Tuberculin skin test
The primary screening test for TB in cattle in the UK is the single intradermal comparative cervical tuberculin test (SICCT). This is commonly known as the tuberculin skin test, which is used throughout the world in its various formats to screen cattle, buffalo, bison, other animals, and people for TB. It is the internationally accepted standard for detecting M. bovis infection in live animals.
Rather than look for the bacterium itself, the skin test in cattle relies on measuring the immune response of the animal to injections of tuberculin. Tuberculin is a complex mix of proteins extracted from cultures of M. bovis grown in the laboratory and killed by heat.
The skin test is comparative as the animal’s immune response to injections of both bovine and avian (bird) tuberculin is measured and compared. By simultaneously injecting two types of tuberculin into the deep layers of the skin of the neck, the test can better distinguish between animals infected with M. bovis and animals previously exposed to or infected with other types of mycobacteria found in the environment which do not cause bovine TB.
If an animal’s immune system has previously been ‘sensitised’ (primed) by infection with M. bovis or exposure to other mycobacteria, then the injection of tuberculin triggers an inflammatory response and a reaction (swelling or ‘lump’) develops at the injection site. The intensity of the reaction is greatest at 48-72 hours after injection, and subsides rapidly afterwards. Cattle that are infected with M. bovis tend to show a greater reaction to bovine tuberculin than avian tuberculin.
The size and nature of any reactions at the avian and bovine injection sites are measured and compared. Depending on the degree of reaction to the skin test and the interpretation of the test, the animal is classified as;
- Clear – negative result
- Fail – reactor or positive result
- Inconclusive reactor (IR) – the animal shows a reaction to bovine tuberculin greater than the avian, but not strong enough to be classified as a reactor. IRs must be isolated and re-tested after 60 days. Animals that have an inconclusive result at two consecutive skin tests are considered reactors
The tuberculin skin test is carried on two days;
- Day one – injection of avian and bovine tuberculin.
- Day two – reading of the test 72 (+/- four) hours later.
Day 1 (TT1)
- The ear tag number of the animal to be tested is reconciled with the list of animals on the test chart (or hand held device if applicable).
- The two injection sites are marked on the animal’s neck, usually by clipping the hair away with clippers or curved scissors.
- The skin thickness at each site is measured using callipers and the measurements are recorded against the animal.
- Avian tuberculin is then injected into the skin of the upper site and bovine tuberculin into the skin of the lower site.
Day 2 (TT2)
- The animal’s ear tag number is confirmed against the testing record from day one.
- Each injection site is visually inspected and felt with the fingers for any reactions (swellings).
- Where there is any detectable reaction at either site, both sites are re-measured with callipers and the measurements and type of reaction recorded against the animal, along with the test result after interpretation.
Yes. Needles should be cleaned and disinfected between each animal using surgical spirit or another suitable disinfectant. This is usually achieved by placing cotton wool moistened with disinfectant at the bottom of the syringe holsters used by most vets carrying out TB testing. Needles should be changed immediately if they become contaminated with blood or other material during testing. The requirement for disinfection of needles between individual cattle does not relate specifically to any risk of transmission of TB, but rather the potential for transmission of other infectious agents such as bluetongue virus and Schmallenberg virus. There is no evidence to indicate that transmission of TB occurs via the correct use of multi-use needles used during TB testing.
No, as long as the injection is intradermal, the depth is unimportant. It is more important and more difficult to ensure the injection is intradermal in thin skinned animals.
The skin test can be interpreted at either ‘standard’ or ‘severe’ interpretation. Standard interpretation is the default used for all routine surveillance testing, whilst severe interpretation is used in circumstances where TB is strongly suspected or confirmed e.g. for testing in TB breakdown herds. Interpreting the test at severe interpretation involves lowering the cut-off point for an animal to be classified as a reactor. What this means in practice is that some animals considered inconclusive reactors at standard will become reactors at severe interpretation. The severe interpretation used in England and Scotland is slightly different to the one used in Wales, where some clear animals at standard interpretation may become IRs at severe interpretation.
No diagnostic test is perfectly accurate and there is a trade-off between sensitivity and specificity.
Sensitivity is the probability that a test will correctly identify an infected animal as positive. The higher the sensitivity of the test, the lower the probability of incorrectly classifying an infected animal as uninfected (a false negative result).
