Proceedings of the 10th International Colloquium on Paratuberculosis

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#177 Associations between CARD15 polymorphisms, MAP DNA in blood and lactase persistence in a Crohn’s disease case-control study in North-Spain Ramon A Juste, Natalia Elguezabal, Susana Chamorro, Elena Molina, Joseba M Garrido, Sabino Riestra, Ruth de Francisco, Luis Rodrigo NEIKER-Tecnalia, Spain; Hospital Universitario Central de Asturias, Spain Polymorphisms in CARD15 gene have been associated with Crohn’s disease (CD). Also an epidemiologic association between national CD incidence and lactase persistence frequencies has been reported. CARD15 polymorphisms and presence <strong>of</strong> MAP DNA in blood results are controversial. Here we test <strong>the</strong> association <strong>of</strong> R702W, G908R and L1007fs polymorphisms in CARD15, as well as CT-13910 polymorphism in <strong>the</strong> lactase gene with <strong>the</strong> presence <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) DNA in blood and <strong>the</strong> diagnosis <strong>of</strong> CD. Blood samples from 150 CD patients and 65 blood donors from Asturias (Spain) were genotyped by RTi-PCR for CARD15 and by PCR and restriction enzyme digestion for lactase. MAP was detected by nested PCR. A marginally significant association between CD and MAP in blood was found (controls: 37.7%, CD: 25.6%, p=0.0598). NOD2 R709W variant was associated with higher rate <strong>of</strong> MAP detection in blood (60.0% versus 27.8%, p=0.0048). Associations between CD and genotypes were weak, but were partially confirmed by comparing allelic frequencies. The T allele in lactase gene was more frequent in CD patients (61.9%) than in controls (47.1%)(p=0.0275), while only marginal differences were observed for CARD15 at R709W (5.0% vs. 6.4%, p=0.3641) and L1007fs (3.9% vs. 0.8%, p=0.0792). Logistic regression analysis yielded odds ratios <strong>of</strong> over 4 between both genes and MAP in blood. These results confirm an association <strong>of</strong> genetic factors to CD and to MAP infection in humans. The observation <strong>of</strong> higher frequencies <strong>of</strong> MAP in controls than in patients is controversial, but has been explained as <strong>the</strong> result <strong>of</strong> bacterial reduction by <strong>the</strong> antibiotic effect <strong>of</strong> standard CD <strong>the</strong>rapy. Alternatively, it could represent evidence <strong>of</strong> a more continuous and efficient boosting <strong>of</strong> protective immune mechanisms in slow infections. This hypo<strong>the</strong>sis matches better with observation <strong>of</strong> more frequent MAP-haemia in herds without paratuberculosis clinical cases and in vaccinated animals. 269
#207 Nicotinic and Salicylic acids and α & β nicotinamide adenine dinucleotide (NAD) cause dose dependant enhancement, and iso-nicotinic acid (INH) and para-amino-salicylic acid (PAS) cause dose dependant inhibition <strong>of</strong> M. avium subspecies paratuberculosis (MAP) Robert John Greenstein, Liya Su, Sheldon Thomas Brown VAMC Bronx NY, USA Background: There is increasing concern that MAP may be zoonotic, responsible for, at a minimum, Crohn’s disease. Tobacco usage may exacerbate Crohn’s disease and tobacco cessation results in clinical improvement. We hypo<strong>the</strong>sized that nicotine, its structural analogs or possible metabolites, may influence <strong>the</strong> growth <strong>of</strong> MAP. Methods: Nicotine, nicotinic acid, α & β NAD, INH, and as experimental controls salicylic acid and PAS were evaluated in 14CO radiometric culture (Bactec 460 2 ® .) Strains studied included MAP isolated from cattle (ATCC 19698 & 303), or humans with Crohn’s disease (Dominic & UCF-4), M. avium subspecies avium (ATCC 25291 & 101) and BCG (a level II Biosafety surrogate for M. tb.) Growth inhibition or enhancement is indicated by “percent decrease (-) or increase (+) in cumulative Growth Index” (%-/+ΔcGI) from control. All data in this Abstract are at 64µg/ml.) Results: Responses vary amongst sub-species and strains. Salicylic and nicotinic acids and α & β-NAD may cause dose dependant enhancement. Nicotinic acid: Dominic; +82%: UCF-4; +58%: MAP 303; +62%: Salicylic Acid: 303; +66%: 25291; +104%: BCG; +95%: α-NAD; Dominic +120%. β-NAD: Dominic +101% Both PAS and INH may cause dose dependant inhibition. INH: Dominic -74%: UCF-4; -89%: 25291; -99%: PAS: 19698 -74%: 303; -80%: 25291; -98%: BCG; -98%. In contrast, Nicotine has no demonstrable effect on any strain tested. Conclusions: We show dose dependant mycobacterial growth enhancement by nicotinic and salicylic acids and α & β NAD. Additionally we show anticipated dose dependant inhibition by <strong>the</strong>ir spatially modified analogs, <strong>the</strong> antibiotics INH and PAS. In contrast, we find no effect <strong>of</strong> pure nicotine. Our data indicate that, at <strong>the</strong> doses tested, pure nicotine has no growth enhancement effect on MAP but that its structural analogs pr<strong>of</strong>oundly influence MAP’s growth kinetics. 270
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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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Kari Røste Lybeck, Anne Kristine S
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1120-1140 #88: Influence of
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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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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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#178 Age structure of</stro
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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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Proceedings of the 10th International Colloquium on Paratuberculosis

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