Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.
In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.
Taddei
R
R
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Centro di Referenza Nazionale Paratubercolosi, Sezione di Piacenza, Strada Faggiola 1, Gariga di Podenzano, Piacenza, Italy. roberta.taddei@bs.izs.it
Barbieri
I
I
Pacciarini
M L
ML
Fallacara
F
F
Belletti
G L
GL
Arrigoni
N
N
D000818:Animals; D001431:Bacteriological Techniques; D001483:Base Sequence; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D008892:Milk / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial