ARTICLE IN PRESS Research in Veterinary Science ■■ (2014) ■■–■■

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Current ante-mortem techniques for diagnosis of bovine tuberculosis Javier Bezos a,b,*, Carmen Casal b, Beatriz Romero b, Bjoern Schroeder c, Roland Hardegger c, Alex J. Raeber c, Lissette López d, Paloma Rueda d, Lucas Domínguez b,e a b c d e

MAEVA SERVET SL, C/ de la Fragua 3, 28749, Alameda del Valle, Madrid, Spain Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain Prionics AG, Wagistrasse 27A, 8952 Schlieren-Zürich, Switzerland INGENASA, C/ Hermanos García Noblejas 39, 28037, Madrid, Spain Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

A R T I C L E

I N F O

Article history: Received 28 November 2013 Accepted 5 April 2014 Keywords: Tuberculosis Cattle Tuberculin test Gamma-interferon assay Serology

A B S T R A C T

Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Diagnosis of bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is still a challenge since available diagnostic tools have limitations regarding sensitivity (Se) and specificity (Sp) that affect the detection of infected animals and the advance, in part, of the TB eradication programmes towards their final objective. Current bovine TB eradication programmes are based on a screening and slaughter policy using mainly the intradermal tuberculin test. The intradermal tuberculin test is recognised by the World Organisation of Animal Health (OIE) and the European Commission as the primary screening test for detection of tuberculosis in cattle (Karolemeas et al., 2012; Schiller et al., 2010). Other diagnostic tools have been also developed over the years as the in vitro interferon-gamma (IFN-γ), lymphocyte proliferation or serological assays showing some benefits or lacks regarding Se and Sp in comparison with the intradermal test. IFN-γ assay is approved for use in the European Union since 2002 [Council Directive 64/432/EEC, amended by (EC) 1226/ 2002], received approval by the United States Department of Agriculture (USDA) in 2003 (USDA:APHIS, 2005), and was accredited by the Standing Committee on Agriculture as an official diagnostic test for bovine TB in Australia in 1991. In Europe, the single and comparative intradermal tuberculin (SIT and SCIT respectively) tests at

* Corresponding author. Tel.: +34 91 394 40 96; fax: +34 91 394 37 95. E-mail address: [email protected] (J. Bezos).

cervical site are used. The intradermal test in the caudal fold is used in the United Stated and New Zealand and was also used in Australia during their bovine TB eradication campaign (Good and Duignan, 2011). Advantages of the intradermal tuberculin test and reasons for its wide use are low cost, low logistical demands, well-documented use and, for a long time, lack of alternative methods to detect bovine TB. Still, this test has many known limitations including difficulties in administration and interpretation of results, need for a secondstep visit, low degree of standardisation and imperfect test accuracy (Rua-Domenech et al., 2006). The fact that a significant proportion of outbreaks are detected in the slaughterhouse suggest that the intradermal test has some limitations or it is not performed permanently in the most adequate way. In this sense, an appropriate training of the veterinary practitioners is essential to ensure that the results are reliable (Working Document on Eradication of Bovine Tuberculosis in the EU accepted by the Bovine tuberculosis subgroup of the Task Force on monitoring animal disease eradication, SANCO/10067/2013). Although the IFN-γ assay overcomes many of the disadvantages associated with the skin test, its use as a primary test has been limited only to certain countries (Flores-Villalva et al., 2012) or to difficult TB situations (Keck et al., 2010). However, recently the European Commission requested the European Food Safety Authority (EFSA) to issue a scientific opinion on the suitability of the IFN-γ test for inclusion in Directive 64/432/EEC as an official primary or stand-alone test and as equivalent to the intradermal test to define

http://dx.doi.org/10.1016/j.rvsc.2014.04.002 0034-5288/© 2014 Elsevier Ltd. All rights reserved.

