A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.     

Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system.

Three methods of harvesting DNA from broth culture tubes for quantitative real-time polymerase chain reaction (qrtPCR) confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) were evaluated. A commercial DNA extraction kit, the boil method (boiling for 5 minutes), or direct addition of broth culture media to the PCR reaction mix were tested. Samples were evaluated at 8 or 11 days of incubation and at the time of instrument-signal culture-positive. In total, when tested at time to instrument signal positive, 10/10 (100%) of samples extracted by the commercial method were positive on qrtPCR, whereas 9/10 (90%) were positive after the boil method, and 6/10 (60%) were positive after the direct method. Increased volumes of egg-yolk emulsion added to the culture tubes prolonged the number of cycles to threshold positive for the samples that were not subjected to commercial extraction or boiling. Samples were not reliably positive when tested at 8 or 11 days of incubation. The boil method appears to represent a reasonable time- and money-saving method to harvest DNA for qrtPCR confirmation of MAP in broth culture at time to instrument signal positive.     

Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction

Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect >/=10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.     

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.     

Real-time polymerase chain reaction testing for the detection of Mycobacterium genavense and Mycobacterium avium complex species in avian samples

Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR. In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products. The TaqMan PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan PCR products. The TaqMan PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.     

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.     

Polymerase chain reaction detection of Salmonella spp. in fecal samples of pet birds

The aims of this study were 1) to determine the prevalence of Salmonella in clinically ill birds in aviaries in Ankara, Turkey, and 2) to compare conventional culture and polymerase chain reaction (PCR) for detection of Salmonella in feces from clinically ill pet birds. In the study, 185 fecal samples (feces and/or swabs) collected from the pet birds kept in the seven different aviaries in the city of Ankara were investigated for the existence of Salmonella spp. by bacterial isolation and PCR. The conventional isolation and identification methods were performed for Salmonella isolation from fecal cultures. Suspected colonies were confirmed with the Salmonella polyvalent O antiserum and serogrouped with Salmonella group-specific antiserum. PCR was performed after the fecal swabs were incubated for 18 hr in 10 ml of tetrathionate broth. Three (1.63%) out of 185 fecal samples were found to harbor Salmonella spp. by conventional identification tests and were found to belong to serogroup B. Five (2.7%) swab samples were found to harbor Salmonella DNA by PCR tests. As a conclusion, PCR following incubation of clinical samples in pre-enrichment broth seemed to be a fast and practicable method for Salmonella spp. diagnosis when compared to protracted labor-intensive conventional culture techniques.     

A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.     

Direct polymerase chain reaction detection of Campylobacter spp. in poultry hatchery samples

A rapid, sensitive, and specific polymerase chain reaction (PCR) assay was developed for the direct detection of Campylobacter in environmental samples from hatcheries. PCR, with a set of primers specific for the Campylobacter flaA short variable region (SVR), detected the presence of Campylobacter in both fluff and eggshell samples; however, a determination of whether the organism was living or dead could not be made. Conventional cultural methods detected no Campylobacter from the same samples. An additional benefit of the direct PCR assay is it allows for the production of a product that can be sequenced to provide further epidemiologic information.     

Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II.

The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.     

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis

SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.     

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.     

Comparison of blood polymerase chain reaction and enzyme-linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep.

A study was carried out to compare the performance of enzyme-linked immunosorbent assay (ELISA) and blood polymerase chain reaction (PCR) for diagnosis of paratuberculosis in cattle and sheep. For cattle, a set of 278 samples from 1 paratuberculosis-affected Friesian farm was used; it included 80 ELISA-positive samples and 198 ELISA-negative samples from an age-matched group. Ninety-four samples were from heifers and 184 were from 2-5-year-old cows. The overall analysis showed a clear association (Fisher exact test [FET] P = 0.0049) but a weak negative agreement (45.3%, kappa = -0.1665 +/- 0.0994) between the 2 tests. It reflected a moderate agreement among heifers (87.7%, kappa = 0.4471 +/- 0.2435) and a moderate disagreement among cows (62.7%, kappa = -0.3670 +/- 0.1057). For sheep, 496 blood samples from 53 Latxa dairy flocks were used; 180 of the blood samples were from dam/offspring pairs. The overall association between the 2 tests on ovine samples was strong (FET, P = 0.0005), whereas the agreement was low (kappa = 0.1622 +/- 0.1188). There was slightly better agreement for ewes (kappa = 0.2135 +/- 0.1992) than for lambs (kappa = 0.1193 +/- 0.1301). There was also a highly unlikely proportion of dam/offspring positive results (FET, P < 0.0001, kappa = 0.6269 +/- 0.1854). Four of 6 lambs that were necropsied 1 year after testing had paratuberculosis microscopic lesions in the ileocecal valve (3 lambs) or a PCR-positive result (4 lambs). These results suggest that blood PCR testing might be a potentially useful new approach in paratuberculosis diagnosis, especially in young animals.     

