Introduction

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a zoonotic disease that is endemic in many countries worldwide, impacting on animal health, welfare, productivity and trade. In Great Britain (GB), despite a compulsory test-and-slaughter programme introduced in 1950, the disease is now endemic in West and South-west England and also in Wales (although Scotland was declared officially bTB free in 2009). Bovine TB has been described as GB’s “biggest endemic animal health issue” [1] and in 2010–2011, the Department for the Environment, Food and Rural Affairs (Defra) spent over £91 million on bTB control in England [2]. The imperfect sensitivity (the probability that an infected animal is detected by the test) of the current diagnostic field test is one plausible contributor to the sustained increase in the incidence of bTB in GB over the last 25 years.

The intradermal tuberculin test is recognised by the World Organisation for Animal Health (OIE) and the European Commission as the primary screening test for detection of bTB in cattle [3], [4]. The application of this test, supplemented with slaughterhouse surveillance and movement controls on infected and suspect herds, underpins many bTB control programmes worldwide and, in the absence of wildlife reservoirs for M. bovis, has led to the successful eradication of bTB in many countries [4]–[6].

Two variants of the intradermal tuberculin test are in use today: the single intradermal tuberculin test, which measures a cell-mediated delayed-type hypersensitivity response to injection of M. bovis tuberculin in the caudal skin fold (e.g. New Zealand, USA) or the skin of the mid-cervical region (e.g. most European Union Member States); and the single intradermal comparative cervical tuberculin (SICCT) test, which compares immune responses to M. bovis (bovine) and M. avium (avian) tuberculins in the cervical region. In GB, Ireland and Portugal, the more specific, but less sensitive, SICCT test is used to minimise the likelihood of false positive results caused by exposure to cross-reacting mycobacteria [7], [8].

Nearly half of all cattle herds in GB are tested for bTB every year, 41% are tested every four years and the remainder tested at 2- or 3-yearly intervals (Defra, unpublished data, 2012). Cattle with a positive SICCT test result (known as “reactors”), or those from which M. bovis is cultured from suspect bTB lesions detected during commercial slaughter, trigger a bTB herd “breakdown”. All reactors are compulsorily slaughtered, whilst the affected herd is placed under movement restrictions and then re-tested at a minimum interval of 60 days (short-interval testing) until a series of tests with negative results is obtained. The number of short-interval tests, and their interpretation, is largely (but not exclusively) dictated by the detection of evidence of M. bovis infection at post-mortem examination (VLs) or on culture.

The sensitivity of the SICCT test is a measure of its ability to detect infected animals. Knowledge of SICCT test sensitivity is fundamental to understanding the potential success and limitations of bTB control programmes. Reduced test sensitivity increases the risk of within-herd persistence of infection, recurrent breakdowns, onward transmission through cattle movements and, potentially, infection spill-over into local wildlife reservoirs. Given the chronic nature of bTB and the relative insensitivity of post-mortem techniques used to confirm the presence of M. bovis, the true infection status of every animal cannot be determined and thus sensitivities have been estimated relative to post-mortem confirmation methods, namely detection of typical lesions of bTB (VLs) and/or laboratory culture of M. bovis.

SICCT test sensitivities in different countries and settings have been reported to range from 75.0–95.5% [8] and 77–95% [9], but for the majority of the studies from which these estimates were reported or derived, post-mortem data for a representative group of SICCT test-negative animals from the same population were lacking. Indeed, post-mortem data are rarely available for SICCT test-negatives, as normally only reactor animals are compulsorily slaughtered during herd breakdowns. Conducting experimental studies to collect such data would be expensive, labour-intensive and suffer from problems of extrapolation to the field situation. Without these data, the true proportion of infected animals not detectable by the test (false negatives) remains unknown, which could substantially impact on estimated values.