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the infectious status. In response to this request, EFSA published a scientific opinion in which EFSA acknowledged that the performance of the PPD-based IFN-γ test is comparable to that of the intradermal tests. In this sense, they reported that the IFN-γ test should be considered after harmonisation for inclusion in the official tests for the purpose of granting and retaining official TB-free herd status and for certification for intra-European Union trade of cattle. Nevertheless, the EFSA expert panel recognised that in certain conditions, the Sp of the IFN-γ test might not be as high as the SIT test. Therefore, and in consideration of ongoing research on improved antigens, it was recommended to further evaluate the performance of specific antigens to be used for blood stimulation in the IFN-γ assay with the aim to improve its Sp (EFSA, 2012). As the TB infection progresses, a shift from the predominant cellmediated immune response towards humoral response occurs (Pollock and Neill, 2002). These antibodies (Abs) are generally targeted at immunodominant antigens that elicit a humoral response, notably MPB70 and MPB83 released in large amounts by M. bovis in the later stages of the disease. In general, the profile of the humoral response varied individually and among animal species affecting significantly to the Se achieved using these assays. For these reasons, the detection of Abs continues being a bottleneck and it has not played an important role in eradication programmes yet since it is not included as an official diagnostic tool. A variety of ELISA tests have been developed that rely on the detection of circulating Abs against the immunodominant antigens of M. bovis (Green et al., 2009; Waters et al., 2011; Whelan et al., 2008). In general, these assays are simple, rapid and inexpensive although they have shown lower Se than the assays based on cell mediated immune response. 2. Intradermal tuberculin test 2.1. Historical background The tuberculin test has been used for ante-mortem diagnosis of latent and active TB in man and animals for more than 100 years (Good and Duignan, 2011; Monaghan et al., 1994). Finland was the first country in the late 1890s to start a TB eradication campaign using the tuberculin test (Francis, 1958). Once bovine TB programme based on a test and slaughter policy began, the incidence of clinical TB decreased since a high proportion of infected cattle was removed. Other countries gradually applied eradication programmes using different methodologies of the tuberculin test (ophthalmic and palpebral test, Stormont test, vulval test, etc.) that are discarded nowadays. Neck was finally selected as the site of tuberculin injection due to the higher Se and Sp values in cattle in comparison with the caudal fold (Good and Duignan, 2011). Moreover, different studies were carried out to determine which part of the neck was more suitable for tuberculin injection. Good et al. (2011a) reported that, in a practical sense, location of the PPD sites is of great importance and the middle and anterior third of the neck is recommended. However, the avian and bovine PPD measurements were not significantly different when sites anterior or posterior to this were chosen (Good et al., 2011a). The Koch’s old tuberculin described in 1890 is replaced nowadays by the PPD tuberculin prepared after a heat-treatment and lysis of M. bovis AN5 (bovine PPD) and M. avium D4ER or TB56 (avian PPD). The current PPD tuberculins consist of a mixture of small watersoluble proteins and lack of some non-specific components of the Koch’s tuberculin (Tameni et al., 1998). 2.2. Potency of PPD tuberculins Tuberculin potency is critical for the outcome of the intradermal test since a significant difference in the number of reactors detected using high and low potency tuberculins has been reported