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection.

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.     

Development of quantitative real-time polymerase chain reaction for duck enteritis virus DNA

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, we introduce a quantitative real-time polymerase chain reaction (PCR) assay for DEV DNA using TaqMan technology and a two-step protocol. It was confirmed to be rapid, sensitive, and specific for DEV detection. The primers and probe were designed and directed to the DNA polymerase gene of DEV. The method will provide a valuable tool for rapid laboratory diagnosis of DEV infection. By virtue of its high-throughput format and its ability to accurately quantify the viral DNA, the method may be useful for large epidemiological surveys and clarification of pathogenesis, such as latency and reactivation of the virus.     

Detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples: comparison of three polymerase chain reaction-based diagnostic tests with a conventional culture method.

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.     

Quantitation of sheep-associated malignant catarrhal fever viral DNA by competitive polymerase chain reaction.

A single-step, competitive polymerase chain reaction technique was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed coamplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the 2 primer binding sites. The assay yielded a linear response (r = 0.98) for DNA measurement within the range of 30-300,000 copies. Amplification efficiency analysis by coamplification of target and competitor in equal copy numbers for various numbers of cycles showed that the relative abundance of the coamplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA-MCF.     

Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction

Few studies were conducted on the diagnosis and control of small ruminants’ leptospirosis. Thirteen goat herds and seven sheep flocks located in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity by MAT (Microscopic Agglutination Test), 19 and 40 seropositive goats and sheep, respectively, were selected, and urine samples were collected for bacteriology and PCR. For both species of animals, the most prevalent reactions were due to serogroups Sejroe and Shermani. Although leptospires were observed by darkfield microscopy in eight samples, pure isolates were obtained by bacteriological culture from only two samples. However, twelve urine samples (six goats and six sheep) were positive by PCR. Based on these findings, we consider that the combined use of MAT as a screening test followed by urine PCR for the direct detection of Leptospira spp. DNA was adequate for the identification of carrier animals among goats and sheep. These are valuable tools for the control of leptospirosis in small ruminants.     

Development of a real-time polymerase chain reaction assay for rapid diagnosis of neuropathogenic strains of equine herpesvirus-1.

This communication reports the development and performance assessment of a rapid diagnostic test for identifying horses actively infected with the neurovirulent pathotype of equine herpesvirus-1 (EHV-1). The test is a real-time polymerase chain reaction (PCR)-based assay that uses EHV-1 pathotype-specific TaqMan(R) reporter probes for discrimination between neuropathogenic and non-neuropathogenic strains of EHV-1 in equine blood or nasal swabs. The diagnostic performance of the new technique was evaluated by testing specimens collected from 234 horses involved in recent outbreaks of EHV-1 myeloencephalopathy at three separate thoroughbred racetracks and one large riding/boarding stable. Side-by-side comparison of the EHV-1 pathotyping results yielded by the new single-step, PCR-based allelic discrimination technique (24-hour turn-around-time) with those generated by a multi-step, conventional nested PCR followed by nucleotide sequencing of the amplified DNA (4-day turn-around-time) revealed complete agreement between the 2 test methods. The ability to rapidly identify horses infected with neuropathogenic strains of EHV-1 using a single-step, PCR-based method has significant implications for future diagnostic evaluation of suspect animals.