Monoclonal antibody-based immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues.

An immunohistochemistry technique was developed for the diagnosis of porcine epidemic diarrhea virus (PEDV). The technique was tested on formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. Five different monoclonal antibodies (MAbs) were tested in this study. PEDV antigen was consistently detected in the PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in phosphate-buffered saline)-fixed PEDV-infected Vero cells or formalin-fixed, paraffin-embedded intestinal tissues from piglets naturally infected with PEDV. The C9-2-2 MAb gave the strongest reactivity and least background staining, detecting 10 of 10 infected pigs. The positive reaction was cytoplasmic. Positive enterocytes were distributed over the tip and along the sides of atrophied or fused villi in the jejunum and ileum. Positive-staining cells were not detected in the crypts. No staining was observed in cecum and colon. No positive cells were observed when the C9-2-2 MAb was reacted with the tissue sections from noninfected piglets or from transmissible gastroenteritus virus (TGEV)- and rotavirus-infected piglets. The selected anti-PEDV MAbs tested on formalin-fixed, paraffin-embedded tissue sections are useful for diagnosis when virus isolation is not available. This method would be of particular value in countries where both PEDV and TGEV are epizootic and would aid in differentiating between PEDV and TGEV infection.     

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.     

Diagnosis of malignant catarrhal fever by PCR using formalin-fixed, paraffin-embedded tissues.

A previously described polymerase chain reaction (PCR) assay (amplification of a 238-bp fragment of ovine herpesvirus 2 [OHV-2] genomic DNA) for diagnosis of sheep-associated malignant catarrhal fever (MCF) was adapted for use on formalin-fixed, paraffin-embedded tissues. Variables affecting its use were examined. Archived tissues from cattle, white-tailed deer (Odocoileus virginianus), and bison (Bison bison) diagnosed with MCF by clinical signs or histologic lesions were obtained from 2 veterinary diagnostic laboratories. Tissues from healthy animals and from animals diagnosed with other common bovine viral diseases were examined as controls. A total of 86 blocks from 37 suspect MCF cases were examined. Forty-one blocks from healthy animals and animals with unrelated viral diseases were examined as controls. The assay was specific for sheep-associated MCF and did not yield false-positive signals from healthy animals or from cases of infectious bovine rhinotracheitis, bovine virus diarrhea, mucosal disease, or parainfluenza-3 virus infection. A wide variety of tissues were suitable substrates, including spleen, lymph node, intestine, brain, lung, and kidney. Extracted DNA provided a more suitable target than did unextracted tissue lysate. The highest levels of viral DNA were present in lymphoid organs and intestine, but the data indicate that in acute clinical cases, most organs contain sufficient viral DNA to serve as a suitable diagnostic specimen. Fixation of 0.5-cm3 blocks of tissue in 10% neutral buffered formalin was deleterious to the target DNA, and PCR signals progressively diminished after fixation for >45 days. Detection of genomic DNA of OHV-2 by PCR was successful for archived tissues that were 15 years old.     

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.     

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.     

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for polyomavirus DNA and antigen

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain polyomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for polyomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-murine polyomavirus polyclonal antiserum as the primary antibody. The DNA was extracted from sections of each tissue block, and a polymerase chain reaction assay was performed, using primers designed to amplify regions of the bovine polyomavirus genome and consensus polyomavirus primers designed to detect unknown polyomaviruses. RESULTS: Polyomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that polyomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.     

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.     

Evaluation of formalin-fixed paraffin-embedded tissues from vaccine site-associated sarcomas of cats for papillomavirus DNA and antigen

OBJECTIVE: To determine whether vaccine site-associated sarcomas (VSS) from cats contain papillomavirus antigen or DNA. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded tissue blocks of VSS from cats. PROCEDURE: Sections from each tissue block were evaluated for papillomavirus antigen by use of an avidin-biotin-complex immunohistochemical staining method, using rabbit anti-bovine papillomavirus type-1 antibody. The DNA was extracted from sections of each tissue block, and polymerase chain reaction assays were performed, using primers designed to amplify regions of the E5 gene of bovine papillomavirus and consensus primers designed to amplify a region of the L1 gene of animal papillomaviruses. Sections from 20 of the tissue blocks were evaluated by use of nonradioactive in situ hybridization for bovine papillomavirus DNA. RESULTS: Papillomavirus antigen and DNA were not detected in any of the VSS. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that papillomaviruses likely do not have any direct involvement in the pathogenesis of VSS in cats.     