(Good et al., 2011a). Production of PPD tuberculins are standardised and regulated by the EU. Manufacture must fulfil the Good Manufacturing Practice conditions and comply with the European Pharmacopeia and OIE requirements (OIE, 2009; Good and Duignan, 2011). The protein content of the tuberculins is not correlated with the biological activity and therefore, potency assays of the tuberculin batches must be performed in guinea pigs and cattle (Haagsma, 1986). The requirement to check the potency in the bovine bioassay was included in the original Directive 64/432/EEC and was recommended in the OIE technical reports. However, this requirement was modified mainly due to the high cost and logistical demands of the assays and removed from the Directive in 2002 (Good and Duignan, 2011). Nowadays it is rarely conducted and only some laboratories, including the European Reference Laboratory for Bovine TB are carrying out potency tests of bovine PPDs from different manufacturers in cattle. In these experiments, potency of the tuberculins is compared to an international standard (National Institute for Biological Standards and Control-NIBSC, UK) with an established potency of 32,500 IU/mg. The standardisation is based on an eight-point and fourpoint assay which use four and two different dilutions in guinea pigs and cattle respectively (OIE, 2009). Assessment of tuberculin potency in cattle requires naturally infected animals that are reactors in the intradermal test whereas guineas pigs are experimentally infected 5–7 weeks prior to the assay using a low dose of M. bovis. The results are statistically evaluated using the parallel-line assays according to Finney (OIE, 2009). A bovine tuberculin is considered acceptable for diagnosis in the eradication programmes if it has a minimum potency of 2,000 IU per dose and if the estimated potency is between the 66% and the 150% of the potency stated by the manufacturer on the label. Potency estimations in guinea pigs can be imprecise due to the inherent variability of the tuberculin PPD and the biological variations of the in vivo models. An imprecision of the potency estimated in the guinea pigs bio-assay has been reported although the potency test in cattle may have the same lacks (Good et al., 2011a). Moreover, according to current legislation, a reduction in the use of experimental animals and the use of in vitro alternative assays when possible is mandatory (Directive 2010/63/ EU) and, for this reason, some in vitro methodologies are being developed with the aim of replacing the potency test in guinea pigs in the future (Ho et al., 2006). 2.3. Performance of the intradermal test The intradermal tuberculin test measures dermal swelling primarily because of a cell-mediated immune response (CMI) 72 hours after intradermal injection of purified protein derivative (PPD) in the skin of the neck or the caudal fold. The skin of the neck is considered more sensitive to a tuberculin-related hypersensitivity reaction than the skin of the caudal fold and, therefore, higher doses of PPD may be used in the caudal fold to compensate this difference (Schiller et al., 2010). Two approaches for the intradermal tuberculin test are currently in use. The SIT test measures the cell-mediated delayed type hypersensitivity against bovine PPD injected in the mid-cervical region (Member States) or in the caudal skin fold (United States, Canada and New Zealand). The SCIT test compares the response against bovine PPD and avian PPD in the cervical region with the aim of increasing the Sp. The test procedure are described by the OIE (2009) and the Animal Plant Health Inspection Service of the USDA (USDA: APHIS, 2005). According to the protocols, the injection site should be clipped and cleansed. Afterwards skin fold thickness should be measured using a caliper. The tuberculin can be injected using different syringes: the most frequently used are McLintock (Bar Knight McLintock Limited, UK) and Dermojet (Akra Dermojet, France) syringes. The main difference between them is

