Study of Restriction Fragment Length Polymorphism Analysis and Spoligotyping for Epidemiological Investigation of Mycobacterium bovis Infection

Abstract

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.

Snapshot of Mycobacterium bovis and Mycobacterium caprae Infections in Livestock in an Area with a Low Incidence of Bovine Tuberculosis

“…A low incidence of bTB has been observed until now in Portugal, where a comprehensive eradication scheme has been implemented. Epidemiological surveys carried out in several countries suggest that test-and-slaughter policies may reduce strain diversity, favoring clonal expansion as a result of a bovine population bottleneck (2,6). A remarkable exception to this observation was the first epidemiological analysis carried out in Portugal (3), which highlighted the apparent high genotype diversity of Mycobacterium bovis, possibly due to the low prevalence rates and the absence of major epidemics favoring ongoing transmission of the same strain(s).…”

Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements

“…A proportion of M. tuberculosis strains with marked differences in their IS 6110 RFLP patterns exhibit identical spoligo patterns [108, 154]. Also the discriminatory power of spoligotyping to distinguish between M. bovis isolates is less than that of PGRS‐ or DR‐based RFLP typing [118, 119, 123, 156–159]. Nevertheless, spoligotyping can be used for prescreening.…”

“…IS 6110 RFLP typing is not the most suitable method for investigating the transmission of M. bovis [46, 118, 119, 124, 158, 159], although this method serves to divide M. bovis isolates into broad groups [46, 119, 159, 170]. Most isolates from cows and, hence, a significant proportion of M. bovis strains isolated from humans, contain only one copy of the IS 6110 element at a fixed genomic position [39, 46, 119, 158, 159, 171, 172]. M. bovis isolates from other domesticated and wild animals frequently contain a higher number of IS 6110 elements [46, 119, 124].…”

“…PGRS‐RFLP typing appears to have a higher resolution amongst M. bovis isolates [46, 99, 117, 119, 156, 157, 159]. Recently, other DNA fingerprinting methods have been introduced for typing of M. bovis , such as spoligotyping and variable numbers of tandem repeats (VNTR) typing [118, 123, 125, 140, 156–159]. VNTR typing, a method that exploits the polymorphism of six exact tandem repeat loci and the major polymorphic tandem repeat, is particularly promising [18, 140].…”

Scientific review on Tuberculosis in wildlife in the EU

“…In Ireland, direct repeat restriction fragment length polymorphism (DR-RFLP) analysis has shown isolates of the same strain in feral deer that are common in cattle (Skuce et al, 1996). Spoligotyping and RFLP analysis has been used to show spatial associations between common strain types in cattle, badgers and deer in Ireland, suggesting that transmission is occurring between these species (Costello et al, 1999). Restriction endonuclease analysis (REA) was used in an epidemiological investigation of an outbreak in farmed fallow deer in Sweden and demonstrated transmission within deer populations (Bölske et al, 1995)).…”