欧洲型猪繁殖与呼吸综合征病毒感染仔猪病理学特征及其组织内病毒分布

为了研究仔猪感染欧洲型猪繁殖与呼吸综合征(PRRS)的病理学特征及其病毒在组织内的分布特点,用辽宁分离毒株EU-PRRSV LNEU12接种健康仔猪14d后,扑杀所有仔猪,观察病理学特征并采用免疫组织化学方法分析组织内抗原分布情况。结果表明,欧洲型PRRS主要的病理变化是多发性间质性肺炎和淋巴结炎;病毒主要分布在肺脏、淋巴结、脾脏和脑组织中,其次为肝脏和肾脏。证明PRRS典型病理变化的产生与PRRSV分布情况具有明显的相关性。     

猪细小病毒分离株感染初孕母猪的病毒组织分布和病理学观察

本研究旨在研究猪细小病毒分离株感染初孕母猪后母猪和胎猪的病毒组织分布和组织病理学变化情况。试验选用妊娠35d的初孕母猪接种PPV—BQ株第15代细胞毒,以接种RPMI1640的母猪作为对照。攻毒40d后剖杀,采集母猪的心脏、肝脏、脾脏、肺脏、肾脏、扁桃体、子宫内膜和腹部淋巴结,胎猪的心脏、肝脏、脾脏、肺脏、肾脏、脑和小肠,进行组织病毒载量测定和组织病理学检测。实时定量PCR检测结果显示,攻毒组母猪的心脏、脾脏、肺脏、肾脏和子宫内膜存在病毒复制,子宫内病毒含量最高;胎猪的心脏、脾脏、肺脏中存在病毒复制。对照组脏器中无病毒复制。病理切片结果显示,攻毒组母猪肺脏出现细支气管性肺炎,脾脏出现淋巴细胞减少,心脏、肝脏、肾脏、扁桃体、子宫内膜和腹部淋巴结未见明显的组织病理学变化;胎猪的脏器未见明显的病理损伤。对照组未发现组织病理学变化。     

PRRSV SC-1株在人工感染仔猪体内的分布

为研究猪繁殖与呼吸综合征病毒(PRRSV)在感染猪体内各器官组织的分布,用PRRSV SC-1株人工感染健康断奶仔猪,接毒后23 d,无菌采集试验猪器官组织,用RT-PCR检测其中病毒核酸分布情况。结果显示,心脏、脾脏、肺脏、肾脏、支气管淋巴结、肠系膜淋巴结、腹股沟淋巴结、髂内淋巴结内有病毒核酸,在所有试验猪的肺脏、腹股沟淋巴结都检测到了病毒核酸。本研究结果为PRRS诊断及病毒分离鉴定提供了一定参考依据。     

间接免疫酶组织化学法检测猪繁殖与呼吸综合征病毒感染和抗原定位

以蔗糖密度梯度离心法提纯的猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome,PRRS）病毒SC1株作为抗原,免疫家兔制备兔抗PRRS病毒IgG,成功建立了检测PRRS病毒抗原的间接免疫酶组织化学法。该方法只与PRRS病毒感染猪组织呈现阳性反应,与猪瘟病毒、猪细小病毒、猪伪狂犬病毒、猪乙脑病毒人工感染并致死仔猪的肝脏组织呈现阴性反应。用该方法检测PRRS SC-1株人工感染28日龄仔猪,在感染后7 d即可在肺门淋巴结、胸腺、扁桃体、十二指肠、肺、大脑和肾脏检测到PRRS病毒抗原。PRRS病毒抗原主要分布于胸腺和十二指肠感染细胞的胞浆内、肺门淋巴结小梁周围的淋巴窦和弥散的淋巴组织、扁桃体淋巴结的隐窝及其周边。该法具有特异、直观和敏感的特点,可用于PRRS病毒感染的实验室诊断、抗原定位及甲醛固定样本的回顾性诊断。     

