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Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.

ABSTRACT: A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

SUBMITTER: Tasara T

PROVIDER: S-EPMC1266021 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.

Tasara T T Stephan R R

Applied and environmental microbiology 20051001 10

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratubercu ...[more]

PMID: 16204510

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Project description:BackgroundJohne's disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle.ResultsThe qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher's exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.ConclusionsThis study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.

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.

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.

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.

Dataset Information

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.

Publications

Development of an F57 sequence-based real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk.

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