Brief description of current bTB status and diagnostic strategies in various countries
In the EU, the overall prevalence of bTB is currently slightly increasing. In 2007, 0.53% of cattle herds were bTB positive compared to 0.48% in 2006 (Anon, 2009c). Both officially bTB-free (OTF) and non-OTF countries reported an increase in the proportion of bTB positive cattle herds. Ireland and the United Kingdom are currently facing the highest herd prevalences in Europe (4.37 and 3.27%, respectively, Anon, 2009c). In Great Britain, the incidence of bTB is increasing (Anon, 2009e). Eradication of bTB is expected to take at least 20 years (Anon, 2009b). In face of a reservoir of infection in badgers, vaccination will likely become a major component of Great Britain`s bTB eradication strategy. Recently, a field trial evaluating the practicalities of delivering an injectable M. bovis BCG vaccine for badgers has been initiated in England, with licensing expected in mid-2010 and that of an oral vaccine probably in 2014 (Anon, 2009b). Vaccine strategies for cattle in the United Kingdom are also under consideration (Anon, 2007a). Complete eradication is not only difficult to achieve in high prevalence countries but also in countries with low proportions of bTB-infected herds (Anon, 2009c). Even in some OTF countries bTB prevalences are currently slightly rising. Trade and wildlife reservoirs both in non-OTF and OTF countries are major factors for re-infections of and spillover to livestock.
New Zealand undertook a great effort over the last 15 years to eradicate bTB, and the herd point prevalence was reduced from 2.4% in 1993 to 0.35% in 2004 (Ryan et al., 2006). Complete eradication, however, has failed so far, most likely because of the presence of wildlife reservoirs. Attempts to halt disease transmission from wildlife to livestock by oral vaccination of possums with lipid-formulated M. bovis BCG are currently under evaluation (Ramsey et al., 2009). A recent field trial demonstrated that oral vaccination with BCG significantly protected possums against natural exposure to M. bovis (Tompkins et al., 2009). Vaccination is being considered as a possible adjunct to the current practice of reducing the population of possums (annual costs of possum control accounting for US$ 30 million; Anon, 2005) to hasten the eradication of the disease from wildlife reservoirs.
In the USA, the bTB eradication campaign has been highly successful. The prevalence of bTB has decreased from 5% in 1917 to <0.001% in 2009. Eradication, however, remains elusive and new strategies are proposed to specifically address the continued challenges of imported cases of bTB from Mexico and the presence of a wildlife reservoir (i.e. white-tailed deer) in Michigan and potentially Minnesota. These strategies include more stringent testing of cattle entering from Mexico, enhanced efforts to mitigate risks from wildlife, accelerating diagnostic test development to support increased surveillance, transition from a state-based to a regional or zoning classification system for designation of bTB status, and increasing the options for managing bTB-affected herds. These changes will most likely result in several opportunities for application of new testing strategies, particularly with pre-movement testing – both at the USA/Mexico border and for interstate shipment. Additionally, increased numbers of bTB-affected herds will be managed via a test and slaughter as opposed to a whole herd depopulation strategy.
In Canada, eradication efforts are ongoing based upon slaughter surveillance and test/removal strategies, similar to the USA. As with other countries, spillover from bTB-infected wildlife (e.g. elk in Riding Mountain National Park and bison in Wood Buffalo National Park) hinders eradication efforts (Nishi et al., 2006).
In Mexico, procedures equivalent to the USA bTB program were implemented in 2001 to enhance the capacity for export of beef cattle to the USA; however, focal areas of high prevalence still exist especially in dairy cattle. Additionally, bTB program activity varies greatly in Mexican states.
The current status of control efforts in Central and South America, Indonesia, Africa, and Eastern European countries is not clearly defined, yet bTB is a prominent concern for the livestock industries of many of these countries.
