Detection of viral antigens in bluetongue virus-infected ovine tissues, using the peroxidase-antiperoxidase technique

An indirect immunoperoxidase procedure was developed to detect viral antigens in bluetongue virus (BTV)-infected tissues. Embryonating chicken eggs were infected with BTV serotypes 10, 11, 13, or 17, and the chorioallantoic membranes were subsequently fixed in formalin and embedded in paraffin. The peroxidase-antiperoxidase (PAP) system was used to examine the infected membranes for the presence of viral antigens. Sheep antisera raised against BTV serotypes 10, 11, 13, and 17 served as the primary antibodies in the PAP procedure. Specific staining was observed when each of these antisera was applied to membranes expressing antigens of homologous and heterologous BTV serotypes. The PAP method was rapid, reliable, and specific in its detection of BTV.     

Detection of bovine virus diarrhea virus in cell culture using an immunoperoxidase technique

An indirect immunoperoxidase staining technique was developed for identifying cell cultures infected with bovine virus diarrhea virus. Infected cell monolayers stained intensely while uninfected monolayers remained colorless. Immunoperoxidase staining was as sensitive as direct immunofluorescence in detecting endpoint dilutions of virus suspensions. Using the immunoperoxidase technique, infected monolayers were detectable by macroscopic, as well as microscopic, observation.     

Detection of canine distemper viral antigen in formalin-fixed and paraffin-embedded tissue of a fitch (Mustela putorius), using an immunoperoxidase technique

The present study describes how a naturally infected fitch (Mustela putorius) caused an outbreak of canine distemper in a colony of insufficiently vaccinated dogs. The detection of canine distemper virus (CDV) on paraffin sections of different organs of the fitch and one of the dead dogs was achieved using a monoclonal antibody against the nucleocapsid protein (NP) of CDV and the avidin-biotin-peroxidase complex (ABC) technique.     

Detection of leptospires in tissue using an immunoperoxidase staining procedure

An immunoperoxidase technique for the localisation of leptospires in sections of formalin fixed paraffin embedded kidney tissue is described. The procedure utilises a two-layered antibody sandwich with rabbit anti-leptospiral immunoglobin. Using antiserum to specific leptospiral serovars the presence and distribution of specific serovar in the tissue could be determined. The technique was also used to detect leptospires of given serovars in smears made from infected tissues and fluids. There was good correlation between culture results and results of the immunoperoxidase staining method on kidneys infected with leptospires. The diagnostic possibilities of the technique on formalin fixed tissue specimens are discussed.     

Detection of African swine fever viral antigens in paraffin-embedded tissues by use of immunohistologic methods and polyclonal antibodies.

Tissues obtained from pigs inoculated with African swine fever virus (ASFV), fixed by vascular perfusion using glutaraldehyde, and embedded in paraffin or araldite were used for an immunohistologic electron microscopic study. To detect ASFV antigens, 4 methods were used on paraffin sections with or without pretreatment of the tissues. Use of biotinylated anti-ASFV antiserum combined with avidin-biotin complex and peroxidase proved to be the most suitable method, and antigen was detected in tissues infected with 2 ASF viruses of different virulence. Use of the glutaraldehyde fixation method should ensure optimal morphologic (structural and ultrastructural) data while allowing an immunohistologic study, and add to knowledge of the pathogenesis of ASF.     

Validation and standardization of virus neutralizing test using indirect immunoperoxidase technique for the quantification of antibodies to rabies virus

A virus neutralizing test using an indirect immunoperoxidase technique (VNT-IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT-IIP has the advantage that results obtained can be viewed by the naked eye. The purpose of this study was to validate the VNT-IIP and compare it with one of the international standard methods, the fluorescent antibody virus neutralization test (FAVNT). The VNT-IIP showed satisfactory repeatability, high analytical specificity and good accuracy. Regarding the comparison between the VNT-IIP and the FAVNT, the VNT-IIP showed good agreement (91.9%), high sensitivity (92.8%) as well as specificity (87.0%) and good correlation (r = 0.92). As described above, the validation of the VNT-IIP was satisfactory and the performances of the test proved to be equivalent to those of an international standard method.     

