Influence of Weybridge and Lelystad tuberculins

Tuberculin is the name given to extracts of Mycobacterium tuberculosis, M. bovis, or M. avium that is used in skin testing in cattle (and humans) to identify a tuberculosis infection. The skin test involves injecting a small amount of the tuberculin into the skin of the animal. In most cattle infected with M. bovis, this will cause the animal’s immune system to react to the tuberculin and cause a localised allergic reaction (swelling) of the skin a few days after the injection.

In the summer of 2005, after difficulties with the production of tuberculin at VLA Weybridge, Defra began to source paired stocks of bovine and avian tuberculins from ID-Lelystad in The Netherlands. These stocks started to be used in herds across GB from October 2005 and were alternated with Weybridge tuberculins for release to veterinarians on a strict temporal basis, dependent on the shelf life of the available stocks from each manufacturer. The alternate use of both tuberculins continued in GB until the production of Weybridge tuberculin at VLA ceased and stocks eventually ran out in September 2009. Since then (this was written in 19th January 2011), Dutch tuberculin from ID-Lelystad (now owned by Prionics) has been the only antigen used in the UK bovine TB testing programme (and it had also been in use in Ireland for many years before it started to replace Weybridge tuberculin in the UK).¹¹

This section looks at how the number of cattle slaughtered has changed over these years to examine if there may be any dependence on which tuberculin was used.

Compared to previous years, cattle incidence in Great Britain went through large fluctuations between 2005 and 2010 as shown in the graph below. (Data for this graph were sourced here)


A Veterinary Laboratories Agency report¹² reports that between 1st Jan 2005 and 30th Jun 2009, out of 26,363,877 cattle tests, tests using the Weybridge tuberculin found on average that 497 animals tested positive in every 100,000 tests whereas the Lelystad tuberculin only found 391.¹² The ratio given by 497 to 391 closely matches the ratio 0.47 to 0.38 which, as can be seen in the above graph, are the percentage prevalences of TB in cattle in 2008 and 2010 respectively. Although the Weybridge tuberculin was not used exclusively in 2008 (in fact between 60 and 65% of tests in 2008 were performed using it - deduced from Figure 1 in Reference 12), if no Weybridge tuberculin was used in 2010, the switch over to using the Lelystad tuberculin probably made a major contribution to the drop in the reported TB prevalence between 2008 and 2010.

The influence of switching to the Lelystad tuberculin on the risk of an animal testing positive in each month from January 2004 to August 2011 is illustrated in the graph below. Risk was calculated by dividing the number of TB reactors slaughtered¹⁴ by the number of cattle tested¹⁴. The highlighting shows the months in which the Lelystad tuberculin was predominantly used. Reference 12 only shows usage up to 31st June 2009 and it is assumed that the Lelystad tuberculin was predominantly used in each month from July 2009 onwards. Whether or not this assumption is correct will be confirmed when DEFRA is able to send to me the necessary information. However, in the meantime, it may be worth noting that in response to a request for information¹⁵ made in June 2011, the following was said in the reply¹⁶.

The alternate use of Weybridge and Lelystad tuberculins continued in GB until tuberculin production at VLA Weybridge ceased and stocks eventually ran out in September 2009. Thereafter, only tuberculins from Prionics Lelystad have been in use in the bovine TB testing programme in GB and, consequently, there is no equivalent tuberculin skin test data from cattle tested with VLA tuberculin to compare with. VLA (now AHVLA) have not carried out further comparative monitoring/analyses of tuberculins since then, as the only antigens in use are those from Lelystad.


The above section gives some insight into

1. the extent to which use of the different tuberculins is likely to have distorted the picture of how the disease has been progressing in the 5 years since 2005 in Great Britain,
2. the extent to which introduction of the Lelystad tuberculin during this time is likely to have reduced the reported prevalence, and
3. why it is now more important that veterinaries classify herds in accordance with the protocol and resist pressures which are placed on them to classify herds as clear when they should be classified as infected. (This pressure exists because it is not only unpleasant for the farmer but also for the veterinary when the veterinary has to inform the farmer that an animal has reacted to the test. This is because the repercussions of a failed test cause extreme hardship to the farmer¹³. Also the results of many herd tests are close to borderline.)

Under reporting of TB reactors

A study carried out in Northern Ireland found that in-house staff were 1.5 and 1.8 times more likely than Private Veterinary Practitioners to classify a herd as a breakdown herd.⁵ In the Irish Republic, equipment is monitored annually and the performance and results for each testing veterinary surgeon is tested.⁶ However this monitoring may be too simplistic as stated in the following extract from Reference 7.

The government has been concerned about the quality of bTB testing for some time. Countries like Ireland already use a system of performance indicators to audit testers. But these results show that method to be too simplistic and could lead to false comparisons between vets.

Nevertheless such monitoring is probably better than no monitoring at all as in England and Wales for which Reference 8 reports the following.

