Proceedings of the 10th International Colloquium on Paratuberculosis

MATERIALS AND METHODS
Sampling
For <strong>the</strong> UK samples 54 BTM samples were provided from <strong>the</strong> VLA in Sutton Bonington, UK. For
<strong>the</strong> Cyprus survey samples were collected from from each <strong>of</strong> 225 registered dairy farms in
Cyprus.
Combined phage PCR assay
BMT samples were processed by a modified version <strong>of</strong> <strong>the</strong> method described by Stanley et al.
(2007). In <strong>the</strong> assay bacteria in <strong>the</strong> milk samples are collected by centrifugation and <strong>the</strong> cell
pellets resuspended in 2 ml <strong>of</strong> Media Plus. Here a second wash step was included using <strong>the</strong>
same centrifugation conditions and <strong>the</strong>n finally <strong>the</strong> pellet was resuspended in 1 ml Media Plus.
Culture examination and identification <strong>of</strong> mycobacterial isolates
Samples (30 ml) <strong>of</strong> BTM were decontaminated for 5 h with 0.75% hexadecyl-pyridinium chloride
(HPC; Merck KGaA, Germany) and three samples were cultured on Herrold’s Egg Yolk Media
(HEYM) containing 2 �g ml -1 <strong>of</strong> Mycobactin J; two slopes were purchased from Becton Dickinson
(New Jersey, United States) and one slope was prepared as described previously (Ayele et al.,
2005). Slopes were monitored after <strong>the</strong> first week <strong>of</strong> incubation to identify ei<strong>the</strong>r contaminated
cultures or those with fast-growing mycobacteria and <strong>the</strong>n observed at two week intervals until
<strong>the</strong>re was visible colony growth: incubation was carried out for not less than eight months at
37°C. From all primary cultures, presumptive colonies were stained using Ziehl-Neelsen (Z-N)
for <strong>the</strong> presence <strong>of</strong> acid-fast bacilli (AFB). AFB isolates were identified as MAP by PCR as
described previously (IS900, Whittington et al., 1998 and F57, Coetsier et al., 2000). IS1311
REA-PCR was used to distinguishing between cattle and sheep strains <strong>of</strong> MAP with PCR
products being cleaved with HinfI (Marsh et al. 1999).
Total viable count (TVC)
TVC was performed on Milk Count Agar (MCA) using standard methods. Milk was diluted in
MRD (Maximal Recovery Dilluent) and <strong>the</strong> dilutions were plated on MCA and incubated
aerobically at 30 °C for 3 days.
RESULTS AND DISCUSSION
The results from <strong>the</strong> two surveys are presented in Table 1. From a total <strong>of</strong> 54 samples analyzed
in <strong>the</strong> UK survey 19 (35.2%) were positive using <strong>the</strong> phage assay which indicates <strong>the</strong> presence
<strong>of</strong> Mycobacteria spp, but one <strong>of</strong> <strong>the</strong>se samples (1.9%) was identified as MAP following PCR
genotyping. A duplicate <strong>of</strong> <strong>the</strong> samples collected was cultured by <strong>the</strong> VLA and none <strong>of</strong> <strong>the</strong>
samples were positive for MAP. A recent UK survey reported that 75% <strong>of</strong> dairy herds were seropositive
but seroprevalance was only 0.12 and <strong>the</strong>refore levels <strong>of</strong> MAP expected in BTM would
be low (Woodbine et al., 2009). From a total <strong>of</strong> 225 samples analysed for <strong>the</strong> Cyprus survey
218 (96.9%) were Mycobacteria-positive using <strong>the</strong> phage assay and 50 <strong>of</strong> <strong>the</strong>se (22.2%; 95% CI:
17.1% - 28.0%) were identified as MAP by IS900 PCR. In this case only two <strong>of</strong> <strong>the</strong> samples
(0.9%) were culture positive. From <strong>the</strong> 225 cows’ BMT samples that were collected during this
survey 50 (22.2%; 95% CI: 17.1% - 28.0%) were found to be MAP positive using <strong>the</strong> combined
phage-PCR assay. The results <strong>of</strong> Slana et al. (2009) reported that 28.6% (95% CI: 95% CI:
22.5% – 34.3%) were MAP positive by IS900 qPCR, showing a good agreement with <strong>the</strong> level <strong>of</strong>
prevalence recorded in <strong>the</strong> two different surveys undertaken.
All BTM samples collected in <strong>the</strong> Cyprus were also cultured after decontamination and only two
<strong>of</strong> <strong>the</strong>m (0.9%) produced colonies on HEYM slopes that were identified as MAP-positive by both
IS900 and F57 qPCR. Both <strong>of</strong> <strong>the</strong> samples from which viable MAP were cultured were also
phage-PCR positive. The two confirmed MAP isolates were analysed using IS1311 REA PCR
and one <strong>of</strong> <strong>the</strong>se was identified as a MAP cattle strain and <strong>the</strong> o<strong>the</strong>r as a sheep strain.
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