Proceedings of the 10th International Colloquium on Paratuberculosis

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• 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 61
MLVA Analysis (table 3) showed three alleles at locus 1067, two at loci 1658 and 3527 and one at loci 1605 and 3249. The combination <strong>of</strong> <strong>the</strong>se alleles gave five different clusters with a DI <strong>of</strong> 0.667. According to Overduin et al. 7 , <strong>the</strong> most frequent pattern was 22222. One <strong>of</strong> <strong>the</strong> isolates from milk, <strong>the</strong> strain 7, showed an unusual pattern, 22212. This pattern has never been described before, although <strong>the</strong> allele 1 at locus 3527 has been previously found in bovine strains. Table 3. MLVA Typing MLVA No <strong>of</strong> TR motifs Strains locus 1067 locus 1605 locus 1658 locus 3527 locus 3249 1 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 4 2 2 2 2 2 5 3 2 2 2 2 6 2 2 2 2 2 7 2 2 2 1 2 8 1 2 2 2 2 9 2 2 2 2 2 10 2 2 1 2 2 MLSSR technique showed <strong>the</strong> highest DI value (0.978) resulting in nine clusters. We found only one monomorphic locus (SSR 4), while o<strong>the</strong>r loci showed allelic frequency ranging from 0.18 to 0.72, with 2.3 as mean value. Table 4. MLSSR Typing MLSSR No <strong>of</strong> TR motifs Strains SSR1 SSR2 SSR3 SSR4 SSR5 SSR6 SSR7 SSR8 SSR9 SSR10 SSR11 G G CG GC GC GCG CCG GGT TGC GCC CCG 1 8 10 5 5 5 4 6 4 5 5 5 2 8 9 5 5 5 4 6 3 5 5 5 3 7 10 5 5 5 4 5 4 4 5 5 4 7 9 5 5 5 4 6 4 5 5 5 5 7 11 5 5 5 4 6 4 4 5 5 6 7 11 5 5 5 4 5 4 4 5 5 7 7 13 5 5 5 5 5 5 5 5 5 8 >14 13 5 5 5 5 5 5 5 5 5 9 7 11 5 5 5 4 5 4 4 5 5 10 7 9 4 5 6 4 5 3 4 6 4 Combinations <strong>of</strong> <strong>the</strong> methods gave different results: both MIRU+MLVA and VNTR- MIRU+MLVA gave 5 clusters (DI 0.806), MLSSR+MIRU or MLSSR+VNTR-MIRU did not increase <strong>the</strong> DI <strong>of</strong> MLSSR alone (9 clusters), while MLSSR+MLVA gave <strong>the</strong> maximum <strong>the</strong>oretic DI value (10 clusters). CONCLUSIONS Although <strong>the</strong> limited number <strong>of</strong> strains used, our data suggest that <strong>the</strong> combination <strong>of</strong> simple PCR based methods such as MIRU, VNTR-MIRU and MLVA enhanced <strong>the</strong> discriminatory 62
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
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Scott Wells - Planning Committee Ch
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Monday, August 10, 2009 - All sessi
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Page 100 and 101: Table 2. Paratuberculosis PCR resul
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Host Response and Immunology Keynot
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#33 MERKAL AWARD LECTURE No interfe
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#75 Interleukin 17 and interleukin
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#173 Local and systemic roles for b
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#23 Role of Mycoba
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Host Response and Immunology Poster
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#28 Intestinal MAP Infection via Pe
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#73 Age susceptibility of</
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#95 Role of nitric
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#134 Analysis of <
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#157 Study of <str
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#163 Immunohistochemical expression
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#193 Development of</strong
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#209 Murine macrophages carrying an
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#224 Application of</strong
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Table 1 UK and Cyprus MAP survey Re
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#228 Mycobacterium avium ssp. parat
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#145 MERKAL AWARD LECTURE Multinomi
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#50 Assessment of
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Table 1. Posterior median (CrIs) <s
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#55 Birth clusters of</stro
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from their dam, se
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#88 Influence of b
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#104 Relation between faecal shedde
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Epidemiology Poster Abstracts 109 1
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#22 The Prevalence of</stro
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CONCLUSION IS1311-based nested Ma/M
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#42 Seroprevalence survey o
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#77 Characterisation of</st
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Environmental samples were collecte
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REFERENCES 1) Chiodini RJ, Van Krui
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#113 Effect of soi
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#136 Error-adjusted, variance parti
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Latium Region Viterbo districts RES
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#144 The suitability of</st
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A statistically significant lower f
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#183 Sero-prevalence of</st
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#218 Johne’s disease in t
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#244 A survey to estimate t
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Control Programs Keynote Lecture Re
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#202 Milk quality assurance for par
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#141 Control of pa
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#198 Use of small
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Control Programs Poster Abstracts 1
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RESULTS Loss of al
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#40 Managing Bovine Johnes Disease
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#74 Economic analysis of</s
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#87 Evaluation of
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separately for each existing herd <
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#98 An inter-laboratory ring-trial
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#120 Paratuberculosis vaccine inter
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The statement provides: • A stand
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COMPLEMENTARY ASPECTS On-Farm Disea
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#168 Paratuberculosis in Iran: past
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#180 Behavioural change in owners <
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the fact that <str
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#182 An integrated risk based appro
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etween 0 to 10, depending on <stron
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#203 Milk quality assurance for par
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#234 Johne’s disease vaccine: a c
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Pathogenomics and MAP Biology Conve
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#45 Identification of</stro
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#117 Adsorption of
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#222 Atypical structural features <
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Pathogenomics and MAP Biology Persp
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Abstract not available at press tim
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#174 Sharing of
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#177 Associations between CARD15 po
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Perspectives Talk: Public Health My
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A total of 206 qua
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Feller, M., K. Huwiler, R. Stephan,
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In 2008, the Ameri
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#115 Crohn’s disease and ruminant
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International John
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#76 Towards improved reporting stan
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#97 The EU ParaTBTools Project - Th
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#241 US Vaccine Initiative through
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Paratuberculosis ELISA Reliable, si
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PRIONICS AG WAGISTRASSE 27A PHONE +
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