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1.
The aim of this study was to develop and to optimize an immunohistochemistry (IHC) method for PCV2 identification and to compare it with an in situ hybridization (ISH) technique. The results demonstrated that both ISH and IHC successfully detected PCV2 viral antigens or nucleic acid in the examined tissues. Most of the slides identified previously in ISH as PCV2-positive were also positive in IHC. In the case of nearly half of the slides the results of IHC examination revealed an increase in the intensity of staining. IHC presented higher sensitivity and specificity than ISH. No negative impact of the time of paraffin block storage on ISH detection results was observed. In addition, IHC results were easier to interpret due to better image quality after staining. Overall results confirmed IHC was a reliable and useful technique for PMWS diagnosis.  相似文献   

2.
Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.  相似文献   

3.
Viable Haemophilus somnus of reproductive tract origin (OSU-1167) was inoculated transcervically into the uterus of 6 virgin heifers. Five heifers were sham-inoculated (intrauterine) with sterile mycoplasmal medium and served as controls. After inoculation and observation, all heifers had nasal and vaginal vestibular swab specimens and serum obtained periodically for 44 days. Signs of systemic illness were not detected. On the day after inoculation, all inoculated heifers had signs of vulvovaginitis, whereas none of the control heifers had similar signs (P less than 0.002). Haemophilus somnus was not isolated from any nasal or vaginal vestibular swab specimens obtained before inoculation or from any nasal swab specimens obtained after inoculation. During the 44 days after inoculation, H somnus was isolated from 25 of 54 vestibular specimens obtained from inoculated heifers and from 3 of 45 specimens obtained from controls (P less than 0.02). Vulvovaginal lesions were associated with vestibular isolation of H somnus in 23 of 25 (92%) such isolations from inoculated heifers; lesions were never associated with concurrent isolation of H somnus in controls. All heifers had H somnus microagglutination test (MAT) titer less than or equal to 256 against a commercially prepared H somnus antigen at the beginning of the study. Considered as groups, neither inoculated nor control heifers achieved fourfold increases in MAT titer during the 44 days after inoculation. When compared by day of sample collection, inoculated heifers did have significantly (P less than 0.04) lower geometric mean titer at 7 days after inoculation than did control heifers when tested by use of a commercially prepared antigen.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
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   

5.
Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.  相似文献   

6.
The objective of the present study was to evaluate polyclonal- and monoclonal-antibody-based immunohistochemical (IHC) tests for the detection of 2 genotypes of Porcine circovirus type 2 (PCV2), a and b, in formalin-fixed, paraffin-embedded lymph-node tissue from pigs with experimental or natural postweaning multisystemic wasting syndrome and to compare the IHC results with those of in-situ hybridization (ISH) assays. The ISH assays proved more sensitive than the IHC tests for the detection of PCV2a and PCV2b. According to these findings, polyclonal-antibody-based IHC testing is the most practical routine diagnostic method for the detection of PCV2 regardless of genotype because IHC testing is less technically complex than ISH testing. However, ISH assays are useful to differentiate between PCV2a and PCV2b in surveillance programs for the monitoring of PCV2 in swine herds.  相似文献   

7.
Bovine respiratory syncytial virus (BRSV) and Haemophilus somnus are two bovine respiratory pathogens that cause disease singly or as part of a polymicrobial infection. BRSV infection is often associated with a predisposition towards production of a T helper type 2 (Th2) response and IgE production. In contrast, an IgG2 response to H. somnus has been shown to be most important for recovery. An experiment was performed to evaluate the hypothesis that infection with H. somnus on day 6 of experimental BRSV infection would result in disease enhancement and potentially an altered immune response when compared with single infection. Three groups of calves were either dually infected or singly infected with H. somnus or BRSV. Serum and bronchoalveolar lavage fluid (BALF) pathogen specific IgG1, IgG2, IgE, and IgA responses were evaluated by ELISA. TaqMan RT-PCR was used to examine cytokine gene expression by PBMC and BAL cells. Clinical signs were evaluated for 28 days after BRSV infection, followed by necropsy and histological examination of the lungs. In dually infected calves, disease was significantly more severe, H. somnus was isolated from the lungs at necropsy, and high IgE and IgG responses were detected to H. somnus antigens. Cytokine profiles on day 27 were elevated in dually infected calves, but did not reflect a skewed profile. These results contrasted with singly infected calves that were essentially normal by day 10 of infection and lacked both lung pathology and the presence of H. somnus in the lung at necropsy. The increase in IgE antibodies specific for antigens of H. somnus presents a possible mechanism for pathogenesis of the disease enhancement.  相似文献   

