Proceedings of the 10th International Colloquium on Paratuberculosis

could be partly attributed to a small number <strong>of</strong> animals per herd, i.e. <strong>the</strong> family-farm breeding
strategy. In 2008, <strong>the</strong> tested herds comprised on average 5.67 animals (aged >2 years) in
contrast to <strong>the</strong> period 2000-2001 when <strong>the</strong> average was 31 animals (aged >2 years) per
herd. The relatively good present situation could change markedly in <strong>the</strong> near future due to
unlimited trade <strong>of</strong> animals in <strong>the</strong> European Union. Moreover, in-country animal trade
originating from big dairy-cattle herds with Black and white (Holstein-Friesian) breed, which is
most commonly affected, can also contribute to <strong>the</strong> spread <strong>of</strong> paratuberculosis.
Differences in <strong>the</strong> prevalence on both <strong>the</strong> animal and <strong>the</strong> herd level, observed over <strong>the</strong>
years, reflect also <strong>the</strong> different tested populations, <strong>the</strong> number <strong>of</strong> animals and herds included
in <strong>the</strong> test and <strong>the</strong> use <strong>of</strong> ELISA kits with different sensitivities.
In general, our findings contribute to <strong>the</strong> current knowledge on paratuberculosis
prevalence in <strong>the</strong> European countries. Because <strong>of</strong> <strong>the</strong> increasing trade and changes in
animal breeding strategy (decreasing numbers <strong>of</strong> herds and increasing number <strong>of</strong> animals
per herd), <strong>the</strong> decision and policy makers should prepare efficient measures for surveillance
and control <strong>of</strong> <strong>the</strong> infection.
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Cocito C, Gilot P, Coene M, De Kesel M, Poupart P, Vannuffel P, 1994. Paratuberculosis.
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Nielsen SS, T<strong>of</strong>t N, 2009. A review <strong>of</strong> prevalences <strong>of</strong> paratuberculosis in farmed animals in
Europe. Prev. Vet. Med, 88, 1-14.
Ocepek M, Posedi J, Pislak M, 1999. Prevalence <strong>of</strong> bovine paratuberculosis in Slovenia in
1997 and 1998. Zb. Vet. Fak, 36, 111-119.
Ocepek M, Krt B, Pate M, Pogačnik M, 2002. Seroprevalence <strong>of</strong> paratuberculosis in Slovenia between
1999 and 2001. Slo. Vet. Res, 39, 179-185.
#137 Comparative evaluation <strong>of</strong> <strong>the</strong> specificity and % recovery capabilities <strong>of</strong> different
magnetic capture approaches for Mycobacterium avium subsp. paratuberculosis
Antonio Foddai, Chris T Elliott, Irene R Grant
Queen’s University Belfast, United Kingdom
The objective <strong>of</strong> this study was to compare <strong>the</strong> performance <strong>of</strong> two types <strong>of</strong> commercially-available anti-MAP
magnetic beads - Pathatrix-PM50 (polyclonal antibody coated beads, Matrix MicroScience, UK) and AnDiaTec
ParaTub-S ® (monoclonal antibody coated beads, AnDiaTec GmbH, Germany) - and three types <strong>of</strong> in-house
coated magnetic beads - M280-Dynabeads coated with polyclonal sheep anti-rabbit IgG (original IMS approach)
and Pierce paramagnetic beads covalently linked to MAP-specific peptides aMp3 and aMptD (Stratmann
et al. 2002, 2004) – in order to identify <strong>the</strong> best magnetic capture approach for isolation <strong>of</strong> MAP. MAP
broth samples (10 4 CFU/ml) were processed as follows: addition <strong>of</strong> 10 µl beads, 30 min capture at room temperature,
magnetic separation for 10 min, three washes in PBS-0.05% Tween 20, and final resuspension in 1
ml 7H9/OADC broth before culture on Herrold’s egg yolk medium containing 2 µg/ml mycobactin J (HEYM).
Percentage recovery was calculated on <strong>the</strong> basis <strong>of</strong> before and after counts (CFU/ml). Specificity for MAP
was assessed by performing magnetic separation on broths <strong>of</strong> five o<strong>the</strong>r Mycobacterium spp. Pathatrix-PM50
beads and Pierce magnetic beads coated with ei<strong>the</strong>r aMp3 or aMptD showed <strong>the</strong> highest % recoveries <strong>of</strong> MAP
(close to 100%). However, >10% non-specific binding was recorded in all cases. The inclusion <strong>of</strong> a blocking
step before magnetic separation reduced non-specific binding to ≤1%, although this also resulted in a slight
reduction in MAP recovery. In-house polyclonal antibody-coated Dynabeads and AnDiaTec ParaTub-S beads
achieved much lower % recovery <strong>of</strong> MAP (≤10%). Results indicate that not all magnetic capture approaches for
<strong>the</strong> isolation <strong>of</strong> MAP perform equally well. We observed that bead characteristics (composition, size, surface
area) as well as <strong>the</strong> nature <strong>of</strong> coating antigen (polyclonal or monoclonal antibody, or peptide) impact specificity
<strong>of</strong> capture and % recovery.
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