Proceedings of the 10th International Colloquium on Paratuberculosis

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#110 Detection and molecular confirmation <strong>of</strong> Mycobacterium avium subsp. paratuberculosis from intestinal tissue and faecal material <strong>of</strong> hares (Lepus europaeus) Miguel Angel Salgado, Robert Söderlund, Göran Bölske, Sergio Javier Leiva, Monica Viviana Pradenas, Armin Fabian Mella, Juan Kruze Universidad Austral de Chile, Chile; National Veterinary Institute (SVA), Sweden Evidence <strong>of</strong> natural infection with M. avium subsp. paratuberculosis (Map) has previously been reported in <strong>the</strong> European hare (Lepus europaeus), with low infection rate and no corresponding histopathological lesions. This could indicate that no active infection but passive transmission has occurred. Low diagnostic sensitivity could be one explanation for this discrepancy. The aim <strong>of</strong> this study was to detect Map infection in wild hares using <strong>the</strong> BACTEC MGIT 960 system, improving resuscitation and growth <strong>of</strong> Map. A total <strong>of</strong> 385 hares captured in different areas in sou<strong>the</strong>rn Chile were randomly selected and sampled for Map from <strong>the</strong> ileum, mesenteric lymphatic nodes and faeces. All positive MGIT tubes were confirmed by a robust real-time PCR system for IS900 and F57 targets, including effective mechanical cell disruption, DNA extraction and removal <strong>of</strong> PCR inhibitors. Map was detected and confirmed in 22 (5,7 %) ileum, 21 (5.5 %) lymphatic node, and 16 (4.2 %) faecal samples. In 10 hares Map was simultaneously detected and confirmed in tissue and faeces by IS900 and F57. There was no difference between <strong>the</strong> proportion <strong>of</strong> positive results in any type <strong>of</strong> sample (Fisher = 0.089). All positive samples (59) showed on average 33 cycle threshold values (Ct), where <strong>the</strong> Ct cut-<strong>of</strong>f value is above 40 for a negative result. The analytical sensitivity <strong>of</strong> <strong>the</strong> real-time PCR system used here has been estimated to be
#125 Detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis DNA isolated from archival formalin-fixed, paraffin-embedded tissue <strong>of</strong> Crohn’s disease patients Katarina Logar 1 , Anton Cerar 2 , Jera Jeruc 2 , Zvezdana Dolenc – Stražar 2 , Darja Ferme 1 , Mateja Pate 1 , Tanja �vara 3 , Matjaž Ocepek 1 1 Veterinary Faculty, Institute <strong>of</strong> Microbiology and Parasitology, Gerbičeva 60, SI-1000 Ljubljana, 2 Faculty <strong>of</strong> Medicine, Institute <strong>of</strong> Pathology, Korytkova 2, SI-1000 Ljubljana, 3 Veterinary Faculty, Institute <strong>of</strong> Pathology, Forensic and Administrative Veterinary Medicine, Gerbičeva 60, SI-1000 Ljubljana, Slovenia ABSTRACT The objective <strong>of</strong> our study was to examine <strong>the</strong> archival formalin-fixed, paraffin-embedded tissues <strong>of</strong> Slovenian patients with Crohn's disease (CD) for <strong>the</strong> presence <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) by IS900 PCR. One hundred and sixty-four intestinal tissue specimens, taken from 33 CD patients, and 39 control specimens, taken from individuals with intestinal cancer, were processed. After <strong>the</strong> paraffin removal, a commercially available kit was used to extract DNA. For <strong>the</strong> detection <strong>of</strong> Map, specific primers were used to amplify a 298 bp fragment <strong>of</strong> IS900. Map was found in six <strong>of</strong> 164 (4%) specimens obtained from four patients. Three specimens belonged to a single patient and <strong>the</strong> o<strong>the</strong>r three to <strong>the</strong> remaining patients. In total, four out <strong>of</strong> 33 (12%) CD patients had Map DNA detected in <strong>the</strong>ir tissues. All <strong>of</strong> <strong>the</strong> control samples tested negative. With <strong>the</strong> method described in this study we could detect <strong>the</strong> IS900 sequence <strong>of</strong> Map in <strong>the</strong> formalin-fixed and paraffin-embedded tissues <strong>of</strong> CD patients. Our findings contribute to <strong>the</strong> current knowledge about Map being a possible etiological agent <strong>of</strong> CD in Slovenian patients. INTRODUCTION Crohn’s disease (CD) is a chronic, transmural inflammatory bowel disease <strong>of</strong> unknown etiology. Current <strong>the</strong>ories implicate <strong>the</strong> role <strong>of</strong> genetic, microbial, immunologic, environmental, dietary, vascular, and even psychosocial factors as potential causative agents. It has been suggested that patients have an inherited susceptibility for an aberrant immunologic response to one or more <strong>of</strong> <strong>the</strong>se provoking factors. The leading infectious candidate is Mycobacterium avium subsp. paratuberculosis (Map), in part because it causes a very similar disease, Johne's disease, in animals, including primates (Chacon et al., 2004; Shanahan, 2002). For <strong>the</strong> detection <strong>of</strong> Map in CD patients, various specimens including fresh or formalinfixed intestinal mucosa obtained by biopsy or surgical resection, blood etc. have been analyzed using different techniques, e.g. PCR, in situ hybridization, cultivation <strong>of</strong> Map and cultivation <strong>of</strong> Map followed by PCR (Bull et al., 2003; Feller et al., 2007; Romero et al., 2005; Ryan et al., 2002; Schwartz et al., 2000). Different detection methods and diverse clinical material may be one <strong>of</strong> <strong>the</strong> reasons for conflicting results about <strong>the</strong> detection rate, ranging from 0 to 100% (Feller et al., 2007; Quirke, 2001). Considering solely <strong>the</strong> results obtained from archival, formalin- fixed and paraffin-embedded tissue, similar incongruence is observed (Baksh et al., 2004; Cheng et al., 2005; Ryan et al., 2002). The present preliminary study was carried out to determine whe<strong>the</strong>r Map DNA, as an indicator <strong>of</strong> formerly present live bacteria, could be detected in <strong>the</strong> archival formalin-fixed, paraffin-embedded gut tissues <strong>of</strong> Slovenian patients with CD by IS900 PCR. MATERIALS AND METHODS Patients and specimens. A total <strong>of</strong> 203 formalin-fixed, paraffin-embedded archival specimens obtained from <strong>the</strong> Institute <strong>of</strong> Pathology, Faculty <strong>of</strong> Medicine, Ljubljana were included in <strong>the</strong> study. Among <strong>the</strong>m, 164 were taken from 33 CD patients and 39 from patients with colorectal carcinoma without inflammatory bowel disease (IBD). The latter represented a control group. All 33 CD patients had an established diagnosis <strong>of</strong> CD based on clinical presentation, 68
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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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Page 20: Diagnostics and Genotyping Convenor
Page 23 and 24: #229 Detecting Johne’s Disease he
Page 25 and 26: Kalis CHJ, Hesselink JW, Barkema HW
Page 27 and 28: MATERIALS AND METHODS Sampling For
Page 29 and 30: To better determine the</st
Page 31 and 32: #107 Multilocus Short Sequence Repe
Page 33 and 34: Fig. 1. Dendogram (showing genetic
Page 35 and 36: #105 MIRU-VNTR genotyping o
Page 37: Diagnostics and Genotyping Poster A
Page 40 and 41: Missouri). Serum from a cow with do
Page 42 and 43: #7 Significance of
Page 44 and 45: RESULTS Of the 327
Page 46 and 47: Direct fecal nested Map PCR testing
Page 48 and 49: #16 Histopathological and immunohis
Page 50 and 51: #19 Development of
Page 52 and 53: #36 Genetic diversity of</s
Page 54 and 55: #60 Evaluation of
Page 56 and 57: #66 Comparison of
Page 58 and 59: (b) IS900 Nested PCR, using p90-p91
Page 60 and 61: (d) f57 Real Time PCR. The figure o
Page 62 and 63: • MIRU (3 loci); • VNTR-MIRU (7
Page 64 and 65: indexes, compared to each single an
Page 66 and 67: #108 Evaluation of
Page 70 and 71: endoscopic criteria and histopathol
Page 72 and 73: Bull TJ, McMinn EJ, Sidi-Boumedine
Page 74 and 75: #131 Seroprevalence of</str
Page 76 and 77: could be partly attributed to a sma
Page 78 and 79: #146 Analysis of v
Page 84 and 85: #191 Potentiating day-old blood sam
Page 86 and 87: samples from the s
Page 88 and 89: (MAA) (MAP87 and MAP3776) and 1 fro
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
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Page 110 and 111: #276 Elimination of</strong
Page 112 and 113: other projects and
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Page 116 and 117: 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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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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