As part of the European
Commission 6th Framework Programme of research into
paratuberculosis (ParaTBTools) we designed and validated a combined
60mer oligonucleotide microarray for both the MAP and
Mycobacterium avium subspecies avium strain 104 (MAA)
genomes. Using the Inkjet in situ synthesised (IJISS) array system
(Oxford Gene Technologies) and ortholog matching bioinformatics
software we designed 15,000 candidate 60mer reporter probes and
15,000 SNP-containing mismatched pairs spanning both genomes. In
cycles of optimisation against reference genomic DNA, we used these
to prepare a finalised set of oligos having greatest specificity
for their corresponding genes and minimising differences in signal
strength between genes. The resulting array designated MAPAC
comprised 4132 oligos reporting genes shared between MAP and MAA,
218 MAP specific genes, 952 MAA specific genes, 18 genes found only
in MAP sheep strains, 19 MAP MIRU sequence regions and 58 MAP
intergeneic regions, together with 7 genes carried on the MAA
plasmid pVT2. Initially we have used the array to explore genomic
differences between MAP vaccine strains of differing origins and
pedigree's. Weybridge vaccine strains including 316F obtained
directly from the Veterinary Laboratories Agency (VLA), UK and
indirectly from other European centres revealed differences between
strains. Vaccine strain II from VLA was found to have a previously
unreported large contiguous sequence deletion of 32,826bp encoding
33 ORFs. A strain of 316F which had originated from VLA in the
1970's and had subsequently been passed for use in
vaccination between European centres was found to have a contiguous
deletion of 26,830bp encoding 22 ORFs. By contrast, a strain
designated 316F recently obtained from VLA and a human
Crohn's Disease isolate of MAP, lacked this deletion and
appeared to be identical with MAP K10 in agreement with a previous
report (Semret et al. 2006. J Clin Microbiol 44; 881-887). The
deletions found in both MAP strain II and 316F affected regions of
the genome predicted to be involved in pathogenicity. These
findings may have implications for the safety of vaccine strains
and emphasise the importance of comparative genomics in the
definition and monitoring of MAP vaccines. Other MAP vaccine
strains are currently being investigated.