Specificity is the probability that a test will correctly identify an animal that is free from infection as negative. The higher the specificity, the lower the probability of incorrectly classifying an uninfected animal as infected (a false positive result).
The specificity of the skin test used in the UK is very high – approximately 99.98% at standard interpretation. In practical terms, this means that you would expect approximately one false positive result for every 5,000 uninfected cattle tested using standard interpretation. This very high specificity gives us a high degree of confidence that the vast majority of the skin test reactors identified in high incidence areas of England and Wales are animals truly infected with TB, irrespective of whether or not lesions typical of the disease can be detected during post-mortem meat inspection in the slaughterhouse (see the next section on the predictive value of the test). Even in low incidence areas such as Scotland the majority of skin test reactors are likely to be truly infected with TB, irrespective of whether or not lesions typical of TB are found at slaughter.
However, the main limitation of the tuberculin skin test is its sensitivity. Studies have shown that skin test sensitivity in Great Britain lies between 52% and 100% with an average of about 80% sensitivity at standard interpretation and slightly higher at severe interpretation. In practical terms this means that on average 20-25% of TB-infected cattle can be missed by one round of skin testing using standard interpretation.
There are many factors that can affect the sensitivity of the tuberculin skin test i.e. its ability to correctly identify an infected animal as positive. Potential reasons for missing infected animals (false negatives) are:
- Animal is tested in the pre-allergic period i.e. the unreactive period soon after infection when it may not react to the skin test.
- Desensitisation, where an animal’s skin reactivity to tuberculin is reduced for some time after the skin test, which is why there is at least 60 days between skin tests.
- Overwhelming infection with M. bovis, where cattle in the terminal stages of TB can have a depressed immune response that prevents them from reacting to the skin test (anergy).
- Environmental mycobacteria.
- Johne’s disease caused by the bacterium Mycobacterium avium subspecies paratuberculosis, which is closely related to M. bovis.
- Suppression of the immune system, for example due to infection with viruses (e.g. Bovine Viral Diarrhoea), treatment with certain drugs (e.g. steroids), and suppression of the immune system during the early post-calving period.
- Errors when carrying out the skin test.
Potentially, however it is unlikely that it would have enough of an effect to impact the result of the test. If the immune system of an animal is compromised for any reason, it is more susceptible to infection and may be less likely to react to the test (i.e. infected animals may be missed). Reasons for depression of the immune system in cattle include infection with viruses, drug treatment and during the early post-calving period.
TB testing and other official veterinarian (OV) work in England and Wales is undertaken by Veterinary Delivery Partners, who are responsible for allocating local vets and ensuring that TB testing is carried out to a high standard. In Scotland, TB testing is delivered through OV practices. The Animal & Plant Health Agency (APHA) carries out risk-based audits of OVs carrying out skin testing to check that it is being carried out correctly in line with agreed procedures. Further details are available on the APHA website.
No. When TB reactors are found to the tuberculin skin test, it is very likely that those cattle are infected with bovine TB, even if no visible TB lesions are found at post-mortem meat inspection. This is because the skin test is very specific, meaning it is extremely unlikely to generate a false positive result (only 1 false positive for every 5,000-6,000 TB-free cattle tested with the standard test). The skin test can also find TB long before infected cattle have developed clinical signs that would be noticed by an experienced stockman or a vet.
Reactors to the skin test may therefore not have any visible lesions because they are either in the early stages of the disease, or the lesions are too small to be detected by the naked eye during a relatively quick visual inspection of the carcass in a busy slaughter line. Often, even if no visible lesions are found at the slaughterhouse, had the carcass been more carefully examined in a laboratory, microscopic lesions could have been detected. But even if no lesions can be detected a reactor animal is still very likely to be infected with TB. However, whilst non-lesioned reactors can be infectious, animals that are found to have visible lesions are more likely to be frequently excreting TB bacteria and present a higher infection risk to other cattle than the non-lesioned animals. Therefore it is preferable to catch the disease in its earlier stages before visible lesions have had an opportunity to develop, and remove those reactors from the herd before they have had the opportunity to infect other animals and cause a more serious TB herd breakdown.
