Detection of Mycobacterium avium subsp. paratuberculosis in drinking water and biofilms by quantitative PCR.
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ABSTRACT:
SUBMITTER: Beumer A
PROVIDER: S-EPMC2976226 | biostudies-other | 2010 Nov
REPOSITORIES: biostudies-other
Publications
Detection of Mycobacterium avium subsp. paratuberculosis in drinking water and biofilms by quantitative PCR.
Beumer Amy A King Dawn D Donohue Maura M Mistry Jatin J Covert Terry T Pfaller Stacy S
Applied and environmental microbiology 20100903 21
It has been suggested that Mycobacterium avium subspecies paratuberculosis has a role in Crohn's disease. The organism may be acquired but is difficult to culture from the environment. We describe a quantitative PCR (qPCR) method to detect M. avium subsp. paratuberculosis in drinking water and the results of its application to drinking water and faucet biofilm samples collected in the United States. ...[more]
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Project description:Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.
| S-EPMC1352208 | biostudies-literature
Project description:BackgroundJohne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples.ResultsOur confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system.ConclusionWe recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.
| S-EPMC1609169 | biostudies-literature
Project description:In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.
| S-EPMC2394907 | biostudies-literature
Project description:A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.
| S-EPMC1538760 | biostudies-literature
Project description:Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611-622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.
| S-EPMC3957769 | biostudies-literature
Project description:Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.
| S-EPMC4810606 | biostudies-literature
Project description:Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.
| S-EPMC88986 | biostudies-literature
Project description:BackgroundMycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study.ResultsTo characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical.ConclusionsWe present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.
| S-EPMC156888 | biostudies-literature
Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.
| S-EPMC140397 | biostudies-literature
Project description:Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP(A)s) and those only present in the latter (LSP(P)s). We examined the distribution of 3 LSP(A)s and 17 LSP(P)s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP(A)8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP(P)s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.
| S-EPMC1234005 | biostudies-literature