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A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets
P Abiven1, B Blanchard, C Saillard
1Centre National D'Etudes Véterinaires et Alimentaires, Laboratoire Central de Recherches Avicole et Porcine, Ploufragan, France.
Abstract:
Mycoplasma hyopneumoniae is the primary agent of swine enzootic pneumonia. Because of fastidious growth requirements and its serological cross-reactions with other porcine mycoplasmas, we developed a specific DNA probe for its detection. A partial genomic library of M. hyopneumoniae was constructed in plasmid pBR 322 using Hind III chromosomal fragments. The recombinant plasmids were screened by differential hybridization with M. flocculare and M. hyorhinis genomic DNA probes. One non-hybridizing recombinant plasmid was selected and its 1.65 kbp insert (designated I141) tested for specificity against genomic DNA from numerous mycoplasmas, other bacteria species and DNA from lung tissue of specific pathogen free (SPF) piglets. The 32P labelled I141 could detect specifically down to 400 pg of M. hyopneumoniae genomic DNA. To test the suitability of the I141 probe for the laboratory diagnosis of M. hyopneumoniae infections, we used clinical tracheobronchial specimens from piglets which were experimentally infected with M. hyopneumoniae. The results with hybridization on each specimen were compared to findings with an immunofluorescence test. Of the clinical specimen tested, there was agreement in the two tests of 63%.
Insights
A specific DNA probe, I141, was developed to detect Mycoplasma hyopneumoniae, the cause of swine enzootic pneumonia. This probe offers a sensitive method for diagnosing infections in piglets, showing 63% agreement with immunofluorescence tests in clinical specimens.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Swine Disease Research
Background:
- Mycoplasma hyopneumoniae is the primary pathogen causing swine enzootic pneumonia.
- Challenges in diagnosis include fastidious growth requirements and serological cross-reactivity with other porcine mycoplasmas.
Purpose of the Study:
- To develop a specific DNA probe for the accurate detection of Mycoplasma hyopneumoniae.
- To evaluate the diagnostic utility of the developed probe in clinical samples.
Main Methods:
- Construction of a partial genomic library of M. hyopneumoniae in plasmid pBR 322.
- Screening of recombinant plasmids using differential hybridization with related porcine mycoplasma DNA probes.
- Testing the specificity and sensitivity of the selected DNA insert (I141) against various DNA sources and clinical specimens.
Main Results:
- A specific DNA probe, I141 (1.65 kbp insert), was identified and characterized.
- The I141 probe demonstrated high specificity for M. hyopneumoniae, detecting as little as 400 pg of genomic DNA.
- In clinical tracheobronchial specimens, the I141 probe showed 63% agreement with immunofluorescence testing for M. hyopneumoniae infection.
Conclusions:
- The I141 DNA probe is a specific and sensitive tool for detecting Mycoplasma hyopneumoniae.
- This probe shows potential for improving the laboratory diagnosis of swine enzootic pneumonia.