Interferon-gamma blood test
The interferon-gamma test (or gamma test for short) is a supplementary blood test used alongside the tuberculin skin test to maximise the probability of detecting TB-infected animals in cattle herds affected by TB breakdowns. The gamma test was developed in Australia in the early 1990s and approved under EU legislation for use in cattle in 2002. It then underwent extensive evaluation under GB conditions before being officially rolled out in this country in October 2006. The gamma test achieved approval and registration by the OIE (the World Organisation for Animal Health) in 2015. To this day it remains the only blood test approved in the UK and the EU to supplement the skin test for TB in cattle.
The gamma test is carried out in the laboratory using freshly collected blood samples. The test is based on measuring the levels of a cytokine (an immunological hormone or messenger protein) called interferon gamma (IFNγ). IFNγ is released by the white blood cells of cattle infected with TB when stimulated with bovine and avian tuberculins (the same as used in the tuberculin skin test). IFNγ can be measured, and like the skin test, blood samples from TB-infected animals will release greater amounts of IFNγ in response to bovine tuberculin compared to avian tuberculin.
The gamma test is used as a supplementary test alongside the tuberculin skin test in specific TB breakdown herds. The aim of combining the skin and blood tests is two-fold: to shorten the duration of the TB breakdown and reduce the risk of leaving infected animals undetected in the herd by the time movement restrictions are lifted. Any skin test-negative but gamma test-positive animals must be slaughtered to increase the probability of correctly identifying all TB-infected cattle in the affected herd. The gamma test is compulsory for every new TB breakdown herd with lesion and/or culture positive animals in;
- Low incidence areas of England and Wales
- Edge Area of England
In Wales only, the gamma test is also compulsory for certain inconclusive reactors that give an inconclusive result upon re-testing. In England and Wales the gamma test is repeated as long as standard interpretation skin test reactors and/or animals with visible lesions of TB at slaughter continue to be identified in those herds.
Since 1st April 2017, gamma testing is also compulsory for TB breakdowns with lesion and/or culture positive animals in the High Risk Area (HRA) of England where any of the following three criteria are met;
- Criterion 1: The APHA veterinary investigation concludes that the most likely transmission route for the affected herd was contact with infected cattle and measures are in place to prevent further spread of disease from this source;
- Criterion 2: The infected herd is located in one of the areas where at least two years of effective licensed badger culling have been completed;
- Criterion 3: There is clear evidence that repeated skin testing of the herd has failed to resolve a TB breakdown
The gamma test is also used to help resolve persistent TB breakdowns (herds that have been under movement restrictions for more than 18 months) and on a discretionary basis in the HRA in other situations, for example in extensive TB breakdowns with high numbers of reactors to inform decisions about a potential full or partial herd slaughter.
For disease control reasons it is important that the gamma test is carried out as soon as possible after the herd becomes eligible so that infected animals can be identified and removed at the earliest opportunity. The sooner the blood test is deployed, the greater its potential benefits such as reducing the length of time that the herd is under TB restrictions.
Where practical, gamma testing is completed before the first short interval test (SIT) in new TB breakdowns. The Animal & Plant Health Agency (APHA) will discuss the timing of gamma testing with the herd owner before arranging the test. If the gamma test is carried out at the same time as the skin test, the blood sample is taken before the injections of tuberculin on day 1 of the test (TT1) or after the reading of the skin test on day 2 of the test (TT2).
Yes, the gamma test is approved for statutory use in the testing of bovine animals in England, Scotland and Wales and can be used by APHA when it is considered necessary. The keeper has a legal obligation to comply with all reasonable requirements of APHA with a view to facilitating the gamma test. If a keeper fails to comply, APHA may take any steps necessary to facilitate the testing of the animals and may recover the amount of any expenses reasonably incurred. Any non-compliance may constitute an offence which can be referred to the relevant Local Authority for investigation and any enforcement action they deem appropriate.
Usually all animals in the herd over six months of age are eligible for gamma testing. Animals under six months of age are automatically excluded from gamma testing as their immune systems are still developing and this can interfere with the test, leading to false positive results.
Certain herds (e.g. intensive fattening herds) may be eligible for exemption from gamma testing if they fulfil certain specific criteria following individual risk assessment by an APHA vet. Sometimes specific, separate groups of cattle on a TB breakdown holding may be excluded from gamma testing if they are deemed to be at low risk of infection following a veterinary risk assessment by APHA.
