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Published on: July 15, 2011
A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links
Lenka Dvorska1, Tim John Bull, Milan Bartos
1Veterinary Research Institute, Brno, Czech Republic.
Abstract:
A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.
Insights
A new standardized method for Mycobacterium avium typing using restriction fragment length polymorphism (RFLP) was developed. This method helps differentiate virulent and non-virulent strains, aiding in understanding disease transmission.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Mycobacterium avium subsp. avium and M. avium subsp. silvaticum are significant pathogens with diverse hosts.
- Accurate strain differentiation is crucial for epidemiological studies and disease control.
- Existing typing methods may lack the resolution or standardization required for comprehensive analysis.
Purpose of the Study:
- To develop and validate a standardized restriction fragment length polymorphism (RFLP) typing method for Mycobacterium avium.
- To evaluate the discriminatory power of PvuII-PstI-IS901 and PvuII-IS1245 RFLP typing for various M. avium isolates.
- To correlate RFLP profiles with IS901 copy number and virulence in avian models.
Main Methods:
- Developed a standardized PvuII-PstI-IS901 RFLP typing protocol.
- Analyzed 173 M. avium isolates from diverse sources using PvuII-PstI-IS901 and PvuII-IS1245 RFLP.
- Utilized Gel Compar software for DNA fingerprint analysis.
- Performed virulence assays in pullets for IS901-positive isolates.
Main Results:
- Established a standardized PvuII-PstI-IS901 RFLP method yielding 52 distinct profiles.
- Demonstrated stability of RFLP profiles in vitro and in vivo.
- Observed associations between IS901 copy number and isolate source (e.g., birds, humans).
- Identified virulent (83 isolates, 32 RFLP types) and non-virulent (17 isolates, 12 RFLP types) strains through virulence assays.
Conclusions:
- The developed PvuII-PstI-IS901 RFLP method is a stable and discriminatory tool for typing Mycobacterium avium.
- IS901 copy number correlates with specific RFLP profiles and host origins.
- Virulence attenuation in M. avium is multifactorial and not solely determined by IS901 or IS1245 profiles.
