Proceedings of the 10th International Colloquium on Paratuberculosis

#94 A simple liquid culture method for <strong>the</strong> assessment <strong>of</strong> paratuberculosis status in dairy
cattle herds
Elisabetta Stefani, Caterina Dal Ben, Jacek Gwozdz, Anna Valente, Vanessa Benini, Paola Fiorini, Nicola Pozzato
Istituto Zoopr<strong>of</strong>ilattico Sperimentale delle Venezie - Laboratorio di biologia molecolare, Verona, Italy
Automated liquid systems for culture <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) are more sensitive
and rapid than culture on solid media, but <strong>the</strong>y are expensive and require specialised equipment. In this
study an inexpensive, non-automated culture method using <strong>the</strong> liquid 7H9 medium was compared with <strong>the</strong>
conventional method on Herrold’s medium for Map detection in dairy cattle manure. Infected herds A and B
and negative herds C and D were recruited. The seroprevalence <strong>of</strong> paratuberculosis in herds A and B was
10% and 1%, respectively. A mix <strong>of</strong> manure was collected from <strong>the</strong> floor <strong>of</strong> <strong>the</strong> lactating-cow feeding area on
3 occasions at weekly intervals. Three replicates <strong>of</strong> each sample were processed for a direct realtime-PCR
test, conventional Herrold’s culture, and non-automated liquid culture. In <strong>the</strong> latter method, each replicate
sample was inoculated into 2 tubes <strong>of</strong> 7H9 medium that were subsequently tested for growth <strong>of</strong> Map using <strong>the</strong>
realtime-PCR at weekly intervals over 8 weeks. Map was detected in each sample from herd A by each method
applied. Positive samples from herd B varied according to <strong>the</strong> method used: 8/9 with Herrold’s, 5/9 with direct
realtime-PCR and 18/18 with realtime-PCR on liquid cultures. Realtime-PCR confirmed <strong>the</strong> presence <strong>of</strong> Map at
<strong>the</strong> 2nd week on 7H9 in 14/18 tubes for herd A and at <strong>the</strong> 5th week or before in 16/18 tubes for herd B. Herds
C and D gave negative results in all replicates for all <strong>the</strong> methods applied. Our results demonstrated that nonautomated
liquid culture on herd manure increases sensitivity and reduce <strong>the</strong> time with respect to traditional
culture. A protocol based on direct realtime-PCR followed by liquid culture on negative samples, confirmed at
5 weeks and 8 weeks by realtime-PCR, can be used for a semiquantitative assessment <strong>of</strong> paratuberculosis
status in dairy cattle herds.
#99 Rapid assessment <strong>of</strong> <strong>the</strong> impact <strong>of</strong> heat treatments on viability <strong>of</strong> Mycobacterium avium
subsp. paratuberculosis using an optimized phage assay
Antonio Foddai, Chris T Elliott, Irene R Grant, Queen’s University Belfast, United Kingdom
Thermal inactivation studies were carried out to demonstrate <strong>the</strong> utility <strong>of</strong> a recently optimized phage amplification
assay to enumerate viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk. UHT milk
was spiked with 10 6 -10 7 CFU MAP/ml and dispensed in 100 µl aliquots into thin-walled 200µl PCR tubes. A
Primus 96 advanced <strong>the</strong>rmal cycler (Peqlab, Erlangen, Germany) was used to achieve <strong>the</strong> following time/
temperature treatments: a) 63°C for 3, 6 and 9 min; b) 68°C for 20, 40 and 60 s; and c) 72°C for 5, 10, 15 and
25 s. Milk samples (non-heated (time 0) and heat-treated) were centrifuged (16,000 x g for 15 min) to remove
milk components inhibitory to <strong>the</strong> phage assay, and <strong>the</strong> pellet resuspended in 1 ml 7H9 broth plus 10% OADC
and 2 mM CaCl 2 . After overnight incubation <strong>of</strong> <strong>the</strong> samples at 37 o C, appropriate dilutions <strong>of</strong> each sample were
processed through an optimized phage assay (Foddai et al. (2009) AEM). Plaques were counted (PFU/ml)
<strong>the</strong> next day and <strong>the</strong>rmal D values calculated for MAP in milk at 63, 68 and 72 o C; D values <strong>of</strong> 63.7 s, 10.4 s
and 2.9 s, respectively, were obtained enabling calculation <strong>of</strong> a z value for MAP <strong>of</strong> 6.8°C. The plaque counts
obtained using <strong>the</strong> optimized phage assay were similar to colony counts on HEYM, carried out in parallel. In
contrast, phage assay results were available within 48 h ra<strong>the</strong>r than after a minimum <strong>of</strong> 6-8 weeks for conventional
culture on HEYM. The D and z values obtained in this study are similar to previously published D and z
values for MAP obtained using HEYM culture, so we have clearly demonstrated that <strong>the</strong> optimized phage assay
enables rapid and accurate assessment <strong>of</strong> <strong>the</strong> impact <strong>of</strong> heat treatments on MAP viability.
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