This section summarises the advances made by the Animal and Plant Health Agency (APHA) in the validation and adoption of new supplementary serum antibody tests for the detection of Mycobacterium bovis infection (tuberculosis [TB]) in deer. This work builds upon previous work by APHA and elsewhere that has demonstrated the usefulness of serology for the diagnosis and control of TB in different taxonomical groups of mammals. Used in conjunction with the tuberculin skin test whenever possible, antibody tests represent a proportionate, relatively simple and inexpensive way of improving the diagnosis of TB in non-bovine species, where the numbers of affected herds remain relatively small, although severe TB incidents are occasionally detected.

TB in deer is a notifiable disease in Great Britain (GB), which means that there is a legal obligation to report the suspicion of disease to APHA. There is no routine statutory TB surveillance testing programme in GB for captive deer, which have largely been regarded as spillover hosts for M. bovis (Crawshaw et al. 2013). Limited targeted TB surveillance takes place in farmed deer herds that are contiguous to (or co-located on) premises affected by lesion- or laboratory-confirmed TB breakdowns in cattle herds, or more rarely where M. bovis infection has been confirmed in other (non-bovine) farmed animals. Therefore, TB surveillance in deer is generally by postmortem meat inspection in abattoirs (farmed deer) or approved game-handling establishments (park deer). This relies on the detection and notification of suspicious visible lesions (VLs) of TB in culled deer, which are then submitted to the APHA TB reference laboratories for confirmatory testing and mycobacterial speciation. Historically, this has involved mycobacterial culture to identify M. bovis, which can take six or more weeks. From 30 March 2022, a validated polymerase chain reaction (PCR) test (Morris et al. 2023) was rolled out to allow for more rapid identification of the bacterium in non-bovines and the implementation of subsequent TB control measures.

The powers of veterinary inspectors to control TB (infection with M. bovis) in deer are underpinned by various Statutory Instruments: The Tuberculosis in Animals (England) Order 2021; The Tuberculosis in Specified Animals (Scotland) Order 2015; The Tuberculosis (Wales) Order 2011. However, the complexity of rounding up and presenting captive deer (especially park deer herds) for antemortem TB testing has sometimes resulted in TB tests becoming overdue and movement restrictions remaining in place indefinitely. Where there is little or no impact upon the business models of restricted premises, there is also little incentive to proactively engage in TB testing to lift those restrictions.

TB testing for deer, where implemented, e.g. following a confirmed M. bovis infection, has been reliant upon the Single Intradermal Cervical Comparative Tuberculin test (SICCT) only, which compares the response to bovine and avian tuberculins (purified protein derivative bovine [PPDB] and purified protein derivative avian [PPDA], respectively) and provides a result based upon the level of PPDB-bias as a measure of the M. bovis-specific response. The SICCT is not validated for deer under GB conditions, although a scientific opinion of the European Food Safety Authority (EFSA 2008) summarising the SICCT test characteristics suggests a high sensitivity (85.8% [95% CI: 83.4–88.0]) and specificity (97.7% [95% CI: 97.2–098.2]). The need to hold and restrain deer twice (i.e. first for tuberculin injection and then for the reading of any skin reactions 72 hours later) and the inherent difficulty in interpreting a positive, negative or inconclusive test result under often difficult field conditions has led to some mistrust in the SICCT ability to correctly identify infected deer. Consequently, studies have been undertaken to evaluate a range of supplementary blood tests that could increase the sensitivity and/or specificity of infection detection. The need for supplementary blood-based tests for deer in GB was recognised in the review of the Bovine TB Strategy for England (Godfray et al. 2018).

One well-established benefit of the skin test is the transient anamnestic boost to specific antibody titres induced by the intradermal injection of bovine tuberculin (PPDB) in M. bovis-infected individuals. This effect has been observed across species, e.g. cattle, camelids and goats (see summary by de la Rua-Domenech and Rhodes 2021), resulting in an enhanced antibody test sensitivity where blood samples are taken within an optimal ‘window of opportunity’ following the injection of PPDB. To take advantage of this anamnestic boost and achieve the highest possible antibody test sensitivities, blood samples should be taken 10–30 days after an injection of PPDB during skin testing.