Whole herd slaughter (“stamping out” or depopulation) is a last-resort control option in GB, reserved only for severely or chronically infected herds in which repeated SICCT testing (occasionally supplemented since 2003 by interferon-gamma parallel testing [10]) has failed to resolve the breakdown. Depopulated herds provide a rare opportunity to improve estimates of relative SICCT test sensitivity (rSe), since they generate data on both SICCT test-positive and SICCT test-negative animals. In this study we analyse data from 16 such herds in GB, comprising a total of 748 SICCT test-positive and 1031 SICCT test-negative animals slaughtered between 1988 and 2010.

Results

rSe Calculations

The overall rSe of the SICCT test at severe interpretation for the 16 slaughtered herds was estimated to be 85% (95% CI: 78–91%) from the model fitted with herd-level effects, and 84% (95% CI: 81–87%) when calculated assuming no herd-level heterogeneity (Table 1 and Figure 1A). Although data were available on skin test positivity or negativity for all 16 herds, data on exact skin test reaction sizes were only available for 11 of these (the five herds for which these data were not available had a breakdown date prior to September 2000, before which these data were not routinely recorded). When recalculated at the standard interpretation, the overall rSe was estimated to be 81% (95% CI: 70–89%) from the model fitted with herd-level effects, and 78% (95% CI: 74–81%) when calculated without (Table 1 and Figure 1B). This estimate is based on 11 herds only. The numbers of reactors at both interpretations are provided in Tables S2 and S3.

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Figure 1. Overall mean relative Single Intradermal Comparative Cervical Tuberculin test sensitivity (rSe) estimates for each herd.

Estimates are shown for severe and standard test interpretation, derived from logistic regression models fitted with and without herd-level effects. The solid lines represent the mean estimates, and the dashed lines represent the 95% credible intervals. The red lines are from the model including herd-level effects, and the blue lines are from the model excluding herd-level effects.

https://doi.org/10.1371/journal.pone.0043217.g001

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Table 1. Overall mean relative Single Intradermal Comparative Cervical Tuberculin (SICCT) test sensitivity (rSe) estimates (%).

https://doi.org/10.1371/journal.pone.0043217.t001

Figures 1A and 1B show the between-herd heterogeneity in the overall rSe estimates at severe and standard interpretation, obtained from the hierarchical model. Overall adjusted rSe estimates were slightly higher when the herd-level effects were included at both severe and standard interpretation, as a result of accounting for the herd-level heterogeneity. Although exceeding 90% in some herds, the estimated relative sensitivity of the test at severe interpretation was as low as 60% in others (Figure 1A). At the standard interpretation, one herd had a sensitivity estimate as low as 52% (Figure 1B).

Since we do not have SICCT test results for those animals that were detected via slaughterhouse surveillance, we conducted a sensitivity analysis assuming in the first instance that all of these animals would have been SICCT test positive, and in the second case assuming they would have been SICCT test negative. The results from these two extreme scenarios are shown in Table S4 and show negligible impacts on the estimates.

Discussion

The SICCT test remains the primary bTB surveillance tool in several countries worldwide. Elsewhere, it is used as a second-line confirmatory (serial) test of caudal-fold and mid-cervical test reactors. The application of this test is central to restoring the officially TB-free status of infected herds in GB. A dearth of post-mortem data for SICCT test-negative animals has hindered the generation of reliable estimates for SICCT rSe. Depopulation, a radical bTB control method for severely affected herds, generates data for positive and negative-testing animals, providing a rare opportunity to improve current rSe estimates. Data for 16 cattle holdings, depopulated in GB between 1988 and 2010 due to bTB, were analysed calculating rSe relative to post-mortem detection of typical lesions of bTB (VLs) in slaughtered animals. The overall rSe was estimated to be 85% (95% CI: 78–91%) at the severe interpretation (16 herds) and 81% (95% CI: 70–89%) at the standard interpretation (11 herds) after accounting for herd-level heterogeneity.

