中都得到广泛应用,为细胞凋亡研究提供了有效的技术手段。该技术具有简便、快速、多参数分析等优点,可针对细胞在凋亡时产生的一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases 激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质 Ca2 浓度升高,DNA片段化及含量变化等特点,进行定性、定量测试分析,从而实现对细胞凋亡的准确测定。     

致鹅败血症粪肠球菌的分离鉴定

为确定导致通辽某鹅场雏鹅败血症的病原,本研究将从病鹅心、肝、淋巴结和脑组织液中分离的革兰氏阳性球菌纯化培养,进行形态学和生长耐受性观察,菌属区别、分群和菌种鉴定试验,16S rDNA检测、系统进化分析和致病性试验。结果表明,分离菌株(HQ831431)为中等致病力粪肠球菌,具有β型溶血特性,可致小鼠急性败血症。分离菌株与参考菌株ATCC29212及国内外分离的其他15株粪肠球菌16S rDNA的同源性均在99.0%以上,位于系统发育树的同一分支。     

Experimental study on the role of Brachyspira alvinipulli in intestinal spirochaetosis of geese

Ten one-day-old goslings were inoculated orally with a Brachyspira alvinipulli strain isolated from the large intestine of geese that had died of intestinal spirochaetosis (Group A), 10 day-old goslings were inoculated orally with a B. hyodysenteriae strain (Group B), and a third group of 10 goslings (Group C) served as uninfected control. The goslings were observed daily for clinical signs. They were sacrificed on days 7, 14, 21 and 35 days postinfection (PI), and necropsied. Segments of the large intestine were subjected to histopathological, immunohistochemical, electron microscopic (TEM, SEM) and microbiological examinations. Mortality did not occur during the experimental period. However, in both groups the caecum of the goslings killed by bleeding was slightly dilated, in its lumen there was a watery, yellowish and frothy content, and the mucous membrane was slightly swollen. By histopathological, immunohistochemical and electron microscopic examination, B. alvinipulli and B. hyodysenteriae could be detected in the caecum or colon, in the lumen of the glands and sometimes among the glandular epithelial cells in goslings of the respective groups, and could be reisolated from these organs by culturing. A mild inflammation of the intestinal mucosa was also noted. In transverse section of the brachyspirae, numerous (16-22) periplasmic flagella could be detected inside the outer sheath, also depending on the plane of section.     

小鹅瘟病毒分离与初步鉴定

取死于具有小鹅瘟典型症状的 7日龄吉林白鹅脾脏 ,制成乳剂 ,接种于未免疫小鹅瘟疫苗的健康母鹅所产种蛋的 12日龄鹅胚尿囊腔。结果当盲传至第 3代 ,鹅胚死亡 ,电镜负染尿囊液观察到细小病毒样粒子。雏鹅接种试验结果表明 ,试验组雏鹅表现出小鹅瘟临床症状和剖检特征。由此证明 ,该雏鹅死于小鹅瘟     

小鹅瘟病毒的分离鉴定与临床病变观察

从患病雏鹅的病料样本中分离鉴定出一株小鹅瘟野毒。采用鹅胚扩增病毒。以琼脂扩散试验从感染鹅胚尿囊液中检出小鹅瘟病毒抗原,用感染鹅胚尿囊液人工接种8日龄健康雏鹅,雏鹅表现出典型的小鹅瘟临床症状和剖检特征。     

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer''s patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1β mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1β and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-α decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.     

JA1体外诱导HCC细胞凋亡的实验研究

采用噻唑蓝(MTT)还原法,测定了梯度浓度的某植物种子粗提物JA1对人肝癌细胞株HCC增殖作用的影响;同时采用流式细胞术、DNA凝胶电泳和透射电子显微镜技术,在体外观察了JA1对HCC细胞凋亡的诱导作用。结果显示,JA1可显著抑制肝癌细胞HCC的增殖,而且这种抑制有浓度依赖性和时间依赖性;流式细胞仪分析表明,经JA1作用后的肝癌细胞HCC检测标本中有明显的DNA低含量颗粒(“亚G1期”峰);凝胶电泳呈现出典型的DNA梯形条带;电镜下出现细胞凋亡典型的形态学改变。