HP-PRRSV抑制猪肺eNOS表达与猪肺微血栓形成的相关性研究

为了解高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)与肺微血栓病变的关系及内皮型一氧化氮合酶(eNOS)的表达对微血栓的影响,选取50头70日龄的鄂通两头乌猪,4头作为正常对照,46头感染HP-PRRSV。采取50头猪的肺组织,应用HE染色观察组织病理变化并对微血栓病变程度进行等级评分,将46头感染猪分为轻度微血栓组、中度微血栓组、重度微血栓组,每组选取4头猪进行后续研究。应用免疫组化、荧光定量PCR和Western blot技术,明确HP-PRRSV和eNOS在微血栓猪肺中的表达、分布及变化。组织病理学研究结果显示感染猪肺组织肺泡间隔明显增宽,毛细血管有不同程度的淤血、出血,肺泡腔内有巨噬细胞、脱落的肺泡上皮细胞及淋巴细胞,肺泡壁有大量微血栓。HP-PRRSV主要分布于肺泡巨噬细胞及少量的淋巴细胞的细胞质。随着HP-PRRSV病毒含量的增加,微血栓病变越严重。微血栓病变越严重,死亡率越高。eNOS主要分布于肺泡Ⅱ型细胞、巨噬细胞、血管内皮细胞及支气管平滑肌细胞的细胞质,感染猪肺组织的eNOS mRNA和蛋白表达显著低于未感染对照组。研究结果表明HP-PRRSV使eNOS的表达下调,并参与感染猪肺组织微血栓的形成,增加感染猪的死亡率。     

通城猪抗猪高致病性蓝耳病遗传基础研究

高致病性猪繁殖与呼吸综合征是制约养猪业健康发展的严重的传染病,该病由高致病性猪繁殖与呼吸综合征病毒（HP-PRRSV）引起。作者发现通城猪对该病具有一定的抗性。本研究利用通城猪和大白猪为试验对象,通过人工感染后临床症状观察和组织病变程度对两品种的易感性进行评定。用基因芯片方法比较通城猪在人工感染HP-PRRSV前后猪肺泡巨噬细胞中基因表达差异,发现在1．5倍的阈值下感染前后存在321个差异表达基因其中219个上调,102个下调。感染前后的差异表达基因参与了对细胞骨架、细胞分泌、蛋白降解、蛋白折叠、胞内钙离子及锌离子浓度等诸多方面的调节。研究结果为发现与HP-PRRSV免疫应答相关的基因乃至抗病相关基因提供了依据。     

猪繁殖与呼吸综合征病毒在猪呼吸道内的免疫发病机理

促炎细胞因子和抗炎细胞因子之间的平衡可以调节CD163的表达,影响病毒在不同组织中的致病性和复制能力。在猪繁殖与呼吸综合征中,它们之间的不平衡能抑制感染猪肺脏的免疫反应。猪繁殖与呼吸综合征病毒通过削弱猪肺泡巨噬细胞的功能,破坏黏膜纤毛转运系统及诱导免疫细胞的凋亡来抑制肺脏的局部免疫反应,并降低巨噬细胞的灭菌活性,提高巨噬细胞对继发细菌感染的敏感性。     

猪肺疫与猪伪狂犬病混合感染诊治

猪肺疫又被称为锁喉风,致病原是多杀性巴氏杆菌,该种疾病是一种急性热性传染性疾病。猪伪狂犬病是由猪伪狂犬病病毒感染引起的一种急性传染性疾病,是养殖场普遍发生的一类病毒性疾病,主要危害母猪和仔猪,导致母猪出现繁殖障碍,仔猪出现很高的死亡率,感染该种病毒后的猪会引起机体免疫抑制,影响到猪群的正常生长。该文主要分析了猪肺疫与猪伪狂犬病混合感染综合防治,以进一步提高该种疾病的诊断效果。     

