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W G Van Alstine M Popielarczyk S R Albregts 《Journal of veterinary diagnostic investigation》2002,14(6):504-507
The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC. 相似文献
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采用蛋白酶 Ｋ 消化,酚、氯仿抽提的方法提取法氏囊匀浆和细胞培养液中的鸡传染性法氏囊病病毒（ Ｉ Ｂ Ｄ Ｖ）基因组 Ｒ Ｎ Ａ,用特异的寡核苷酸引物对其 Ｖ Ｐ２ 高变区进行反转录套式 Ｐ Ｃ Ｒ 扩增。应用该方法从一个法氏囊匀浆中即可特异地检出 Ｉ Ｂ Ｄ Ｖ Ｒ Ｎ Ａ,得到的扩增产物可用于分子流行病学的进一步分析,而从感染材料的处理到扩增结果的电泳检测,在两个工作日之内可轻松完成。本实验为法氏囊病病毒的诊断和分子流行病学的分析提供了一种快速、简便、可靠的手段。 相似文献
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F S Kibenge 《American journal of veterinary research》1992,53(8):1337-1342
Two nonoverlapping clones, pOH405 and pOH632, containing cDNA inserts in the VP2 coding region of genome segment A were selected from a cDNA library prepared from the double-stranded RNA genome of the OH strain of infectious bursal disease virus (IBDV) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with IBDV strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with IBDV serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-IBDV double-stranded RNA, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 IBDV double-stranded RNA, was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for IBDV serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 IBDV serotypes by nucleic acid hybridization. 相似文献
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Enzyme immunohistochemical staining of formalin-fixed tissues for diagnosis in veterinary pathology 总被引：1，自引：1，他引：0 
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下载免费PDF全文 Haines DM Clark EG 《The Canadian veterinary journal. La revue veterinaire canadienne》1991,32(5):295-302
Disease diagnosis often relies on the detection of specific antigens in tissue specimens. Enzyme-based immunohistochemical stains of formalin-fixed, paraffin-embedded tissues may be used to identify antigens associated with viral, bacterial and protozoal microorganisms, autoimmunity, and neoplasia. The detection of antigens in routinely fixed tissues offers several advantages over other diagnostic techniques. Sample submission is convenient and facilitates safe handling of potential human pathogens. Retrospective studies of stored specimens are possible. The technique is relatively rapid and enables detection of nonviable microorganisms. In addition, the ability to detect antigens in fixed specimens allows simultaneous visualization of the antigen and the histological lesion which may enhance the accuracy of diagnosis. 相似文献
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Magalhães KG Jannotti-Passos LK Caldeira RL Berne ME Muller G Carvalho OS Lenzi HL 《Veterinary parasitology》2008,152(3-4):333-338
Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections. 相似文献
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Margaret A Miller José A Ramos-Vara Steven B Kleiboeker Robert L Larson 《Journal of veterinary diagnostic investigation》2005,17(5):461-463
The effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus (BVDV) antigen were evaluated in skin. Ear-notch specimens from 2 calves persistently infected with BVDV type 1 were handled in 1 of 3 ways: 1) fixed in formalin promptly and processed for immunohistochemistry (IHC) after 3-176 days; 2) held at 3-4degreesC in plastic bags up to 10 days, then fixed in formalin for 2-5 days before processing; or 3) exposed to room air and temperature for 1-5 days before formalin fixation. Immunohistochemical staining intensity was evaluated without the knowledge of specimen handling. Staining of specimens that had been promptly fixed in formalin was moderate to strong at all fixation periods through 36 days, weak or no staining was evident in specimens fixed for 176 days. Refrigerated specimens typically had moderate to strong immunohistochemical staining. Even after 10 days of refrigeration before fixation, all immunohistochemical reactions were positive. However, no immunohistochemical staining was detected in any specimen that was exposed to room air. Results indicate that prompt formalin fixation is optimal for BVDV IHC. Samples can be held in formalin at least 36 days, without loss of reactivity. A 1-day delay in fixation caused no loss of reactivity, provided the specimen was refrigerated and protected from desiccation. 相似文献
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Gutierrez M Forster FI McConnell SA Cassidy JP Pollock JM Bryson DG 《Veterinary immunology and immunopathology》1999,71(3-4):321-334
In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59. 相似文献
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Robert A Kunkle Janice M Miller David P Alt Randall C Cutlip Noelle E Cockett Shiquan Wang Juergen A Richt Bruce V Thomsen S Mark Hall 《Journal of veterinary diagnostic investigation》2006,18(5):443-447
Amino acid polymorphisms of the prion protein (PrP) greatly influence the susceptibility of sheep to scrapie. Selective breeding to increase the prevalence of PrP gene alleles associated with scrapie resistance is a flock management practice that is important for scrapie control programs. Determination of sheep PrP alleles typically has required extraction of DNA from host tissues that are freshly derived or stored frozen. We describe application of a DNA extraction procedure for formalin-fixed, paraffin-embedded tissues (PET) for the purpose of PCR amplification and nucleotide sequencing of relevant codons (136-171) of the sheep PrP gene. Tissues derived from 96 sheep were studied. The DNA sequence identity was confirmed in 87 of 94 matched samples of PET and frozen tissue specimens. DNA from brainstem PET of 2 sheep, from which fresh tissue was not available, was amplified and sequenced after formalin fixation for 7-70 days. This method will allow retrospective analysis of PrP genetics of sheep subsequent to postmortem diagnosis of scrapie when nonfixed tissue is unavailable for DNA extraction; however, it is not recommended that submission of fixed tissue supplant collection of fresh tissues for the purpose of determining PrP gene polymorphisms. 相似文献
