Detection of fibrin deposits in horse tissues by immunohistochemistry

BACKGROUND: Histochemical and immunohistochemical techniques have been used to detect fibrin deposits in different tissues in humans and experimental animal models with disseminated intravascular coagulation (DIC). Fibrin deposits also have been observed in horses with severe ischemic and inflammatory disorders by histochemical stainings (phosphotungstic acid hematoxylin [PTAH]). HYPOTHESIS: Immunohistochemical (IHC) methods can be used to accurately detect fibrin deposits in horses at risk of DIC. ANIMALS: Tissue-organ samples collected on postmortem examination from 87 horses with severe inflammatory and ischemic gastrointestinal disorders. In addition, tissue samples from 13 horses with colic and colonic obstructions or displacements and from 13 slaughter horses were used as controls. METHODS: Tissue samples (kidney, lung, and liver) were stained with PTAH and IHC for blinded histologic examination and comparison. A fibrin score (grades 0 to 4) was established for each tissue sample and for each horse for both techniques. RESULTS: When the IHC method was used, fibrin deposition was observed in 47.1% of the horses with colic with a poor prognosis, compared with 41.4% with PTAH. An agreement of 70% was achieved when both methods were compared, and the lung was confirmed as the most affected organ. Almost none of the colic and slaughter control horses had fibrin deposits in their tissues. CONCLUSIONS AND CLINICAL IMPORTANCE: IHC technique for fibrin antigens was very effective in the detection and identification of fibrin deposits in equine tissues and may be a reliable technique for the postmortem diagnosis of DIC.     

检测猪流行性腹泻病毒的R-PCR方法的建立

根据猪流行性腹泻病毒 (PEDV)的N基因自行设计和合成了一对可扩增长度为 641bp目的片段的引物 ,成功地建立了检测的猪流行性腹泻病毒的RT PCR方法。对猪轮状病毒 (PRV)、猪传染性胃肠炎病毒 (TGEV)的RT PCR检测结果均呈阴性。对PEDV JS株的RT PCR产物的序列分析表明 ,与CV777株的同源性为 97 3 %。     

Induction of aggregation in porcine lymphoid cells by antibodies to CD46

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12 production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent on active cell metabolism and on the presence of divalent cations and required a functional cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b. Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is indicative of a role of protein kinases in the transduction of the signal generated by CD46 engagement.     

Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction.

This report describes the first diagnosis of porcine circovirus (PCV) infection in weaned pigs with postweaning multisystemic wasting syndrome in Korea by immunohistochemistry and polymerase chain reaction. The most unique lesions were multifocal granulomatous inflammation affecting lymph nodes, liver, and spleen, characterized by infiltrates of epithelioid macrophages and multinucleated giant cells. Circoviral antigen was detected in formalin-fixed sections and was usually present in large, round, dendritic cells in the white pulp of spleen and remnants of follicles in lymph nodes. Lymphoid follicles in the tonsils also contained PCV antigen. A 530-bp DNA fragment of circovirus was successfully amplified from all tested lymph nodes, liver, and spleen.     

Detection of rabbit haemorrhagic disease virus antigen in tissues by immunohistochemistry

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.     

食源性动物组织中CSFV和PRRSV双重双色荧光定量RT-PCR检测方法的建立及应用

为建立同时检检测食源性动物组织中猪瘟病毒(CSFV)和猪蓝耳病病毒(PRRSV)的双色荧光定量RT-PCR方法。本研究根据SCFV和PRRSV基因序列设计特异性的引物和不同荧光标记的TaqMan荧光探针,通过优化反应的体系和扩增条件,建立了能够检测食源性动物组织中SCFV和PRRSV的双重双色荧光定量PCR的方法。其检测下限为1×102拷贝/μL,而且与其他一些猪病病毒无交叉反应,具有良好的特异性。该方法重复性和稳定性试验表明其组内和组间的变异系数最高分别为4.2和4.5。对比试验表明,该方法对CSFV和PRRSV检验的敏感性为常规RT-PCR方法的200倍;该方法的建立为食源性动物组织中CSFV和PRRSV提供了有效手段,该方法特异性和敏感性较好,能够应用于临床检测。     