There is a lack of supervision and monitoring of Local Veterinary Inspector performance by the State Veterinary Service. Not all divisions carry out supervised tests before they are given a permanent appointment and there is no systematic monitoring of subsequent performance.

According to Reference 9, one infected animal will on average affect five others over a year. If TB reactors are being under-reported, this may be substantially increasing the opportunity for cattle-to-cattle transmission. Unfortunately the UK appears to place more reliance on this routine skin testing than New Zealand due to different protocols used in slaughter houses during post-mortem inspection. The following is an extract from Reference 9.

We observe the difference in cattle detection rates between the UK and New Zealand, which is presumably due to different post-mortem protocols. In New Zealand slaughter houses specifically examine lung tissue for Tb, with an 85% accuracy rate (AHB pers. comm.), whereas a different protocol appears to be used in the UK. Based on the known failure rate of the tuberculin Tb test, we would expect 20% of Tb cases to be detected at slaughter, on a risk adjusted basis, compared to the 32% observed for cattle in New Zealand. The difference is probably explained by under-reporting of Tb in Reactors which has been observed in the UK (ISG 2007).

Is the skin test at best only 80% accurate?

On the One Show broadcasted on the 30th September 2011 on BBC1, Brain May was featured presenting an initiative to bring Jan Rowe who is a spokesman for the National Farmers Union into talks with the Badger Trust. Both Jan Rowe and Jeff Hayden, who is the Finance Director of the Badger Trust, were shown being interviewed by Brian May. The following shows the course of the dialogue (as broadcasted on the One Show) between Brian May and Jeff Hayden.

Brian May said:
Animal welfare group the Badger Trust reject the theory that badgers are the main cause of the transmission of the disease. They believe infected cattle flick?/flip?/flit? through the TB testing and infect the rest of the herd.

Jeff Hayden said:
The so called skin test that they use to test cattle at the moment is at best only 80% accurate so we hear this figure from the farmers about 25,000 cattle being slaughtered last year in England alone and we feel as sorry about that as we do about the death of badgers but what this means is that there are over 6,000 infected cattle left in the national herd. That's the single biggest problem.

If animals were slaughtered based on a method which was only 80% accurate at best the situation would be very dire. In theory, this is not the case because interpretation rules which give 80% are usually only applied to detect an infected herd. If a herd is confirmed to be infected by postmortem examination, another set of stricter rules are then applied to identify infected cattle. It is this stricter, more severe, set of rules which are used to remove infected cattle from the herd, if the herd is confirmed to be infected, which then has to pass 2 consecutive tests at 60 day intervals before restrictions are removed.

The sensitivity of the cattle skin test when using severe interpretation

Reference 4 states the following.

Several studies from various countries have reported estimates of sensitivity for the comparative and other variants of the tuberculin skin test. Test sensitivity (and specificity) is independent of the prevalence of infection in the population and is frequently assumed to be constant across different populations. In practice, however, it can be influenced by a host of other factors including the test procedure, cut-off point for a positive result, tuberculin potency, the stage of infection in the host, other inter-current infections and prevalence of cross-reacting organisms in the locality. It is thus very difficult to quote a single sensitivity estimate for the comparative skin test that would apply to all herds in GB at all times.

Studies evaluating the sensitivity of the test suggest that its sensitivity lies between 52% and 100%, with median values of 80% and 93.5% for standard and severe interpretations, respectively.

It would appear from this that the 'accuracy' (sensitivity) of the skin test when removing infected cattle from the herd using severe interpretation is about 93.5%. Although this is better than 80% 'accuracy' (sensitivity) which would result in one in every 5 tests failing to identify an infected animal, 93.5% still means that one in about every 15 tests fail to detect infection and this is not good. In addition to this, replacement of the tuberculin with a less potent tuberculin, which has been used in the last 5 years since 2005, implies that sensitivities have been reduced to less than those reported above. However the tuberculin(s) used to arrive at the 80% sensititivity is not currently known by the author of this page and enquiries to DEFRA are currently in progress.

The risk of false negatives and positives when testing cattle

In the extract below quoted specificities imply that in Great Britain the blood test is on average about 40 times more likely than the skin test to indicate that a healthy animal is infected. Average specificity of the skin test is quoted to be 99.9% (1 in 1000 wrongly classified as a reactor) whereas that of the blood test is quoted to be 96% (4 in 100 wrongly classified as a reactor).

The extract below is taken from a reply received on 27th March 2009 sent by DEFRA in response to the FOI request shown in Reference 1.

" There are two immunologically based diagnostic tests used in Great Britain (GB) for bTB screening of cattle herds. The primary screening test is the single intradermal comparative cervical tuberculin (SICCT) test, which is commonly known as the comparative tuberculin skin test. Additionally, the ancillary gamma-interferon (γ-IFN) diagnostic blood test has been used since 2002 alongside the skin test in certain prescribed circumstances.