类猪圆环病毒因子P1在自然感染仔猪体内的组织分布

选用3头自然感染仔猪和1头阴性对照猪,剖杀后采集心脏、肝脏、脾脏、肺脏、肾脏、胰脏、脑、空肠、扁桃体、胸腺、淋巴结、膀胱、性腺等组织,用荧光定量PCR方法检测病毒在组织中的分布及病毒载量.结果显示,类猪圆环病毒因子P1分布在仔猪的心脏、肝脏、肺脏、胰脏、脑和膀胱等组织,病毒载量以胰脏、脑和膀胱为最高,可达105拷贝/g以上；对照猪脏器中没有检测到病毒核酸.该研究为P1的诊断及致病机制奠定了基础.     

鸭源流感病毒人工感染鸡的病毒抗原定位动态

研究了由南京地区分离的鸭源流感病毒A/duck/Nanjing/21/95（H9N2？）感染商品来航鸡后，各组织脏器中病毒分离情况和病毒抗原分布。结果表明，禽流感病毒（AIV）可以从肾脏、肺脏、脾脏和心脏中分离出来，接种后3d(PI3），病毒在组织中的分离率最高，且又以肾脏的分离率最高。病毒抗原主要分布在肾小管上皮细胞、呼吸系统单核细胞和少量上皮细胞的胞核内，PI3时病毒抗原的检出率最高。这些结果表明，肾脏是该毒株复制的主要部位，肾脏的病变是病毒直接损伤的结果，而且该株AIV具备在呼吸系统内复制的潜力。     

小反刍兽疫病毒在自然感染羊体内的病毒分布

为了解小反刍兽疫病毒在自然感染羊体内的分布情况,平行采集临床发病羊的脾脏、肺脏、心脏、肝脏、肾脏、大肠、淋巴结以及眼拭子、鼻拭子、肛门拭子、血清等样品,应用荧光RT-PCR方法对各样品进行平行检测,比较各样品检测Ct值,评估病毒分布和各样品中的病毒含量,结果显示,所有样品小反刍兽疫病毒核酸均为阳性,其中大肠、肺脏、脾脏和淋巴结的检测Ct值较小,表明小反刍兽疫病毒在自然感染羊体内呈全身分布,但在大肠、肺、脾和淋巴结等组织中的含量较高。     

猪繁殖与呼吸综合征病毒SJ株(天津分离株)感染仔猪的病理学

选用10～15日龄健康仔猪20头，随机分为攻毒组（15头）与对照组（5头），攻毒组滴鼻接种猪繁殖与呼吸综合征病毒（PRRSV）SJ株，3mL／头；对照组滴鼻接种无PRRSV的细胞培养物，3mL／头。攻毒组的主要临床表现为眼睑水肿、打喷嚏、呼吸急促、嗜睡、体温略升高。攻毒组与对照组在试验开始后24h、7d、14d、21d、28d分别剖杀3头和1头仔猪，采集肺、脾、肾、扁桃体及各部位淋巴结作免疫组化及病理组织学观察；迅速采集肺组织，制作超薄切片，透射电镜观察。试验结果表明，PRRSV SJ株主要感染仔猪的肺脏与脾脏。免疫组化观察，攻毒后24h～7d，肺门淋巴结和扁桃体PRRSV抗原阳性反应最强；攻毒后14～28d，牌睦PRRSV抗原阳性反应最强。攻毒后14～28d，显微病变可见肺组织发生弥漫性、局灶性或间质性肺炎，肺泡隔因巨噬细胞、淋巴细胞及Ⅱ型上皮细胞增生而明显增厚。超微病变主要表现为，肺泡间隔中单核细胞增生，核孔消失，核内有异染色质聚集，Ⅱ型肺泡细胞高度变性等。     

