Methodology

The test sensitivity was assessed using serum samples from cattle that were positive by the SICCT test, IFNg test, presence of visible lesions at post mortem, culture of M. bovis, or a combination of these. The blood samples were taken approximately 10-30 days after SICCT to take advantage of the known ‘boosting’ effect of the injection of PPD. A high sensitivity and high specificity settings of the test were established by adjusting cut-offs for eradication or screening purposes respectively. The specificity was determined using sera from herds that were deemed to be free of bTB on the basis tuberculin test history, long term freedom from bTB.

Results

Diagnostic specificity:

Does tuberculin testing affect the specificity of the test?

When the antibody test is performed without any prior skin test, it relies on the presence of detectable antibody in already present in blood, and therefore unlike the skin and interferon gamma tests it is not being used as a memory recall test. However, it has been found that antibody responses usually receive a boost from the PPD-B given in following skin testing. This is most probably due to B memory cells (B cells are the lymphocytes that secrete antibody) becoming activated by the PPD-B “boost” to secrete antibody. In this way, the test is converted into a “memory recall test” and the sensitivity of the test can be increased accordingly.

There has been a postulated concern that the administration of tuberculin itself induces the production of antibody by the animal and therefore animals that have not met the disease could be found as positive by antibody tests. We have conducted a large trial on 160 cattle to check whether this concern has any basis and have found NO effect of the multiple administration of tuberculin (4 short interval tests) on the likelihood that a bovine Tb free animal will test positive, i.e. there is no effect on the specificity of the test ¹.

Results from trial on use of tuberculin on test specificity

Cycle 1: 100% specificity at both TT2 and TT20 (161 animals)
Cycle 2: 1% specificity at both TT2 and TT20 (160 animals)
Cycle 3: 4% specificity at both TT2 and TT20 (159 animals)
Cycle 4: 4% specificity at both TT2 and TT20 (159 animals)

Potential for cross reaction with Mycobacterium avium subsp. paratuberculosis

To analyse the cross-reactivity between MAP and M. bovis antibodies in the Enferplex Bovine TB antibody test, samples were obtained from animals naturally infected with MAP from Bovine TB free herds in UK, Ireland, Netherlands, Spain and USA. Negative controls samples (n = 1118) were obtained from Bovine TB free herds in the UK including Scotland (OTF Status) that were antibody negative by the ID.vet ID Screen Paratuberculosis Indirect ELISA. Negative samples were also obtained from MAP antibody negative animals (n = 438) from Norway

The results are consistent with what would be expected when using the Enferplex Bovine TB antibody test at the high sensitivity setting (97.8%) and high specificity setting (99.8%) for the UK and Ireland in a Bovine TB-free population and indicate that there is minimal or no cross-reaction between MAP antibodies and M. bovis antigens used in the Enferplex Bovine TB antibody test.

Diagnostic sensitivity:

Kappa analysis

Relative Sensitivity in SICCT or IFNg negative in contact animals from bTB infected herds

Diagnostic likelihood ratio analysis

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).

Relationship between the number of antigens recognised by antibodies and VL in cattle

The results show:

The diagnostic specificity is 98.4% and 99.7% using the high sensitivity and high specificity settings of the test respectively.
The diagnostic sensitivity is 93.9% in M. bovis culture positive animals and the relative sensitivity was between 90.0 – 96.1% depending on the comparator used at the high sensitivity setting.
Kappa values showed almost perfect agreement between the test results and diagnostic comparators used.
Positive and negative diagnostic likelihood ratios of 56.4 – 58.8 and 0.060 – 0.100 were obtained respectively using three different comparators indicating that the test provides good diagnostic lesions evidence of the infection being either present or absent respectively (Caraguel & Colling, 2021).
The test detects infected animals missed by the SICCT and IFNg tests.
There is a good correlation between the number of antigens recognised by antibody and the presence of visible lesions in SICCT positive, IFNg positive or in-contact SICCT negative animals.
The use of different antigen rules might allow a risk based approach to managing bTB infection in breakdown herds, similar to current methods based on high antibody titres used to control Johne’s Disease.

Significance/Impact:

Enferplex Bovine TB Antibody test shows high sensitivity and specificity and detects infected animals which are missed by SICCT and IFNg tests. This, along with the ease of use of the test compared to skin or IFNg tests, could prove useful in managing bTB outbreaks.
The correlation between the number of antigens recognised by antibody and the presence of visible lesions suggests that a risk based approach could be adopted to identify animals mostly likely to be shedding bacteria. This could be used to remove selectively high risk animals in a way that is sustainable when there are large numbers of seropositive animals.

Reference: Caraguel & Colling, 2021. Rev. Sci. Tech. Off. Int. Epiz., 40 (1), 299-309).