高致病性猪繁殖与呼吸综合征病毒感染对仔猪单核巨噬细胞系统影响的研究

为研究高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)感染对仔猪单核巨噬细胞系统的影响,本实验采用HP-PRRSV Hu N4株人工感染30日龄的健康断奶仔猪。接种后的第7 d、10 d和14 d,分别通过组织学、免疫组织化学以及免疫荧光组织化学方法对脾脏、淋巴结和肺脏组织中巨噬细胞的数量进行研究,分析病毒感染后单核巨噬细胞变化的规律。本研究实验结果显示,感染后仔猪脾脏以及淋巴结中的单核巨噬细胞逐渐增多,第14 d达到峰值;免疫组化增殖细胞核抗原(PCNA)的结果显示,在感染HP-PRRSV的仔猪肺组织和脾组织中出现大量巨噬细胞的增殖。脾组织的免疫荧光双重染色的结果显示PRRSV出现在巨噬细胞中,表明脾脏巨噬细胞是HP-PRRSV的主要靶细胞,并且诱导脾中巨噬细胞的增生。本研究对进一步研究宿主感染HP-PRRSV的天然免疫反应及阐明病毒的致病机制奠定了基础。     

猪繁殖与呼吸综合征病毒在猪肺组织精细切片中感染特性的研究

为研究猪繁殖与呼吸综合征病毒(PRRSV)在猪肺脏组织中的感染特性,本研究建立了高度分化的猪呼吸系统体外培养模型-猪肺组织精细切片(PCLS)。该培养体系包含肺脏组织中多种相关细胞并能够体现出肺脏组织的生理结构和生物学功能。本研究针对制备的猪PCLS进行生物学活性的鉴定,利用评估支气管上皮细胞纤毛摆动百分比的方法检测支气管上皮细胞纤毛活性,结果显示猪PCLS制备良好,在制备10 d以后仍保持95%以上的纤毛活性;利用活/死细胞染色法测定制备的猪PCLS体外培养后活细胞的比例,结果显示制备的猪PCLS在体外培养7 d后支气管上皮细胞和肺泡细胞仍为活细胞;利用间接免疫荧光试验测定猪PCLS中上皮细胞、杯状细胞和肺泡巨噬细胞(PAM)的完整性与分布情况,结果显示制备的猪PCLS上皮细胞和杯状细胞保存完整且猪PCLS中包含丰富的PAM。利用6株不同PRRSV分离株(HuN4、XD-15、WK-34、WK-38、LCL-75和DL-1510)以2.5×10~5TCID_(50)/片的剂量感染猪PCLS,检测不同PRRSV分离株在猪PCLS中的感染特性,结果表明不同PRRSV分离株在该培养体系中表现出不同的增殖能力。本研究表明猪PCLS可用于PRRSV的体外感染试验。本研究为PRRSV的体外研究提供了新的模型与方法,同时也为其它猪呼吸道病原体提供了新的研究平台。     

猪肺疫的防治

1发病情况 2003年秋冬季以来，我市一些镇发生数起“猪肺疫”疫情。特别是养猪专业户尹某，共饲养母猪20头、育肥猪58头，于2003年11月5日该猪场发生猪肺疫，共死亡母猪12头、育肥猪27头，经济损失惨重。此次流行的猪肺疫主要以最急性“锁喉疯”为主，一旦发病，数小时到一日内死亡，发病率高达80％以上，死亡率也在50％以上。病猪呈犬坐姿势，体温突然升高(41～42℃)，     

治疗急性猪肺疫的体会

81年11月至82年2月,我县城关地区猪肺疫暴发流行,大小猪均可被感染,死亡率高,损失严重,仅县食品公司生猪仓库就死猪700余头,占库存数的53.8％。若按常规治疗,患猪一经出现喘气症状,即迅速恶化,口腔、鼻孔一出现泡沫,则很快就会死亡。     

猪肺疫的诊断与中西医防治

<正>猪肺疫(swine pasteurellosis)广泛分布于世界各地,是猪的一种常见传染病,发病率达40%,死亡率在5%左右。随着我国养猪业的不断发展,该病感染的危害性日益凸显,我国猪群以散发为主。近年来,随着我国集约化养猪业的迅猛发展,猪肺疫的报道逐渐增多。     