间接酶免疫组化检测DPV在感染鸭体内细胞定位的研究和应用

以超速离心浓缩结合蔗糖密度梯度离心法提纯的鸭瘟病毒（DPV）作为抗原免疫家兔制备兔抗DPV血清,用饱和硫酸铵沉淀结合DEAE-Sephedax-A-50离子交换柱层析提纯兔抗DPV IgG,建立了检测甲醛固定鸭体组织石蜡切片上DPV抗原的间接酶免疫组化法。该法能特异地检测出DPV感染鸭组织中的DPV而对鸭疫里默氏菌、鸭源多杀性巴氏杆菌（5∶A）、鸭源大肠杆菌（O1）感染致死的鸭组织以及正常鸭组织呈阴性反应,检测DPV感染死亡鸭的肝、十二指肠、脑、脾和胸腺的结果与PCR检测结果相同。其敏感性高于原位杂交。对DPV CH强毒株人工感染致死鸭的检测结果表明,该法可特异检测到肝、肺、肾、脑、十二指肠、空肠、回肠、直肠、法氏囊、脾脏、腺胃以及食管中的DPV。DPV主要分布于这些器官的上皮细胞和巨噬细胞。对1992-2004年经10%福尔马林保存的鸭瘟临床病例的肝脏检测结果均为阳性。该法可用于DPV感染鸭的诊断和定位检测,也可用于对甲醛固定组织进行回顾性诊断检测。     

Detection of bovine herpesvirus type 1 via an immunoperoxidase method, using monoclonal antibodies

The sensitivity and specificity of the immunoperoxidase technique, using monoclonal antibodies, for the detection of bovine herpesvirus type 1 (BHV-1) was assessed and compared with viral isolation methods. In this study, BHV-1 antigens were detected in impression smears of brain obtained from calves in which BHV-1 was isolated. False-positive results were not observed after double-blind examination. Preliminary identification of isolates as antigenic variants was possible by use of 3 monoclonal antibodies reactive with neurotropic and/or pneumotropic strains of BHV-1. Results were consistent with previous work in which characterization was performed by use of immunofluorescense and ELISA. The immunoperoxidase technique, using monoclonal antibodies, was determined to be specific and sensitive, compared with viral isolation, for the diagnosis of BHV-1 encephalitis. In addition, it has operative advantages in that the assay does not require tissue culture facilities, and results can be obtained within hours after specimens are obtained.     

Identification of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining

This paper describes the demonstration of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining. An indirect immunoperoxidase method was applied to the organs of 20 dogs in which leishmaniasis was previously diagnosed. Haemosiderin pigment was eliminated with 5 per cent oxalic acid. Amastigotes of L donovani appeared as dark brown stained bodies which contrasted with haematoxylin stained host cells. No positively stained amastigotes could be seen in any of the sections incubated with control serum. The organs which more frequently showed leishmanids were: skin (macrophages and fibroblasts), liver, spleen, lymph nodes and bone marrow. In a few cases amastigotes were seen in kidneys, gut, adrenal glands, eyes and testicles. This technique is simple to perform, gives consistent results and allows unequivocal histopathological diagnosis of canine leishmaniasis.     

Detection of antibody to Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells by immunoperoxidase techniques

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-l-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron-microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.     

Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroenteritis (TGEV).

An immunoperoxidase technique performed on the TGEV-infected cells was developed for detection of virus-induced antigens. The presence of M antigen of TGEV on the surface of infected cells was demonstrated by this technique. This finding is in contrast to the M protein of murine hepatitis coronavirus which migrates to the Golgi apparatus but is not transported to the plasma membrane. The time course of appearance M and S antigens on the surface of TGEV-infected cell can be studied by this technique.     

Use of an immunoperoxidase stain for the demonstration of bovine viral diarrhea virus by light and electron microscopies

Bovine viral diarrhea (BVD) virus-specific antiserum eluted from an immunoabsorbent column was conjugated with horseradish peroxidase. The immunoperoxidase (IP) conjugate was used to stain BVD virus-inoculated and control tissue cultures processed for light and electron microscopies. Infected cells (in the inoculated cultures) viewed by light microscopy had diffuse staining of the cytoplasm of nonvacuolated cells and a more predominant staining associated with vacuoles as vacuolation occurred. The peroxidase label as demonstrated by electron microscopy was associated primarily with nonenveloped and membrane-associated viral particles. Viral particles 20 to greater than 100 nm (diam) were observed. Some of the particles which were greater than 30 mm in diameter contained multiple nuclear cores. The most intensely stained viral particles were outside of the cells.     

Detection of Akabane viral antigens in spontaneous lymphohistiocytic encephalomyelitis in cattle.