用间接免疫荧光染色法检测鸭病毒性肠炎病毒在人工感染鸭体内的侵染过程和分布规律

用鸭病毒性肠炎病毒（DEV）CHv强毒株感染成年鸭复制鸭病毒性肠炎急性病例,分别于接种后不同时间,取心、肝、脾、肺、肾、胸腺、食道、十二指肠、胰腺、法氏囊和脑组织,制作切片,应用间接免疫荧光染色法（IFA）检测DEV在鸭体内的侵染过程和分布规律。结果显示：感染后4 h可在脾脏、胸腺和法氏囊中检测到DEV抗原;感染后6 h可在肝脏、食道、十二指肠、直肠及肺脏检测到DEV抗原;IFA对各组织器官中DEV的平均检出率为肝脏46/50、脾脏48/50、肺脏46/50、肾脏0/50、肠道46/50、法氏囊46/50、胸腺47/50、胰腺0/50、大脑0/50、食道44/50、心脏0/50。研究表明：在急性病例中,脾脏、法氏囊、胸腺、食道、肠道、肝脏和肺脏为DEV的主要靶器官;接种后,病毒首先在脾脏、胸腺、法氏囊中出现,然后病毒迅速传播到肝脏、消化道和肺脏中;IFA检测石蜡切片中DEV的方法具有直观、特异性强的优点,是对DEV进行检测和抗原定位的较好方法。     

PRRSV在仔猪体内的分布规律及其对组织的病理性损伤

为了研究猪繁殖与呼吸综合征病毒在仔猪体内的分布规律及其对组织的病理性损伤,试验将3头60日龄的健康仔猪随机分为2组,A组2头,B组l头.A组滴鼻接种PRRSV LC毒株(TC:ID50为1×10-5.25/0.1 mL)3 mL/头,B组为对照组,观察临床症状.攻毒25 d后剖杀,采集淋巴结、脾脏、肺脏和肝脏各2份,其中一份进行中性福尔马林固定,制作石蜡切片,观察组织病理学变化;另一份在-70℃保存,利用RT-PCR检测PRRSV.病理学检查结果表明:肺脏出现明显的间质性肺炎,肺泡渗出物增多,可见局灶性出血;脾脏和淋巴结可见出血、增生;肝脏中央静脉及门管区静脉可见明显的淤血.利用RT-PCR方法在淋巴结、脾脏、肺脏和肝脏中都检测到了PRRSV.     

流行性乙型脑炎病毒GI型毒株SD12在C57BL/6小鼠体内的动态分布研究

为了解流行性乙型脑炎病毒基因I型毒株SD12在感染C57BL/6小鼠体内的病毒分布,应用qRT-PCR方法检测该毒株在小鼠体内各组织的动态分布情况.结果显示腹腔感染组中2 dpi仅能在心脏和脾脏检测出病毒核酸,5～7 dpi能够在心脏、肝脏、脑部、盲肠和扁桃体检测到病毒核酸.血脑屏障受损的腹腔感染组中最早2 dpi在肠...     

不同毒力猪繁殖与呼吸综合征病毒对仔猪肺和外周免疫器官损伤的研究

为研究不同毒力的PRRSV对仔猪肺脏和外周免疫器官损伤的差异,本实验分别采用PRRSV变异株(HuN4株)和PRRSV经典株(CH-1a株)感染35日龄健康的断奶仔猪,并在感染后0 d、3 d、7 d、10 d和14 d各迫杀3头,检测肺、颌下淋巴结、肠系淋巴结、腹股沟淋巴结、扁桃体和脾脏的病毒载量及病理变化情况,同时检测血清中抗PRRSV的抗体水平。结果表明:感染后3 d肺脏及各免疫器官可检测到病毒,HuN4感染组病毒载量比CH-1a感染组病毒载量高1 000倍;HuN4感染组病毒载量峰值出现在感染后10 d,而CH-1a感染组维持着较低水平的病毒载量。组织病理学检测显示HuN4感染组淋巴结内淋巴细胞显著减少,呈空泡状;CH-1a感染组淋巴结内淋巴细胞轻度减少,呈星隙状。本实验表明HuN4株比CH-1a株对肺和外周免疫器官造成更严重的损伤。     