中西医治疗猪肺疫

正猪肺疫又称猪巴氏杆菌病(清水喉或锁喉风),是由多杀性巴氏杆菌引起的一种急性呼吸道传染病。该病常与猪瘟、猪丹毒一起混合感染。由于该病常呈地方性散发,防疫工作易被一些基层工作者忽视,但实际上猪肺疫还时有发生。1发病情况2014年3月,我镇某养殖户从外地购入50~60日龄仔猪118头,卖方承诺已注射猪口蹄疫、猪瘟-猪肺疫-猪丹毒三联苗、高致病性猪蓝耳病疫苗。进猪5d后,陆续有11头     

PRRSV感染仔猪肾组织的超微病理变化分析

猪繁殖与呼吸综合征病毒（PRRSV）是能引起仔猪高死亡率的疾病,给养猪业带来极大的经济损失。本研究对PRRSV感染仔猪肾组织的病毒定位及产生的超微病理变化进行观察,以期了解PRRSV对肾组织细胞以及血液循环的影响。主要采用透射电镜技术结合血清生化检测、免疫组织化学染色（IHC）,对仔猪感染PRRSV后肾组织进行超微病理学观察及分析。结果发现,PRRSV感染后引起肾组织损伤,病毒主要分布于肾小球、坏死的肾小管上皮细胞胞质及巨噬细胞胞质内;肾小球内足细胞足突广泛融合或微绒毛化,内皮细胞肿胀;肾小管上皮细胞线粒体肿胀、嵴断裂、溶解;间质炎性细胞浸润。PRRSV通过巨噬细胞内复制随血液扩散至肾组织,引起损伤;根据透射电镜观察结果,足细胞足突融合,肾小球血管内皮细胞损伤引起微血栓形成,说明PRRSV感染仔猪后影响了肾小球滤过率,同时,对肾血液微循环系统造成损伤。本研究为阐明PRRSV的感染致病机制提供理论基础。     

藏獒和松狮犬犬瘟热的病理学观察

对藏獒和松狮犬的犬瘟热自然感染病例进行了临床观察、尸体剖检以及病理组织学检查.结果显示,病毒主要侵害犬的呼吸、消化、神经、心血管和免疫系统.肺脏呈严重的间质性肺炎和支气管肺炎的变化,肺泡壁结构消失,肺泡腔内有大量巨噬细胞;胃、肠黏膜充血、水肿,上皮细胞坏死、脱落;脑膜和实质充血、水肿,可见微血栓形成;心脏明显扩张,右心肥大,心室腔内有鸡脂样凝血块;免疫器官受损严重,脾脏和淋巴结均呈明显退行性病变.在胃腺上皮细胞、肝细胞、肺泡上皮细胞和细支气管上皮细胞内发现嗜酸性的核内或胞浆包涵体.     

Pathological, ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (PEARS))

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6-day-old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re-isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum. Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Pathological,ultrastructural, and immunohistochemical changes caused by Lelystad virus in experimentally induced infections of mystery swine disease (synonym: Porcine epidemic abortion and respiratory syndrome (PEARS))

Summary

The pathogenicity and pathogenesis of Lelystad virus was studied in six 6‐day‐old SPF piglets. A third passage of the agent was propagated on porcine alveolar macrophages and intranasally inoculated into pigs. Pigs were killed at hours 24, 48, 60, and 72, and on days 6 and 8 after inoculation. From day 2 on pigs developed diffuse interstitial pneumonia with focal areas of catarrhal pneumonia, and from this day on splenic red pulp macrophages were enlarged and vacuolated. Lelystad virus was re‐isolated from the lungs of infected pigs from day 2 after inoculation. Lelystad virus antigens were detected by immunohistochemical techniques in bronchiolar epithelium and alveolar cells, and in spleen cells of infected pigs from day 2 after inoculation. Ultrastructural examination of tissues by electron microscopy revealed degenerating alveolar macrophages and epithelial cells in lungs and nasal mucosa, with excessive vacuolation of the endoplasmic reticulum.

Although the respiratory tract seems to be the target organ for this virus, macrophages in other organs, such as the spleen, can also be infected. This preference for macrophages may impair immunological defences.     

Ultrastructural Observation of Nasal and Pulmonary Intracellular Pasteurella multocida A:3 in Rabbits

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multocida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the cilated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.     