Previously-reported median sensitivity estimates of the SICCT test at the standard (77.5%) and severe (92.2%) interpretation, were derived from a range of published studies that varied in location (some outside GB), tuberculin source and dosage [8] (these values were reported as 80% and 94% respectively; however, when the Costello et al. estimate [11] was re-examined, it was noted that this should have been included in the estimate for the severe interpretation, and thus the values have been recalculated to take account of this). For the five studies [11]–[15] where the test was performed as in GB, estimates ranged from 75.0–95.5% at the standard interpretation [8]. When the estimate from Costello et al. (1997) is excluded (since it was not interpreted at standard interpretation–as discussed previously), the range remains the same. SICCT test sensitivity ranged from 77–95% in an earlier review [9]: three of these studies [13]–[15] were included in the review by de la Rua-Domenech et al. (2006), and the remaining four studies [16]–[19] were not comparable, employing tuberculin extracted from M. tuberculosis that was replaced in GB by the current (M. bovis-derived) tuberculin in 1975 [9], [13].

Critically, the majority of these previous studies have lacked post-mortem data for SICCT-negative animals, which could plausibly have inflated the estimated sensitivities by reducing the denominator. However, one study carried out in Ireland [11], did slaughter the entire SICCT-tested study population (2528 animals), including all test-negatives, and also calculated sensitivity relative to VL detection (rSe). The rSe reported was 90.9% (severe interpretation), which is higher than our overall estimate although just inside our upper credible interval limit (overall mean 85%, 95% CI: 78–91%). The avian and bovine tuberculins used in the Costello study, manufactured in Lelystad (Netherlands), have been shown to be associated with a slightly higher probability of confirmation (VLs and/or culture) when compared with tuberculin manufactured in Weybridge (UK) [20], the SICCT test antigen used in the majority (69%) of the herds in our study (for the remaining herds, 25% herds used Lelystad and 6% herds [1 herd] had the tuberculin type classified as undetermined). Costello et al. also calculated sensitivity relative to VL detection, and although their higher rSe could have been due to a reportedly more thorough post-mortem inspection, this was not reflected in the percentage of reactors that had VLs: 41% of reactors and IRs in their study compared with 40% of reactors nationally in GB [21], although inclusion of IRs here could have artificially lowered this figure due to the proportion of VL animals being lower in IRs than in reactors (in both GB and Ireland).

The true infection status of all individual animals is impossible to determine. Sensitivities calculated in this study from naturally-infected herds were relative to detection of gross bTB lesions post-mortem. While in some respects our estimates may provide a lower bound for the true sensitivity, the impact of well-developed disease on lesion visibility in some of these herds is hard to determine as there may be biases in the depopulation data when compared to other situations, which could influence the rSe estimate in an unpredictable manner.

Any culture data that were available from the selected herds were not combined with the post-mortem VL data for rSe calculations, as there may have been bias towards reactors being more likely to be cultured than direct contacts. Sensitivity of VL detection, relative to culture positivity, was explored for two depopulated herds that had VL/NVL and culture data for >90% of animals slaughtered. The overall rSe derived from a logistic regression model was 90%, suggesting that VL detection is a good measure of infection status, as defined by detection by culture. This is supported by the fact that, in GB, an estimated 95% of all VL reactors yield M. bovis on culture [21].

Although in our calculations we do not assume that visible lesions are synonymous with infection, the majority of suspect lesions do indeed culture M. bovis. In 2011, around 75% of suspect granulomas detected during routine slaughterhouse surveillance (i.e. not in reactor animals identified during a breakdown) cultured M. bovis (Defra, unpublished, 2012). However, this proportion of culture-positive lesions is likely to be much higher in severe bTB breakdowns with previously confirmed infection and a high prevalence of within-herd infection. VLs have been shown to develop as early as 17 days in experimentally-infected calves infected with doses to mimic naturally-occurring infection [22], (albeit in an experimental situation where postmortem inspection for visible lesions was conducted at a higher level of detail with serial slicing of target organs and lymph nodes), which is earlier than the minimum time taken to develop a detectable SICCT test response, reported to be 3–6 weeks after infection [8].