猪生殖—呼吸道综合征病毒抗原在死胎及新生仔猪体内的分布

应用抗猪生殖－呼吸道综合征病毒（ＰＲＲＳＶ）单克隆抗体所做的间接免疫荧光抗体试验（ＩＦＡ）,对人工接种ＰＲＲＳＶ的３头妊娠母猪所产９头死胎和２头新生仔猪体内的ＰＲＲＳＶ抗原分布进行了观察。结果表明,ＰＲＲＳＶ抗原在死胎主要分布于脾脏（９／９）和淋巴结（３／４）的巨噬细胞内,而少见于肺（２／９）和肾（０／９）等其他脏器。但新生仔猪则仅在肺脏（２／２）的巨噬细胞内检出了ＰＲＲＳＶ抗原。此结果说明ＰＲＲＳＶ可通过胎盘屏障垂直传播给胎儿,而且ＰＲＲＳＶ对组织的嗜性在胎儿和新生仔猪之间存在差异     

应用灭光定量RT-PCR方法检测PRRSV变异株在仔猪体内动态分布

为进一步了解高致病性猪繁殖与呼吸综合征病毒(PRRSV)变异株(HuN4株)在体内的动态分布规律和特点,探讨病毒对组织器官的嗜性,本研究将HuN4株和细胞传代致弱毒株HuN4-F65人工感染40日龄健康仔猪,分别于感染后1 d、3 d、5 d、7 d、10 d、14 d、21 d和28 d各迫杀2头,并应用建立的荧光定量RT-PCR方法对脑、颌下淋巴结、扁桃体、肺脏、肝脏和十二指肠等组织及血清中的PRRSV进行定量检测.结果显示:感染后HuN4组各器官病毒载量比HuN4-F65组高100倍,病毒栽量峰值期出现在感染后7 d,随后呈下降趋势.HuN4株在仔猪体内分布广泛,在21 d的试验期内持续存在,并且各器官的病毒载量呈正态分布规律;而HuN4-F65株血清病毒载量较低,并且集中在单核巨噬细胞相对密集的扁桃体和颌下淋巴结,与HuN4株主要嗜性器官肺脏相比发生了改变.     

猪繁殖与呼吸综合征病毒和猪瘟病毒混合感染的检测

2005年3月,江苏某猪场仔猪发生体温升高,呼吸困难,四肢末端、耳尖发绀,站立不稳和淋巴结出血为主要症状的疾病。4头发病仔猪的淋巴结、脾、肺脏组织用RT-PCR方法分别检测猪繁殖与呼吸综合征病毒和猪瘟病毒为阳性,用猪瘟ELISA试剂盒检测猪瘟病毒野毒为阳性。结合本病的临床症状和病理剖检,病例确诊为猪繁殖与呼吸综合征和猪瘟野毒的混合感染。     

巴马小型猪对高致病性猪繁殖与呼吸综合征病毒的敏感性评价

为评价巴马小型猪对高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的敏感性,本研究选择PRRSV抗体阴性的16头巴马小型猪和17头本地二元杂交猪作为研究对象,分别分为低剂量接毒组、高剂量接毒组、对照组1和对照组2,低剂量、高剂量接毒组肌肉注射接种PRRSV NVDC-JXA1强毒株,对照组1和接毒组混合饲养,对照组2分开饲养作为空白对照。接毒后每天测量体温,观察精神、食欲、死亡等情况,死亡动物剖检病变,攻毒后每隔7d采血用RT-PCR法检测病原,连续观察21d。结果绝大部分猪体温升高到41℃以上,且有3个以上温次,所有接种及混养动物都死亡,巴马小型猪死亡时间在8d~17d,二元杂交猪在7d~13d,死亡动物出现肺充血、出血、实变,扁桃体、淋巴结、肝脏、肾脏、皮下有出血等症状,病原RTPCR检测为阳性,说明攻毒动物死于HP-PRRS,表明巴马小型猪对PRRSV NVDC-JXA1强毒株非常敏感,可用于PRRS活疫苗的检验。     

