Proceedings of the 10th International Colloquium on Paratuberculosis

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een recognized that <strong>the</strong> use <strong>of</strong> longitudinal studies using several herds in multiple states is essential to apply MLSSR technique in understanding <strong>the</strong> epidemiology <strong>of</strong> Johne’s disease (Harris et al., 2006). In this study, by using MLSSR straintyping technique in combination with observed MAP shedding levels, we aimed to evaluate: (i) whe<strong>the</strong>r low shedders <strong>of</strong> MAP were passive shedding (pass-through) animals or whe<strong>the</strong>r <strong>the</strong>y were truly infected and (ii) whe<strong>the</strong>r <strong>the</strong>se animals were possibly infected by <strong>the</strong> super-shedders. MATERIALS AND METHODS Isolates used in this study were obtained from three commercial dairy farms in <strong>the</strong> nor<strong>the</strong>astern United States: farm A in New York State, farm B in Pennsylvania, and farm C in Vermont (Pradhan et al., 2009). In this study, selected isolates were collected from all animals that were culture-positive at <strong>the</strong> same time super-shedders were present in <strong>the</strong> herds, from supershedders, and from slaughter house samples available for <strong>the</strong>se animals. We used <strong>the</strong> following criteria to evaluate <strong>the</strong> infection status <strong>of</strong> animals with positive fecal culture test results: (i) to be considered passive shedding (pass-through), an animal may shed only once with low level <strong>of</strong> shedding (i.e., � 21 cfu <strong>of</strong> MAP/g) with subsequent fecal samples testing culture-negative, shedding <strong>the</strong> same strain as contemporary super-shedder(s) and subsequently culture-negative tissues and (ii) to be considered being truly infected, an animal shed low level <strong>of</strong> shedding, which may occur more than once and subsequently culture-positive in tissues at slaughter. DNA from bacterial cells was extracted using QiAamp DNA Mini kit (Qiagen Inc., Valencia, CA, USA). For MLLSR analyses, loci 1, 2, 8, and 9 that were associated with <strong>the</strong> highest Simpson’s diversity indices and identified as <strong>the</strong> most discriminatory and informative SSR loci were used for fingerprinting (Amonsin et al., 2004; Harris et al. 2006). PCR amplification was performed using <strong>the</strong> previously published primers (Amonsin et al., 2004). The amplification conditions consisted <strong>of</strong> an initial denaturation at 94°C for 2 min, followed by 35 cycles <strong>of</strong> denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. Electrophoresed gels were stained with ethidium bromide for 2 min followed by de-staining in water for about 1 hr. The gels were visualized for quality check through UV transillumination with <strong>the</strong> Molecular Imager Gel Doc XR system and Quantity One s<strong>of</strong>tware (Bio-Rad, Hercules, CA, USA). The PCR products were purified with a PureLink PCR Purification kit (Invitrogen Corp., Carlsbad, CA, USA). The PCR amplicons were sequenced at <strong>the</strong> Cornell University Life Sciences Core Laboratories Center. For each locus, allele numbers were assigned to reflect <strong>the</strong> number <strong>of</strong> copies represented in <strong>the</strong> SSR sequence. On <strong>the</strong> basis <strong>of</strong> <strong>the</strong> unique combination <strong>of</strong> alleles for each locus, MLSSR types were <strong>the</strong>n assigned. RESULTS AND DISCUSSION We found 15 different strains (i.e., genotypes or MLSSR types) on three farms from a total <strong>of</strong> 142 isolates (from fecal and tissue samples; 81, 9, and 52 isolates from farms A, B, and, C, respectively); 9 types on farm A, 7 types on farm B, and 6 types on farm C (Fig. 1). The results indicated herd-specific infections; a clonal infection on farm C with 89% <strong>of</strong> isolates shared <strong>the</strong> same strain (type 2) and different strains on farms A and B (Fig. 2). On farm A, type 4 was <strong>the</strong> most predominant one with about 59% <strong>of</strong> isolates belonged to this strain. On farm B, we found a variety <strong>of</strong> strains (7 types) from a limited number <strong>of</strong> isolates (9 isolates) and all <strong>the</strong>se isolates were obtained from <strong>the</strong> animals that were purchased from different sources. 31
Fig. 1. Dendogram (showing genetic relationships among all MAP isolates) was generated by <strong>the</strong> unweighted pair-group method with arithmetic averages (UPGMA) with <strong>the</strong> PAUP program. On each farm, <strong>the</strong> numbers <strong>of</strong> isolates (n) belong to a particular type and <strong>the</strong> number <strong>of</strong> isolates from super-shedders (nss) belong to a particular type (if any) are shown to <strong>the</strong> right <strong>of</strong> <strong>the</strong> dendogram (‘n’ includes ‘nss’). 2.5% 1.2% 1.2% 1.2% 1.2% 7.4% 14.8% 59.3% 11.1% Farm A Type 1 Type 2 Type 3 Type 4 Type 5 Type 6 Type 7 Type 9 Type 11 11.1% 11.1% 11.1% 11.1% 11.1% Farm B Fig. 2. Pie-charts showing <strong>the</strong> percentage <strong>of</strong> each type from a total <strong>of</strong> 81, 9, and 52 isolates (fecal and tissues) on farms A, B, and C, respectively. We also determined <strong>the</strong> shedding level (cfu/g) over time for individual animals at each culture-positive occasion. For example, in <strong>the</strong> first visit on farm A, animal 693 was <strong>the</strong> only animal with super-shedder status, with an observed shedding level <strong>of</strong> 462,000 cfu/g, which was shedding strain type 4 in <strong>the</strong> feces. The same type was found in <strong>the</strong> fecal samples <strong>of</strong> five o<strong>the</strong>r animals on that visit. In <strong>the</strong> first visit on farm C, one animal with super-shedder status (cow 152) was present in <strong>the</strong> herd with <strong>the</strong> observed shedding level <strong>of</strong> 1.26 millions MAP/g; type 2 was found in her fecal sample and <strong>the</strong> same type was found in her fecal sample at slaughter as well as in 4 tissues collected. During that first visit, all 17 culture-positive animals on farm C were shedding <strong>the</strong> same strain (type 2). 32 MLSSR Genotype Farm A Farm B Farm C Type n; nss n; nss n; nss 22.2% 22.2% 1 7-9-5-5 1 - 2 2 7-9-6-5 6 - 46; 18 3 7-10-5-5 9; 3 2 - 4 7-10-6-5 48; 12 - 1 6 7-11-5-5 2 2; 2 - 7 7-11-6-5 12 - 1 8 7-12-5-5 - 1 1 14 14-12-5-5 - 1 - 11 8-11-5-5 1 - - 12 12-11-5-5 - 1 - 13 13-10-5-5 - 1 - 15 16-10-5-5 - 1; 1 - 10 8-9-6-5 - - 1; 1 5 7-11-4-4 1 - - 9 7-14-4-4 1 - - Type 3 Type 6 Type 8 Type 12 Type 13 Type 14 Type 15 1.9% 1.9% 1.9% 1.9% 3.8% 88.5% Farm C Type 1 Type 2 Type 4 Type 7 Type 8 Type 10
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
Page 12 and 13: Kari Røste Lybeck, Anne Kristine S
Page 14 and 15: 1120-1140 #88: Influence of
Page 16 and 17: Concurrent Session B - Rm 175 Wille
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 and 28: MATERIALS AND METHODS Sampling For
Page 29 and 30: To better determine the</st
Page 31: #107 Multilocus Short Sequence Repe
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 68 and 69: #110 Detection and molecular confir
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 80 and 81: #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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247
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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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273
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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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