Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.The development of molecular techniques for differentiation of Mycobacterium bovis isolates has been of considerable benefit in epidemiological studies. Typing methods that have been commonly used include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (7,19).RFLP analysis of M. bovis isolates has commonly utilized polymorphism of the insertion sequence IS6110 and repetitive DNA elements such as the polymorphic GC-rich sequence (PGRS) and the direct-repeat (DR) region. Analysis of polymorphism of IS6110, the PGRS, and the DR region in combination has provided a high level of discrimination between strains (7, 19). REA has been widely used in New Zealand and has also given excellent resolution of strains (4). However, both RFLP analysis and REA require relatively large quantities of DNA and are laborious and time-consuming procedures. Complex banding patterns make analysis and interlaboratory comparisons difficult. Spoligotyping is a PCR-based typing method that reveals the presence or absence of unique spacer sequences located between the direct-repeat sequences of the DR region (12). It is a relatively easy procedure to perform, and the results can be expressed in a digital format. However, spoligotyping does not differentiate M. bovis strains t...

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Background In Ireland, meat by-products (MBP) harvested at knackeries from farmed animals that have not died of an infectious or systemic disease are legally permitted to be fed to dogs in kennels and packs of hounds. There is limited information available on the risks of spreading foodborne bacteria or antimicrobial resistant (AMR) determinants to dogs, their handlers or the associated environment. The aim of this study was to investigate the distribution of Salmonella serovars, Listeria monocytogenes, Campylobacter species, enterococci, their associated AMR determinants and the level of Escherichia coli in samples of MBP from knackeries and associated equipment and kennels. For this purpose, 313 fresh and 208 frozen MBP samples from 22 knackeries, 16 swabs of mincing equipment from two of the knackeries and 138 swabs from kennels adjacent to seven of the knackeries were collected and processed over a 12-month period. Results From the 521 MBP samples analysed, a total of 77 Salmonella (14.8%), 101 L. monocytogenes (19.4%), 12 Campylobacter (2.3%), 271 Enterococcus faecalis (52.0%) and 127 Enterococcus faecium (24.4%) strains were recovered. The 154 analysed environmental samples from kennels and mincing equipment yielded 194 isolates (3 Salmonella, 85 E. coli, 76 E. faecalis and 30 E. faecium.). E. coli was quantifiable in 423 of the 521 MBP samples with log counts per gram ranging between 1 and 6. AMR characterisation of 168 E. coli, enterococci and Salmonella isolates from MBP and environmental samples showed high levels of AMR including multi-drug resistance (MDR) with 63.6%, 9.1%, 29% and 45.8% of E. coli, Salmonella, E. faecalis and E. faecium isolates, respectively showing resistance to three or more antimicrobials (MDR) Conclusions The findings of this survey confirm that MBP from fallen animals contain high levels of zoonotic and AMR-harbouring bacteria that pose a risk of transmission to dogs, their handlers, and the environment.