实验性水貂链球菌病研究

给水貂人工接种从患病水貂分离的链球菌ＭＳＺ株（Ｃ群）后，采集心、肝、脾、肾、脑和肌肉等器官组织制成标本，用光镜和荧光显微镜观察的结果为，本菌定位的靶器官为脾、肝、肺和心。     

Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs

OBJECTIVE: To evaluate the influences of animal age, bacterial coinfection, and porcine reproductive and respiratory syndrome virus (PRRSV) isolate pathogenicity on virus concentration in pigs. ANIMALS: Twenty-one 2-month-old pigs and eighteen 6-month-old pigs. PROCEDURE: Pigs were grouped according to age and infected with mildly virulent or virulent isolates of PRRSV. The role of concurrent bacterial infection was assessed by infecting selected pigs with Mycoplasma hyopneumoniae 21 days prior to inoculation with PRRSV. On alternating days, blood and swab specimens of nasal secretions and oropharyngeal secretions were collected. On day 21 after inoculation with PRRSV, selected tissues were harvested. Concentrations of PRRSV were determined by use of quantitative real-time PCR and expressed in units of TCID(50) per milliliter (sera and swab specimens) or TCID(50) per gram (tissue specimens). RESULTS: Concentrations of virus were higher in blood and tonsils of pigs infected with virulent PRRSV. Pigs infected with virulent PRRSV and M hyopneumoniae had significantly higher concentrations of viral RNA in lymphoid and tonsillar tissue. Coinfection with M hyopneumoniae resulted in a higher viral load in oropharyngeal swab specimens and blood samples, independent of virulence of the PRRSV isolate. Two-month-old pigs had significantly higher viral loads in lymph nodes, lungs, and tracheal swab specimens than did 6-month-old pigs, independent of virulence of the PRRSV isolate. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple factors affect PRRSV concentration in pigs, including pathogenicity of the PRRSV isolate, age, and concurrent infection with M hyopneumoniae.     

Interferon-alpha-producing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (TGEV) infected piglets

Transmissible gastroenteritis virus (TGEV) infection of piglets results in a very rapid and massive release of IFN-alpha in serum and secretions. The objective of this work was to characterize the IFN-alpha-producing cells (IPC) in tissues of TGEV-infected piglets. Caesarean-derived colostrum-deprived piglets were infected orally with the TGEV virulent Miller strain and IPC were characterized in situ by immunohistochemistry, using a rabbit anti-pig IFN-alpha antiserum. IPC were almost exclusively detected in intestinal tissues and mesenteric lymph nodes (MLN), as early as 6 h post inoculation (p.i.), with a peak at 12-18 h. They disappeared by 24 h. IPC were localized between enterocytes in the small intestine epithelial layer, in the lamina propria, around the Peyer's patches and, at highest frequency, in MLN. Very few IPC were present in the spleen and popliteal lymph nodes of infected piglets. Double immunohistochemical staining for IFN-alpha and leukocyte markers on MLN cryosections showed that IPC were mainly Swine Leukocyte Antigen (SLA) class II positive, and were not stained by an anti-macrophage (SWC3a) MAb. In addition, double staining with anti-TGEV and anti-IFN-alpha MAbs showed that viral antigens were present in MLN, close to IPC. These results show for the first time the presence of IPC in gut mucosa and gut-associated lymphoid tissues in response to an enteropathogenic virus. Moreover, this work shows that IFN-alpha released in serum is likely to originate almost exclusively from gut IPC triggered locally by viral antigens to produce IFN-alpha, since there were very few IPC in spleen or peripheral lymph nodes. MHC class II molecule expression by gut-associated IPC suggests that these cells may be the in vivo mucosal counterparts of the dendritic cells recently shown to produce IFN-alpha after in vitro viral induction.     

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.