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that Dermojet is a needle-less syringe that inject intradermally the tuberculin through high pressure (100 bar). A maximum volume of 0.2 ml containing a minimum of 2000 IU of bovine and avian PPDs is injected at cervical site (0.1 ml of USDA PPD at 1mg/ml into either side of the caudal fold). A small pea-like swelling in each site should be palpated by the veterinary practitioner to confirm the correct intradermal injection. When both avian and bovine PPDs are injected in the same animal, the site for injection of avian PPD shall be about 10 cm from the crest of the neck and the site for the injection of bovine PPD and about 12.5 cm lower on a line roughly parallel with the line of the shoulder or on different sides of the neck. In young animals in which there is not room to separate the sites on one side of the neck, one injection shall be made on each side of the neck at identical sites in the centre of the middle third of the neck. These injection sites are recommended and to ensure equivalent test Se at both avian and bovine sites, it has been suggested that both PPDs should be located on a line that is parallel to the angle of the shoulder (Good et al., 2011b). After 72 hours (±4 hours), the skin fold thickness at each injection site should be remeasured by the same veterinary. The standard interpretation of the test is described in the Annex B of EU Directive 64/432/EEC. It is based on observation of clinical signs and the recorded increases in skin-fold thickness 72 hours after injection of PPDs. Non-officially TB-free (OTF)-Member States use SIT or SCIT tests for diagnosis in their eradication programmes based on the epidemiological conditions (Eradication, Control and Monitoring Programmes, European Commission; http://ec.europa.eu/ food/animal/diseases/index_en.htm). In this sense, the SIT test is the main diagnostic assay in Spain, Italy or Croatia whereas in UK, Ireland or Portugal SCIT test is used. In both cases results can be interpreted using different interpretation criteria (standard or severe) depending on the epidemiological conditions. For example, severe interpretation (considering inconclusive reactors as positives) of the SIT test is used in infected herds or high prevalence regions in Spain in order to maximise Se to the detriment of certain Sp. On the other hand, for surveillance testing and in any TB breakdowns without post-mortem evidence of M. bovis infection (non-confirmed breakdowns) a standard interpretation of SCIT test is used in UK. In confirmed breakdowns, a severe interpretation of the SCIT test is used. Consequently, inconclusive reactors with the strongest bovine– avian tuberculin reaction differences are culled as reactors under the severe interpretation of the SCIT test. Therefore, severe interpretation is applied in an effort to accelerate the detection of infected cattle by increasing the Se at the expense of the Sp (Karolemeas et al., 2012). Some immunological factors (early infection, anergy or concurrent immunosuppression), factors related to the PPDs (expired product, product stored under inappropriate conditions, manufacturing errors, low potency) or to the methodology (doses, site of injection, inexperience) might cause false negative results (Humblet et al., 2011; Rua-Domenech et al., 2006). On the other hand, coinfection or pre-exposure to other related non-tuberculous mycobacteria is a potential cause of a false positive result due to the similar antigenic composition of these bacteria (Humblet et al., 2011).

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fore, the type of intradermal test to select might depend on the prevalence of TB and exposure to other sensitising mycobacteria (Monaghan et al., 1994). The caudal fold SIT test is routinely used in the United States, Canada and New Zealand, and the SCIT test is subsequently used as an ancillary serial test for the inconclusive reactors detected in the caudal fold (Tweddle and Livingstone, 1994). Reactivity against the tuberculin usually develops between 3 and 6 weeks post-infection; some studies have pointed out the preallergic phase as the cause of false negative reactions and therefore, the lack of Se in recently infected animals (Buddle et al., 1995; Lilenbaum et al., 1999b). Moreover, anergic cattle have been reported due to advanced or generalised TB or being temporarily under stress (Pollock and Neill, 2002). Due to the subjective nature of intradermal test, a number of factors can affect its accuracy what has led to a very wide range of Se and Sp reported values: Se of the SIT test reported in some previous studies in cattle ranged between 80.2% and 100% although they were performed in different epidemiological circumstances (Rua-Domenech et al., 2006). Recently, a Bayesian approach has been used to estimate the Se and Sp of the SIT test showing a Se between 53% (27.3–81.5, 95% CI) and 69.4% (40.1– 92.2, 95% CI) depending on the interpretation criteria used (Álvarez et al., 2012). The results from this study highlighted the low Se of the intradermal test after the disclosure test. Nevertheless, the inclusion in this study of a large number of bullfighting cattle in which SIT test may be particularly difficult to perform, could be also the cause of such a low Se. Reported Sp of the SIT test in different previous studies ranged between 55.1% and 99% showing a median value over 95% (Rua-Domenech et al., 2006; Schiller et al., 2010) although in a recent study a Sp over 99% has been estimated (Álvarez et al., 2012). Overall Se of the SCIT test is considered lower than that achieved using the SIT test with reported values between 52% and 100% using different interpretation criteria and doses of PPD (Rua-Domenech et al., 2006; Schiller et al., 2010) and with a reported median value of 83.9% (Rua-Domenech et al., 2006). A recent study conducted in Great Britain reported a Se of 81% (70–89, 95% CI) using the standard interpretation and a Bayesian model including also postmortem data from SCIT test negative cattle (Karolemeas et al., 2012). In contrast to the SIT test, comparative interpretation increases the Sp (median of 99.5%) (Rua-Domenech et al., 2006; Schiller et al., 2010) although it is necessary to remark that in absence of significant interference factors, the Sp of the SIT test is almost as high as the estimated using the SCIT test (Álvarez et al., 2012). The caudal fold SIT test is not used in Europe although it is widely applied in other countries such as the United States or New Zealand and it was used to eradicate bovine TB in Australia (Cousins, 2001; Norby et al., 2005; Schiller et al., 2010). A meta-analysis carried out by Farnham et al. (2012) showed that the Se ranged from 80.4% to 93% and the Sp from 89.2% to 95.2% (Farnham et al., 2012). To increase overall test Sp, the USDA adopted the comparative cervical test as a follow-up for reactors to the caudal test in 1973. In that case, the estimated Se ranged from 74.4% to 88.4% and the Sp from 97.3% to 98.6% showing to be an adequate methodology to increase the Sp but still achieving lower values than those achieved using the SCIT test in Europe.