8.
Forty-five cases of bovine abortion were examined using in situ hybridization (ISH) with a biotinylated DNA probe specific for bovine herpesvirus-1 (BHV-1). Of the 45 cases, 16 were diagnosed as due to BHV-1, 15 were determined to be due to other causes, and 14 were of undetermined etiology. Direct comparisons between ISH and an immunoperoxidase (IP) test specific for BHV-1 were performed on formalin-fixed, paraffin-embedded tissue sections of lung, liver, kidney, spleen, thymus, and placenta; fluorescent antibody tests for BHV-1 and virus isolation were performed on fresh lung and liver. In comparison to these routine BHV-1 detection techniques, ISH had an overall sensitivity of 88.2% and a specificity of 89.3% in detecting BHV-1 in aborted fetuses. Immunoperoxidase was more sensitive than ISH with tissue sections from lung (87.5% vs. 69%), liver (92% vs. 17%), spleen, and placenta; results of the tests on tissue sections from kidney were concordant. Liver sections presented special problems in that nonspecific reactions were frequently observed with hybridization. With thymus sections, the rate of detection was higher by hybridization than by IP, but the specificity of some of these reactions could not be confirmed.  相似文献   

9.
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10.
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.  相似文献   

11.
Immunoperoxidase assays were performed on 21 archived formalin-fixed, paraffin-embedded tissues from from American bison (Bison bison) with bronchopneumonia. Seven of the 21 bison had positive staining for Haemophilus somnus in alveolar exudate, visceral pleura, lung parenchyma, and chronic necrotic lesions, and H. somnus was isolated from tissues from 1 of these 7 animals. Results suggest that H. somnus is a respiratory pathogen in bison.  相似文献   

12.
During 1 year, the association between microbiological and pathological findings in 72 lungs from calves submitted to the Danish Veterinary Laboratory for diagnostic purposes was studied. All cases were evaluated pathologically and bacteriologically, whereas only 68 cases were examined for the presence of bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI-3 virus) and bovine coronavirus, 62 cases for bovine viral diarrhoea virus (BVD), 45 cases for bovine adenovirus and 51 cases for mycoplasmas. Based on histopathological examination, the cases were diagnosed as fibrinous and/or necrotizing bronchopneumonia, suppurative bronchopneumonia, embolic pneumonia and others. The diagnoses were based on the dominating and most severe lesions in each lung. Haemophilus somnus, Pasteurella multocida, Actinomyces pyogenes, P. haemolytica and BRSV were the most commonly found bacterial and viral lung pathogens, respectively. Pasteurella spp. and H. somnus were often associated with the more severe fibrinonecrotizing type of bronchopneumonia, whereas BRSV was primarily detected in cases of suppurative bronchopneumonia. Mycoplasma bovis was isolated from one case only, whereas M. dispar, M. bovirhinis and Ureaplasma diversum were present, often concomitantly, in the majority of cases. Aspergillus fumigatus was isolated from one case.  相似文献   

13.
Haemophilus (H.) somnus strains were isolated from nasopharyngeal swabs collected from buffalo calves showing respiratory symptoms as well from pneumonic lung tissue samples, the incidence being 4.7% and 10.4%, respectively. The organism was not recovered from samples obtained from apparently healthy buffalo calves. All isolated strains were highly virulent to mice, causing acute septicaemia and death within 3-5 days from intraperitoneal inoculation with 7.5 x 10(6) viable organisms. All tested strains were resistant to tetracycline and sulphafurazole, but most strains were highly sensitive to gentamicin, ampicillin, penicillin G, and colistin sulphate. H. somnus must not be neglected as a causative agent of respiratory disorders in buffalo calves, in addition to other incriminated organisms.  相似文献   

14.
Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.  相似文献   

15.
Chronic, antibiotic-resistant pneumonia, sometimes with concurrent polyarthritis, occurs in feedlot cattle in western Canada. The prevalence of Mycoplasma bovis, bovine viral diarrhea virus, and Haemophilus somnus was determined by using immunohistochemical staining of lung and heart tissue from 2 groups of animals with this history. Mycoplasma bovis antigen was present in 44/48 cases submitted between 1995 and 1998 (retrospective group) and 15/16 of cases from 1999 (prospective group), and was associated with pulmonary necrosis. Bovine viral diarrhea virus antigen was present in association with microscopic vascular lesions in 31/48 retrospective and 9/16 of prospective cases. Types Ib and II bovine viral diarrhea virus were isolated from 4/16 prospective cases. Haemophilus somnus antigen was present in heart, lung, or both of 15/48 retrospective and 8/16 prospective cases. The results suggest that there may be synergism between bovine viral diarrhea virus and M. bovis in this pneumonia with arthritis syndrome.  相似文献   

