Genetic material (DNA) is found in all living organisms, including bacteria. Each M. bovis bacterium contains unique DNA which carries the genetic instructions for its development, function, growth and reproduction. Whole genome sequencing (WGS) is a molecular technique used to characterise the entire DNA content of an organism.
Genotyping was the method of genetic typing previously used by APHA to characterise M. bovis isolates. It involved a combination of two techniques; spoligotyping and variable number tandem repeat (VNTR) typing. The techniques targeted specific regions of the bacterium’s DNA sequence in order to characterise it and assign a genotype. Spoligotyping is a rapid, PCR-based method that checks for the presence or absence of 43 unique short DNA sequences (or spacers) in a particular region of the genome of M. bovis (and related bacteria) containing repetitive sequences. Each pattern found has a spoligotype number associated to it such as 9, 17, 35, etc. VNTR typing is based on the number of repetitions of short DNA sequences found in six specific regions of the bacterial genome. Each unique spoligotype and VNTR pattern combination is defined by a number followed by a letter, such as 9:a, 9:b,17:b, 25:a, 25:b, 35:a, 10:u etc., where the letter denotes the frequency in decreasing order of that particular VNTR pattern within a spoligotype in GB (9:a being more common than 9:b, etc.). WGS is a relatively new technique used by APHA which has now replaced genotyping of M. bovis isolates. Instead of targeting specific regions of the bacterium’s genome, WGS involves the analysis of its entire DNA sequence. This allows a greater degree of differentiation (‘granularity’) between M. bovis isolates. The technique involves breaking the bacterial genome into smaller fragments which are sequenced individually and then put back together in the correct order.
Yes. APHA implemented routine use of WGS for typing of M. bovis isolates on 19 April 2021. WGS has replaced genotyping which was the previous technique used.
The main benefit of WGS is that it provides greater and more accurate differentiation between M. bovis isolates when compared to genotyping. This allows assessment of genetic relatedness between isolates and provides additional information on evolutionary relationships. In practice, APHA can use this information to better assess transmission pathways of M. bovis to understand how and where it has spread.
APHA uses WGS to characterise isolates of M. bovis cultured from cattle slaughtered for TB control. This provides information about the genetic relatedness of M. bovis strains, where they have come from and how they have evolved. WGS is an important tool used by APHA for investigating TB breakdowns and possible transmission pathways between cattle herds. WGS supports study of the spread of TB in the local and national cattle population and the factors that affect it over time. At farm level, WGS helps APHA field vets to identify the most likely source of TB infection for a breakdown herd, and also whether it has spread to other cattle herds. Once APHA is aware of the likely origin of a breakdown, they can advise farmers on measures they could take to reduce the risk of further infection entering the herd.
Instead of genotypes, WGS clade labels are used to denote related strains of M. bovis, for example “B6-11”. As WGS provides greater differentiation between strains of M. bovis, some genotypes can be split further into multiple WGS clades e.g. genotype 9:d is split into eight distinct WGS clades. This means that 9:d isolates can be quite different across their genomes. Conversely, a few genotypes are assigned to the same WGS clade. This means that isolates with these genotypes are quite similar across their genomes and therefore more closely related than suggested by their genotype. The majority of genotypes are assigned to a single WGS clade however 1:1 correlation between the two schemes is not always possible. A conversion table is available to APHA staff to allow comparison of WGS clades with genotypes.
Yes. Records of historical genotypes of M. bovis isolates are still available to APHA staff involved in the control of bTB and its epidemiology.
Like all genetic typing methods, you need to isolate the bacterium in the first place to be able to carry out WGS. So, like genotyping, WGS can only be carried out once the TB bacterium has been isolated by microbiological culture. It takes a minimum of six weeks to culture M. bovis, and then WGS is carried out on the isolate. For a small proportion of isolates, the quality of the sample does not allow WGS to be carried out. WGS has only been used by APHA since June 2017, and not many historic M. bovis isolates have been sequenced. This means that the current WGS database which new isolates are compared against is limited, however this will improve over time now that WGS is being used routinely. Although WGS can provide more information about genetic relatedness of isolates than genotyping, there are limits to the inferences that can be made when interpreting WGS data. For example it’s often not possible to determine the direction of transmission of M. bovis between different species.
The number of reactor animals sampled for culture and WGS depends on the status of the breakdown (whether the herd’s officially TB free status is suspended or withdrawn), the number of reactors, and previous post-mortem examination and culture results if applicable.
Yes. APHA routinely uses WGS on M. bovis isolates from non-bovine animals such as South American camelids (alpacas, llamas), goats, pigs, and captive deer when investigating TB incidents affecting these species.
Yes. WGS is also used to analyse M. bovis isolates from wildlife species (e.g. road killed badgers) and compare them with isolates from local TB breakdowns in cattle herds and other farmed animals. This allows APHA to assess whether cattle and badgers from the same geographical area are affected by the same strain of the TB bacterium, and to assess the spread of TB strains between geographical areas and across time. For instance, WGS is being used to support TB surveillance in cattle and badgers and understand the epidemiology of bTB in the confirmed hotspot area in east Cumbria (HS21), where a link between cattle and badger TB infection was first identified in 2017. Visit GOV.UK to find out more about TB surveillance in wildlife.