Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis,Streptococcus suis,and Mycoplasma hyorhinis in formalin-fixed,paraffin-embedded tissues from pigs with polyserositis

An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.     

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.     

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.     

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.     

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.     

Feline leukemia virus detection by immunohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissue from cats with lymphosarcoma.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

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.     

Development of an oligonucleotide-specific capture plate hybridization assay for detection of Haemophilus parasuis.

An oligonucleotide-specific capture plate hybridization assay has been developed to rapidly, specifically, and sensitively detect Haemophilus parasuis from nasal swabs. Several in vitro studies have been performed to determine the sensitivity and specificity of the test, and in vivo studies have validated this technique in pigs. Results suggest that the assay detects <100 colony-forming units/ml in a pure culture and gives a positive result when H. parasuis is present in a ratio of 1:10(3)-10(4) in a mixed culture, and the probe does not hybridize with other related species found in the upper respiratory tract. This assay is more sensitive than culture for detection of the microorganism from nasal swabs and lesions.     

Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction

Chicken anaemia virus was detectable from various samples such as cell-free virus, infected cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded ( ) tissues from experimental or field infected chicks using assay. The detection limit of the first PCR assay was 1 infected cell or 10^−1·5, ₅₀ of cell-free virus (strain A2). The nested assay increased the sensitivity 10- or 100-fold. was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested assay was very sensitive. However, was detectable in most field samples using the first assay. Therefore, the nested assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect in tissues or formalin-fixed material. Use of the assay in detection from tissues may be most valuable in diagnosis of diseases caused by or associated with , because it allows detection of both microscopic lesions and .     

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.     

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.     

Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.     

Evaluation of a real-time polymerase chain reaction assay of the outer membrane protein P2 gene for the detection of Haemophilus parasuis in clinical samples

A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively.     

Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization.

An adult domestic shorthair cat had severe chemosis due to purulent and necrotizing blepharitis and conjunctivitis. Purulent rhinitis, necrotizing glossitis, and dermatitis were also diagnosed. The cat was positive for feline immunodeficiency virus and feline leukemia virus. Histologically, intranuclear Cowdry type A inclusions were found within numerous epithelial cells adjacent to the lesions in skin, conjunctiva, and tongue. Electron microscopic examination revealed herpesviral particles within the lesions. Paraffin-embedded skin and tongue tissues were processed in a polymerase chain reaction, using primers to amplify a 306-bp region of the thymidine kinase gene of feline herpesvirus type 1, resulting in a distinct amplification product of the predicted size. The distribution of feline herpesvirus was demonstrated by immunohistochemistry and nonradioactive in situ hybridization. Positive immunostaining was found in nuclei and cytoplasm of numerous epithelial cells within and next to the lesions, whereas in situ hybridization, performed with a digoxigenin-labeled double-stranded DNA probe, revealed hybridization signal only in nuclei of intact epithelial cells. Neither immunohistochemistry nor in situ hybridization showed feline herpesvirus type 1 in tissues of lungs, liver, spleen, intestine, or brain.