TB breakdown herds eligible for gamma testing will initially undergo one round of blood testing. After this the potential need for further gamma tests is assessed by an APHA vet using specific criteria relating to the results of subsequent tests and post mortem/culture results. In England and Wales, the test is repeated as long as standard interpretation skin test reactors and/or animals with visible lesions of TB at slaughter continue to be identified in those herds. The number of gamma tests will vary between TB breakdown herds according to their individual circumstances.
The option of private gamma testing was made available in April 2016 in England only. With prior approval from the Animal & Plant Health Agency (APHA), private vets are able to submit blood samples for gamma testing to an APHA laboratory at the owner’s expense. Private gamma testing is limited to a small number of specific scenarios outside of the government funded testing programme, where owners seek additional assurances as to the TB-free status of animals over and above statutory testing. Private gamma testing can be used in the following situations;
- To supplement pre- or post-movement testing of animals that are not subject to, or have passed a compulsory skin test.
- To screen animals joining high-value herds, including pedigree bulls entering semen collection centres.
- To test animals following a negative routine or tracing skin test.
- As a marketing tool to add value to herds/animals intended for sale.
- Rapid retesting of inconclusive skin test reactors (before or after the skin re-test) where no government funded gamma test is planned.
Cattle not eligible for private gamma testing include reactors and other cattle awaiting slaughter for TB control purposes, cattle from TB breakdown herds undergoing government-funded gamma testing and herds under restrictions for overdue skin testing. If a positive result is received, the animal is likely to be compulsorily slaughtered with compensation paid. The herd would be placed under movement restrictions and normal breakdown procedures followed. In specific circumstances some low-risk animals giving a positive result may be re-tested at government discretion and expense. Cattle keepers wishing to carry out private gamma testing should discuss this with their private vet. For more information about private gamma testing see the guidance for private vets on the APHA website.
The Animal & Plant Health Agency (APHA) will contact the herd owner to arrange a date and time to carry out the gamma test and discuss animal handling facilities and logistics. Blood sampling is carried out by trained Animal Health Officers and they usually work in pairs or teams for all but the smallest herds. Government-funded gamma testing is currently only carried out by APHA staff and not private vets. Blood samples are taken most commonly from the tail vein, or sometimes from the jugular vein in the neck. It is important that the cattle handling facilities allow safe, easy access to the tail area of the animals so that blood samples can be taken quickly and efficiently.
The blood samples must be handled carefully and kept warm; specifically between 17ºC and 27ºC. The blood samples are stored in temperature-controlled packing systems to protect them from extremes of temperature. If the temperature of the samples is too hot or too cold on arrival at the laboratory, the test cannot be completed and the samples will be rejected. The risk of rejection is higher in very cold weather and APHA will sometimes postpone or re-schedule gamma testing if this is considered a significant risk.
Gamma test results are reported to the owner within 10 working days after the blood samples are taken. APHA will usually contact the herd owner by telephone to discuss the results and in all cases a letter is sent detailing the results.
There are four possible outcomes for each blood sample subjected to the gamma test;
- Positive – the animal has failed the test and is classified as a reactor (gamma reactor). The animal is compulsorily slaughtered and compensation is paid.
- Negative – the animal has passed the test.
- Re-sample – when a blood sample has a quality control fail, and the same sample is re-tested and fails a second time, the lab will request a new sample from the same animal (APHA will advise the herd owner accordingly). An animal is only resampled once. The average resample rate for samples in GB is approximately 5%.
- Rejected – the sample was not tested by the laboratory and another blood sample will need to be collected from the animal.
The APHA laboratory may reject blood samples where any of the following occurs:
- Insufficient volume of blood taken.
- Blood extensively clotted in the tube.
- Temperature of the blood samples on arrival at the laboratory falls outside of the recommended range of 17- 27°C.
- Incorrect, broken or cracked blood tubes used.
- Incorrectly labelled or unlabelled blood tubes.
- Samples received too late at the laboratory.
No diagnostic test is perfectly accurate and there is a trade-off between sensitivity and specificity.
Sensitivity is the probability that a test will correctly identify an infected animal as positive. The higher the sensitivity of the test, the lower the probability of incorrectly classifying an infected animal as uninfected (a false negative result). The gamma test is more sensitive than the skin test (~90% compared to ~81%), i.e. it is less likely to miss infected animals.