一步法多重RT-PCR检测猪圆环病毒2型和猪繁殖与呼吸道综合征病毒

根据猪圆环病毒2型（PCV2）的ORF2和猪繁殖与呼吸道综合征病毒（PRRSV）ORF7相对保守区序列分别设计了两对引物PH1／PH2和PT1／PT2，将PCV2和PRRSV细胞毒按1：1混合，设为模拟样品，采用病毒基因组DNA／RNA提取试剂盒同时提取PCV2 DNA和PRRSVRNA核酸，利用2对引物进行一步法多重RT-PCR。结果同时得到与实验设计相符的560bp（PCV2）和398bp（PRRSV）特异性扩增条带，而对其他5种猪病原的扩增均为阴性。敏感性试验表明，建立的一步法多重RT-PCR方法可检测出10ngPCV2 DNA和5ngPRRSVRNA。     

CD34 protein is expressed in murine,canine, and porcine lungs

The cell surface protein CD34 is expressed in various human tissues and cells, including hematopoietic stem cells, vascular endothelial cells, mucosal dendritic cells, mast cells, eosinophils, microglia, fibrocytes, muscle satellite cells, and platelets. There is a lack of data on the expression of CD34 in canine and porcine tissues. Therefore, we designed a series of immunoblotting, immunohistochemistry, and immunofluorescence experiments to observe CD34 expression in murine, canine, and porcine lungs. We used a rabbit antibody (clone EP373Y) to target the conserved human CD34 C-terminal region and validated its immunoreactivity against mouse lung homogenates. The data showed diffuse bronchiolar and alveolar epithelial localization of CD34 protein in normal murine, canine, and porcine lungs. At 9 or 24 h after bacterial endotoxin exposure, murine CD34 protein shifted to specific bronchoalveolar cells with a punctate pattern, as quantified by CD34 fluorescence. Specific porcine bronchoalveolar cells and leukocytes had significant CD34-positive immunostaining after H3N1 influenza infection. Thus, our study provides fundamental data on the expression of CD34 in lungs and validates an antibody for use in further experiments in these animal species.     

荧光定量分析猪睾丸组织中miR-34b的表达规律

运用荧光定量分析方法检测军牧1号白猪不同发育阶段睾丸组织miRNA-34b的表达量差异,结果:miRNA-34b在3月龄前的表达量都非常低,从5月龄开始表达量呈明显上升,7月龄达到峰值后开始明显下降.在猪的发育过程中,miRNA-34b的表达趋势曲线呈先增后降.结果表明miRNA 34b可能与猪雄性生殖发育相关.     

猪脂肪组织中UCP-2 mRNA表达产物的半定量分析

解偶联蛋白基因是新近发现的能够增加能量消耗，与脂肪代谢和能量调控密切相关的一组基因。本实验室建立了以18s rRNA为内标的RT—PCR法，半定量分析了猪脂肪组织中UCP-2 mRNA的表达水平。提取猪脂肪组织总RNA，经反转录和PCR扩增，PCR产物的电泳条带于VDS摄像系统下扫描灰度找出PCR线性扩增范围，确定RT—PCR的最佳循环数和Mg^2＋浓度。通过UCP-2 mRNA与18s rRNA PCR产物的灰度之比，即可计算出UCP-2 mRNA的相对含量。     

Detection of porcine epidemic diarrhea virus by immunohistochemistry with recombinant antibody produced in phages

Several diagnostic methods including immunofluorescence, enzyme-linked immunosorbent assay, polymerase chain reaction and immunohistochemistry have been developed for the detection of porcine epidemic diarrhea virus (PEDV). An immunohistochemical method using a new recombinant antibody produced by a phage antibody system (PAS16) kit was investigated and compared with that using a monoclonal antibody for PEDV detection in PEDV-infected piglets. In both the immunohistochemical methods, PEDV antigens were detected in the cytoplasm of villous enterocytes and in the macrophages infiltrated in the lamina propria at 18 to 110 hr post inoculation. The positive signals with the recombinant PAS16 antibody were similar to those with the monoclonal antibody. This result suggests that the recombinant PAS16 antibody can be applicable for the rapid immunohistochemical diagnosis of PEDV infection.     