Primary screening for bTB in live cattle is performed using one of the variants of the TST: the caudal fold test (CFT), the (mid) cervical intradermal test (CIT), or the comparative cervical test (CCT). In addition, the interferon gamma (IFN-γ) assay is applied either as a confirmatory test of reactors to the CFT or CIT (serial testing), or in alongside TSTs to increase diagnostic sensitivity (parallel testing). Diagnostic strategies including requirements for removal of individual animals testing positive, whole herd depopulation, and pre-movement testing differ largely between countries and regions and are summarized in Table 1. In OTF countries, control programs are usually limited to passive surveillance by post-mortem examination of all slaughtered cattle. The following section gives an overview of current ante- and post-mortem testing tools and indicates modifications which could contribute to optimize disease control and eradication.
Tuberculin skin test (TST)
The TST represents the OIE prescribed test for international trade and constitutes a delayed type hypersensitivity test (Anon, 2008a). It measures dermal swelling primarily because of a cell-mediated immune response (CMI) three days after intradermal injection of purified protein derivative (PPD) in the skin of the caudal fold (CFT) or neck (CIT), respectively. The skin of the neck is regarded to be more sensitive to a tuberculin-related hypersensitivity reaction than the skin of the caudal fold. To compensate for this difference, higher doses of PPD may be used in the caudal fold. Because animals are frequently exposed to or infected with various non-tuberculous mycobacteria, cross-reactive responses to PPD-B may occur as many antigens contained within PPD-B are shared between non-tuberculous and tuberculous mycobacteria. The CCT is used to differentiate between animals infected with M. bovis and those sensitized to PPD-B as a result of exposure to other mycobacteria. Responses to bovine and avian tuberculins are compared according to OIE guidelines or those developed by national eradication programs (e.g. use of a scattergram in the USA).
Historically, the TST has been the primary antemortem test available to support bTB eradication campaigns. Advantages of the TST and reasons for its wide use are low costs, high availability, long history of use and, for a long time, the lack of alternative methods to detect bTB. Still, this test has many known limitations including difficulties in administration and interpretation of results, need for a second-step visit, low degree of standardization, and imperfect test accuracy (De la Rua-Domenech et al., 2006).
Performance of TST techniques may vary because of differences in tuberculin doses, PPD preparations, site of application, and interpretation schemes. In fact, disparate performances have been found in many international studies (Monaghan et al., 1994): 68–96.8% sensitivity and 96–98.8% specificity for CFT, 80–91% sensitivity and 75.5–96.8% specificity for CIT, 55.1–93.5% sensitivity and 88.8–100% specificity for CCT. Recent data from some countries or regions indicate a markedly reduced sensitivity of TST in certain situations. A study in Northern Ireland showed that as few as 59% of animals with confirmed M. bovis infection were detected by CCT in some herds (Welsh, M., and McNair, J., unpublished results). Similarly, herd (n = 42) sensitivity of CCT in fighting bulls in Camargue, France, was 58% (10.6% sensitivity for individuals, n ≥ 9000) with more than 80% of bTB cases detected upon subsequent slaughter inspection (Keck, N., unpublished results). Twenty-eight of the 42 herds contained at least one culture-positive animal and 142 animals had tuberculous lesions upon slaughter inspection. Immunosuppression caused by stress (handling of fighting bulls) or anergy because of an advanced stage of bTB possibly account for the low sensitivity of TST in some circumstances. These data may represent the lower end of the performance spectrum of TST and not average values. Still, a low sensitivity constitutes a serious problem for the sustainable success of control and eradication programs.
