Proceedings of the 10th International Colloquium on Paratuberculosis

• MIRU (3 loci);
• VNTR-MIRU (7 loci);
• MLVA (5 loci);
• MLSSR (11 loci).
All PCR reactions were carried out in a final volume <strong>of</strong> 25 μl with Taq DNA Polymerase
(Qiagen). For MLSSR, we sequenced <strong>the</strong> obtained amplicons using ABI Prism 310 DNA
genetic analyser (Applied Biosystems, Monza, Italy). The sequences were analysed using
<strong>the</strong> SeqMan Module <strong>of</strong> Lasergene Package (DNA Star, Madison UK). Finally, <strong>the</strong>
discriminatory index (DI) was calculated as reported by Hunter and Gaston 9 .
RESULTS
The results for MIRU and VNTR-MIRU are shown respectively in table 1 and 2. Both
methods gave two clusters (DI 0.556) with two alleles respectively at locus 2 and 292.
According to Bull et al. 5 , MIRU typing showed two pr<strong>of</strong>iles, pr<strong>of</strong>ile I and II. Combination <strong>of</strong>
MIRU and VNTR-MIRU did not improve <strong>the</strong> DI. The two pr<strong>of</strong>iles identified by <strong>the</strong> association
<strong>of</strong> <strong>the</strong>se methods have been previously described by Möbius et al. 10 .
Table 1. MIRU Typing
Strains Source Matrix
MIRU No. <strong>of</strong> TR motifs
locus 1 locus 2 locus 3 MIRU Pr<strong>of</strong>ile a
1 cattle faeces 3 9 5 II
2 cattle faeces 3 9 5 II
3 cattle faeces 3 7 5 I
4 cattle faeces 3 9 5 II
5 cattle faeces 3 9 5 II
6 cattle milk 3 7 5 I
7 cattle milk 3 7 5 I
8 goat faeces 3 7 5 I
9 cattle faeces 3 9 5 I
10 goat tissue 3 ND ND
a 4
According to Bull et Al. . ND: not detectable
Table 2. VNTR-MIRU Typing
MIRU- VNTR No. <strong>of</strong> TR motifs
Strains locus 32 locus 25 locus 292 locus 47 locus 3 locus 7 locus X3
1 8 3 4 3 2 2 2
2 8 3 4 3 2 2 2
3 8 3 3 3 2 2 2
4 8 3 4 3 2 2 2
5 8 3 4 3 2 2 2
6 8 3 3 3 2 2 2
7 8 3 3 3 2 2 2
8 8 3 3 3 2 2 2
9 8 3 4 3 2 2 2
10 ND ND ND ND ND ND ND
ND: not detectable
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