Polymerase chain reaction detection of Mycobacterium tuberculosis complex and Mycobacterium avium organisms in formalin-fixed tissues from culture-negative ruminants

In the US eradication program for bovine tuberculosis, a definitive diagnosis depends on the isolation of Mycobacterium bovis. However, in some cases bacterial culture is unsuccessful, even though the tissue is considered suspicious by histopathology because granulomatous lesions and acid-fast organisms are present. The purpose of this study was to determine if polymerase chain reaction (PCR) tests on formalin-fixed tissue would successfully identify the organisms observed in suspect lesions from culture-negative animals. Diagnostic laboratory records were used to select paraffin blocks of tissue from 102 ruminants that had suspect microscopic lesions but no bacterial isolation. Sections from these blocks were examined with PCR primers for IS6110 to detect Mycobacterium tuberculosis complex infection, or with 16S ribosomal RNA and IS900 primers for detection of Mycobacterium avium. The PCR tests successfully identified a mycobacterial infection in 58 of 102 tissues, including 41 M. tuberculosis complex and 17 M. avium (11 subspecies paratuberculosis). These results demonstrate that PCR testing of formalin-fixed tissue, in combination with bacterial culture, may increase the effectiveness of laboratory diagnostic efforts to detect and identify the most common mycobacterial diseases of ruminants.     

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5′ non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.     

Use of the polymerase chain reaction assay for the detection of Mycobacterium avium subspecies paratuberculosis in blood and liver biopsies from experimentally infected sheep

OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.     

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.     

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.     

Systemic Mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat

Dogs may be infected by Mycobacterium (M.) tuberculosis, M. bovis, and M. avium complex, and the clinical signs associated with each of these infections may be indistinguishable. Rapid speciation of the infecting organism is desirable because of the public health concerns associated with M. bovis and M. tuberculosis infections. A mycobacterial infection was suspected in the dog of this report based on acid-fast staining of organisms in macrophages obtained from liver aspirates and buffy-coat preparations. Polymerase chain reaction (PCR) analysis of a buffy-coat preparation identified M. avium.     

A comparison of routine culture with polymerase chain reaction technology for the detection of Mycoplasma species in feline nasal samples.

Nasal flush samples were collected from 20 cats and submitted for Mycoplasma culture and polymerase chain reaction (PCR). Nasal biopsy samples were also obtained from each cat and simultaneously evaluated for Mycoplasma by standard culture and PCR. Concordance of the test results was determined through calculation of the kappa statistic. In 6 cats, nasal flush samples were culture positive for Mycoplasma. PCR was positive in each culture-positive cat and also positive in 1 flush sample that was culture negative. DNA sequencing of the PCR product from the culture negative flush sample identified the organism as Mycoplasma arginini. All other flush samples that were culture negative were also PCR negative (kappa = 0.89). Nasal biopsy samples from 7 cats were culture positive for Mycoplasma, and all were PCR positive. Biopsy samples that were culture negative for Mycoplasma were also PCR negative (kappa = 1.0). Results of culture and PCR for both nasal flush and biopsy were concordant in 19 of 20 cats, and PCR was able to identify an unusual Mycoplasma species that did not grow in culture. In most cats, organisms could be detected in either nasal flush or biopsy samples. In this study, PCR provided rapid and sensitive detection of Mycoplasma species in nasal samples from cats and detected 1 organism that did not grow in culture.     

Use of pooled samples for the detection of Salmonella in feces by polymerase chain reaction.

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.     

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.     

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.     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

Real-time polymerase chain reaction for Mycoplasma gallisepticum in chicken trachea

In this work, we describe a rapid detection procedure for Mycoplasma gallisepticum from chicken tracheal swabs by real-time polymerase chain reaction (PCR) by LightCycler system, where we were able to monitor the amplification of the newly synthesized M. gallisepticum-specific PCR product as a proportionally increasing fluorescent signal by using the double-stranded DNA binding dye SYBR Green I and have identified M. gallisepticum-specific PCR products by DNA melting curve analysis by plotting the first negative derivative (-d[F1]dT) of fluorescence over temperature. Detection limits of the PCR were found to be 3 and 3000 colony-forming units ml(-1) with pure culture of M. gallisepticum and artificially spiked samples, respectively. Out of 96 tracheal swabs, 68 were taken from live chickens and 28 were taken by scraping the mucosal surface of the trachea (SMST) of necropsied chickens. All of the 18 PCR-positive results were from the swabs taken by the SMST method, whereas all of the samples taken from live chickens were negative. Thus, the PCR with the SMST method had a sensitivity and a specificity of 64.2% (18 of 28 chickens) and 100%, respectively. The total time required for template preparation from tracheal swab samples and real-time PCR was approximately 65 min. These results indicate that real-time PCR with the LightCycler technology is a rapid and sensitive test to identify M. gallisepticum-infected flocks if a proper sampling is applied.     

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.     

RT-PCR技术在家禽传染病检测中的应用

传染性疾病已成为危害家禽业安全生产的主要因素,病毒性传染病变异性强、容易传播、难以控制;因此,建立快速、准确的检测方法对于病毒性传染病的防控非常重要。利用聚合酶链式反应(PCR)技术对病毒基因组进行诊断鉴定,与传统病毒检测方法相比具有快速、简便、特异性强的优势。目前,在常规PCR基础上已发展出套式PCR、实时荧光定量PCR、一步法多重PCR等,笔者对PCR技术在家禽病毒性传染病检测中的应用进行了分析与归纳,希望能对实验室研究和临床诊断提供帮助。     

Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.     

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.     

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

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.