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In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa. 相似文献
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Protective immunity against infectious bursal disease virus in chickens in the absence of virus-specific antibodies 总被引：1，自引：0，他引：1
The role of cell-mediated immunity (CMI) in pathogenesis of infectious bursal disease virus (IBDV) was investigated. One-day-old specific pathogen-free chickens were treated with 3mg of cyclophosphamide (Cy) per chicken for 4 consecutive days and, 3 weeks later, infected with the IBDV-IM strain. Chickens were examined for: (a) mitogenic response of splenocytes to ConA, as an indicator of T-cell functions in vitro, (b) antibody against IBDV by ELISA, (c) IBDV genome in various tissues by RT-PCR and (d) immunological memory. At the time of IBDV infection, Cy-treated chickens had depleted bursal tissue (an avian primary B-cell lymphoid organ), severely compromised antibody-producing ability, but normal T-cell response to ConA. In primary infection, no detectable antibody against IBDV antigen in Cy-treated, IBDV-infected chickens was observed up to 28 days post-infection (PI), while IBDV genome was detected by RT-PCR in spleen, thymus, liver and blood until 10 days PI. Like intact control chickens infected with IBDV, Cy-treated, IBDV-infected chickens suppressed splenocytes responses to ConA from 5 to 10 days PI, suggesting that intact control as well as Cy-treated chickens responded similarly to IBDV infection in the early phase. Following re-infection with IBDV, no detectable secondary antibody response to IBDV as well as IBDV genome in tissues were observed in Cy-treated chickens, while intact control chickens developed vigorous secondary antibody response. Similar to intact control chickens infected with IBDV, Cy-treated chickens after second infection with IBDV did not suppress splenocyte response to ConA. These results suggested that in the absence of detectable anti-IBDV antibodies, protection of Cy-treated chickens from IBDV infection may occur via immunological memory mediated by CMI. We concluded that under normal conditions, IBDV induces a protective antibody response, however, in the absence of antibody, CMI alone is adequate in protecting birds against virulent IBDV. 相似文献
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Haematopoietic necrosis in a goldfish (Carassius auratus) associated with an agent morphologically similar to herpesvirus 总被引：1，自引：0，他引：1
One of 14 goldfish (Carassius auratus) died 4 weeks after purchase and was investigated by necropsy and histological examination. Routine formalin fixation of the goldfish was followed by histopathology. Formalin fixed spleen and kidney from the fish was further processed by embedding in epoxy resin and examined by transmission electron microscopy (EM). Severe, diffuse necrosis of haematopoietic tissue in the spleen, thymus and kidney and severe, diffuse hyperplasia in the gill epithelial cells were seen. In the spleen there was severe, diffuse necrosis of lymphocytes and many nuclei with marginated chromatin and intranuclear inclusions were scattered throughout the necrotic tissue. EM of affected tissues demonstrated intranuclear particles morphologically similar to herpesvirus. The presence of an agent similar to a herpesvirus in a goldfish with severe haematopoietic necrosis suggests that the herpesvirus responsible for haematopoietic necrosis in cyprinid species throughout the world has entered the goldfish population in Australia. 相似文献
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G M Allan M S McNulty D Bryson D Mackie M Platten 《Research in veterinary science》1989,46(3):416-418
The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining. 相似文献
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Association of porcine circovirus 2 with reproductive failure in pigs: a retrospective study, 1995-1998 总被引：7，自引：0，他引：7 下载免费PDF全文
下载免费PDF全文 Bogdan J West K Clark E Konoby C Haines D Allan G McNeilly F Meehan B Krakowka S Ellis JA 《The Canadian veterinary journal. La revue veterinaire canadienne》2001,42(7):548-550
In order to determine if vertically transmitted porcine circovirus (PCV) has played a role in reproductive failure in pigs in areas of endemic infection, archival fixed tissues were examined by polymerase chain reaction (PCR) and immunohistochemistry. Tissues tested were from routine cases of abortion or reproductive failure submitted between 1995 and 1998 to the diagnostic laboratory at the Western College of Veterinary Medicine, Saskatoon. They originated from 29 high-health herds in the provinces of Alberta and Saskatchewan and comprised a total of 36 individual submissions. Porcine circovirus type 1 (PCV1) was not detected by PCR in any submitted tissues. Porcine circovirus type 2 (PCV2) was not detected by PCR or immunohistochemistry in any of the submitted tissue. The effect of extended formalin fixation on the detection of PCV2 by PCR was assessed and fixation for up to one week had no gross effect on sensitivity of detection using this PCR technique. Failure to detect porcine circoviruses in cases of reproductive failure prior to 1999 in areas of endemic infections, suggests that reproductive disease may be a new clinical manifestation of PCV2 infection, and that vertical transmission may not have been the primary mechanism of initial dissemination of the virus in the pig population. 相似文献