Proceedings of the 10th International Colloquium on Paratuberculosis

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#101 Using liquid and agar-based methods to investigate <strong>the</strong> bacteriostatic activity <strong>of</strong> lactic acid bacteria and <strong>the</strong>ir metabolites against Mycobacterium avium subsp. paratuberculosis John Anthony Donaghy, Jane Johnston, Michael Rowe Food Microbiology Branch, Agri-Food & Biosciences Institute, Belfast, N. Ireland, United Kingdom Mycobacterium avium subsp. paratuberculosis (Map) may not be completely inactivated by pasteurisation and o<strong>the</strong>r sub-pasteurisation heat treatments applied during <strong>the</strong> processing <strong>of</strong> milk and manufacture <strong>of</strong> o<strong>the</strong>r dairy products respectively. Consequently, <strong>the</strong> inactivation <strong>of</strong> Map may rely on <strong>the</strong> product’s manufacturing process or intrinsic properties, inter alia <strong>the</strong> presence lactic acid bacteria (LAB) and <strong>the</strong>ir concomitant metabolic products such as lactic acid, hydrogen peroxide, acetic acid and antimicrobial peptides. The objective <strong>of</strong> this study was to develop assays to investigate <strong>the</strong> in vitro inhibition <strong>of</strong> Map by commercial probiotic LAB, cheese LAB and <strong>the</strong>ir metabolites. LAB supernatants, cultured in MRS broth and milk was screened for bacteriostatic activity by supplementation into BACTEC 12B medium inoculated with Map cells (approx. 10 4-5 cfu/ml). The effect on Map growth was monitored throughout a 12 wk incubation period. An agar plate screening assay was developed to monitor <strong>the</strong> effect <strong>of</strong> LAB and <strong>the</strong>ir metabolic products on Map growth. Two commercially available probiotic strains elicited a substantial inhibitory effect (50-60 day growth delay) on Map. Concentrated supernatant from a number <strong>of</strong> Cheddar cheese LAB isolates exhibited bacteriostatic activity towards Map in <strong>the</strong> liquid culture and agar-screening assays. This effect was not demonstrated by D, L, D/L lactic acid (0.1-10%), H 2 O 2 (0.1-1%), nisin or low pH (HCL; < 2.5) conditions. The agar screening assay could also be used for <strong>the</strong> determination <strong>of</strong> minimum inhibitory concentrations <strong>of</strong> antibiotics as part <strong>of</strong> culture method development. The results <strong>of</strong> this study suggest <strong>the</strong> in vitro inhibitory effect <strong>of</strong> some lactobacilli on Map growth may be due to factors o<strong>the</strong>r than lactic/acetic acid production. Irrespective <strong>of</strong> <strong>the</strong> mechanism <strong>of</strong> inhibition, <strong>the</strong> possibility exists for <strong>the</strong> inclusion <strong>of</strong> such strains in dairy products for <strong>the</strong> in situ inhibition <strong>of</strong> Map or fur<strong>the</strong>rmore <strong>the</strong>ir use as bio<strong>the</strong>rapeutic agents against Map. 29
#107 Multilocus Short Sequence Repeat Analysis <strong>of</strong> Mycobacterium avium subsp. paratuberculosis Isolates from Dairy Herds in Nor<strong>the</strong>astern United States <strong>of</strong> a Longitudinal Study Indicates Low Shedders are Truly Infected Pradhan AK, 1* Kramer AJ, 2 Mitchell RM, 1 Whitlock RH, 3 Smith JM, 4 Hovingh E, 5 Van Kessel JS, 6 Karns JS, 6 and Schukken YH 1 1Quality Milk Production Services, Department <strong>of</strong> Population Medicine and Diagnostic Sciences, College <strong>of</strong> Veterinary Medicine, Cornell University, Ithaca, NY, USA; 2 Faculty <strong>of</strong> Veterinary Medicine, Utrecht University, The Ne<strong>the</strong>rlands; 3 New Bolton Center, Department <strong>of</strong> Clinical Studies, School <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Pennsylvania, Kennett Square, PA, USA; 4 Department <strong>of</strong> Animal Science, University <strong>of</strong> Vermont, Burlington, VT, USA; 5 Department <strong>of</strong> Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA, USA; 6 Environmental Microbial and Food Safety Laboratory, ANRI, USDA-ARS, Beltsville, MD, USA ABSTRACT The objectives <strong>of</strong> this study were to evaluate whe<strong>the</strong>r low shedders <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) were pass-through animals or whe<strong>the</strong>r <strong>the</strong>y were truly infected. We also evaluated whe<strong>the</strong>r <strong>the</strong>se animals were possibly infected by <strong>the</strong> super-shedders. The MAP isolates were obtained from a longitudinal study that involved three different herds in <strong>the</strong> nor<strong>the</strong>astern US. The shedding levels <strong>of</strong> animals at each culture-positive occasion were determined. 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 and from super-shedders. Using a multilocus short sequence repeat (MLSSR) approach we found 15 different strains from a total <strong>of</strong> 142 isolates analyzed. The results indicated herd-specific infections; a clonal infection in herd C with 89% <strong>of</strong> animals sharing <strong>the</strong> same strain, different strains in herds A and B. In herd C, 100% and in herd A, 17 to 70% <strong>of</strong> cows shed <strong>the</strong> same strain as that <strong>of</strong> contemporary supershedders at a given collection date. About 82% <strong>of</strong> available tissue samples were culture-positive indicating a true infection. Taken toge<strong>the</strong>r <strong>the</strong> results <strong>of</strong> MAP strain-typing and shedding levels, we conclude that at least 50% <strong>of</strong> low shedders have same strain as that <strong>of</strong> a contemporary super-shedder. The results <strong>of</strong> this study indicate that very few cows had characteristics <strong>of</strong> a possible pass-through animal; many more cows were actively infected. The sharing <strong>of</strong> same strain <strong>of</strong> low shedders with <strong>the</strong> contemporary super-shedders suggests that low shedders may be infected as adults by <strong>the</strong> super-shedders. Key words: Mycobacterium avium subsp. paratuberculosis, MLSSR analysis, longitudinal study, low shedders, truly infected INTRODUCTION Mycobacterium avium subsp. paratuberculosis is <strong>the</strong> causative agent <strong>of</strong> paratuberculosis (or Johne’s disease), a debilitating chronic gastroenteritis in ruminants (Möbius et al., 2008) and is characterized by a very long incubation period (up to several years). Shedding <strong>of</strong> MAP can occur without showing clinical signs. Recently, using fur<strong>the</strong>r serial dilutions to determining <strong>the</strong> range <strong>of</strong> shedding in animals, super-shedders were identified and defined as cattle shedding more than 10,000 MAP/g <strong>of</strong> manure (Whitlock et al., 2005). It has been indicated that supershedders represent <strong>the</strong> greatest risk to spread <strong>of</strong> Johne’s disease (Whitlock et al., 2005). Although several molecular subtyping techniques have been previously applied to investigate genetic variation in MAP, MLSSR approach has recently gained much attention and been applied in different studies (Amonsin et al., 2004; Harris et al., 2006). While only a limited number <strong>of</strong> cross-sectional studies have used this method with a restricted set <strong>of</strong> isolates, it has 30
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: To better determine the</st
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 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
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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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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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