2.4. Sensitivity and specificity of the intradermal test 2.5. Alternative antigens to the PPDs used in the intradermal test Numerous studies have been carried out in the last decades to evaluate Se and Sp of the intradermal test in cattle under different epidemiological situations using different antigens (Álvarez et al., 2012; Aagaard et al., 2010; Coad et al., 2013; Farnham et al., 2012; Karolemeas et al., 2012; Rua-Domenech et al., 2006). In general, the SCIT test shows a higher Sp than the SIT test in detriment of the Se since it can distinguish animals infected with non-tuberculous mycobacteria or those vaccinated against paratuberculosis (PTB) (Aagaard et al., 2010; Aranaz et al., 2006; Coad et al., 2013). There-

Bovine PPDs consist in a poorly defined mix of proteins, lipids and carbohydrates obtained from M. bovis AN5 (Casal et al., 2012; Tameni et al., 1998). Since some of these components are present in non-pathogenic environmental mycobacteria a lack of Sp has been attributed to the PPDs (Hope et al., 2005). In this sense, the use of alternative antigens to increase the Sp of the diagnostic assays has been previously suggested (Flores-Villalva et al., 2012). The most relevant alternative antigens to the PPDs used in the intradermal test

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are the early secretory antigenic target-6kDa (ESAT-6), the culture filtrate protein 10 (CFP-10) and the Rv3615c (Mb3645c) or combination/s of them with other antigens (Buddle et al., 2003a; Casal et al., 2012; Flores-Villalva et al., 2012; Whelan et al., 2010a; Xin et al., 2013). Recently, Jones and collaborators demonstrated that addition of the Rv3020c improves diagnostic Se without compromising Sp (Jones et al., 2012). The aim of these studies assessing different antigens and replacing the PPDs has been the development of assays for differentiating vaccinated from infected animals (DIVA) that is an essential prerequisite to consider the inclusion of BCGbased vaccination as part of the bovine TB control programmes in certain countries. Most of these DIVA tests have been carried out in vitro using the IFN-γ assay (see section 3.2) although they have also been used in the intradermal test (Casal et al., 2012; Xin et al., 2013). Times of reading and doses using these new antigens have to be optimised since previous studies suggested greater times of reading (96 or 120 hours) and the necessity of high doses (≥400 µg) (Pollock et al., 2003). Certain studies report similar skin-fold thickness measures to those observed using the PPDs although, in general, a lower Se is attributed to these antigenic cocktails compared with the PPDs (Casal et al., 2012; Pollock et al., 2003; Whelan et al., 2003, 2010a). Infection with M. avium subsp. paratuberculosis (MAP) mycobacteria has been suggested as interference factor in the diagnosis of bovine TB using PPDs (Aranaz et al., 2006). Previous studies have demonstrated that the presence of PTB or other infections by environmental mycobacteria did not affect to the ESAT-6/CFP-10 protein cocktail performance, unlike when bovine PPD was used as antigen, since the animals sensitised animals to environmental mycobacteria, infected with MAP did not respond to the protein cocktail. For this reason, in the absence of TB infection, the using of these antigenic cocktails could be useful to increase the Sp (Flores-Villalva et al., 2012). 3. IFN-γ assay The in vitro IFN-γ assay developed in Australia in the late 1980s (Wood and Jones, 2001) is recommended by the OIE since 1996 (OIE Terrestrial Manual) as ancillary laboratory-based test to the tuberculin intradermal test. Most of the bovine TB control programmes rely on the use of BOVIGAM® (Prionics, Switzerland) as parallel test to the intradermal test to maximise the detection of TB-infected animals. The assay is accepted for use as ancillary test to the intradermal test in the European Union since 2002 [Council Directive 64/ 432/EEC, amended by (EC) 1226/2002]. 3.1. Performance of the IFN-γ assay The test is carried out in two stages. In the first stage, blood is collected in heparinised tubes and transported to the laboratory where the blood samples are incubated in the presence of antigens such as tuberculin PPD or antigen cocktails to stimulate the release of IFN-γ by sensitised T lymphocytes. Several parameters of the blood incubation have recently been optimised to increase the flexibility and ease of use of the assay (Schiller et al., 2009b). The second stage involves harvesting of the plasma from the blood samples and the detection of the released IFN-γ in the plasma by an enzyme sandwich immunoassay such as the BOVIGAM® IFN-γ assay. Based on the resulting optical density (OD) readings, interpretation criteria for a positive test result for the BOVIGAM® IFN-γ assay after stimulation with bovine PPD (PPDb), avian PPD (PPDa) and PBS (Nil) as control are described by the manufacturer (Prionics AG) as: PPDbOD − NilOD ⩾ 0.1 and PPDbOD − PPDaOD ⩾ 0.1. One of the advantages of the IFN-γ assay is the fact that interpretation criteria can be adapted to suit local conditions such as epidemiological situation, disease prevalence and the stage of the bovine TB control

