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The Use of Spoligotyping in TB Case Management

01 April 2016, at 12:00am

Post mortem samples are taken from reactors and sent for culture, from every TB breakdown. For every case where TB is confirmed by the culture of mycobacterium bovis, further testing is carried out to establish the m.bovis spoligotype infecting that herd.

Rapid advances in genome sequencing since the 1980s have led to the development of techniques for identification of strains of M. bovis. Using these, over 180 distinct strains have been found in the current bovine TB epidemic in Great Britain. The various strains are differentiated by analysing two areas of repetitive DNA in the genome, the spoligotype and Variable Number of Tandem Repeat (VNTR) loci. The spoligotype loci are part of the natural anti-viral defence system of the bacterium and the VNTR loci have effects on gene expression.  However, the pattern of clinical disease appears to be the same with the different strains, though further research may yet highlight some differences.

In spoligotyping (spacer oligotyping), classification of strains is based on an area of the genome where a 36 base-pair DR (direct repeat) sequence is repeated many times. Between adjacent DRs lie unique and identifiable ‘spacer’ sequences, each of 35-41 base-pairs. Strains are differentiated by the presence or absence of each of 43  different spacers in the DR locus in M bovis.

Each spoligotype is further subtyped on the basis of the specific pattern of Variable Number of Tandem Repeats (VNTR). These VNTR loci are found in a different region of the genome from the spoligotype loci. There are several different VNTR loci, each with up to 79 base-pairs, each repeated a number of times in tandem. The VNTR type (e.g 7-5-5-5*-3-3.1) gives the number of repeats for each locus, an asterisk or decimal point indicate that there is a partial repeat at this locus.   

The VNTR subtype of the spoligotype is shown by a suffix ‘a’ ‘b’ ‘c’ …. etc, ‘a’ being the most common subtype. As an example, spoligotype 9 has many VNTR subtypes, ‘a’ being that most frequently found, and the isolate in this case is identified as genotype ‘9:a’.   Bacteria in the M. tuberculosis complex, which includes M. bovis, are unusual in that there appears to be no exchange of genes between individuals.  This means that strains bearing the same genotype are a set of individuals descended by clonal replication from a single common ancestral cell.

New genotypes may arise on occasion; changes in spoligotype are seen less frequently than changes in VNTR type. 

Geographical Pattern of Strains in the Current Epidemic

APHA Weybridge has a database of genotypes built up from breakdowns since the mid-1970s. When these are mapped it becomes clear that, in general, the sites where any particular genotype is found are clustered closely together. The map above of the geographical distribution of the M. bovis isolates from breakdowns in 2014 shows this clustering in the High Risk Area (HRA) clearly.   

The clustering seen in the HRA is not apparent in the occasional breakdowns seen in the Low Risk Area (LRA) where a range of genotypes are found, often reflecting direct movement from the HRA.

The geographical clustering observed has been formalised in a set of ‘homerange’ maps. In producing these a 5km square is considered to be part of a genotype homerange if there have been three different breakdowns of that genotype in at least two holdings within a 5 year period.  Each 5km square is given a 10 km buffer to generate the homerange for that genotype.

The example below shows the homeranges of the most common spoligotype 9 subtypes (9:a, 9:b, 9:c etc). Each of these occupies a different area of the HRA:  9:a in Devon and Cornwall, 9:b in Carmarthan, 9:c in N Powys and Welsh borders and so on, though some overlap does occur eg 9:a and 9:e in the SW.   

M. bovis isolates from other farmed and domestic species and also from wild mammals typically match those found in the cattle in the same area.

