Proceedings of the 10th International Colloquium on Paratuberculosis

#196 Inter- and intra-subtype genotypic differences that divide Mycobacterium avium
subspecies paratuberculosis strains
Iker Sevilla, Franck Biet, Thierry Cochard, Joseba M. Garrido, Ian Heron, Ramón A. Juste, Joyce McLuckie,
Philip Supply, Virginie C. Thibault, Karen Stevenson, NEIKER-Tecnalia, Spain; INRA, Infectiologie Animale,
Santé Publique, Nouzilly, France; Moredun Research Institute, United Kingdom; INSERM U629 and Institut Pasteur
de Lille, France
Mycobacterium avium subspecies paratuberculosis (Map) strains are <strong>of</strong> two major types known as Sheep type
(also called type I) and Cattle type (type II), but an Intermediate subtype (type III) that seems to cluster within
<strong>the</strong> Sheep group according to several genotypic and phenotypic features has been described also. This study
was undertaken to genotype a panel <strong>of</strong> type I or III small ruminant isolates from different origins with known
pulsed-field gel electrophoresis (PFGE) pr<strong>of</strong>iles and to compare <strong>the</strong>m with a panel <strong>of</strong> type II isolates and a
well documented type II isolate collection. Methods used included <strong>the</strong> multiple-locus variable-number tandem
repeat analysis (MLVA) which is based on genetic elements called mycobacterial interspersed repetitive units
(MIRUs), <strong>the</strong> analysis <strong>of</strong> large sequence polymorphism (LSP), <strong>the</strong> single nucleotide polymorphism (SNP)
based analysis <strong>of</strong> gyr genes and <strong>the</strong> IS900-restriction fragment length polymorphism (IS900-RFLP) analysis.
Seventeen type I or III isolates and 24 type II isolates were analyzed. Eight different IS900-RFLP pr<strong>of</strong>iles were
identified among type I or III isolates, some <strong>of</strong> <strong>the</strong>m not previously published. Five belonged to <strong>the</strong> Intermediate
type (or type III) and 3 were <strong>of</strong> <strong>the</strong> Sheep type (or type I). Some novel types were found amongst <strong>the</strong> 6 MLVA
genotypes identified in <strong>the</strong>se isolates. No amplification was observed for type I strains (pigmented) in LSP20
while type III isolates (pigmented and non-pigmented) gave a negative result, compared to <strong>the</strong> positive result
recorded for type II strains. LSPA4 was positive for all type I or III isolates and negative for all type II isolates.
Pigmented type III isolates have been found. Our results suggest that <strong>the</strong> techniques used correlate well.
These findings support <strong>the</strong> division <strong>of</strong> Map strains into type II (Cattle lineage including Cattle types) and Type I
and III (Sheep lineage subdivided in Sheep and Intermediate types).
#197 Mycobacterium avium subsp. paratuberculosis detected and quantified using different
DNA extraction and real-time amplification methods in artificially inoculated fecal
samples from cattle
Iker Sevilla, Joseba M. Garrido, Isbene Sánchez, Elena Molina, Felix Bastida, Ramón A. Juste
NEIKER-Tecnalia, Spain; Vacunek, Bizkaia, Spain
The aim <strong>of</strong> this study was to identify <strong>the</strong> best combinations <strong>of</strong> DNA extraction and real-time amplification
methods available using different genomic targets for a sensitive and specific PCR detection <strong>of</strong> Mycobacterium
avium subsp. paratuberculosis (Map). Homogenized fecal samples from a paratuberculosis-free cow were
spiked with 10 to 107 bacteria per gram as assessed by direct microscopic count in a Neubauer Improved
chamber <strong>of</strong> two Map cultures (K10 reference strain and 764 field isolate). Serial dilutions were plated to assess
viable Map cells in <strong>the</strong> inocula. Six different commercially available DNA extraction kits were employed on triplicate
samples <strong>of</strong> each level <strong>of</strong> inoculation with both reference and field strains. DNA extracts from all kits were
submitted to two triplex real-time PCR amplification assays with an internal amplification control to rule out
inhibition. Two extraction kits included <strong>the</strong>ir own PCR method that was used with extracts from <strong>the</strong>ir respective
kit only. Map targets used in <strong>the</strong>se assays were <strong>the</strong> multi-copy insertion elements IS900, ISMav2, ISMAP02
and <strong>the</strong> single copy element F57. In addition, a quantitative real-time PCR kit based on sequence F57 was
used to quantify <strong>the</strong> mycobacterial DNA present in extracts. Culture <strong>of</strong> inoculated feces to compare isolation
and PCR detection sensitivities is also in progress. Two extraction methods have been discarded because <strong>of</strong>
low efficiency. Two appeared to have <strong>the</strong> highest sensitivity according to quantification results. The quantitative
PCR showed an excellent correlation between Map estimated number <strong>of</strong> copies and actual cell concentration.
Most combinations <strong>of</strong> extraction and amplification reliably detected 10,000 cells per gram <strong>of</strong> feces according to
direct counts or, 1,240 CFUs and 223.75 CFUs per gram according to plate counts both for field and reference
strain inocula.
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