Worldwide, the M. bovis strain AN5 is used for bovine tuberculin production (details and also concerns are summarized in the supporting information). Tuberculin potency is estimated by biological methods (in vivo testing of tuberculin batches in sensitized guinea pigs or cattle against an international reference standard; Anon, 2008b). Standardization of tuberculins is critical for test accuracy. Variations in PPD production methods may lead to different antigenic profiles and therefore to qualitative differences between PPDs (Seibert and DuFour, 1940; Landi and McClure, 1969; Haagsma et al., 1982; Tameni et al., 1998). Moreover, the in vivo potency tests are inherently variable and of questionable precision, demonstrated by studies with repeated batch testing (Good et al., 2008). Importantly, a recent comparison of commercially available tuberculins has shown that if applied in a dose of 1 mg/ml, the majority of these tuberculins would not meet the required minimum dose of 2000 International Units (Bakker et al., 2009). In this study, potencies largely varied among the bovine tuberculins and, to a lesser degree, among the avian tuberculins. Marked variations were also found with regard to the relative potencies of tuberculin pairs (PPD-B and PPD-A) from the different manufacturers. As these tuberculins are used in current control and eradication programs, the marked variability of potencies may have direct implications for the diagnosis of bTB cases.
Improvements in the production and quality control of tuberculins would have the potential to enhance sensitivity and specificity of TST. In fact, improved and extended control of PPD activity after production by standardized methods resulting in higher reproducibility than in vivo guinea pig potency testing could markedly improve TST. Furthermore, more active and more specific tuberculins would be desirable. To date, limited progress has been achieved in this field, mainly because of the ill-defined nature of the antigens present in tuberculins as well as the complexity of PPD production.
Attempts to improve the TST by the use of defined antigens such as ESAT-6 (Pollock et al., 2003) have resulted in increased specificity compared to the PPD-based test (100 compared to 90%) but decreased sensitivity (82 compared to 86%). Only a high dose of the recombinant protein (400 μg) was able to induce these responses, which would make the test unaffordable. Co-administration of a synthetic lipopeptide resulted in induction of antigen-specific CMI using lower concentrations of ESAT-6. However, an adjuvant application harbours the risk of sensitization and constitutes, therefore, not a practical solution (Whelan et al., 2003). The combined use of ESAT-6, CFP-10, Rv3615c, and MPB83 in a cocktail of recombinant proteins with low protein doses (10 μg each) showed comparable sensitivity levels as the PPD-based TST in naturally infected cattle (Whelan et al., 2009). Encouragingly, a peptide cocktail containing 21 overlapping peptides of the same antigens (10 μg each) also enhanced sensitivity compared to recombinant proteins (Whelan et al., 2009). Additionally, an improved antigen cocktail (ESAT-6 and CFP-10 based) is currently being tested for use in humans (P. Anderson, personal communication) and application of these antigens to the TST for cattle is also currently under investigation (R. Waters, P. Anderson and K. Lyashchenko; research in progress). However, these promising results will need to be validated in large-scale field trials before such antigens can be used as part of control strategies.
Alternative tools for the read out of TST may improve its ease of use. Infrared thermography (IRT) is currently being evaluated in the USA to determine skin test reactivity without the need for direct measurement of skin thickness (Johnson and Dunbar, 2008). Preliminary data from 15 cattle sensitized with PPD-B, PPD-A, or nothing (control) indicate that 86% of cattle were correctly classified with infrared thermography compared to 80% with skin thickness measurements using callipers. In addition, infrared thermography may be performed 24 h after PPD injection when compared to 72 h with traditional reading; and, importantly, this technique may represent a more objective method. However, further studies are required to determine the specificity of the 24-h vs 72-h readings of infrared thermography as early arthus-type reactions may be falsely interpreted as positive at 24 h.
Interferon gamma assay
The IFN-γ assay (Rothel et al., 1990) is OIE listed as an alternative test for international trade (Anon, 2008a) and approved as a complementary bTB test by the United States Animal Health Association as described in USDA, APHIS, Bovine Tuberculosis Eradication, Uniform Methods and Rules (Anon, 2004a), and by the European Union (Anon, 2002). This in vitro assay is a laboratory-based test detecting specific cell-mediated immune responses by circulating lymphocytes. Briefly, the assay consists of two stages. First, heparinized whole blood is incubated with antigens (i.e., PPDs, specific antigens) for ∼18–24 h. Antigenic stimulation induces production and release of IFN-γ by predominantly T lymphocytes. Second, IFN-γ present in the plasma supernatants is quantified in a sandwich ELISA. With PPDs, a differential optical density (OD) value is determined by subtracting the OD value achieved with PPD-A stimulation from that of PPD-B, thereby, analogous to an in vitro comparative skin test. In general, most laboratories also include a no stimulation negative control (e.g. PBS or media) and a mitogen or superantigen positive control [e.g. pokeweed mitogen (PWM) or staphylococcal enterotoxin B (SEB)].
