PCR test for detection of M. bovis in post-mortem tissue samples

The Animal & Plant Health Agency (APHA) plays a central role in delivering the bovine tuberculosis (bTB) eradication programmes in England and Wales and the bTB surveillance programme in Scotland. Diagnostic and confirmatory testing is the cornerstone of any disease eradication programme and APHA is responsible for all the laboratory based bTB testing to support this effort. 

The “gold standard” for bTB diagnostic testing from tissue samples (both bovine and non-bovine farmed animals) is traditional microbiological culture. Although well established, the major disadvantage of this approach is the length of time (6-22 weeks) it typically takes to obtain a result. APHA has validated a new polymerase chain reaction (PCR) test which can detect the bacterium responsible for bovine TB, Mycobacterium bovis (M. bovis) directly from tissue samples collected at post-mortem inspection. The major advantage of this method is that it typically takes only three weeks to report a result to the keeper.

The PCR test became operational on 30 March 2022 and is initially only being used in Great Britain (GB) for tissue samples from carcases of:

  • TB slaughterhouse cases in cattle and non-bovines i.e. animals routinely sent for private commercial slaughter that have suspicious lesions of TB at routine meat inspection;
  • Non-bovine animals such as camelids, goats, pigs, sheep and farmed deer that are removed as TB test reactors, direct contacts or clinical TB suspects, and cases where TB lesions are identified on diagnostic post-mortem examination in a veterinary laboratory; and
  • Domestic pets (cats and dogs) and exotic species of animals (e.g. in zoological collections) submitted to APHA for laboratory investigation.

The PCR test reduces the time taken by the APHA laboratory to confirm whether M. bovis is present in tissue samples taken from carcases at post-mortem inspection. This means that in certain situations (i.e. slaughterhouse cases), if the PCR test results are negative, and check testing has been completed where required, APHA can lift herd movement restrictions sooner than the previous protocols using microbiological culture, which typically take 6-22 weeks. If the PCR test results are positive, APHA can inform the keeper and their private vet quickly, so that further action can be taken as soon as possible to control TB in the affected herd.

Q&A

Polymerase chain reaction (PCR) is a molecular-based laboratory technique that involves detecting small amounts of the genetic material (DNA) that is contained within all living organisms. This is done in the laboratory by making millions of copies of a specific sequence of the target DNA, a process called ‘amplification’.

Each M. bovis bacterium contains unique DNA which carries the genetic instructions for its development, function, growth and reproduction. PCR can detect tiny amounts of the DNA present within these bacteria and amplify it to produce a quantity which is then detectable. By looking for targeted bacterial DNA, the APHA laboratory is able to rapidly detect and differentiate the presence of M. bovis bacteria in tissue samples taken from carcases at post-mortem inspection. Critically, this detection is significantly quicker than the microbiological culture method.  

PCR test results are typically available within three weeks. Depending on the sample type submitted, traditional culture can take 6-22 weeks for confirmation.

No. The PCR test is designed to be used on tissue samples taken from the animal’s carcase at post-mortem examination or meat inspection. It is not tissue- or organ-specific and is applicable irrespective of the anatomical location of the TB lesions.

Yes. APHA requires all new diagnostic tests to be fully validated in accordance with OIE (World Animal Health Organisation) guidelines to provide assurance in the results generated. Validation involves multiple steps to assess the diagnostic performance characteristics of the test and show that it is specific (it only detects the organism of choice) and it is sensitive (it will have a high probability of detecting the organism if present). To further ensure the highest quality standards possible, APHA will also apply to have the diagnostic method accredited in accordance with ISO17025 certification, issued and audited by an independent third party.

In a validation study carried out by APHA, the M. bovis PCR test has been shown to produce equivalent results to the traditional microbiological culture method, for both bovine and non-bovine tissue samples.

In a very small proportion of cases it may not be possible to obtain a valid PCR test result. In those instances, APHA will use bacteriological culture to attempt to grow the TB bacterium. APHA will inform keepers if this applies to samples from their herd/flock.