The Use of Genotypes in Field Investigations of Breakdowns in the High Risk Area (HRA)

A critical factor in understanding a particular TB breakdown is whether infection entered the herd through purchase of an already-infected animal or whether it was of 
‘local origin’. The M. bovis strain can often be helpful in the investigation of the most likely origin.
As an example, a farm in SW Shropshire had a single reactor at the breakdown test. This was in 2002 when the area in which the farm is situated was not yet considered endemic for TB and there were few cases locally. However it lay close to areas where genotypes 9:c, 17:a and 35:a were found.   
At post-mortem the heifer had lesions in the broncho-mediastinal lymph nodes from which M. bovis genotype 25:a was cultured.  Further investigation showed that the heifer had been purchased a year before from a farm in Staffordshire where genotype 25:a was the most common strain. Our conclusion in this case was that the breakdown was most likely of purchased origin.
Some years later, in 2007, the same farm had another breakdown and by this time the area had gradually become endemic for TB with many cases in the area, almost all of genotype 9:c. The single reactor in this breakdown was a homebred animal with retropharyngeal lymph node lesions from which M. bovis with genotype 9:c was isolated. In this case the indication was that this was a locally acquired infection, either from local wildlife or from other cattle in the area; something that would be investigated  in more detail. 

The Low Risk Area (LRA)

The Low Risk Area covers large parts of the north and east of England. It has a low and stable incidence of bTB (~0.1% herd prevalence due to non-indigenous infection) and no recognised significant reservoir of the disease in wildlife. 

One of the objectives in the Government’s bovine TB (bTB) strategy for England, published in April 2014, is to progressively achieve Officially TB-Free (OTF) status for counties in the LRA in the North by 2019, with a target of all the counties in the current LRA becoming OTF by 2025.

This is possible because the vast majority of TB breakdowns in the LRA can be attributed directly to the introductions of infected cattle from the endemic bTB areas of GB and following this introduction, have not resulted in spread to other cattle herds in the locality of the breakdown. The following two examples illustrate typical LRA breakdowns.

Example 1 A dairy farm in west Lancashire had confirmed TB resulting from a slaughterhouse case in 2010. The strain was subsequently confirmed as genotype 25:a.  This area of the county had never had bovine TB before.   The animal in question had been purchased 5 months earlier direct from a farm dispersal sale near Manchester, along with 5 other cows.  An immediate herd check test following confirmation, revealed another reactor from this purchased group which had visible lesions on slaughter. Tracing and subsequent testing of the other cattle from the dispersal sale revealed several more reactors on separate farms, all of which were also confirmed as 25:a.   An examination of the movement records for the Manchester farm, indicated a cattle movement from Cheshire, where 25:a is common. Extensive testing around the affected farms in west Lancashire and Manchester over an 18 month period did not reveal any more cases.

A second OTFW breakdown on this farm in 2015 was confirmed as genotype 17:a. With different genotypes identified in the two breakdowns we can be certain that this second breakdown was not a recrudescence of the previous infection.

Example 2 A small beef fattening herd in mid Cumbria had confirmed TB in 2014 and M bovis genotype 9:a was identified. There had been no previous TB breakdowns in the vicinity. This farm regularly bought from farms in the south west of England where 9:a is a common genotype.   Subsequent tracing back to the natal farms of these reactors, confirmed the presence of this genotype on these farms.

A radial testing regime has been instigated around the farm in mid Cumbria. To date, there is no evidence of any spread from this farm into the neighbouring herds.

In both of these examples it is apparent that knowledge of genotypes was critically important in establishing the purchased origin of the breakdowns and demonstrating that the LRA remains free of endemic infection.   

Use in Case Management

Once APHA case vets are aware of the likely origin of a breakdown they can advise farmers and their OVs on measures they might take to reduce the risk of further infection entering the herd.

If the breakdown appears to be of local wildlife origin there are a range of measures which we can suggest to reduce the risk of further infection. These include advice on biosecurity at pasture and in farm buildings and feed stores, identification of areas of badger activity on the farm and consideration of other wildlife species that may spread infection.  Information on these is available on the TB Hub (http://www.tbhub.co.uk/tb-policy/england/).   

Where the origin appears to be from purchase of infected cattle, measures to help reduce the risk may include riskbased trading, pre and post movement testing, on-farm isolation before entry to the herd and farm cleansing and disinfection.   Information on these is also available on the TB Hub.

Article written by Jonathan Kahn, VO at Shrewsbury AHO, and Elaine McArthur, VO at Preston AHO. 

We would like to thank Dr Noel H. Smith of the Bovine TB Genotyping Group and Dr Jenny Broughan of the Department of Epidemiological Sciences at APHA Weybridge for providing figures for this article. We would also like to thank Dr Noel H Smith for critically reading the manuscript.