Proceedings of the 10th International Colloquium on Paratuberculosis

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#95 Role <strong>of</strong> nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis Judith R. Stabel, Mohammad S. Khalifeh, Ahmad M Al-Majali USDA-ARS-NADC, USA; Jordan University <strong>of</strong> Science and Technology, Jordan Nitric oxide (NO) is a crucial mediator in host defense and is one <strong>of</strong> <strong>the</strong> major killing mechanisms within macrophages. Its induction is highly affected by <strong>the</strong> types <strong>of</strong> cytokines and <strong>the</strong> infectious agents present. In <strong>the</strong> current study, NO production was evaluated after in vitro infection <strong>of</strong> unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 hr, 3 and 6 days <strong>of</strong> culture for cows in different stages <strong>of</strong> disease. In addition, <strong>the</strong> effects <strong>of</strong> in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as <strong>the</strong> pro-inflammatory cytokine IFN-γ were correlated with <strong>the</strong> level <strong>of</strong> NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation <strong>of</strong> NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to <strong>the</strong> addition <strong>of</strong> MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was fur<strong>the</strong>r boosted with <strong>the</strong> combination <strong>of</strong> both in vitro MAP infection and IFN-γ stimulation. Finally, <strong>the</strong> in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in <strong>the</strong> downregulation <strong>of</strong> this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level <strong>of</strong> NO production was associated with cows in <strong>the</strong> subclinical stage <strong>of</strong> MAP infection. #127 Multiplication <strong>of</strong> Mycobacterium avium subsp. paratuberculosis is higher in macrophages induced by M-CSF than GM-CSF Kazuhiro Yoshihara, Reiko Nagata, Yasuyuki Mori National Institute <strong>of</strong> Animal Health, National Agriculture and Food Research Organization, Japan Mycobacterium avium subsp. paratuberculosis (MAP) strain ATCC 19698 was grown in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 2 micrograms/ml mycobactin J and 12.5% OADC Enrichment for several weeks. One milliliter <strong>of</strong> bacteria culture was collected in a microtube and centrifuged. After removal <strong>of</strong> <strong>the</strong> supernatant, <strong>the</strong> pellet was suspended in 0.5 ml PBS, and <strong>the</strong>n <strong>the</strong> bacteria were dispersed for 5 seconds with a homogenizer. The microtubes were allowed to settle for 3 minutes, and several suspensions were pooled and passed through a 5-micrometer-pore-size filter. Twenty milliliters <strong>of</strong> bovine blood was infected with MAP (3x10 8 cells). After 90 minutes <strong>of</strong> incubation, peripheral blood mononuclear cells (PBMC) were isolated by use <strong>of</strong> Ficoll-Paque PLUS density gradient centrifugation. Monocytes were collected from <strong>the</strong> PBMC by a magnetic cell separation system (MACS) with anti human CD14 antibody- coupled microbeads. Monocytes were incubated at 2x10 5 /well in 96-well tissue culture plates with 10% FCS RPMI1640 including ei<strong>the</strong>r M-CSF or GM-CSF. One, two and three weeks later, macrophages induced by M-CSF or GM-CSF were collected into microtubes by using 0.2% EDTA and 5% FCS in PBS and genomic DNAs were extracted with an UltraClean Microbial DNA Isolation kit. Multiplication <strong>of</strong> MAP was estimated by <strong>the</strong> amount <strong>of</strong> MAP DNA measured by realtime PCR. The amount <strong>of</strong> MAP DNA phagocytosed by monocytes in blood was 14.5pg/well (day 0). The amounts <strong>of</strong> MAP DNA within macrophages incubated with M-CSF were 40.1 pg/well (day 7), 48.3 (day 14) and 23.1 (day 14). Meanwhile, DNA volumes <strong>of</strong> MAP in GM-CSF-stimulated macrophages were 29.5 pg/well (day 7), 23.7 (day 14) and 8.9 (day 21). These results suggest that MAP multiplies in high number in macrophages induced by M-CSF as compared to GM-CSF. For <strong>the</strong> next step, we will investigate cytokine pr<strong>of</strong>iles <strong>of</strong> <strong>the</strong>se macrophages. 131