The IFN-γ assay (Bovigam®, Prionics, Switzerland) is being incorporated into bTB eradication programs in many countries (reviewed by De la Rua-Domenech et al., 2006; Vordermeier et al., 2008), either in a serial testing regime as confirmatory test after CFT to enhance specificity or in a parallel testing regime to enhance sensitivity of TSTs (Table 1). Advantages of the IFN-γ assay are its increased sensitivity, the possibility of more rapid repeat testing, no need for a second visit to the farm and more objective test procedures and interpretation in comparison to the TST. Limitations comprise a reduced specificity, high logistical demands (culture start is required within 24 h after blood sampling), an increased likelihood of non-specific response in young animals (owing to natural killer (NK) cell activity) and its high costs (reviewed by De la Rua-Domenech et al., 2006; Vordermeier et al., 2006), as well as the difficulties in the standardization of tuberculins already discussed in relation to the TST.
The combined use of the in vivo and in vitro CMI assays raises the question whether the IFN-γ responses are influenced by injection of PPDs for TST. Briefly, no significant effect can be observed after CCT (Doherty et al., 1995; Gormley et al., 2004; Coad et al., 2010). In contrast, injection of PPD-B for CFT boosts IFN-γ responses in cattle experimentally infected with M. bovis (Palmer et al., 2006). The boost was shorter than previously observed with sensitized cattle (Whipple et al., 2001) and lasted only from 3 to 7 days after the CFT. Importantly, there was no effect on the differential response to tuberculin and therefore no interference with the final assay interpretation. In naturally infected cattle, however, CFT-related boosting selectively increased the in vitro M. bovis PPD (PPD-B) response 3 days after CFT, resulting in an increased PPD-B response relative to the response to PPD-A (Coad et al., 2010). Additional studies are warranted to evaluate the influence of CFT on the IFN-γ responses (i) with tuberculous cattle exhibiting weak IFN-γ responses, (ii) with uninfected cattle, and (iii) to evaluate the effect in view of a possible depression of IFN-γ production.
Sensitivity and specificity of the IFN-γ assay have been estimated in a great number of international studies (reviewed by De la Rua-Domenech et al., 2006). Estimates of test sensitivity range from 73.0 to 100%, with a median value of 87.6%, and specificity from 85.0 to 99.6%, with a median of 96.6%. Variations in assay protocols may have resulted in disparate results in test accuracy between studies. More standardized procedures would be needed to strengthen reliability of screening tools within TB programs. In recent studies, parameters of the IFN-γ assay have been analyzed in view of defining a range of possible conditions (summarized in the supporting information). Importantly, assay conditions which have been identified to be of significant influence on the IFN-γ test performance and which have already been defined to optimize the assay (e.g. sample and culture conditions; validity controls and their interpretations) should be integrated in the commercial assay. Thus, users could refer to consolidated, standardized procedures at the implementation of the assay in their laboratory quality system. This would also increase the likelihood that the assay will be used in a more standardized way and it would facilitate national approvals of the IFN-γ (as it would not be efficient to repeat the validation of all relevant assay parameters in each country).