Specificity is the probability that a test will correctly identify an animal that is free from infection as negative. The higher the specificity, the lower the probability of incorrectly classifying an uninfected animal as infected (a false positive result). On average, the gamma test has a specificity of 96.6%, which in practical terms means 3-4 false positives per 100 disease-free animals tested. The tuberculin skin test has a higher specificity of 99.98% which equates to one false positive per 5,000 disease-free animals tested.
The gamma test is not as specific as the tuberculin skin test which means that more false positives occur. That is why the gamma test is, in most cases, only applied to lesion and/or culture positive TB breakdown herds. In these herds the risk of taking out low numbers of false positives is outweighed by the need to identify and remove all infected cattle, and a test-positive animal is in any case more likely to be infected.
The skin test can miss 20-25% of TB-infected cattle at standard interpretation and likewise the gamma test cannot identify every infected animal. The two tests identify slightly different sub populations of TB-infected animals; for example the gamma test can identify cattle at an earlier stage of infection, as well as infected animals that simply fail to respond to the skin test. Hence using both tests together increases the probability of detecting all infected animals in the herd. Also, by applying the gamma test at the beginning of a new TB breakdown, we can shorten the duration of movement restrictions applied on herds and reduce the cost of breakdowns.
Of approximately 490,000 skin test-negative animals from TB breakdown herds that have been gamma tested since the test was introduced in 2006, over 29,000 gamma reactors have been identified that otherwise might have been missed. This highlights the benefits of supplementing skin testing with the gamma test to maximise the chances of removing all TB-infected animals and reducing the risk of leaving undetected residual infection in the herd.
All cattle compulsorily slaughtered for TB control purposes undergo post mortem meat inspection to check for lesions suspicious of TB in the carcase. The result of the post mortem inspection is reported as either;
- VL (visible lesions); or
- NVL (no visible lesions)
A relatively small proportion of gamma test reactors show visible lesions typical of TB at post mortem inspection. It is important to recognise that this is due to the insensitive methods of confirming infection with M. bovis at slaughter, and not a failure of the gamma test. Furthermore, the gamma test is always used after the TB breakdown herd has been subjected to at least one round of skin testing. This means that, by the time the blood test is deployed, many of the worst affected animals (i.e. those with the most extensive lesions of TB) will have been removed from the herd. A gamma test reactor that shows no visible lesions at post mortem inspection can occur for one of the following reasons;
- The animal was in the early stages of infection with M. bovis when it was detected by the test, at which point the TB lesions were too small or localised to be seen by the naked eye. This is particularly relevant for the gamma test, as it tends to identify animals earlier in the course of infection than the skin test and before any visible lesions have developed.
- The animal had visible lesions at post mortem inspection, but they were missed during a relatively quick post-mortem inspection at the slaughterhouse.
- The gamma test result was a genuine false-positive. As stated above, we can expect this to happen in approximately 3.5% of TB-free animals.
The use of the gamma test by APHA is limited to herds that are suffering a TB breakdown and (nearly always) following confirmation of M. bovis infection by post-mortem or laboratory culture in one or more skin test reactors or slaughterhouse cases from those herds. Therefore, NVL gamma test reactors can represent a combination of the three scenarios described above. However, the fact that TB has previously been detected in the herds that are subjected to gamma testing gives us more confidence that the majority of NVL test reactors are animals truly infected with M. bovis.
Additionally, published studies in GB, the Republic of Ireland and Northern Ireland show that gamma test-positive animals left in herds have a significantly higher risk of disease progression compared with test-negative animals from the same herds. In that sense, the early detection and compulsory removal of all gamma-positive animals from a TB breakdown herd, before they can reach the more infectious (VL) stages of the disease, should be regarded as one of the key benefits of the gamma test.
Potentially, however it is unlikely that it would have enough of an effect to impact the result of the test. If the immune system of an animal is compromised for any reason, it is more susceptible to infection and may be less likely to react to the test (i.e. infected animals may be missed). Reasons for depression of the immune system in cattle include infection with viruses e.g. Bovine Viral Diarrhoea (BVD), drug treatment (e.g. steroids) and during the early post-calving period.
