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Proceedings of the 10th International Colloquium on Paratuberculosis

Proceedings of the 10th International Colloquium on Paratuberculosis

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MATERIALS AND METHODS<br />

Sampling<br />

For <str<strong>on</strong>g>the</str<strong>on</strong>g> UK samples 54 BTM samples were provided from <str<strong>on</strong>g>the</str<strong>on</strong>g> VLA in Sutt<strong>on</strong> B<strong>on</strong>ingt<strong>on</strong>, UK. For<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g> Cyprus survey samples were collected from from each <str<strong>on</strong>g>of</str<strong>on</strong>g> 225 registered dairy farms in<br />

Cyprus.<br />

Combined phage PCR assay<br />

BMT samples were processed by a modified versi<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> method described by Stanley et al.<br />

(2007). In <str<strong>on</strong>g>the</str<strong>on</strong>g> assay bacteria in <str<strong>on</strong>g>the</str<strong>on</strong>g> milk samples are collected by centrifugati<strong>on</strong> and <str<strong>on</strong>g>the</str<strong>on</strong>g> cell<br />

pellets resuspended in 2 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> Media Plus. Here a sec<strong>on</strong>d wash step was included using <str<strong>on</strong>g>the</str<strong>on</strong>g><br />

same centrifugati<strong>on</strong> c<strong>on</strong>diti<strong>on</strong>s and <str<strong>on</strong>g>the</str<strong>on</strong>g>n finally <str<strong>on</strong>g>the</str<strong>on</strong>g> pellet was resuspended in 1 ml Media Plus.<br />

Culture examinati<strong>on</strong> and identificati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> mycobacterial isolates<br />

Samples (30 ml) <str<strong>on</strong>g>of</str<strong>on</strong>g> BTM were dec<strong>on</strong>taminated for 5 h with 0.75% hexadecyl-pyridinium chloride<br />

(HPC; Merck KGaA, Germany) and three samples were cultured <strong>on</strong> Herrold’s Egg Yolk Media<br />

(HEYM) c<strong>on</strong>taining 2 �g ml -1 <str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobactin J; two slopes were purchased from Bect<strong>on</strong> Dickins<strong>on</strong><br />

(New Jersey, United States) and <strong>on</strong>e slope was prepared as described previously (Ayele et al.,<br />

2005). Slopes were m<strong>on</strong>itored after <str<strong>on</strong>g>the</str<strong>on</strong>g> first week <str<strong>on</strong>g>of</str<strong>on</strong>g> incubati<strong>on</strong> to identify ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r c<strong>on</strong>taminated<br />

cultures or those with fast-growing mycobacteria and <str<strong>on</strong>g>the</str<strong>on</strong>g>n observed at two week intervals until<br />

<str<strong>on</strong>g>the</str<strong>on</strong>g>re was visible col<strong>on</strong>y growth: incubati<strong>on</strong> was carried out for not less than eight m<strong>on</strong>ths at<br />

37°C. From all primary cultures, presumptive col<strong>on</strong>ies were stained using Ziehl-Neelsen (Z-N)<br />

for <str<strong>on</strong>g>the</str<strong>on</strong>g> presence <str<strong>on</strong>g>of</str<strong>on</strong>g> acid-fast bacilli (AFB). AFB isolates were identified as MAP by PCR as<br />

described previously (IS900, Whittingt<strong>on</strong> et al., 1998 and F57, Coetsier et al., 2000). IS1311<br />

REA-PCR was used to distinguishing between cattle and sheep strains <str<strong>on</strong>g>of</str<strong>on</strong>g> MAP with PCR<br />

products being cleaved with HinfI (Marsh et al. 1999).<br />

Total viable count (TVC)<br />

TVC was performed <strong>on</strong> Milk Count Agar (MCA) using standard methods. Milk was diluted in<br />

MRD (Maximal Recovery Dilluent) and <str<strong>on</strong>g>the</str<strong>on</strong>g> diluti<strong>on</strong>s were plated <strong>on</strong> MCA and incubated<br />

aerobically at 30 °C for 3 days.<br />

RESULTS AND DISCUSSION<br />

The results from <str<strong>on</strong>g>the</str<strong>on</strong>g> two surveys are presented in Table 1. From a total <str<strong>on</strong>g>of</str<strong>on</strong>g> 54 samples analyzed<br />

in <str<strong>on</strong>g>the</str<strong>on</strong>g> UK survey 19 (35.2%) were positive using <str<strong>on</strong>g>the</str<strong>on</strong>g> phage assay which indicates <str<strong>on</strong>g>the</str<strong>on</strong>g> presence<br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> Mycobacteria spp, but <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se samples (1.9%) was identified as MAP following PCR<br />

genotyping. A duplicate <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> samples collected was cultured by <str<strong>on</strong>g>the</str<strong>on</strong>g> VLA and n<strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g><br />

samples were positive for MAP. A recent UK survey reported that 75% <str<strong>on</strong>g>of</str<strong>on</strong>g> dairy herds were seropositive<br />

but seroprevalance was <strong>on</strong>ly 0.12 and <str<strong>on</strong>g>the</str<strong>on</strong>g>refore levels <str<strong>on</strong>g>of</str<strong>on</strong>g> MAP expected in BTM would<br />

be low (Woodbine et al., 2009). From a total <str<strong>on</strong>g>of</str<strong>on</strong>g> 225 samples analysed for <str<strong>on</strong>g>the</str<strong>on</strong>g> Cyprus survey<br />

218 (96.9%) were Mycobacteria-positive using <str<strong>on</strong>g>the</str<strong>on</strong>g> phage assay and 50 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se (22.2%; 95% CI:<br />

17.1% - 28.0%) were identified as MAP by IS900 PCR. In this case <strong>on</strong>ly two <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> samples<br />

(0.9%) were culture positive. From <str<strong>on</strong>g>the</str<strong>on</strong>g> 225 cows’ BMT samples that were collected during this<br />

survey 50 (22.2%; 95% CI: 17.1% - 28.0%) were found to be MAP positive using <str<strong>on</strong>g>the</str<strong>on</strong>g> combined<br />

phage-PCR assay. The results <str<strong>on</strong>g>of</str<strong>on</strong>g> Slana et al. (2009) reported that 28.6% (95% CI: 95% CI:<br />

22.5% – 34.3%) were MAP positive by IS900 qPCR, showing a good agreement with <str<strong>on</strong>g>the</str<strong>on</strong>g> level <str<strong>on</strong>g>of</str<strong>on</strong>g><br />

prevalence recorded in <str<strong>on</strong>g>the</str<strong>on</strong>g> two different surveys undertaken.<br />

All BTM samples collected in <str<strong>on</strong>g>the</str<strong>on</strong>g> Cyprus were also cultured after dec<strong>on</strong>taminati<strong>on</strong> and <strong>on</strong>ly two<br />

<str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>m (0.9%) produced col<strong>on</strong>ies <strong>on</strong> HEYM slopes that were identified as MAP-positive by both<br />

IS900 and F57 qPCR. Both <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> samples from which viable MAP were cultured were also<br />

phage-PCR positive. The two c<strong>on</strong>firmed MAP isolates were analysed using IS1311 REA PCR<br />

and <strong>on</strong>e <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se was identified as a MAP cattle strain and <str<strong>on</strong>g>the</str<strong>on</strong>g> o<str<strong>on</strong>g>the</str<strong>on</strong>g>r as a sheep strain.<br />

144

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