Many studies have demonstrated that the sensitivity of the IFN-γ assay is superior to that of the skin test (data from most relevant published studies and additional unpublished data are summarized in the supporting information). Concerns, however, exist about Bovigam specificity (Lauzi et al., 2000; Pollock et al., 2000; Palmer et al., 2006). The replacement of PPDs by defined antigens offers the greatest potential for the improvement of specificity (Pollock et al., 2000; Buddle et al., 2001). ESAT-6 and CFP-10 are outstanding diagnostic target proteins in the whole blood IFN-γ assay (Pollock et al., 2000; Vordermeier et al., 2001; Aagaard et al., 2003; Buddle et al., 2003; Waters et al., 2004; Vordermeier et al., 2005; Aagaard et al., 2006; Cockle et al., 2006). Results from a recent field trial in Great Britain indicate that a peptide cocktail composed of peptides from ESAT-6 and CFP-10 (E/C) achieved only a slightly better specificity (97.0%; Vordermeier, H., unpublished results) than the conventional PPD-based IFN-γ assay (96.6%; Anon, 2008f); IFN-γ sensitivities in this field trial were 91.0% for PPDs and 81.0% for E/C. The gain in specificity by the use of E/C may be especially striking in individual animals and in regions with a low bTB prevalence and in OTF countries. A study performed in Switzerland (OTF) testing a selected herd (n = 69) with a high percentage of non-specific reactors (i.e. false positives) in the PPD-based assay (specificity of 66.7%) resulted in an E/C-specificity of 97.1% (Vordermeier, H., and Schiller, I.; unpublished results), again, demonstrating the potential for improved specificity of the test when specific antigens are used in place of PPDs.
Defined mycobacterial antigens have not only the potential to increase specificity but also sensitivity: in a PPD-based IFN-γ assay the diagnosis of bTB may be masked by a high response to avian tuberculin superseding that to PPD-B, as may occur early after infection (Schiller et al., 2009c) or in animals co-infected with non-tuberculous mycobacteria such as M. avium ssp. paratuberculosis (Aranaz et al., 2006). As indicated earlier, however, overall results of current studies with defined antigens using ESAT-6 and CFP-10 demonstrated a reduced sensitivity compared to tuberculins, reduced by approximately 10% (Pollock et al., 2000; Buddle et al., 2003; Vordermeier et al., 2006; Vordermeier, H., unpublished results). Therefore, a wider range of antigens in addition to ESAT-6 and CFP-10 is needed for improving the sensitivity of specific antigen-based IFN-γ assays. In recent studies, promising antigen combinations for the IFN-γ assay resulting in improved sensitivity and specificity have been described (Cockle et al., 2006; Sidders et al., 2008; Schiller et al., 2009b). Importantly, Rv3615c and OmpATb were found to detect cattle with confirmed bTB but not responding to E/C. Recent data indicate a sensitivity of 89.0% and specificity of 97.0% for E/C combined with Rv3615c (Sidders et al., 2008; Vordermeier et al., 2009). These data have been achieved after 24-h storage of blood samples. A shortening of pre-culture time, however, has the potential to further increase sensitivity of a peptide-based IFN-γ assay. As shown with E/C in experimentally infected cattle, 24-h storage of blood samples resulted in slightly decreased OD values and reduced predictive outcome using E/C compared to 8 h storage (Whelan et al., 2004). This warrants further examination in field reactors and with additional defined antigens.
High logistical demands limit the use of the IFN-γ assay. Currently, blood samples need to be transported to a laboratory and to be processed within 24 h. An “in-tube” or “in-plate” stimulation device allowing rapid stimulation of lymphocytes after blood collection would possibly have a positive impact on diagnostic sensitivity using defined antigens for stimulation. Such a device would surely be beneficial to overcome logistical difficulties and to reduce costs now required for “express transportation”.
Current costs of the IFN-γ assay constitute a major disadvantage in relation to TST. Regarding full cost calculations of TST (e.g. $7-10 in the USA (M. Dutcher, personal communication, 2007) and £7.5 in GB (R. De la Rua-Domenech, personal communication, 2007), the difference between in vitro and in vivo CMI assays might be smaller than anticipated, particularly considering rising costs associated with on-farm visits. Yet constraints in terms of costs should be mitigated to make the IFN-γ assay more widely available. Increased use of the assay might allow high scale production at a lower price. Importantly, additional cost reduction could be achieved by applying automation at production and application (i.e. in diagnostic laboratories). Modification of the ELISA as a rapid ELISA would allow further reduction of laboratory costs.