IDEXX laboratories (Maine, USA) have developed a diagnostic blood test for TB in cattle known as the IDEXX antibody test. As the name suggests, it detects specific antibodies produced by the animal in response to infection with Mycobacterium bovis, the bacterium that causes bovine TB. The IDEXX antibody test is not yet recognised as an official test for TB in cattle in EU legislation, however it is approved by the OIE (World Animal Health Organisation) to supplement the tuberculin skin test in cattle. This means that although it cannot be used as a statutory TB test in Great Britain, APHA can use it in exceptional circumstances with prior consent from the herd owner. In Wales, the IDEXX antibody test is considered a relevant test under TB legislation, which means that APHA does not need permission from the herd owner to use the test or to remove test-positive animals.
It is a third line test used on cattle in chronic TB breakdown herds where repeated tuberculin skin testing and use of the interferon-gamma blood test has already occurred. The IDEXX antibody test is also one of the three blood tests officially approved by Defra and Welsh Government for voluntary and statutory TB testing of South American camelids in England and Wales.
No diagnostic test is perfectly accurate and there is a trade-off between sensitivity and specificity.
Sensitivity is the probability that a test will correctly identify an infected animal as positive. The higher the sensitivity of the test, the lower the probability of incorrectly classifying an infected animal as uninfected i.e. a false negative result.
Specificity is the probability that a test will correctly identify an animal that is free from infection as negative. The higher the specificity, the lower the probability of incorrectly classifying an uninfected animal as infected i.e. a false positive result.
OIE data suggest an overall moderate test sensitivity of 65% and a specificity of 98% for the IDEXX antibody test in cattle.
To maximise the sensitivity of the test, a prior tuberculin skin test is required to boost M. bovis-specific antibody levels in TB-infected cattle. For this reason, APHA recommends that blood samples for the IDEXX antibody test are taken within 10 to 30 days of a prior skin test. Cattle of any age can be tested. Data from Great Britain show that antibody tests are less sensitive overall compared with the tuberculin skin test and interferon-gamma blood test. However they can be useful for identifying small numbers of infected cattle that are skin and interferon-gamma test negative; approximately 3-5% of cattle from TB breakdown herds with lesion and/or culture positive animals in Defra study SE3263 “Validation of new serology tests for bovine tuberculosis”.
Use of the IDEXX antibody test by APHA may be considered in multiple-reactor herds with confirmed chronic infection where, despite repeated skin testing and use of the interferon-gamma blood test, the APHA case vet suspects ongoing cattle to cattle transmission. In these cases, residual infection in the herd may be due to infected cattle which produce little or no response to either the skin and interferon-gamma tests, but produce high levels of M. bovis-specific antibody which can be detected by the IDEXX test. The test is used by APHA in exceptional circumstances, and eligibility is determined on a case-by-case basis. In England only, the IDEXX antibody test is available for private use at the herd owner’s expense, however prior permission from APHA must be obtained by the private vet.
In England, before the IDEXX antibody test can be used, the herd (or management group) in question must have undergone interferon-gamma blood testing if this is required under the current Defra policy. The herd owner must agree to surrender any test-positive animals for slaughter, with compensation paid by Defra. In Wales, use of the IDEXX antibody test does not require consent from the herd owner and test-positive animals are removed with compensation paid by the Welsh Government.
In England only, herd owners wishing to use the IDEXX antibody test privately should first discuss this with their private vet who will need to seek permission from APHA. The private vet must also obtain informed written consent from the herd owner for use of the test. Before the IDEXX antibody test can be used privately in a TB breakdown herd, the herd (or management group) in question must have undergone interferon-gamma blood testing if this is required under the current Defra policy. For privately-funded IDEXX antibody tests, Defra will not pay compensation for any test-positive animals slaughtered.
Where the IDEXX antibody test is instructed by APHA and carried out according to Defra policy, compensation will be payable for the removal of any test-positive animals as normal. In England only, for privately-funded IDEXX antibody tests, all results must be shared with APHA by the private vet. Any test-positive animals will need to be slaughtered before APHA can lift movement restrictions from the herd and restore its officially TB free (OTF) status. Defra will not remove and pay compensation for any test-positive animals where the test was privately-funded. The removal and slaughter costs are the responsibility of the herd owner.