Proceedings of the 10th International Colloquium on Paratuberculosis

#121 Detection <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (MAP) using culture
from blood during early, subclinical and clinical stages <strong>of</strong> disease
Kate Bower, Douglas Begg, Richard Whittington,
Faculty <strong>of</strong> Veterinary Science, University <strong>of</strong> Sydney, Australia
MAP organisms have been found in infected animals at sites that are distant from <strong>the</strong> gut including culture <strong>of</strong>
MAP from milk, liver, mammary tissue, spleen, foetal tissues, reproductive tissue, and extra intestinal lymph
nodes. These studies, in association with detection <strong>of</strong> MAP DNA in <strong>the</strong> blood, indicate that at some stage <strong>of</strong> <strong>the</strong>
disease a bacteraemia may occur.
Demonstration <strong>of</strong> <strong>the</strong> organism in blood from infected sheep and cattle by PCR has led to <strong>the</strong> question
<strong>of</strong> <strong>the</strong> temporal pattern <strong>of</strong> <strong>the</strong> bacteraemia during <strong>the</strong> course <strong>of</strong> paratuberculosis infection. This study aims
to determine when bacteraemia is detectable by culture during <strong>the</strong> time course <strong>of</strong> paratuberculosis infection,
using an optimised method <strong>of</strong> culturing S-strain <strong>of</strong> MAP from blood samples.
One hundred and eleven Merino or Merino-cross lambs aged 3-4 months were used in four experimental
infection trials. The lambs were shown to be free from detectable MAP infection prior to <strong>the</strong> study. Lambs were
inoculated with ei<strong>the</strong>r a pure culture <strong>of</strong> MAP strain Telford 9.2 or a gut homogenate from a clinically infected
sheep.
Blood was collected from all animals prior to inoculation with MAP, every 3-4 months throughout <strong>the</strong> trial
and <strong>the</strong>n prior to necropsy. Additionally, in one trial, blood was sampled monthly during <strong>the</strong> first 6 months. Naturally
infected animals were also studied, with blood collected from animals from heavily infected flocks prior
to necropsy. Infection status <strong>of</strong> all animals was assessed by culture <strong>of</strong> gut and faeces, histology and antibody
ELISA.
Results from both naturally and experimentally infected animals indicate that MAP can be isolated from <strong>the</strong>
blood <strong>of</strong> a low proportion <strong>of</strong> sheep at late stages <strong>of</strong> <strong>the</strong> disease. Though preliminary, <strong>the</strong>se studies provide some
indications <strong>of</strong> <strong>the</strong> nature <strong>of</strong> <strong>the</strong> bacteraemia and identify <strong>the</strong> difficulties associated with culture from blood.
#132 Full genome sequence <strong>of</strong> a Danish isolate <strong>of</strong> Mycobacterium avium subspecies
paratuberculosis, strain Ejlskov2007
Mamuna Afzal, Soad Abidi, Heidi Mikkelsen, Sheila Tuyet Tang, Ole Lund, Morten Nielsen, Claus Lundegaard,
Gregers Jungersen, Dave Ussery
Center for Biological Sequence Analysis, Dept Systems Biology, Technical University <strong>of</strong> Denmark, Denmark;
National Veterinary Institute, Technical University <strong>of</strong> Denmark, Denmark
We have sequenced a Danish isolate <strong>of</strong> Mycobacterium avium subspecies paratuberculosis, strain Ejlskov2007.
The strain was isolated from faecal material <strong>of</strong> a 48 month old second parity Danish Holstein cow, with
clinical symptoms <strong>of</strong> chronic diarrhoea and emaciation. The cultures were grown on Löwenstein-Jensen media
by standard procedures and passed once to new tubes before DNA extraction and being sequenced on a
454 FLX machine. Currently, <strong>the</strong> genome has been assembled into 70 contiguous pieces, for a total <strong>of</strong> around
5.0 Mbp, with a 63% GC content. We have predicted a total <strong>of</strong> 4687 proteins, consisting <strong>of</strong> 4317 unique gene
families. Comparison with M. avium paratuberculosis strain K10 revealed only 3436 genes in common (~70%).
We have used GenomeAtlases to show conserved (and unique) regions along <strong>the</strong> Ejlskov2007 chromosome,
compared to 2 o<strong>the</strong>r Mycobacterium avium sequenced genomes. Pan-genome analyses <strong>of</strong> <strong>the</strong> sequenced
Mycobacterium genomes reveal a surprisingly open and diverse set <strong>of</strong> genes for this bacterial genera.
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