Proceedings of the 10th International Colloquium on Paratuberculosis

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#213 Construction <strong>the</strong> auxotrophic leuD mutant <strong>of</strong> Mycobacterium avium subspecies paratuberculosis K10 as a live attenuated vaccine candidate Jenn-Wei Chen, Subhash Chandra, Tzu-Yi Ma, Maria A. P. Moreira, Yung-Fu Chang, College <strong>of</strong> Veterinary Medicine, Cornell University, USA Background: Mycobacterium avium subspecies paratuberculosis is an obligate pathogenic bacterium in <strong>the</strong> genus Mycobacteria. It is intracellular pathogens and reside inside host cells primarily macrophage. MAP causes Johne’s disease in cattle and o<strong>the</strong>r ruminants, and is responsible for an economy loss to dairy industry. It has long been suspected as a causative agent in Crohn’s disease in humans, although this connection is controversial. It is urgently needed to develop a vaccine with a better protection and fewer side effects. We have constructed a live attenuated MAP leuD mutant as a live vaccine candidate. Methods: A phage-mediated allelic exchange technique was used to delete leuD gene. The MAP leuD gene deletion was confirmed by both PCR and DNA sequence. The complementation <strong>of</strong> leuD by pVV16-leuD vector abolished its leucine requirement for growth. Mice were used to perform a vaccine trial. Result: C57BL/6 mice were used to determine <strong>the</strong> pathogenicity <strong>of</strong> MAP LeuD mutant. We found that mice infected with wild type <strong>of</strong> MAP with a larger liver and spleen than that <strong>of</strong> leuD mutant. Histopathological examination also showed <strong>the</strong> leuD mutant strain infected mice have a mild inflammation in spleen and liver than that <strong>of</strong> wild type. Preliminary data also indicated that <strong>the</strong> leuD mutant could induce protection against challenge in a mouse model. Conclusion: Management <strong>of</strong> Johne’s disease has been a challenge to farmers. We constructed auxotrophic leuD mutant <strong>of</strong> MAP K10 as a live attenuated vaccine candidate which could induce a better protection against MAP infection. Based on our preliminary mouse study, this new vaccine candidate may be used as a live attenuated vaccine against Johne’s disease. 257
#219 Mycobacterium avium subsp. paratuberculosis PPE protein MAP1152 and conserved protein MAP1156 are antigenic in experimentally and naturally infected cattle Avery Lee Paulson 1 , John P Bannantine 2 , Ofelia Chacon 1 , Robert J Fenton, Denise K Zinniel 1 , D. Scott McVey, David R Smith, Charles J Czuprynski 3 , Raul G Barletta 1,1 Department <strong>of</strong> Veterinary and Biomedical Sciences, University <strong>of</strong> Nebraska, Lincoln, USA; 2 National Animal Disease Center, USDA-ARS, Ames, Iowa, USA; 3 School <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Wisconsin-Madison, USA Mycobacterium avium subsp. paratuberculosis causes Johne’s Disease (JD) in ruminants. A transposon insertion upstream from <strong>the</strong> MAP1152-MAP1156 genomic region causes a change in colony morphotype and results in an attenuated phenotype in bovine macrophages. Bioinformatic analysis indicates that MAP1152 encodes a PPE protein, while MAP1156 is a member <strong>of</strong> <strong>the</strong> uncharacterized protein family UPF0089. Transcriptional analysis suggests that <strong>the</strong> corresponding genes are organized in two overlapping operons. Maltosebinding protein tagged recombinant proteins were overproduced and purified from E. coli. The antigenicity <strong>of</strong> MAP1152 and MAP1156 was examined against sera <strong>of</strong> experimentally infected mice, rabbits; and experimentally and naturally infected cattle. MAP1156 yielded a stronger positive Western blot signal than MAP1152 against sera from cattle with JD. Four positive and four negative sera, previously classified by culture and <strong>the</strong> ELISA Idexx R assay, were re-tested in triplicate by ELISA using <strong>the</strong> Idexx R reference antigen and <strong>the</strong> recombinant proteins. MAP1152 displayed 2- to 3-fold greater reactivity (optical density values) against positive sera as compared to negative controls (p < 0.001). Likewise, <strong>the</strong> reactivity <strong>of</strong> MAP1156 was 2 to 4-fold greater for positive sera (p < 0.05.). In contrast, <strong>the</strong> Idexx R antigen reacted 5 to 12-fold greater with positive than negative sera (p < 0.0001). Classification <strong>of</strong> positive and negative status was determined by cut<strong>of</strong>f value determined as <strong>the</strong> average OD <strong>of</strong> <strong>the</strong> negative sera ± 3 SD. Positive/negative classification <strong>of</strong> sera with MAP1152 was in exact agreement with <strong>the</strong> IdexxR antigen (k= 0.96). However, reaction with MAP1156 resulted in a disagreement with 1 <strong>of</strong> 4 presumed negative sera testing positive (k= 0.68). The data presented suggest that MAP1152 and MAP1156 are antigenic and hold strong potential as vaccine candidates or differential diagnostic antigens if used in conjunction with <strong>the</strong> corresponding attenuated mutants. 258
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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
Page 208 and 209: #40 Managing Bovine Johnes Disease
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Page 214 and 215: separately for each existing herd <
Page 216 and 217: #98 An inter-laboratory ring-trial
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Page 238 and 239: Pathogenomics and MAP Biology Conve
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Page 246 and 247: Pathogenomics and MAP Biology Persp
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Page 276 and 277: A total of 206 qua
Page 278 and 279: Feller, M., K. Huwiler, R. Stephan,
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Page 282 and 283: #115 Crohn’s disease and ruminant
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Page 286 and 287: #76 Towards improved reporting stan
Page 288 and 289: #97 The EU ParaTBTools Project - Th
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Page 292 and 293: Paratuberculosis ELISA Reliable, si
Page 294 and 295: PRIONICS AG WAGISTRASSE 27A PHONE +
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Proceedings of the 10th International Colloquium on Paratuberculosis

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