The diagnostic sensitivity and specificity ranges for these tests have been evaluated in several articles and reports published in the veterinary scientific literature over the years. These are in the public domain and for more information you are kindly referred to those on our website at


http://www.defra.gov.uk/animalh/tb/pdf/gifn_trialfinalreport.pdf
http://www.defra.gov.uk/animalh/tb/pdf/gifn_specificityreport.pdf

To summarise, the published estimates for the animal-level sensitivity of the SICCT test in various countries, if correctly performed, range from 52 to 100%, with a median value of 80% at standard interpretation. In other words, this test can be expected to miss about 2 in every 10 infected cattle on a single round of testing (a 20% false negative probability).

Other studies carried out in TB-free cattle populations have found the animal-level specificity of this test to lie between 78.8% and 100%, with a median value of 99.5%. In particular, the SICCT test applied to cattle in bTB-free herds in GB is believed to have a specificity of 99.9%, which is equivalent to a 0.1% probability of false positives, or a one in 1,000 chance that a non-infected animal will be wrongly classified as a reactor.

With regard to the γ-IFN blood test, performance evaluation carried out in a number of countries shows that at the laboratory cut offs used in GB, it has a sensitivity of between 73 and 100%, with a median value of about 87% (i.e. a false negative probability of 10 to 15%, which is slightly lower than that of the SICCT test). Because the two tests detect slightly different sub-groups of infected cattle, by combining the two tests a higher overall sensitivity can be achieved.

A trial conducted in GB to evaluate the specificity of the γ-IFN blood test confirmed the findings of previous studies in other countries, in that it estimated its specificity to be about 96% (i.e. a 4% probability of false positive reactors). This is higher than the false positive probability for the SICCT test and it is one of the reasons why the γ-IFN blood test cannot be used for routine bTB surveillance.

Nevertheless, there is no consensus with regard to the most appropriate values of these classical test characteristics that currently apply to GB. It is important to appreciate that the sensitivity and specificity estimates cited above are indicative averages. The actual performance of a screening test in a particular herd under field conditions is, of course, dependent on a range of variables, such as the diligence of the tester, in adhering to the correct testing procedure, the within-herd prevalence of cattle sensitised to other non-TB environmental mycobacteria, and factors that may alter the immune response to tuberculin of individual animals (e.g. nutritional status, pregnancy, stress levels, concurrent infections, etc). Additionally, for all diagnostic tests there is a trade-off between sensitivity and specificity, so that different interpretations of the test can be used under different disease situations. Sensitivity is enhanced in herds with post-mortem or cultural evidence of TB infection by application of the so-called severe interpretation. "

The reason why it is necessary to slaughter TB reactors which are not confirmed to be infected in post-mortem inspections and tests

5-10% of latently infected humans develop clinical tuberculosis during their lifetime through re-activation of the latent infection (re-activation tuberculosis). The argument that latently infected individuals (culturenegative NVL, skin test reactors for example) constitute a continuous and unpredictable source of reinfection, is equally valid for cattle as it is for human TB. At the early stages of infection, or in latently infected cattle, a period will occur when M. bovis appears to be absent because the bacillary load is not large enough to be detected by culture. In addition, the pathological changes caused by the bacilli are not yet profound enough to be detected during routine abattoir inspection. However, cellular immune responses will be detectable in these animals at an earlier stage of infection than the pathological changes caused by the disease (e.g.visible lesions), or before the bacterial loads exceed the numbers necessary to be able to culture M. bovis from tissue samples. To designate these animals as 'false-positive' is inappropriate as they can harbour bacilli, and become infectious to other cattle, specifically when the disease has progressed further.¹⁰

References

1. Information request: False negatives and positives associated with bovine TB tests
2. Review of the gamma interferon testing policy
3. Review of TB testing procedures: Report for Defra and the Welsh Assembly Government report no.: 22115204-02 Final Report rev 3, 12th June 2006
4. Bovine tuberculosis in England: Towards eradication. Final Report of the Bovine TB Advisory Group. Presented to Defra on 8 April 2009.
5. The Control of Bovine Tuberculosis in Northern Ireland
6. Bovine tuberculosis eradication in Ireland
7. Bovine TB testing under scrutiny
8. Review of TB testing procedures: Report for Defra and the Welsh Assembly Government report no.: 22115204-02 Final Report rev 3, 12th June 2006
9. Bovine Tb Strategy - Review of Costs
10. Government Veterinary Journal
11. Usage of Weybridge and Lelystad tuberculins in bovine TB testing in Great Britain
12. Tuberculin Usage for testing for bovine TB in Great Britain. Report for 1st January 2005 to 30th June 2009.
13. A farmer's utter despair over bovine TB
14. TB in Cattle in Great Britain
15. Tuberculin usage from 1956 to 2011 - Request
16. Tuberculin usage from 1956 to 2011