#133 Immunological and pathophysiological characteristics <strong>of</strong> Mycobacterium avium subspecies paratuberculosis infection in guinea pigs Reiko Nagata, Manabu Yamada, Kazuhiro Yoshihara, Shogo Tanaka, Eiichi Momotani, Kei Nishimori, Yasuyuki Mori National Institute <strong>of</strong> Animal Health, Japan In order to elucidate <strong>the</strong> subject why pr<strong>of</strong>ound chronic enteritis is caused by Mycobacterium avium subspecies paratuberculosis (Map) in ruminants but not by o<strong>the</strong>r M. avium subspecies which have great similarity in genetical and antigenical characteristics, we have tried to find differences that may characterize Map infection from those <strong>of</strong> o<strong>the</strong>r mycobcaterial infections. In preliminary experiments using guinea pigs, shedding <strong>of</strong> intraperitoneally inoculated Mycobacterium organisms into feces was detected with surprising speed <strong>of</strong> within 24 hours after inoculation, <strong>the</strong>refore <strong>the</strong> experiments were divided into two different observation periods <strong>of</strong> 24 hours and two months after inoculation. Four-week-old female Hartley guinea pigs were intraperitoneally inoculated with 5 different species or subspecies <strong>of</strong> live mycobacterium (Map, M. avium subspecies avium, M. intracellulare M. scr<strong>of</strong>ulaceum, and M. bovis). After 24 hours or two months, whole intestine and principal organs were collected and performed bacteriological and histopathological examinations with bacterial culture, quantification <strong>of</strong> bacteria by realtime PCR and immuno-pathological staining using anti CD68 monoclonal antibody. Results obtained from our experiments are as follows: 1) Common findings among all Mycobacterial infections: Granuloma formation in liver and spleen; Thickening <strong>of</strong> duodenum because <strong>of</strong> infiltration with inflammatory cells; Bacterial shedding within 24 hours after intraperitoneal inoculation. 2) Characteristics in Map and o<strong>the</strong>r M. avium subspecies infections: Jejunoileal inflammation consisted <strong>of</strong> macrophages, plasma cells, and eosinophils at 2 months postinoculation. 3) Characteristics exclusively observed in Map infection: Significantly lower number <strong>of</strong> organisms in <strong>the</strong> duodenum at 24 hours after inoculation than those <strong>of</strong> o<strong>the</strong>rs; - Scattered distribution <strong>of</strong> CD68 + macrophages in <strong>the</strong> lamina propria mucosae <strong>of</strong> duodenal villi contrary to focused distribution <strong>of</strong> those cells at <strong>the</strong> tip <strong>of</strong> villi in o<strong>the</strong>r Mycobacterial infections. These observational data generated from guinea pig infections may help us to understand <strong>the</strong> pathogenesis <strong>of</strong> Map infection. 132
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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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Page 84 and 85: #191 Potentiating day-old blood sam
Page 86 and 87: samples from the s
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Page 90 and 91: #196 Inter- and intra-subtype genot
Page 92 and 93: RESULTS In 601 (29%) of</st
Page 94 and 95: #211 Culture of my
Page 96 and 97: #242 Application and field validati
Page 98 and 99: #251 Diagnosis of
Page 100 and 101: Table 2. Paratuberculosis PCR resul
Page 102 and 103: #254 Programmes on Paratuberculosis
Page 104 and 105: Fig. 1. European countries specifyi
Page 106 and 107: A bulk milk quality assurance progr
Page 108 and 109: Republic of Lithua
Page 110 and 111: #276 Elimination of</strong
Page 112 and 113: other projects and
Page 114 and 115: Woodbine KA, Schukken YH, Green LE,
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
Page 122 and 123: #173 Local and systemic roles for b
Page 124 and 125: #23 Role of Mycoba
Page 126 and 127: Host Response and Immunology Poster
Page 128 and 129: #28 Intestinal MAP Infection via Pe
Page 130 and 131: #73 Age susceptibility of</
Page 134 and 135: #134 Analysis of <
Page 136 and 137: #157 Study of <str
Page 138 and 139: #163 Immunohistochemical expression
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Page 144 and 145: #224 Application of</strong
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
Page 152 and 153: #50 Assessment of
Page 154 and 155: Table 1. Posterior median (CrIs) <s
Page 156 and 157: #55 Birth clusters of</stro
Page 158 and 159: from their dam, se
Page 160 and 161: #88 Influence of b
Page 162 and 163: #104 Relation between faecal shedde
Page 164 and 165: Epidemiology Poster Abstracts 109 1
Page 166 and 167: #22 The Prevalence of</stro
Page 168 and 169: CONCLUSION IS1311-based nested Ma/M
Page 170 and 171: #42 Seroprevalence survey o
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Page 174 and 175: Environmental samples were collecte
Page 176 and 177: REFERENCES 1) Chiodini RJ, Van Krui
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Page 180 and 181: #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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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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