programme. In addition to its EU-approved use as a parallel test to the skin test to maximise Se in a control programme, many countries have additionally adopted protocols for the use of the IFN-γ assay as a serial test to the skin test in order to increase the Sp. Applications for such uses of the BOVIGAM® IFN-γ assay are the retesting of suspected unspecific reactors to the skin test in regions with a very low bovine TB prevalence in France using decisional cutoff values for PPDs as well as for the specific antigens ESAT-6 and CFP-10 (Faye et al., 2011). An overview of the different cut-off criteria for the IFN-γ assay in the European Union was recently published by the EFSA (2012). Numerous field studies, conducted worldwide since 1991 to compare the diagnostic performance of the tuberculin intradermal test and BOVIGAM®, have shown that the IFN-γ test has a higher Se than the tuberculin intradermal test, while the Sp is similar or slightly lower than that of the SIT, and lower than the Sp of the SCIT test. Based on a meta-analysis of 15 field studies conducted between 1991 and 2006, an estimated median Se of 87.6% with a range between 73% and 100% and a Sp of 96.6% with a range of 85% and 99.6% were reported for the BOVIGAM® IFN-γ assay (Rua-Domenech et al., 2006). The higher Se of the IFN-γ test compared to the skin test is likely due to the fact that the IFN-γ test detects TB infected animals as early as 14 days following infection (Buddle et al., 1995) and 60–120 days earlier than the SCIT test (Lilenbaum et al., 1999b). More importantly, several studies in the UK (Coad et al., 2008) and Ireland (Gormley et al., 2006) have shown that intradermal test negative but IFN-γ positive animals are more likely to be infected with M. bovis than intradermal test and IFN-γ negative cattle and that removal of all animals reacting positive to either of the two tests is critical to controlling bovine TB outbreaks. 3.2. Alternative antigens to the PPDs used in the IFN-γ test The development of more specific antigens that can be used for blood stimulation in the IFN-γ assay has been the “holy grail” of research efforts for improved diagnostic tools for bovine TB. Identification of specific antigens that are present only in M. bovis but are absent from environmental mycobacteria can significantly increase the Sp of diagnostic tests. In addition, specific antigens from the region of difference 1 (RD1) such as the highly immunogenic ESAT-6 (Pollock and Andersen, 1997) and CFP-10 (Berthet et al., 1998) are absent from many Bacillus Calmette-Guérin (BCG) vaccine strains (Harboe et al., 1996) and can therefore serve as preferred targets for a DIVA test (Buddle et al., 1999; Vordermeier et al., 1999). As shown in Table 1, when used as single antigen, ESAT-6 showed Se between 69% and 88% (Aagaard et al., 2006; Buddle et al., 2001; Pollock et al., 2000). Other mycobacterial antigens showing promising results as diagnostic marker that were tested as single antigen include CFP-10, MPB-70, Rv3615c and Rv0899 with reported Se of 68%–78%, 36%–49%, 37% and 50%–85% respectively (Aagaard et al., 2006; Buddle et al., 2003b; Pollock et al., 2000; Schiller et al., 2009b; Sidders et al., 2008). For all of the studies summarised in Table 1, no antigen when tested singularly gave equivalent Se compared to the PPD-based BOVIGAM® assay. However, the use of single mycobacterial antigens minimised the amount of false positive reactors in uninfected animals as shown in Table 1 with test Sp to single antigens was greater than that observed for the corresponding PPDB – PPDA readout. In order to increase the Se of the IFN-γ test with specific antigens while maintaining its high Sp, selected antigens were combined and formulated either as a mixture of recombinant proteins or as cocktails of peptides (Vordermeier et al., 1999). Two studies which directly compared Se and Sp of ESAT-6 and CFP10 individually and in combination showed an enhanced Se of the cocktail which supports the notion that a combination of epitopes of different antigens increases the diagnostic Se without compromising Sp (Table 1) (Aagaard et al., 2006; Buddle et al., 2003b). Cockle

