Proceedings of the 10th International Colloquium on Paratuberculosis

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#163 Immunohistochemical expression <strong>of</strong> iNOS in different types or paratuberculosis granulomatous lesions Maria Muñoz, Laetitia Delgado, Andrea Verna, Carlos Garcia-Pariente, M Carmen Ferreras, J Francisco Garcia-Marin, Valentin Perez Universidad de Leon, Spain Objective: To assess <strong>the</strong> immunohistochemical expression <strong>of</strong> <strong>the</strong> “inducible nitric oxide synthase” (iNOS), an enzyme which catalyzes <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> NO -a radical produced by macrophages and toxic for intracellular bacteria-, in different types <strong>of</strong> lesions associated with Map infection. Materials and Methods: iNOS expression was evaluated in 69 samples <strong>of</strong> intestine (ileum and jejunum with and without lymphoid tissue, and ileocaecal valve) and lymph nodes, from natural an experimental cases <strong>of</strong> ovine and bovine paratuberculosis. Lesions were categorized as focal (located in <strong>the</strong> intestinal lymphoid tissue or lymph nodes,with none <strong>of</strong> scant bacteria), multifocal (focal granulomas in <strong>the</strong> lamina propria) and diffuse with abundant mycobacteria (multibacillary) or scant (paucibacillary). Immunohistochemical staining was performed using a polyclonal antibody raised against iNOS (Upstate) and intensity subjectively scored and compared with <strong>the</strong> presence <strong>of</strong> Map in <strong>the</strong> lesions, demonstrated by Ziehl-Neelsen. Results: The most common pattern <strong>of</strong> staining observed, both in bovine and ovine samples and regardless <strong>the</strong> tissue sample, consisted <strong>of</strong> a marked immunolabelling <strong>of</strong> macrophages and giant cells forming <strong>the</strong> granulomas found in focal and multifocal lesions, associated with small amounts <strong>of</strong> bacteria, whereas in diffuse multibacillary lesions <strong>the</strong> staining was weak <strong>of</strong> negative. Conclusions: These results suggest that iNOS may play an important role in <strong>the</strong> pathogenesis <strong>of</strong> paratuberculosis. The expression <strong>of</strong> high levels <strong>of</strong> iNOS in lesions with small amounts <strong>of</strong> mycobacteria, would suggest a higher ability <strong>of</strong> macrophages to control Map multiplication. #171 Phagocytosis <strong>of</strong> M. paratuberculosis by human monocytic THP-1 cells Peter Willemsen, Ruth Bossers-de Vries, Arie Kant, Diane Houben, Douwe Bakker Central Veterinary Institute <strong>of</strong> Wageningen UR, The Ne<strong>the</strong>rlands; The Ne<strong>the</strong>rlands Cancer Institute, The Ne<strong>the</strong>rlands Thus far conflicting experimental data originating from different experimental approaches to prove or to disprove <strong>the</strong> existence <strong>of</strong> a causative link between M. paratuberculosis (MAP) and Crohn’s disease in humans, have led to a lack <strong>of</strong> clarity on <strong>the</strong> role <strong>of</strong> MAP in <strong>the</strong> etiology <strong>of</strong> Crohn’s disease. To be able to assess <strong>the</strong> possible causal role <strong>of</strong> MAP a better understanding <strong>of</strong> <strong>the</strong> interaction between MAP and <strong>the</strong> human host will be essential. Whereas <strong>the</strong> interaction <strong>of</strong> M. tuberculosis and M. bovis, both proliferating in macrophages, with <strong>the</strong>ir respective hosts have been well studied, <strong>the</strong> interaction <strong>of</strong> MAP with human macrophages is less well characterized. In this present study this interaction is studied by comparing <strong>the</strong> interaction <strong>of</strong> M. bovis and MAP with <strong>the</strong> human macrophage using in vitro infection by both strains <strong>of</strong> <strong>the</strong> human monocytic cell line THP-1. Within a 96-hour time frame this infection is monitored using in parallel immun<strong>of</strong>luorescent microscopy, electronmicroscopy as well as microarray-assisted mRNA pr<strong>of</strong>iling <strong>of</strong> <strong>the</strong> host. The obtained data showed that in contrast to <strong>the</strong> less efficient phagocytosis <strong>of</strong> MAP, <strong>the</strong> infection by MAP elicited a much stronger and more complex host response than infection by M. bovis. Fur<strong>the</strong>rmore, whereas M. bovis was able to proliferate in <strong>the</strong> human macrophages MAP only showed a limited proliferation and was more susceptible to degradation. These results indicate that MAP shows a human macrophage interaction remarkably different from M. bovis and support <strong>the</strong> need for fur<strong>the</strong>r study <strong>of</strong> <strong>the</strong> interaction <strong>of</strong> MAP with <strong>the</strong> (human) macrophage. 137
#175 Differential activation <strong>of</strong> γδ+ and CD8+ T cells in <strong>the</strong> early innate immune response to MAP infection Julianne Zickovich, Emily Kimmel, Philip Fox, C. Amy Eibs, kerri Rask, Kirk Lubick, Kathy Gauss, Mark Jutila, Jay Radke, Colorado State University, USA We used carboxyfluorescein succinimidyl ester (CFSE) labeling <strong>of</strong> bovine PBMCs isolated from naïve animals, antibody staining <strong>of</strong> distinct T cell subsets and 2-color FACS to show suppression <strong>of</strong> innate proliferative responses <strong>of</strong> gd + and CD8 + T cell subsets after exposure to bovine monocytes that have taken up M. avium ssp. paratuberculosis. Consistent with differential activation, <strong>the</strong> CD8 T cell population showed little IFN-g production when compared with cells exposed to monocytes that had taken up M. avium ssp. avium. The CD4 + subset remained unchanged after exposure to ei<strong>the</strong>r subspecies. Results were similar when comparing T cell responses to infected allogeneic or syngeneic (autologous) monocytes. We have fur<strong>the</strong>r shown that