Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus in formalin-fixed, paraffin-embedded tissues.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.     

Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues.

Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.     

Immunohistochemical detection of swine influenza A virus in formalin-fixed and paraffin-embedded tissues.

An avidin-biotin complex (ABC) immunohistochemical method utilizing a commercially-available polyclonal antiserum to human influenza A virus was used to detect antigens of influenza A virus in formalin-fixed, paraffin-embedded tissues of swine. Influenza A antigens were immunohistochemically detected in 28/30 cases in which influenza A virus was demonstrated by virus isolation and in 5/22 cases suspected to be influenza A-infected by clinical and histological criteria, but from which the virus was not isolated. Viral antigen was not demonstrated in 30/30 cases not suspected clinically or histologically to be associated with influenza infection. This method is a convenient, sensitive, and specific means of influenza A virus detection and is applicable to both routine diagnosis of influenza A virus infection and to retrospective and prospective studies of the occurrence and the pathogenesis of this virus in pigs.     

Comparison of a new synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test with in situ hybridisation for the detection of swine hepatitis E virus in formalin-fixed, paraffin-embedded tissues

A synthetic, peptide-derived, polyclonal antibody-based, immunohistochemical test was developed to detect swine hepatitis E virus (HEV) and was compared with in situ hybridisation for the detection of HEV in formalin-fixed, paraffin-embedded tissues from experimentally infected pigs. Solid-phase peptide synthesis was used to generate peptides from swine HEV open reading frame 2, and the purified peptides were injected into rabbits to produce polyclonal antibodies. The specificity and sensitivity of the test were both 100%. Liver was most consistently positive for swine HEV antigen and RNA by immunohistochemistry and in situ hybridisation, respectively, but both were detected much less frequently in extrahepatic tissues such as lymph node, tonsil, spleen, and intestine. Swine HEV antigen and RNA showed a similar distribution in virus-infected hepatocytes in serial sections. The novel test developed in this study is suitable for consistently detecting swine HEV antigen in formalin-fixed, paraffin-embedded tissues.     

Identification of porcine macrophages with monoclonal antibodies in formalin-fixed,paraffin-embedded tissues

Here we report the successful use of monoclonal antibodies (mAbs) BL1H7, BA1C11 (anti-SWC3) and 4E9 for immunohistochemical identification of macrophages in formalin-fixed, paraffin-embedded porcine tissues. Retrieval of antigen reactivity was achieved by heating the slides in a domestic pressure cooker, which makes the technique suitable for the routine laboratory. This method allows to perform retrospective studies in routinely processed tissues and may be useful to investigate the role of macrophages in different pathological processes.     

Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.     

Immunohistochemical detection of porcine teschovirus antigen in the formalin-fixed paraffin-embedded specimens from pigs experimentally infected with porcine teschovirus

Porcine teschovirus (PTV) antigens were detected by a streptavidin-biotin complex method in formalin-fixed paraffin-embedded tissues of 3-week-old pigs that had been inoculated intravenously with PTV Talfan strain. PTV antigens were detected in cytoplasm of nerve cells, glial cells and endothelial cells in the cerebellar nuclei, the grey matter of the midbrain, pons and medulla oblongata and the ventral horn of the spinal cord and of ganglion cells in the spinal ganglion corresponding to those lesions characterized as non-suppurative encephalomyelitis and ganglionitis. The results of this study suggest that nerve cells of the brain stem and spinal cord and ganglion cells of the spinal ganglion permit PTV replication and represent the main target cell population of PTV. This is the first study to demonstrate PTV antigen by immunohistochemistry in formalin-fixed paraffin-embedded tissue specimens from pigs infected with PTV.     

Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers.

Formalin fixation produces cross-links between the proteins and the fixative that alter the ability of some antibodies to recognize antigens. We used formalin-fixed, paraffin-embedded tissues to compare two different antigen retrieval methods for 63 antibodies used in the diagnosis of infectious and neoplastic diseases of animal species. Eighty-four percent of the antibodies needed some type of antigen retrieval for optimal results. Of those antibodies, 67.7% were monoclonal and 32.3% were polyclonal. Steam heat was the method of choice for 31 antibodies. Ten antibodies reacted only with steam heat, but 9 antibodies did not react when steam heat was used. Optimal results were obtained with enzyme digestion for 22 antibodies. Only 10 antibodies yielded optimal results without antigen retrieval; 64% of these antibodies were polyclonal. All antibodies against cytokeratins were optimally retrieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded tissues.     

