Detection of Mycobacterium bovis in post-mortem tissue samples

The Animal and Plant Health Agency (APHA) plays a central role in delivering the bovine tuberculosis (bTB) eradication programmes throughout England and Wales, as well as the bTB surveillance programme in Scotland. Accurate diagnostic and confirmatory testing are the cornerstones of any disease eradication programme, and APHA is responsible for all the laboratory-based bTB testing to support this effort.

The current gold standard method for bTB diagnostic testing from tissue samples (for both bovine and non-bovine farmed animals) remains traditional microbiological culture. Although well established, this process takes between 6 and 22 weeks to obtain a final result, which is a major disadvantage. However, APHA has recently validated a new polymerase chain reaction (PCR) test which can detect Mycobacterium bovis directly from tissue samples collected at post-mortem inspection. The major advantage of this method is that results will be reported to livestock keepers much quicker, typically within three weeks.

APHA has recently validated a new polymerase chain reaction (PCR) test which can detect Mycobacterium bovis directly from tissue samples collected at post-mortem inspection

From 30 March 2022, this new PCR test will be used to diagnose bTB in post-mortem tissue samples.

How will the new M. bovis PCR test be used by APHA?

The PCR test will initially only be used in Great Britain for tissue samples from carcasses of:

• Tuberculosis (TB) slaughterhouse cases in cattle and non-bovines, ie animals routinely sent for private commercial slaughter that have typical 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, as well as cases where suspicious TB lesions are identified on diagnostic post-mortem examination in a veterinary laboratory
• Domestic pets (cats and dogs) and exotic species of animals (eg in zoological collections) submitted to APHA for laboratory investigation

From 30 March 2022, the M. bovis PCR test will be routinely used in the above prescribed scenarios. After a period of operation and subsequent review of the test’s performance, APHA will consider wider use of the test, for example in routine detection of TB in post-mortem samples taken from skin and blood test positive cattle.

What are the benefits of the M. bovis PCR test?

The main benefit of the PCR test is rapid detection of M. bovis directly from post-mortem tissue samples, as results will typically be reported within three weeks (quicker than the current gold standard). Rapid test results will have significant advantages where a quicker diagnosis allows more effective case management by APHA, for example in TB slaughterhouse cases.

Rapid test results will have significant advantages where a quicker diagnosis allows more effective case management by APHA

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, while also minimising contamination and maximising traceability of samples.

How does the accuracy of the PCR test compare with microbiological culture?

In APHA’s validation study, the M. bovis PCR test produced equivalent results to the traditional microbiological culture method for both bovine and non-bovine tissue samples. However, in a very small proportion of cases it may not be possible to obtain a valid PCR test result. In these instances, APHA will revert to bacteriological culture for disease confirmation, and APHA will inform keepers if this applies to samples from their herd or flock.

How will the PCR test improve APHA’s management of TB incidents?

Bovines

When a TB slaughterhouse case is identified, the officially TB free status of the herd of origin is suspended, pending the final test results from the laboratory. During this time, whole herd movement restrictions are in place and animals cannot move on or off the affected holding, except under a licence issued by APHA. Currently, tissue samples are taken from the carcass and submitted for microbiological culture which can take up to 22 weeks for a result. In the meantime, the herd remains under movement restrictions and a skin test (check test) of the herd may be carried out pending the final culture result.

[The PCR test] will limit the negative impact of movement restrictions on cattle herds and (in England and Scotland only) eliminate, in most cases, the need for a check test

Instead of relying on culture results for confirmation or negation of M. bovis infection, PCR testing will be carried out on the lesions sampled from the carcass of the slaughterhouse case. PCR test results will typically be reported to the livestock keeper within three weeks; and if negative, APHA could revoke the herd movement restrictions sooner instead of waiting for culture results. This will limit the negative impact of movement restrictions on cattle herds and (in England and Scotland only) eliminate, in most cases, the need for a check test.

Farmed non-bovine animals

Similarly to bovines, the new PCR test will also allow rapid detection of M. bovis in tissue samples from carcasses of non-bovine animals. This will be to confirm or negate 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 regarding 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 or flock of origin in most cases.

Once implemented, the new PCR test will be used as the confirmatory test, with M. bovis-positive samples that meet certain criteria sent out for culture and WGS testing

Currently, samples are sent for culture and whole genome sequencing (WGS) if M. bovis is suspected. If M. bovis is not isolated, movement restrictions are lifted without further testing in most suspect cases. Once implemented, the new PCR test will be used as the confirmatory test, with M. bovis-positive samples that meet certain criteria submitted for culture and WGS testing. If the PCR test result is negative, APHA may be able to revoke herd movement restrictions sooner due to the quicker turnover rate. In the rare cases where a valid PCR result cannot be obtained, movement restrictions will remain in force until negative culture results are received.

The PCR test will also be 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.

Are there any limitations of the M. bovis PCR test?

An important limitation of this test is that it is not possible to apply WGS directly on the M. bovis target DNA sequence that is amplified through PCR testing (ie the PCR product). As WGS is only possible with DNA obtained from a pure microbial culture, APHA will not be able to rely exclusively on the PCR test for case management and epidemiological analyses of TB breakdowns. Culture will only be 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, that is 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 regarding the management of the TB breakdown.