16.
The objective of this study was to compare the detection rate of bacterial agents in bronchoalveolar lavage fluid (BALF), taken without visual control, to that in affected lung tissue obtained from the same pig at necropsy. BALF and affected lung tissue were examined for Mycoplasma hyopneumoniae using PCR, and standard cultural methods were used for Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida and Streptococcus suis. All pigs with a history of respiratory symptoms were submitted as live animals for routine diagnostic examination. In each animal the site of lavage, marked by injecting methylene blue, differed from the site of pneumonic lesions. M. hyopneumoniae was detected more frequently in lung tissue than in BALF in cases with moderate or severe lung lesions. The detection rates of M. hyopneumoniae were higher in the BALF of pigs with mild lesions. Cultural examination of BALF was at least as satisfactory as affected lung tissue for detecting B. bronchiseptica, H. parasuis and P. multocida.  相似文献   

17.
The initial lung lesions in two calves intrabronchially inoculated with Haemophilus somnus are described. The animals were euthanized within 7 h after challenge. The in situ location of H. somnus and accompanying lesions were examined by light microscopy, immunohistochemistry and transmission electron microscopy (TEM). Inoculation with H. somnus resulted in the development of acute pulmonary lesions within 3.5 h. H. somnus antigen was demonstrated only within the luminal spaces of the airways and in one area of bronchio-associated lymphoid tissue (BALT). As observed by TEM, the bacteria were phagocytized by both neutrophils and alveolar macrophages. Antigen was never demonstrated in the pulmonary intravascular macrophages.  相似文献   

18.
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19.
The pathogenesis of 4 isolates of turkey-origin reovirus (NC/SEP-R44/03, NC/98, TX/98, and NC/85) and 1 chicken-origin reovirus (1733) was examined by infecting specific pathogen free (SPF) poults. These turkey-origin reovirus (TRV) isolates were collected from turkey flocks experiencing poult enteritis and are genetically distinct from previously reported avian reoviruses. Microscopic examination of the tissues collected from the TRV-infected poults revealed different degrees of bursal atrophy characterized by lymphoid depletion and increased fibroplasia between the bursal follicles. To understand the relationship between virus spread and replication, and the induction of lesions, immunohistochemical staining (IHC) for viral antigen, in situ hybridization (ISH) for the detection of viral RNA, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the detection of apoptosis in affected tissues was performed. Both IHC and ISH revealed viral antigen and RNA in the surface epithelial cells of the bursa, in macrophages in the interstitium of the bursa and, to lesser degree, in splenic red pulp macrophages and intestinal epithelial cells. Increased apoptosis of bursal lymphocytes and macrophages was observed at 2 and 5 days postinoculation. No lesions were found in tissues from poults inoculated with the virulent chicken-origin strain, however viral antigen was detected in the bursa and the intestine. Although all TRVs studied displayed similar tissue tropism, there were substantial differences in the severity of the lesions produced. Poults inoculated with NC/SEP-R44/03 or NC/98 had moderate to severe bursal atrophy, whereas poults inoculated with TX/98 or NC/85 presented a mild to moderate bursal lymphoid depletion. The lymphoid depletion observed in the bursa appears to be the effect of an indirectly induced apoptosis and would most likely result in immune dysfunction in poults infected with TRV.  相似文献   

20.
Immunohistochemistry (IHC) is used routinely to detect porcine reproductive and respiratory syndrome virus (PRRSV) in the lung of nursery and grow/finish pigs with respiratory disease and has been reported to be highly specific (100%) but only moderately sensitive (67%). When multiple sections of lung are examined from field cases of porcine pneumonia, it is common to detect PRRSV antigen in only 1 or 2 of the sections. This study was undertaken to determine the impact of the number of lung sections evaluated on the diagnostic sensitivity of IHC for the detection of PRRSV in vaccinated and unvaccinated swine. Five anterioventral sections of lung from animals experimentally challenged with PRRSV were evaluated on a single IHC slide. Utilizing a beta binomial model, observed results were used to calculate the probability of detecting PRRSV with IHC as a function of the number of lung sections assessed. Results demonstrate that the diagnostic sensitivity of PRRSV IHC is dependent on the number of lung sections examined. In unvaccinated pigs, a beta binomial model predicts that if a single lung section were evaluated, PRRSV would likely be confirmed in only 48% of infected animals, and at least 5 sections of anterioventral lung would need to be assessed to detect >90% of PRRSV-infected pigs. Vaccination resulted in significantly lower gross and microscopic lung lesion scores and significantly fewer antigen-positive cells. In vaccinated swine, the calculated probability of detecting a PRRSV-infected pig with IHC when a single lung section is evaluated was only 14%. If PRRSV is a primary concern, diagnosticians should collect at least 5 anterioventral sections of lung from each pig to be evaluated on a single IHC slide. This approach will diminish the number of false-negative results obtained with this method of antigen detection.  相似文献   

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