Simultaneous Detection of <i>Brachyspira hyodysenteriae, Brachyspira pilosicoli</i> and <i>Lawsonia intracellularis</i> in Porcine Faeces and Tissue Samples by Multiplex‐PCR

Oliveira, Celso José Bruno de; Wurm, Melanie; Beilage, Elisabeth große; Givisiez, Patrícia Emília Naves

doi:10.1111/j.1439-0442.2007.00995.x

Cited by 18 publications

(16 citation statements)

References 24 publications

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“…Amplification products sized 525 bp were then purified using the QIAquick ® DNA purification kit (Qiagen). Cloning and quantification of plasmids containing specific genome equivalents (GE) of L. intracellularis were performed as previously described (Nathues et al. 2007).…”

Section: Methodsmentioning

confidence: 99%

Quantification ofLawsonia intracellularisin porcine faeces by real-time PCR

Nathues

Holthaus

Beilage

2009

Journal of Applied Microbiology

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Aim: To develop and to validate a method for the quantification of Lawsonia intracellularis in porcine faeces by real‐time PCR. Methods and Results: A real‐time PCR including a calibrator based on plasmid DNA for quantification by means of ΔΔCt method was evaluated. The parameters specificity, detection limit, quantification limit, linearity, range, repeatability, precision and recovery were validated. The detection limit of the agent was 1 copy per reaction, and quantification was reliable between 101 and 107 copies per μl reaction volume. The linearity calculated by logistic regression revealed a slope of −3·329 reflecting an efficiency of 99·7% for the assay. Moreover, it was shown that storage of samples and repetition of tests including DNA isolation by same or other investigators did not influence the outcome. Conclusion: The quantification method described herein revealed consistent results for the quantitation of L. intracellularis in porcine faeces samples. Significance and Impact of the Study: In contrast to common PCR in combination with gel electrophoresis, this validated quantification method based on real‐time PCR enhances a reliable quantification and is even more sensitive.

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Section: Methodsmentioning

confidence: 99%

Quantification ofLawsonia intracellularisin porcine faeces by real-time PCR

Nathues

Holthaus

Beilage

2009

Journal of Applied Microbiology

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“…These organisms were detected through multiplex PCR (Su and Song 2005;Nathues et al 2007;Song and Hampson 2009;Willems and Reiner 2010).…”

Section: Introductionmentioning

confidence: 99%

Prevalence of Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli infection in hunted wild boars (Sus scrofa) in Germany

2010

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Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli are important pathogens in domestic pig production, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. They are widely distributed among pig-producing units around the world, and transmission is accomplished by relatively weak immunity, long shedding intervals, sequential shedding, and actual environmental survival. Little information is available on occurrence, prevalence, and quantity of these pathogens in free-ranging wild boars. The aim of the present study was to evaluate L. intracellularis, B. hyodysenteriae, and B. pilosicoli infections in wild boars in Germany. Tissue samples from ileocaecal mucosa of 165 wild boars from 18 hunting grounds situated in 14 of the 16 federal states of Germany were examined by conventional PCR and quantified by multiplex real-time PCR. None of the wild boars did show any gross pathological signs of enteritis. The overall prevalence for L. intracellularis, B. hyodysenteriae, and B. pilosicoli was 20.6%, 2.4%, and 12.1%, respectively. None of the three agents was detected in 68.5% of the wild boars and in 11.1% of the hunting grounds. Numbers of bacteria per sample were below the limit of quantification (100 cells/PCR reaction). This is the first study on L. intracellularis and Brachyspira spp. in free-ranging wild boars. The study revealed colonised animals without signs of disease. The meaning of these findings remains unclear, and we do not know whether and to what extent these three pathogens are exchanged between wild boars and domestic pigs. Further research is needed to get insight into the epidemiological impact of the results.

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“…2003; Suh and Song 2005; La et al. 2006; Nathues et al. 2007); however, culture is still needed to obtain isolates for strain typing and determining antimicrobial susceptibility in certain cases.…”

Section: Introductionmentioning

confidence: 99%

“…in faecal samples by using Polymerase chain reaction (PCR). These new methods have detection limits of 10 3 -10 4 spirochaete cells ml )1 (La et al 2003;Suh and Song 2005;La et al 2006;Nathues et al 2007); however, culture is still needed to obtain isolates for strain typing and determining antimicrobial susceptibility in certain cases.…”

Section: Introductionmentioning

confidence: 99%

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces

Lugsomya

Tummaruk

Hampson

et al. 2012

Letters in Applied Microbiology

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Aims: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. Methods and Results: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤1 μg ml−1, while 16 μg ml−1 of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre‐enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre‐enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 μg ml−1), S (400 μg ml−1), R (30 μg ml−1) and colistin (C, 100 U ml−1) (assigning as BAM‐CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. Conclusion: BAM‐CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. Significance and Impact of the Study: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.

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Simultaneous Detection of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in Porcine Faeces and Tissue Samples by Multiplex‐PCR

Cited by 18 publications

References 24 publications

Quantification ofLawsonia intracellularisin porcine faeces by real-time PCR

Quantification ofLawsonia intracellularisin porcine faeces by real-time PCR

Prevalence of Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli infection in hunted wild boars (Sus scrofa) in Germany

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces

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