The current interpretation of the Bovigam assay relies exclusively on the OD values of specific (PPD-based) stimulation and non-stimulation (PBS), without considering standards correcting possible plate-to-plate variations. The kit positive control exhibits a strong positive control and may produce a wide range of OD readings (Jungersen et al., 2002; Robbe-Austerman et al., 2006). Therefore, a different control would be needed to calibrate plate-to-plate variations. Alternative read-outs (such as percentage positivity) based on a standardized positive control should be considered. An alternative calculation of the cut-off may contribute to improve test accuracy.
The IFN-γ assay is generally applied to animals of more than 6 months of age. The reason for this age restriction is a high likelihood of non-specific responses to mycobacterial antigens in young animals triggered mainly by natural killer (NK) cells (Olsen et al., 2005). This phenomenon may possibly be overcome by the depletion of NK cells, thus allowing application of the IFN-γ assay with samples from animals <6 months of age (H.M. Vordermeier and A. Storset, research in progress).
In summary, improving specificity by the replacement of PPDs by defined antigens for stimulation, their application as an in-tube/in-plate stimulation device, in combination with a modified interpretation/cut-off and cost reduction may represent useful developments for the IFN-γ assay. Thus, the assay may be adapted to provide a highly specific and sensitive screening test for use as a stand-alone test or in conjunction with other screening tests. In addition, a multispecies IFN-γ assay for non-bovine species, such as camelids, cervids, dogs, and cats, would be a welcome tool for bTB screening and control in those species and for overall bTB control.
Post-mortem diagnosis of bTB
Abattoir surveillance with lesion detection during commercial slaughter is used as cost-efficient method for passive surveillance of bTB both in OTF and in non-OTF countries, in the latter to supplement live cattle testing. The finding of a tuberculous animal at slaughter initiates an investigation through TST of the herd of origin and any other potentially exposed animals (Whipple et al., 1996; Olea-Popelka et al., 2008). The success of such investigations is highly variable. In the USA, because of the lack of uniform animal identification regulations combined with inconsistent record-keeping, only 50–70% of such investigations result in identification of the herd of origin and in finding all exposed animals (Kaneene et al., 2006). In Australia, which has successfully eradicated bTB, meticulous animal identification was credited as a major factor in successful eradication (Cousins and Roberts, 2001; Radunz, 2006). In the British Isles, national cattle tracing systems allow accurate back-tracing following identification by meat inspectors of any suspect granuloma in commercially slaughtered cattle. The herds of origin of such animals are placed under precautionary movement restrictions pending confirmation of bTB in slaughter lesions by histopathology, culture and/or PCR.
Generally speaking, lesion detection exhibits a major lack in sensitivity. The sensitivity of abattoir surveillance may be as low as 28.5%, as indicated by a recent study in the USA (Anon, 2009a). Possibilities to improve the sensitivity of post-mortem detection of bTB may be limited. Continuous education and training of slaughter inspectors are certainly of major importance. In addition, the combined use of liquid and solid culture media has been reported to improve culture sensitivity (Hines et al., 2006). The addition of assays such as PCR detection of M. bovis DNA from formalin-fixed specimens has further enhanced some surveillance programs (Miller et al., 2002). PCR assays to detect MTB complex bacteria are currently less sensitive than culture techniques. Therefore, important further steps would be to improve PCR sensitivity and to standardize PCR methods.
Genotyping of bacterial isolates or PCR products is increasingly becoming a standard tool for epidemiological disease control and eradication. Distinguishing M. bovis strains on a molecular basis provides important insights into the sources of infection and identification of practices or environments which may aid the spread and maintenance of tuberculosis. Importantly, transmission routes between livestock and wildlife may be identified by strain typing. In addition, transmission routes of bTB within livestock via animal movements become evident, a prerequisite for targeted disease control aiming at testing all potentially exposed animals. More details for post-mortem diagnosis of bTB (lesion detection, culture, PCR, and strain typing) are included in the supporting information.