The main benefit is rapid detection of M. bovis directly from post-mortem tissue samples. PCR test results are reported to the keeper typically within three weeks. This is quicker than the gold standard of microbiological culture to grow the M. bovis bacterium in the laboratory, which typically takes 6-22 weeks for a result. Rapid test results have significant advantages where a quicker diagnosis allows more effective case management, for example slaughterhouse cases. Another benefit is that the PCR test is cheaper to run per sample than microbiological culture. The process is semi-automated and exploits laboratory robotics to facilitate high throughput, whilst also minimising contamination and maximising traceability of samples.

The one important limitation of this test is that it is not possible to apply whole genome sequencing (WGS) directly on the M. bovis target DNA sequence that is amplified through PCR testing (i.e. the PCR product). WGS is only possible with DNA obtained from a pure microbial culture. WGS is a molecular technique that characterises the entire DNA content of an M. bovis isolate. This technique has replaced genotyping, which only looked at certain sections of the bacterium’s DNA. WGS allows differentiation of the ‘strains’ of the M. bovis bacterium at greater resolution than genotyping. WGS is now routinely carried out by APHA on M. bovis isolates from TB breakdown herds to help identify the probable source of infection. APHA cannot rely exclusively on the PCR test for case management and epidemiological analyses of TB breakdowns as culture still has to be carried out for WGS to be performed. Culture is only carried out where WGS is required to allow full analyses of the breakdown and identify the probable source of infection. APHA will not report culture results unless they are used for the confirmation or negation of M. bovis infection i.e. in the rare instances where the PCR test has not provided a valid result. The additional WGS analysis of positive PCR samples will not interfere with or delay APHA’s decisions to manage the TB breakdown.

After a period of operation and review of the test’s performance, APHA will consider wider use of the PCR test, e.g. for routine detection of M. bovis in post-mortem samples taken from skin and blood test positive cattle.

A slaughterhouse case means that typical lesions of TB have been unexpectedly found at routine meat inspection of the carcase of a bovine animal sent for private commercial slaughter. When a slaughterhouse case is identified, the herd of origin’s officially TB free status is suspended (OTFS), which means that bovines can’t move on or off the holding except under a licence issued by APHA. Previously, tissue samples were taken from the carcase and submitted for microbiological culture which can take up to 22 weeks for a result. In the meantime, the herd remained under movement restrictions and a skin test (check test) of the herd may have been carried out pending the final culture result. Now that PCR testing is operational, instead of relying on culture results for confirmation or negation of M. bovis infection, PCR testing is carried out on the lesions sampled from the carcase of the slaughterhouse case. PCR test results are typically reported to the keeper within three weeks and, if negative, herd movement restrictions could be lifted sooner instead of waiting for culture results. This limits the negative impact of movement restrictions on cattle herds and, in England and Scotland only, eliminates in most cases, the need for a check test. PCR test positive results allow for more timely deployment of additional disease control measures, such as source or spread tracing and application of interferon-gamma testing where applicable.

The PCR test allows rapid detection of M. bovis in tissue samples from carcases of non-bovine animals to confirm or negate TB infection in suspected cases of TB identified at post-mortem examination in the laboratory, or during routine post-mortem meat inspection in the slaughterhouse. When APHA receives notification from the Food Standards Agency of a slaughterhouse case of TB in a non-bovine animal, or a suspect case following post-mortem examination of an animal with clinical signs consistent with TB, movement restrictions are automatically applied to the herd/flock of origin in most cases. Previously, samples were sent for culture and WGS if M. bovis was suspected. If M. bovis was not isolated, in the majority of cases movement restrictions were lifted without further testing. The PCR test is now used for this purpose, with positive samples that meet certain criteria being sent for culture and WGS. PCR test results are typically reported to the keeper within three weeks, which means that if the result is negative APHA may be able to lift herd movement restrictions sooner. This has particular advantages for farms on which movement restrictions have the most impact, e.g. commercial pig farms and herds that rely on regular sales of breeding and fattening stock to other farms. In the rare cases where a valid PCR test result cannot be obtained (see above), movement restrictions will be maintained until negative culture results are received. The PCR test is also used by APHA to detect M. bovis in non-bovine animals that have been removed as TB test reactors or direct contacts, whenever APHA considers that the procedure is necessary in addition to a post-mortem examination. In these cases, even if no typical TB lesions are identified at post-mortem examination and the PCR test result is negative, additional testing will still be required before movement restrictions can be lifted.