Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization

Pakarinen, Jaakko; Nieminen, Tea; Tirkkonen, Taneli; Tsitko, Irina; Ali-Vehmas, Terhi; Neubauer, Peter; Salkinoja‐Salonen, Mirja

doi:10.1016/j.vetmic.2006.10.016

Cited by 18 publications

(16 citation statements)

References 18 publications

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“…This low detection limit was comparable to that obtained by Zerihun et al (33), who used a TaqMan probe, and slightly better than that obtained by Salati et al (34) with a nested-PCR test. It is also comparable to the detection levels described by Pakarinen et al (35), whose results showed that the use of a hybridization probe did not significantly improve the assay sensitivity. Moreover, the ability of species identification was not affected by the initial DNA template amount, as melting curves obtained from 10-fold dilutions of genomic DNA (corresponding to 10 6 to 10 genome equivalents) could be superimposed (Fig.…”

Section: Discussionsupporting

confidence: 87%

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Phung

Caruso

Godreuil

et al. 2013

Appl Environ Microbiol

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e Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.

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Section: Discussionsupporting

confidence: 87%

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Phung

Caruso

Godreuil

et al. 2013

Appl Environ Microbiol

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“…DNA was isolated from 30–50 mg of dust and DNA concentration was measured as described elsewhere [16]. …”

Section: Methodsmentioning

confidence: 99%

Contrasting Immunological Effects of Two Disparate Dusts – Preliminary Observations

Alenius

Pakarinen²,

Saris

et al. 2008

Int Arch Allergy Immunol

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Background: Modern lifestyle and urbanization have been associated with a raised risk for atopic diseases whereas early and long-term exposure to a farm environment confers protection against atopic sensitization. Immunomodulatory potential and microbiological characteristics of settled airborne dust from an urban house and a barn were examined. Methods: Pulmonary inflammation was induced in mice by repeated intranasal administration of dusts. Monocyte-derived human dendritic cells (moDCs) were exposed to dusts followed by coculture with purified naïve T cells. Cytokine/chemokine mRNA and protein levels were analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay and flow cytometry. The dusts were analyzed by cloning and sequencing of 16S rRNA genes (290 sequences) for DNA, lipids, endotoxin and β-glucan, by live-dead staining, viable counting, isolation and identification of pure cultures (n = 76). Results: Repeated exposure to house dust elicited pulmonary eosinophilia in mice whereas exposure to barn dust elicited neutrophilic and lymphocytic airway inflammation. Stimulation of moDCs with urban house dust elicited expression of Th2-promoting OX40L and Jagged-1 costimulatory molecules. Dendritic cells (DCs) exposed to house dust directed naïve T cells towards Th2 responses. Exposure of DCs to barn dust elicited the development of Th1-dominated immune responses. Urban house dust contained bacterial debris almost exclusively of human commensal species (corynebacteria, streptococci) whereas barn dust comprised mainly intact, viable bacteria of high diversity and no commensal species. Conclusion: Contact to debris originating from human commensal bacteria in urban house dust elicited a Th2-type response whereas barn dust with high bacterial diversity directed the cells towards a Th1 response.

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“…With the development of nucleic acid-based amplification assays, the identification of difficult to grow microorganisms in tissues, including M. leprae , has become routine [11]–[16]. These assays have enhanced our awareness of clinical disease processes, and in some cases have produced new ways to diagnose and monitor mycobacterial infections.…”

Section: Introductionmentioning

confidence: 99%

Enumeration of Mycobacterium leprae Using Real-Time PCR

et al. 2008

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Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

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Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization

Cited by 18 publications

References 18 publications

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Contrasting Immunological Effects of Two Disparate Dusts – Preliminary Observations

Enumeration of Mycobacterium leprae Using Real-Time PCR

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