The Enferplex Bovine TB Antibody Test identifies the presence of antibodies to M bovis, the cause of Bovine Tuberculosis (bTB). It is a serological test that detects the presence of antibodies within blood to fifteen individual M bovis antigens arranged in eleven ‘spots’ alongside an additional ‘control’ spot.
The fifteen antigens used in the test have been carefully selected to maximise test sensitivity without compromise to specificity.
If antibody to bTB is present in the blood sample being tested, the antibody will bind with the relevant antigen.
The resultant reaction produces a chemiluminescent reaction, the light from which can be measured and quantified.
Figure 1 : Images from three individual testing wells. Each well shows individual antigen spots that have bound antibody and are therefore luminescent. The light is measured to produce a quantitative result for each antigen spot.
The whiter/brighter the spot(s), the higher the level of antibodies. Each spot is measured to asses the amount of bTB antibody in the sample.
Thresholds are set for each individual antigen. If the level of Relative Light Units (RLU) is above this threshold, a positive reaction is deemed to have occurred. A positive test result is when two or more antigens are shown to be above the threshold set for the individual antigen spot.
There are two different thresholds set for each antigen, a high sensitivity* setting and a high specificity** setting.
The high specificity setting requires a higher threshold to be reached to define a positive reaction.
*Sensitivity: the ability of a test to correctly identify individuals with the disease.
**Specificity: the ability of a test to correctly identify individuals without the disease.
To identify
True positive: the individual has the disease and the test result is positive.
True negative: the individual does not have the disease and the test result is negative.
The timing of sampling after a Single Intradermal Comparative Cervical Tuberculin (SICCT) Test (M bovis surveillance test) significantly affects the sensitivity of detection of antibody. Samples taken approximately 5-30 days post—SICCT benefit from an antibody ‘boosting’ effect due to an anamnestic (enhanced) response to Purified Protein Derivative (PPD) antigens (injected for the SICCT Test) in animals infected with bTB. Samples taken outside this ‘window’ (non-boosted) will have lower levels of antibody and the test will be less sensitive.
Performance of the test
The Performance of the test at high sensitivity and high specificity settings has been clearly demonstrated in both bTB positive and negative populations in UK and Ireland. Any data noted as being ‘Boosted’ indicates that samples were collected and analysed within the 5—30 day window post PPD injection on day 1 of a SICCT test and thereby would be expected to have an improved sensitivity compared to non-boosted samples. This window gives the highest test sensitivity and would normally be expected to be used, so the sensitivities displayed are appropriate to the expected performance of the test.
Specificity
Sensitivity
The following are the sensitivities found when samples are tested against a defined sample set, e.g. sera from animals confirmed to have bTB by M bovis culture. It is therefore stating the sensitivity against that defined population.
M.bovis culture positive animals
Animals positive for bTB by the presence of Visible Lesions (VL) in UK and Ireland
Animals positive to the SICCT from the UK and Ireland
Detecting animals missed by SICCT and IFNg test
(Single Intradermal Comparative Cervical Tuberculin and Gamma -Interferon Tests)
The diagnostic data for serum that shows the test can detect M. bovis infected animals not detected by SICCT or IFNg are shown in Tables 6 and 7.
These data indicate that the Enferplex Bovine TB Antibody Test can be used to detect M bovis infected animals not detected by SICCT or IFNg.
Relationship between Number of Antigens recognised and the Presence of Visible Lesions (VL) in animals seropositive (antibody positive) by Enferplex Bovine TB
As with Johne’s disease, it is well accepted that in bovine TB, as disease progresses, the production of antibody increases and the strength of the cell mediated immune response wanes. We have analysed the correlation between antibodies, specifically the number of bTB antigens recognised by antibody-positive sera and the presence of VL at post-mortem examination. In total, 2076 SICCT test-positive UK and Irish animals were tested using the Enferplex Bovine TB antibody test at the high sensitivity setting.
The results were analysed with animals being classified as to how many antigens they recognised, e.g. 2 or more, 3 or more, etc., and this was correlated with the percentage with VL and as a percentage of the total VL animals in the population. The results showed that just under 90% of all Visible Lesions detected were in animals that had 5 or more antigens recognised by the Enferplex Bovine TB antibody test.
This shows that the Enferplex Bovine TB antibody test, as with the MAP serological tests, can aid in the identification of epidemiologically important bTB positive animals as those with lesions tend to be those which are excreting bacteria and are therefore infectious (Casal et al, 2014).
Enferplex Bovine TB Antibody Test Practicalities
Animals
- The test can be used in animals available for the SICCT test.
- The test can only be used on those farms that have undergone a Gamma Interferon test.
- The test identifies a different population of positive animals compared to the SICCT and Gamma Interferon tests. These are the animals that can cause continuation of TB testing and chronically infected herds.
Samples
- Clotted blood, ideally taken 5-30 days after the skin test are the most sensitive samples for the test.
- Blood samples should be taken with a plain red top or plain gold top vacutainer. A minimum of 3-4 ml. Clot the blood at room temperature (20 +/- 4 0C) for 2-3 hours to allow good clot formation and initial clot retraction. Beware taking samples over prolonged period of time in very cold temperatures as this can result in a problem with clot retraction. Samples if not immediately despatched should be refrigerated.
- Samples should be sent to Surefarm for onward delivery to the testing laboratory. We aim to turnaround results within 14 days of receipt although it may occasionally take longer than this.
What happens with Positives?
- Despite OIE Validation, Enferplex is not yet recognised for use by DEFRA. DEFRA will NOT pay any compensation for Enferplex Test positive animals. Farmers wish to choose to use the test so they can identify high risk animals to guide decision making and management to better control the disease.
- Any animals that test positive to non-validated tests must be restricted to the holding for life by the herd owner and can only move off to slaughter.
- All results are shared with the case vet at APHA so that APHA is fully informed and support the farmer throughout the process.
How does a farmer get permission to use the test?
- Once a farmer has decided to test their animals, please let Surefarm know TEL: +44 (0)1935 83203 or email office@surefarm.co.uk).
- They will need to inform their APHA case vet. Additionally, their own vet will need to ask permission from APHA to bleed the animals and use the test.
- Currently the test can only be used in herds which have undergone a gamma-interferon test.
Cost
- Please contact the office for current costs on +44 (0)1935 83203 or email office@surefarm.co.uk
