75 16 79% 7 12% 52 21 28 3 0 16 16 2 11 5 0 2 96% 2 0 0 0 0 1 2 1 24 14 7 1 2 92 40 57% 9 17% 43 22 20 1 0 23 1 2 16 1 0 0 100% 0 0 1 0 2 1 5 4 0 7 23 0 0 SSH 1967 v 11168 ST-42 cattle AluI/DraI 98 24 76% 12 16% 62 14 37 9 2 8 39 1 12 1 0 1 98% 2 0 6 6 4 4 7 18 1 0 12 3 1 ST-45 rabbit RsaI 98 53 46% 28 53% 17 0 4 4 9 4 4 1 5 1 2 0 88% 0 1 3 1 1 0 3 4 0 0 2 3 0 http://www.biomedcentral.com/1471-2164/8/110 670 v 11168 504 v 11168 629 v 2 drivers 961 v 2 drivers ST-21 duck AluI/DraI ST-21 cattle AluI/DraI ST-61 rabbit RsaI 145 60 59% 64 75% 21 1 5 2 13 4 0 0 17 0 0 0 100% 1 2 2 6 4 0 3 0 0 2 1 3 0 BMC Genomics 2007, 8:110 Table 1: Summary of SSH experiments. Tester MLST group Tester source SSH restriction enzymes clones sequenced unsubtracted %subtracted repeats %repeats subtracted (SSH) sequences SSH sequence length range <200 bp 200–400 bp 400–600 bp >600 bp Best BLASTX match by strain RM1221 (chicken) 81–176 (human) 11168 (human) other C. jejuni other Campylobacter sp. other genera no significant match % Campylobacter Campylobacter 50–80% ID Campylobacter <50% ID Best BLASTX by function capsule LOS flagella/motility restriction-modification membrane/transport metabolism/biosynthesis plasmid bacteriophage hypothetical or unknown others no significant match The number of isolates fitting into each category is presented for each of the five subtractions. ID; protein sequence identity Of the three subtractions conducted using AluI/DraI- digested DNAs and a single driver strain (NCTC11168), two were intra-clonal complex and one was between clonal complexes. The numbers of subtracted sequences obtained did not vary significantly between these subtrac- tions (Table 1). However, there were some variations in the putative functions of subtracted sequences. Plasmid and bacteriophage-associated sequences accounted for 73% of those sequences differing between the two ST-21 complex strains 670 and NCTC11168, and 16% of those sequences differing between the two ST-21 complex strains 504 and NCTC11168, but only 2% of those sequences differing between the ST-42 complex strain 1967 and strain NCTC11168. The largest group of sub- tracted sequences for the inter-clonal complex subtraction were those associated with metabolism/biosynthesis (29%). The SSH data suggest that there are both intra- and inter-clonal complex variations in genes associated with LOS, capsule, flagella/motility, membrane/transport and metabolism (Table 1). It has been demonstrated that SSH is an effective method for analysing genetic differences between related strains. In a previous study using SSH, Agron et al. [22] were able to detect most of the 7% of genomic differences between two closely related, fully sequenced strains of Helicobacter pylori. Unlike in our study, the authors used four parallel subtractions with different restriction enzymes. However, they further demonstrated that as tester-specific sequences became limiting the proportion of repeat fragments Page 3 of 11 (page number not for citation purposes)