Diagnosis of taenia saginata cysticercosis by immunohistochemical test on formalin-fixed and paraffin-embedded bovine lesions.

A new method of diagnosing cysticercus or larval stage of the human tapeworm, Taenia saginata, also known as Cysticercus bovis, in formalin-fixed bovine tissue was developed using a monoclonal antibody to T. saginata and avidin-biotin complex immunohistochemistry. Grossly recognizable viable and degenerate cysts were identifiable after immunohistochemical staining and could be differentiated from Sarcocystis, Actinobacillus, or non-cyst, normal bovine structures. Thenew test should permit laboratory confirmation of suspected T. saginata cysticercus lesions.     

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.     

Detection of avian polyomavirus infection by polymerase chain reaction using formalin-fixed, paraffin-embedded tissues

Avian polyomavirus infection in psittacines was diagnosed in tissues by the use of polymerase chain reaction (PCR) test. The tissues used in the procedure were either formalin-fixed tissues embedded in paraffin blocks or fresh tissues (heart, liver, and spleen) collected from the psittacines during necropsy. DNA was extracted from these tissues and was tested with the published primers for avian polyomavirus VP1 gene in the PCR that yielded an amplicon of 550 base pair size, which was then visualized by electrophoresis. The amplicon size was consistent with avian polyomavirus. The PCR test was found to be an effective method for identifying avian polyomavirus infection in both formalin-fixed, paraffin-embedded and fresh tissues from psittacine birds of different age groups.     

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.     

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.     

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.     

The demonstration of rabies antigen in paraffin-embedded tissues using the peroxidase-antiperoxidase method: a comparative study.

Mice experimentally infected with challenge virus standard rabies virus as well as skunks and foxes experimentally infected with street rabies virus were used to demonstrate rabies viral antigen in paraffin-embedded tissue by the peroxidase-antiperoxidase method. Tissues fixed with different fixatives (10% formalin, Bouin's, acetone, ethanol) for various times and fresh frozen tissues were stained by the fluorescent antibody and the peroxidase-antiperoxidase method. Formalin- and Bouin's-fixed tissues were tested with and without use of digestive enzyme (pepsin). The results demonstrated that a procedure using formalin-fixed paraffin-embedded tissue treated with pepsin and stained by peroxidase-antiperoxidase was the best method for both preservation of morphological details and demonstration of antigen.     

Detection of Mycobacterium avium subspecies avium in formalin-fixed, paraffin-embedded tissues of captive exotic birds using polymerase chain reaction.

A presumptive diagnosis of avian tuberculosis can be made when characteristic histologic lesions and acid-fast bacilli are observed in avian tissue samples. However, a definitive diagnosis requires isolation and identification of the causative organism, a process that can take several weeks to complete. The purpose of the study was to determine whether formalin-fixed, paraffin-embedded archival avian tissues could be tested by polymerase chain reaction (PCR) to reliably and rapidly diagnose avian tuberculosis. Tissues were examined from both presumptive and definitive cases of avian tuberculosis from captive exotic birds obtained over a 14-yr period (1983-1997). The cases chosen consisted of birds that had characteristic histologic lesions with acid-fast bacilli. The primers used for PCR amplified a 180-base-pair fragment of 16S ribosomal RNA, a sequence specific for both Mycobacterium avium subsp. avium and M. avium subsp. paratuberculosis. If a sequence was detected in a sample, it was presumed that M. a. avium was the organism being detected. This M. avium fragment sequence was detected in 26 of the 97 samples (27%). Some of the negative PCR results may be explained by any of several factors that adversely affect nucleic acid integrity, particularly prolonged fixation in formalin. Of the 17 samples that were culture positive for M. avium and were known to have been fixed in formalin for < or = 4 wk, 11 tested positive by PCR (65%). The findings of this study show that PCR can be a rapid indicator of the presence of M. a. avium in formalin-fixed, paraffin-embedded tissues. However, the relatively low detection rate the test demonstrated in this sample set may limit its practical use as a diagnostic tool.     

Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues

Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.     

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.