In utero infection with PRRS virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets

The putative immunosuppressive effect of PRRS virus (PRRSV) on innate immune responses was studied in piglets infected in utero with PRRSV. Phagocytosis and oxidative burst capacities in 2-, 4- and 6-week-old in utero infected piglets were investigated and compared with age-matched control piglets. Phagocytic capacity of blood monocytes against Salmonella bacteria was investigated by flow cytometry. Oxidative burst in blood monocytes and in alveolar lung macrophages was investigated by luminol- and lucigenin-enhanced chemiluminescence, respectively. Decreased phagocytosis against Salmonella was found in blood monocytes from 4- and 6-week-old infected piglets compared to controls. In contrast, 2-week-old infected piglets showed phagocytic responses comparable to age matched control piglets. While oxidative burst capacity was increased in blood (PBMC) from in utero PRRSV infected piglets, the oxidative burst capacity of alveolar lung macrophages was decreased, especially in 2- and 4-week-old piglets, compared to age-matched control piglets. The present results indicate that in utero infection with PRRSV inhibits phagocytosis against Salmonella in blood monocytes as well as the oxidative burst capacity of alveolar macrophages. These observations indicate that PRRSV in utero infection induces at state of immunosuppression in piglets paving the way for enhanced secondary infections.     

Evaluation of ultrastructural changes associated with encephalomyocarditis virus in the myocardium of experimentally infected piglets

OBJECTIVE: To evaluate the ultrastructural changes and localization of encephalomyocarditis virus (EMCV) and viral pathogenesis in the myocardium of experimentally infected piglets. ANIMALS: Eight 20-day-old piglets. PROCEDURE: Six piglets were inoculated oronasally with 5 ml (10(6) median tissue culture infective dose/ml) of EMCV suspension, and 2 were used as uninfected controls. Piglets were euthanatized or died between postinoculation days 1 and 3. Samples of heart tissue from all piglets were evaluated histologically, by virus isolation, and by use of immunohistochemistry and electron microscopy. RESULTS: All infected piglets had gross or microscopic lesions of interstitial myocarditis. immunohistochemically, EMCV antigen was detected in the cytoplasm of cardiac muscle cells, Purkinje fibers, and endothelial cells and in the nucleus of cardiac muscle cells and Purkinje fibers. Ultrastructural lesions were characterized by degeneration and necrosis of cardiac muscle cells and Purkinje fibers. Virus was present intracytoplasmically in cardiac muscle cells, Purkinje fibers, and endothelial cells of capillaries and intranuclearly in cardiac muscle cells. The cell membranes of the Purkinje fibers and endothelial cells had distinct protrusions that contained virus particles. In control piglets, no lesions were found, and no EMCV antigen was detected. CONCLUSIONS: Localization of EMCV intracytoplasmically or intranuclearly in various myocardial cells may well reflect the sites of viral proliferation. The presence of virus particles in cell membrane protrusions and in vacuoles within the lumen of capillaries indicates that virus is released not only by disintegration of the host cell but also via exocytosis.     

Time of appearance of esterase-positive alveolar macrophages in bovine fetal lung

Bovine fetal lung tissue was examined histochemically, using alpha-naphthyl butyrate (nonspecific method), to detect the presence of esterase-positive pulmonary alveolar macrophages (PAM). Positively stained PAM were first seen in the alveolar septa and lumina of the lungs of 4 of 8 fetuses 240 days old. Macrophages (n = 100) in 240-day-old fetuses were 6.74 +/- 0.07 microm, and in most cells, esterase-positive granules were sparsely distributed in the cytoplasm. In 10 fetuses 250 days old, the alveolar septa and lumina of lungs contained positively stained macrophages, as did those of all older fetal lungs examined. Compared with macrophages of the 240-day-old fetuses, those (n = 50) of 250-day-old fetuses were significantly larger, 7.32 +/- 0.10 micron (P less than 0.0001), and esterase-positive granules were heavily distributed throughout the cytoplasm. In all fetal lungs, T lymphocytes contained a single, small, esterase-positive granule, compared with the numerous diffuse esterase-positive granules in PAM. Type-II pneumonocytes with well-developed lamellar bodies had no evidence of positive nonspecific esterase reaction in lungs.     

In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non-BLAD calves.