Proceedings of the 10th International Colloquium on Paratuberculosis

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Table 1 UK and Cyprus MAP survey Results Survey Total Samples Phage Assay Phage - PCR Culture No. Pos. % No. Pos. % No. Pos. % UK 54 54 19 35.2 54 1 1.9 54 0 0 Cyprus 225 225 218 96.9 225 50 22.2 225 2 0.9 The TVC results (Table 2) are separated into groups according to microbial load. No relationship was seen between <strong>the</strong> TVC and samples being phage-PCR MAP positive. This confirms that <strong>the</strong> MAP were not being introduced into <strong>the</strong> milk by faecal contamination and that in <strong>the</strong> Cypriot study we can deduce that MAP is being shed into <strong>the</strong> milk by animals that do not display any clinical symptoms <strong>of</strong> <strong>the</strong> disease (clinical herd data was not available for <strong>the</strong> UK samples). Characterization <strong>of</strong> <strong>the</strong> 2 culture isolates identified one as an S strain and <strong>the</strong> o<strong>the</strong>r as a C strain. The presence <strong>of</strong> <strong>the</strong> S strain in <strong>the</strong> cattle herd population is interesting from an epidemiological perspective since <strong>the</strong> presence <strong>of</strong> this strain has also been found in <strong>the</strong> Cypriot sheep and goat populations (Liapi et al., 2009). The extended culture times required to isolate this organism may explain why it was not previously cultured from bovine milk samples. Table 2 MAP and Viable Count Results Total Viable Count Data Phage Positive samples IS900-PCR positive samples Survey � Log4 cfu/ml Log4- Log5 cfu/ml >Log5.0 1 cfu/ml �Log4 cfu/ml Log4- Log5 cfu/ml >Log5.01 cfu/ml �Log4 cfu/ml Log4- Log5 cfu/ml >Log5.01 cfu/ml UK 21 7 26 15 Cyprus 105 95 25 102 93 23 25 21 4 Prospective analysis <strong>of</strong> <strong>the</strong> phage-PCR data revealed a strong relationship between <strong>the</strong> pfu number and <strong>the</strong> probability <strong>of</strong> that sample being shown to contain MAP by PCR genotyping. Figure 1 shows <strong>the</strong> distribution <strong>of</strong> <strong>the</strong> pfu count <strong>of</strong> <strong>the</strong> MAP IS900 positive and negative samples. An area <strong>of</strong> overlap exists between 15 pfu/50 ml (PCR-positive sample with <strong>the</strong> lowest plaque count) and 75 pfu/50 ml (highest PCR-negative sample). However all samples with >75 plaques were found to contain MAP. The FASTplaqueTB assay gives a cut<strong>of</strong>f value <strong>of</strong> 20 pfu per sputum sample. If a cut<strong>of</strong>f is applied in <strong>the</strong> overlap region to this data, a test with a high Sensitivity (Sn, 98%) but a lower Specificity (Sp, 63.4%) was gained. Fig.1: Relationship between plaque number Fig. 2: ROC analysis <strong>of</strong> plaque cut<strong>of</strong>f and IS900 PCR results 2 27 2 1 - -
To better determine <strong>the</strong> most appropriate cut<strong>of</strong>f value Receiver Operating characteristic (ROC) analysis was used (Figure 2) and showed that a cut<strong>of</strong>f <strong>of</strong>f 59 pfu/50 ml gives Sn and Sp values <strong>of</strong> 90% and 99%, respectively. This cut<strong>of</strong>f value can be refined as more data is generated, but shows <strong>the</strong> clear relationship between <strong>the</strong> number <strong>of</strong> plaques and presence <strong>of</strong> MAP in a sample. As high levels <strong>of</strong> Mycobacteria should not be present in hygienically collected milk, this relationship is to be expected if infected animals are shedding MAP. Hence in this case plaque numbers alone can be used to indicate MAP infection without <strong>the</strong> need for PCR confirmation. The Cyprus cattle population is free from bovine tuberculosis however, since any Mycobacteria shed into milk will result in a plaque, if this were to be repeated in a country with endemic levels <strong>of</strong> TB, <strong>the</strong> plaque results alone could not be used as an indication <strong>of</strong> MAP infection. For instance in this work 26 o<strong>the</strong>r