猪流行性腹泻病毒及感染扩散途径

猪流行性腹泻病毒Porcine Epidemic Diarrhea Viruses(PEDV),是冠状病毒科,冠状病毒属。猪流行性腹泻病(PED)传播速度快,传播范围广。主要原因就是猪流行性腹泻病毒(PEDV)的传播途径多样化。文章就猪流行性腹泻病毒在空气、运输车辆、饲料生产设备、精液的传播途径和经鼻黏膜感染进行讨论。     

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed,paraffin-embedded tissues

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.     

猪流行性腹泻病毒胶体金检测方法的建立

应用胶体金免疫层析技术,建立了一种快速检测猪流行性腹泻病毒(PEDV)的方法。采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗猪流行性腹泻病毒的单克隆抗体m Ab-PEDV1C12和(m Ab)-PEDV2C11,并将m Ab-PEDV2C11和羊抗鼠Ig G抗体喷在硝酸纤维素膜上分别作为检测线和质控线,对pH值、抗体浓度、封闭时间等条件摸索与优化。测试结果表明,该猪流行性腹泻病毒快速检测试纸条的检测下限达到310个TCID50的病毒量,检测时间为10 min,批内和批间重复性为100%。结论:该方法使用简单快速,适合猪场检测猪流行性腹泻病毒。     

Immunohistological localisation of rinderpest virus in formalin-fixed, paraffin-embedded tissues from experimentally infected cattle

Six Freesian steers were subcutaneously inoculated with the virulent rinderpest virus strain Kabete "0" and sacrificed at the height of fever. Sections of formalin-fixed, paraffin-embedded tissues were stained according to the peroxidase anti-peroxidase (PAP) technique. Labelling of viral antigen, both in the cytoplasm and in the nuclei of infected cells, was observed in the epithelial cells of the upper and lower alimentary tract and in lymphoid organs, i.e. spleen, lymph nodes, pharyngeal tonsils, Peyer's patches and thymus. Electron microscopy studies confirmed the results.     

A novel immunohistochemical marker of normal and neoplastic melanocytes in formalin-fixed, paraffin-embedded tissues of albino rats

In albino rats, spontaneous occurrence of melanocytic tumors is rare, with diagnosis difficult. This study evaluated immunoreactivity for PNL2 in normal and neoplastic melanocytes in formalin-fixed and paraffin-embedded tissues of albino rats. The samples consisted of 11 (1.57%) amelanotic melanomas in 700 rats (2 studies), 23 non-melanocytic tumors, and a wide variety of normal tissues. In normal albino rats, PNL2 stained the melanocytes in the iris and choroid of the eyeball and the hair bulb and basal cell layers of the epidermis of the whole body. In amelanotic melanoma, the tumor cells consisted of spindle cells with eosinophilic cytoplasm without melanin granules. PNL2 consistently stained cytoplasm in all amelanotic melanoma cells. In contrast, the nonmelanocytic tumor cells were not labeled. Electron microscopically, neoplastic, and normal melanocytes showed numerous cytoplasmic premelanosomes (stage II melanosome). In conclusion, PNL2 is direct against a fixative- and decalcific-resistant melanocyte-associated antigen, and has high specificity against normal and neoplastic melanocytes of albino rats.     

Immunohistochemical detection of feline calicivirus in formalin-fixed, paraffin-embedded specimens.

An immunohistochemical technique was developed for detection of feline calicivirus (FCV) in formalin-fixed, paraffin-embedded cultured cells and tissues. Initial trials with cultured cells indicated that the indirect immunoperoxidase method using rabbit antiserum to FCV strain 255, and horseradish peroxidase-labelled antibodies to rabbit immunoglobulin G lacked sensitivity and showed excessive diffuse background staining despite trypsin digestion of sections before staining. An amplified indirect immunoperoxidase technique using commercially available biotinylated antirabbit antibodies and avidin-biotin-peroxidase or streptavidin-peroxidase (SP) complexes proved highly successful. When optimal conditions, including those for trypsinization, inactivation of endogenous peroxidase and blocking were determined, the SP technique was preferred. Applied to tissue of cats in the acute phase of FCV infection, the technique provided clear identification of cells containing FCV antigens in sections in which histological detail was well preserved.     

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 %。     

猪流行性腹泻病毒S蛋白抗原表位区基因克隆、分析及原核表达

从患流行性腹泻的病猪小肠中分离猪流行性腹泻病毒(PEDV),设计一对引物用于扩增PEDV S蛋白的抗原表位区,将扩增产物克隆到原核表达载体pET-32a,构建了重组表达质粒pET32a-PEDV-S1;在IPTG的诱导下表达,经SDS-PAGE分析,Western blotting鉴定,结果表明该抗原表位区蛋白得到了重组表达.该研究可为PEDV抗原表位区的相关研究提供参考.