A 5-month-old Japanese black bull calf and twenty-seven 1-27-day-old calves exhibiting neurological signs between August and October 1998 were examined. The bull calf exhibited rapid breathing, fever, hypersensitivity, and ataxia and was euthanized 4 days after the onset of symptoms. The 27 calves primarily exhibited ataxia, and 15 had arthrogryposis. Histological examination of the bull calf revealed perivascular infiltraction by mononuclear cells, diffuse to multifocal gliosis, and neuronal necrosis in the brain and spinal cord. Multiple malacic foci were found in the midbrain in 5 cases. In contrast, in the 15 calves necropsied in October, there were fewer inflammatory changes, but there was neuronal cell loss in the ventral horn and a decrease in myelinated axons in the lateral and ventral funiculi. Immunohistochemical examination using a rabbit antiserum against Akabane virus strain OBE-1 revealed a large amount of viral antigen in the degenerating neurons and glial cells of the bull calf, mainly in the spinal gray matter. Small amounts of viral antigen in swollen axons and a few glial cells were found in 5 of 27 calves. Thirteen of the 27 calves had high neutralization antibody titers against the Akabane virus, whereas there was no significant antibody titer in most of the calves necropsied during August. The present study revealed that viral antigen detection was very useful for the diagnosis of Akabane diseases in the 5-month-old bull calf that was suspected to be infected postnatally, while it had limited usefulness in the other young calves.     

Detection of bluetongue virus in bovine fetuses using the avidin-biotin complex immunoperoxidase method

The avidin-biotin complex immunoperoxidase technique was adapted for use in detecting bluetongue virus (BTV) antigens in BTV serotype 11-infected bovine fetuses. Fetuses were infected with BTV serotype 11 at 120 days of gestation and then removed 20 days later by Cesarean section. Blood and tissue samples were collected from each animal and used for virus isolation in embryonated chicken eggs, the immunofluorescent antibody test, and the avidin-biotin complex test. The avidin-biotin complex method successfully identified BTV antigens in both fresh and autolyzed fetal brains. Thus, the avidin-biotin complex immunoperoxidase method has potential as a possible procedure for diagnosing bluetongue disease in aborted bovine fetuses.     

均质荧光复合微球技术定量检测狂犬病病毒中和抗体方法的建立

为实现对狂犬病病毒中和抗体的绝对定量检测,建立了检测中和抗体的均值荧光复合技术。用狂犬病毒糖蛋白标记带有荧光物质Eu3+的纳米微球载体,并用其特异性结合已捕获的中和抗体,根据荧光强弱判定抗体效价。通过试验我们获得了最佳反应条件;在此条件下,荧光强度与中和抗体效价具有良好的线性关系,相关系数能达到0.99以上;而且检测灵敏度能达到0.01IU/mL;另外此方法对犬温热病毒和犬细小病毒阳性血清不产生交叉反应,具有良好的特异性;并且与金标准FAVN的符合率达到98%。此方法不仅能够用于狂犬病毒中和抗体的特异性检测,而且能够实现绝对定量检测;在狂犬疫苗免疫效果评价和动物的检验检疫等应用中具有重要意义。     

Demonstration of bovine viral diarrhoea virus antigens in cell cultures and in paraffin-embedded tissue sections by the peroxidase-antiperoxidase (PAP) technique using monoclonal antibodies

The diagnosis of both bovine viral diarrhoea (BVD) and mucosal disease (MD) is usually made on the basis of characteristic clinical and pathological findings. The definitive etiological diagnosis by virus isolation is time consuming, expensive and elusive. Isolation of the virus in cell cultures is rather difficult since it has no characteristic cytopathic effect (CPE). Furthermore, many strains have no CPE at all. Due to these uncertainties, virus isolation trials are generally supported by additional tests (Radostits & Littlejohns 1988).     

Detection of transmissible gastroenteritis coronavirus antigens by a sandwich enzyme-linked immunosorbent assay technique

A new sandwich enzyme-linked immunosorbent assay, using monoclonal and polyclonal antibodies, was developed to detect transmissible gastroenteritis virus antigens from cell culture and from intestinal wash or feces obtained from experimentally infected pigs. This technique was shown to be suitable for the detection of virulent field strain unadapted to cell culture. Cross reactions had not been observed with other enteric pathogens, rotavirus, porcine epizootic diarrhea virus, and Escherichia coli.     

Morphologic and immunoperoxidase study of neurologic lesions in naturally acquired rabies of raccoons.

Histopathologic (hematoxylin and eosin [HE]) and immunoperoxidase (streptavidin-biotin complex) methods were used for examination of formalin-fixed tissues of rabid raccoons from an enzootic area of Pennsylvania. Extensive morphologic lesions of rabies encephalitis were present in the cerebrum and the brain stem regions. Negri bodies were detected by both methods and were present in the brain (cerebral cortex, hippocampus, brain stem, cerebellum, and cervical spinal cord) and in the ganglia of the trigeminal nerves. The viral inclusions were also seen in ganglion cells in the tongue, parotid salivary glands, pancreas, intestines, and adrenal glands. These sites were not associated with any inflammatory cellular infiltrate. The immunoperoxidase method was superior to HE for the detection of Negri bodies. Because lesions of rabies encephalitis were consistently observed in the cerebrum, brain stem, and cervical spinal cord regions, these areas of the brain should be included when raccoons are examined by the fluorescent antibody test for rabies.