抗体快速金标检测卡结合RT-PCR技术诊断猪繁殖与呼吸综合征

采用猪蓝耳病抗体快速金标检测卡对贵州省不同发病猪场进行血清学检测,其弱阳性检出率为54%。在此基础上参考GenBank登录的猪繁殖与呼吸综合征病毒(PRRSV)LV和PRRSV VR-2332毒株核苷酸序列,设计一对特异性引物,用提取上述检测的某猪场猪繁殖与呼吸综合征疑似病料(包括血液、肝、肺、肾、淋巴结、心、脾组织)总RNA为模版,应用RT-PCR方法扩增出目的条带,大小为739 bp,与预期的结果相同。分析表明:该核苷酸序列与已报道的Hunan-3株的同源性为100%,与国内最早PRRSV分离株CH-1 a同源性为95%,与北美代表株VR-2332和欧洲代表株LV的同源性分别为89.2%和29.2%。说明采用猪蓝耳病抗体快速金标检测卡结合RT-PCR方法作为猪繁殖与呼吸综合征的诊断方法是准确、可行的。     

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

应用多重RT-PCR方法检测158例猪粪样中的两种冠状病毒

根据GenBank上发表的猪流行性腹泻（PEDV）和猪传染性胃肠炎（TGEV）基因序列，针对其PEDVM（膜蛋白）基因保守区及TGEV的S基因（纤突蛋白基因）5’端保守区，各设计一对引物，可特异扩增出目的条带大小分别为467bp和1062bp。用上述两对引物对同一样品中的PEDV和TGEV可进行鉴别检测，对猪的其它病毒和细菌的PCR扩增结果均为阴性。敏感性测定结果表明：该双重PCR方法能检出PEDV1pg、TGEV0．1pg的模板。同时采用建立的多重RT-PCR及韩国引进的PEDV和TGEV病毒抗原快速诊断试剂盒检测结果显示：此多重RT-PCR方法特异性强，且较快速试剂盒更加敏感。应用多重RT-PCR对158份临床猪粪样的检测结果表明：我国很多猪场普遍存在TGEV和PEDV，尤其以PEDV污染更为严重，感染率达53．2％，但双重感染率较低，仅为4，4％。     

Localization and activities of lysosomal enzymes in jejunal and ileal epithelial cells of the young pig

The localization and the specific activities of lysosomal enzymes in jejunal and ileal epithelium of suckling pigs were determined. Acid phosphatase was found in pinocytotic vesicles and vacoles at both intestinal sites in pigs examined during (less than or equal to 1 day old) or shortly after (2 days old) closure. These pinocytolically active vesicles and vacuoles were determined to be phagolysosomes. In the ileum, phagolysosomes persisted well after closure, at an age (10 days) when ileal epithelium retains the capacity for pinocytotic uptake of immunoglobulin, but does not transport it to blood. Neither jejunum nor ileum contained phagolysosomes at an age (30 days) when the intestine had lost both uptake and transport capacities. The specific activities of acid phosphatase and cathepsin B-1 in jejunum were similar for all age groups. However, the activities of these enzymes in ileum of 10-day-old pigs were three and five times greater than at any other age or site. The results are consistent with phagolysosomes in the ileum of 10-day-old pigs functioning as a "barrier" which prevents transport of the macromolecules which are taken up pinocytotically by this epithelium. The results do not permit conclusions whether (or not) the phagolysosomes which appear in jejunum and ileum of pigs less than or equal to 1 day old contribute to closure, because the transcellular route for immunoglobulin absorption in pigs has not been precisely defined.