when taken up in bovine monocytes, M. avium ssp. paratuberculosis suppressed mRNA(s) encoding select signaling leukocyte activation molecules (SLAM) and confirmed by RT-PCR altered expression <strong>of</strong> mRNA encoding SLAM F5, F7 and F8. SLAM proteins serve as TCR co-receptors that can modulate <strong>the</strong> production <strong>of</strong> IFN-g in T cells. All nine receptors in <strong>the</strong> SLAM family (SLAM F1-9) are differentially expressed on hematopoietic cells. In T cell activation, SLAM cross-linking with its homotypic ligand on <strong>the</strong> surface <strong>of</strong> an infected cell will induce interaction <strong>of</strong> SAP with its cytoplasmic tail and induce IFN-g and IL-2. Increased macrophage SLAMF5 has previously been associated with increased T cell proliferation and secretion <strong>of</strong> IFN-g; and higher levels <strong>of</strong> SLAMF7 with increased killing by natural killer (NK) cells and increased proliferation <strong>of</strong> B cells. There is less known <strong>of</strong> effecter functions in cells where SLAMF8 is increased. The apparent suppression <strong>of</strong> SLAM molecules when comparing virulent M. avium ssp. paratuberculosis infection with that <strong>of</strong> avirulent M. avium ssp. avium is consistent with suppressed T cell activation and provides one potential mechanism for M. avium ssp. paratuberculosis to avoid or dampen a robust early innate response. #184 Development <strong>of</strong> an experimental infection protocol in cattle using a low passage cultured strain <strong>of</strong> MAP Karren Plain, Doug Begg, Anna Waldron, Kumi deSilva, Richard Whittington,University <strong>of</strong> Sydney, Australia Experimental infection <strong>of</strong> animals permits <strong>the</strong> study <strong>of</strong> disease pathogenesis over time. However, many cattle experimental infections use gut homogenates or large doses <strong>of</strong> an unknown passage strain <strong>of</strong> MAP. Though ensuring infection, <strong>the</strong>se can have very different disease progression and clinical outcomes compared to natural infection. A pilot study was performed to assess <strong>the</strong> efficacy <strong>of</strong> a low passage laboratory seed stock culture (CM00/416) <strong>of</strong> a MAP field isolate to infect calves. Highly passaged cultured strains <strong>of</strong> MAP can lose virulence resulting in low numbers <strong>of</strong> infected and clinically diseased animals. Calves were sourced from a dairy farm confirmed to be free <strong>of</strong> Johne’s disease in an area <strong>of</strong> Australia with low disease prevalence in cattle. Three doses <strong>of</strong> MAP were given over 4 weeks with a total dose <strong>of</strong> 4.8 x 10E8 viable bacteria. Calves were necropsied 2-3months post-infection and infection status was assessed by faecal/ tissue culture, Mptb DNA detection in <strong>the</strong> gut tissue and associated lymph nodes, serum ELISA and histology. Tissue culture identified MAP in <strong>the</strong> gut and lymph nodes <strong>of</strong> infected calves 2 months post-infection. Quantitative PCR (QPCR) to detect IS900 MAP DNA was performed. This QPCR assay was designed such that it does not detect environmental mycobacteria and has a high analytical sensitivity (0.001pg <strong>of</strong> MAP genomic DNA). Mptb DNA was detectable in gut and lymph nodes <strong>of</strong> exposed calves 2-3 months post-infection. No visible macroscopic or microscopic lesions were found. This experimental model was designed to be a reproducible and standardizable technique for inducing infection, with advantages over high dose regimes and gut or faecal homogenates that contain variable numbers <strong>of</strong> MAP and contaminants. Confirmation <strong>of</strong> infection with this low passage cattle strain via QPCR and tissue culture has enabled <strong>the</strong> initiation <strong>of</strong> a large scale experimental infection trial using this method. 138
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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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Page 92 and 93: RESULTS In 601 (29%) of</st
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Page 100 and 101: Table 2. Paratuberculosis PCR resul
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Page 110 and 111: #276 Elimination of</strong
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Page 116 and 117: Host Response and Immunology Keynot
Page 118 and 119: #33 MERKAL AWARD LECTURE No interfe
Page 120 and 121: #75 Interleukin 17 and interleukin
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Page 128 and 129: #28 Intestinal MAP Infection via Pe
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Page 132 and 133: #95 Role of nitric
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Page 146 and 147: Table 1 UK and Cyprus MAP survey Re
Page 148 and 149: #228 Mycobacterium avium ssp. parat
Page 150 and 151: #145 MERKAL AWARD LECTURE Multinomi
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Page 154 and 155: Table 1. Posterior median (CrIs) <s
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Page 166 and 167: #22 The Prevalence of</stro
Page 168 and 169: CONCLUSION IS1311-based nested Ma/M
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Page 176 and 177: REFERENCES 1) Chiodini RJ, Van Krui
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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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#52 Is ‘Indian Bison Type’ Myco
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#69 In vitro susceptibility <strong
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#118 Cloning, expression and purifi
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#176 A reference proteomic pr<stron
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#213 Construction the</stro
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#230 Iron concentration dependent s
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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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