isolates were recovered on culture and identified as Mycobacteria spp. by Z-N stain and species-specific PCR (Tevere et al., 1996) and <strong>the</strong>se cells would have given rise to IS900 PCR-negative plaques. While <strong>the</strong> relationship between infection and plaque number is expected to be maintained irrespective <strong>of</strong> <strong>the</strong> identity <strong>of</strong> <strong>the</strong> Mycobacterium being shed into milk, a multiplex PCR genotyping assay would <strong>the</strong>n be required to confirm <strong>the</strong> identity <strong>of</strong> <strong>the</strong> organism being detected (see Stanley et al., 2007). CONCLUSION The combined phage PCR assay was successfully applied to real BTM samples and shown to be able to sensitively and specifically detect MAP within 24 h in a population that is free from TB. REFERENCES Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I, 2004. Mycobacterium avium subsp. paratuberculosis cultured from locally & commercially pasteurized cow’s milk in <strong>the</strong> Czech Republic. Appl. Environ. Microbiol, 71, 1210-1214. Coetsier C, Vannuffel P, Blondeel N, Denef J, Cocito C, Gala J, 2000. Duplex PCR for Differential Identification <strong>of</strong> M. bovis, M. avium, & M. avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues from cattle. J. Clin. Microbiol, 38, 3048-3054. Liapi M, Botsaris G, Economides C, Georgiou K, Loucaides P, Kakoyiannis C, Naseby D, 2009. Prevalence <strong>of</strong> paratuberculosis in sheep & goats in Cyprus. In Saridomichelakis MN, Giannenas I, Solomakos N, F<strong>the</strong>nakis GC (ed’s), Abst. 11th Greek Vet. Congress, Hellenic Vet. Med. Soc., A<strong>the</strong>ns, pp128. Marsh I, Whittington R, Cousins D, 1999. PCR-restriction endonuclease analysis for identification & strain typing <strong>of</strong> Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium based on polymorphisms in IS1311. Mol. Cell. Probes, 13, 115-126. Rees CED, Dodd CER, 2006. Phage for rapid detection and control <strong>of</strong> bacterial pathogens in food. Adv. Appl. Microbiol, 59, 159-186. Slana I, Liapi M, Moravkova M, Kralova A, Pavlik I, 2009. Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by iELISA, culture and quantitative IS900 and F57 Real-Time PCR. Prev. Vet. Med, 89, 223-226. Stanley EC, Mole RJ, Smith RJ, Glenn SM, Barer MR, McGowan M, Rees CED, 2007. Development <strong>of</strong> a new, combined rapid method using phage and PCR for detection & identification <strong>of</strong> viable M. paratuberculosis bacteria within 48 h. Appl. Environ. Microbiol, 73, 1851-1857. Whittington RJ, Marsh I, Turner MJ, McAllister S, Choy E, Eamens GJ, Marshall DJ, Ottaway S, 1998. Rapid detection <strong>of</strong> Mycobacterium paratuberculosis in clinical samples from ruminants & in spiked environmental samples by modified BACTEC 12B radiometric culture & direct confirmation by IS900 PCR. J. Clin. Microbiol, 36, 701-707. Woodbine KA, Schukken YH, Green LE, Ramirez-Villaescusa A, Mason S, Moore SJ, Bilbao C, Swann N, Medley GF, 2009. Seroprevalence and epidemiological characteristics <strong>of</strong> M. paratuberculosis on 114 cattle farms in south west England. Prev. Vet. Med, 89,102–109. 28
Page 1 and 2: Book of Abstracts
Page 4 and 5: Proceedings <stron
Page 6 and 7: Grant Support and Sponsorship <stro
Page 8 and 9: Scott Wells - Planning Committee Ch
Page 10 and 11: Monday, August 10, 2009 - All sessi
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Page 14 and 15: 1120-1140 #88: Influence of
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Page 18 and 19: Thursday, August 13 Thursday, Augus
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: MATERIALS AND METHODS Sampling For
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Page 33 and 34: Fig. 1. Dendogram (showing genetic
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Page 44 and 45: RESULTS Of the 327
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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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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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