A Department of food and rural affairs (Defra)-funded study (SE3315) carried out by APHA in 2019–2021 evaluated antibody tests for M. bovis infection in deer, with the expectation that a suite of validated antibody tests could aid the development of more effective and acceptable TB surveillance strategies for herds of farmed and park deer under TB restriction (Barton et al. 2023).

The tests evaluated by APHA were those with published credentials for TB detection in deer and, importantly, readily accessible with the potential to be incorporated into the deer TB testing portfolio within the short to medium term. The following tests were investigated:

• The lateral flow Dual Path Platform (DPP) VetTB assay for Cervids™ (Chembio Diagnostic Systems, USA).
  
• An in-house comparative PPD enzyme-linked immunosorbent assay (ELISA).
  
• The Enferplex Cervid TB antibody assay (Enfer Scientific Laboratories Inc., Nass, Ireland).
  
• The IDEXX M. bovis ELISA (IDEXX Laboratories Inc., USA).

The DPP VetTB test has a stated overall sensitivity of 74.3% and specificity of 97.4% for cervids in the United States (Palmer 2007; Waters et al. 2011; Nelson et al. 2012; Buddle et al. 2010). Supplementary use of the DPP VetTB test with the skin test on fallow deer (Dama dama) in Spain was shown to increase the overall diagnostic sensitivity from 76% (skin test alone) to 97% (skin test followed by the DPP VetTB test; Boadella et al. 2012). The DPP VetTB test was one of the antibody tests used to resolve a TB incident on a large red deer farm in the north of England (Busch et al. 2017). The test was also applied to postmortem samples collected from culled wild deer on Exmoor National Park, to assess TB seroprevalence (Collard 2023). The DPP VetTB test for cervids is a qualitative test, with a positive test denoted by a visible response to either of two target antigens (MPB83 and ESAT6/CFP10).

In New Zealand, the ELISA TB (ETB) test, essentially a comparative PPD antibody ELISA, is used for serial retesting of farmed deer that react to the single intradermal skin test (SIT) – the SIT having a higher sensitivity but lower specificity relative to the SICCT. The systematic combined application of both tests has been instrumental in the drastic reduction of infected deer herds and test reactor numbers in New Zealand between 2005 and 2021 (see Neill 2022). The ETB test (kindly performed in the New Zealand laboratory) was also part of the enhanced TB testing regime used in a herd in Yorkshire (Busch et al. 2017). For project SE3315, APHA developed an in-house comparative PPD ELISA test based on the paired PPDA and PPDB kit routinely used for skin testing of cattle and other livestock species (Prionics, Thermo Fisher Scientific). Like other comparative PPD tests (skin, gamma-interferon), the M. bovis-specific response is assessed by subtracting the response to PPDA from the response to PPDB (∆PPD) with the ∆PPD values statistically assessed.

Various Enferplex antibody tests (Enfer Scientific, Ireland) have been successfully applied to cattle (Whelan et al. 2010; Casal et al. 2014; O’Brien et al. 2023), goats (O’Brien et al. 2017) and camelids (Rhodes et al. 2012). Here, we included the Enferplex Cervid TB Assay, with tests carried out at the Enfer Scientific Laboratory in Ireland, because the test was not available at APHA. For a positive test result, any 2 of 11 antigens must be positive (Rv2975 synthetic peptide p6; PPDB; recombinant Rv2873; recombinant Rv2975; Bovine TB cocktail; recombinant Rv3875–Rv3874 fusion protein; recombinant Rv3874–Rv3875 fusion protein; recombinant Rv3616c; recombinant Rv3881c; recombinant Rv3803c; recombinant Rv1860). Two test readouts are provided: high sensitivity and high specificity.

The IDEXX M. bovis ELISA is a commercial test kit for cattle, detecting antibodies to a cocktail of MPB83 and MPB70 proteins (Waters et al. 2015). This test, while used by APHA for cattle testing, is also amenable to adaptation to non-bovines including camelids (Rhodes et al. 2012), deer, pigs (Barton et al. 2023) and badgers (APHA 2023; McGill et al. 2022).

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