Mechanisms underlying imperfect skin test sensitivity include factors relating to the tuberculin source and potency, the operator’s adherence to the prescribed test protocol [23]–[25], the stage of M. bovis infection in the tested animal (very early or too advanced to mount a detectable hypersensitivity response to tuberculin) [9], and factors that can mask or suppress the cell-mediated immune response to tuberculin, such as anti-inflammatory drugs and simultaneous infections with other pathogens. In addition, a period of transient and partial desensitisation has been shown to occur shortly after tuberculin injection [26] and, although EU protocols recommend a minimum interval of 60 days between sequential skin tests [27], recent evidence has shown that repeated SICCT testing at 60-day intervals can result in a progressive reduction in the comparative bovine-avian tuberculin response in naturally infected cattle [28].

Although the rSe at standard interpretation calculated in our study is in line with previously reported estimates, the mean rSe at severe interpretation calculated by Costello et al. (1997) only just fell within the upper credible interval limit of our estimate. Although the overall sensitivity generated from our study was lower at standard compared to severe interpretation (as one might expect), the 95% CIs overlapped considerably. This may have been due to the small numbers of animals reclassified during the change in interpretation (possibly limited by the number of herds in the study). Although this may suggest that in the field there may be little additional gain in applying the more severe interpretation, the small number of herds in this study should be considered when attempting to extrapolate the findings. The degree of between–herd heterogeneity for the rSe estimates also suggests that caution should be exercised in estimation from single herd breakdowns.

Veterinarians, farmers and government place rely on the SICCT test to detect bTB through surveillance testing and to clear infection from breakdown herds. It should be noted that our rSe estimates (and those from previous studies) are calculated at the animal-level, which provide a lower bound for the herd-level sensitivity (the ability to detect infected herds as part of a surveillance programme). The results suggest that even with the increased sensitivity at the more severe interpretation, undetected infected cattle may remain in some breakdown herds that are officially released from movement restrictions.

Almost a quarter of all breakdowns that are de-restricted after a programme of repeated skin testing at 60-day intervals with negative results recur in the same herd within 12 months, and 38% recur within 24 months [29]. Re-infection from badgers has been postulated to be a key mechanism behind persistence of infection in cattle herds in areas of endemic bTB incidence [30] and is anecdotally thought to be responsible for recurrent breakdowns in those areas. However, the failure of the SICCT test to detect every infected animal should also be recognised, as well as considering how the test is applied, both nationally as well at as the individual-herd level.

In many parts of GB the skin test has worked well as a bTB surveillance and eradication tool, with Scotland, for instance, gaining officially TB-free status in 2009 [31] in the apparent absence of a wildlife reservoir of M. bovis. As herds became attested and more counties became accredited TB free in the 1960s and 1970s, TB herd testing frequency in GB was gradually relaxed since the blanket enforcement of annual testing in the 1950s. It is only over the last 10–15 years that the herd testing coverage has been increasing year-on-year, with the whole of Wales and West and SW England now on yearly testing, reflecting the endemic bTB situation in those regions of GB [32]. The sensitivity of the overall bTB testing regime in GB has recently been boosted by the wider deployment of the interferon-gamma blood test in known infected herds and the introduction of compulsory pre-movement skin testing since 2006 [33], by the earlier removal of cattle with repeat inconclusive SICCT test results since 2010 [34] and by additional testing of high risk breakdown herds since 2011. Substantial efforts continue to be made to address gaps in the bTB eradication programme in GB and enhance the overall ability to detect and control infection (including training and auditing of operators conducting skin tests on farms).

Materials and Methods

A national animal disease surveillance database (VetNet) recorded information relating to bTB testing and herd breakdowns in GB [35]. (This has now been replaced by a new IT system [Sam] since September 2011.) For each herd, this includes the ante-mortem SICCT test results for animals classed as reactors (R: positive), or inconclusive (IR: borderline result) during a bTB breakdown, and the post-mortem results i.e. whether typical lesions were detected on post-mortem inspection (or during further laboratory examination) for all cattle slaughtered to control the infection. In addition, these data are also available for at-risk cattle compulsorily removed as SICCT test-negatives (direct contacts), as is the case in partial or whole-herd depopulations.

Supporting Information

Acknowledgments

The authors are grateful to the Animal Health and Veterinary Laboratories Agency for compilation and provision of VetNet data and for systematic recording of bTB test and post-mortem examination data on VetNet.