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Table 1 Sensitivity and specificity of the IFN-γ assay (BOVIGAM®) in different studies in cattle using different recombinant proteins or peptides or a combination of them. Antigens

ESAT-6 MPB70 PPDB-PPDA ESAT-6 PPDB-PPBA ESAT-6/CFP-10 PPDB – PPDA ESAT-6 CFP-10 MPB70 ESAT-6 CFP-10 ESAT-6/CFP-10 PPDB-PPDA ESAT-6 CFP-10 MPB70 ESAT-6/CFP-10 ESAT-6/CFP-10 ESAT-6/CFP-10/Rv3019c/Rv0288/Rv3879c/Rv3873 PPDB – PPDA RV3615c ESAT-6/CFP-10 ESAT-6/CFP-10 & Rv3615c Rv0899 Rv0899 ESAT-6/CFP-10 ESAT-6/CFP-10/ Rv0899 PPDB – PPDA

Recombinant protein

Peptide

X X X X X X X X X X X X X X X X X X X X X X X

X X X

et al. (2006) set out to combine additional highly expressed mycobacterial antigens with ESAT-6 and CFP-10, and they showed that a peptide cocktail consisting of 32 peptides derived from ESAT-6, CFP-10 and four other mycobacterial genes resulted in a Se of 97% at a Sp of 93% (Cockle et al., 2006). These results encouraged the refinement of tools for antigen mining to identify novel diagnostic markers for TB infection. Using microarray mRNA expression analysis data (Sidders et al., 2007), a set of 14 highly expressed antigens in M. bovis were studied for their ability to be used as antigen in the IFN-γ assay, and it was demonstrated that the antigen Rv3615c detected a significant portion of TB infected animals that were negative to the ESAT-6/CFP-10 peptide cocktail. The combined calculated performance values for an antigen cocktail consisting of overlapping peptides from all three antigens were reported with 91% Se and 100% Sp (Sidders et al., 2008). In a similar animal study, Schiller et al. (2009a) demonstrated that the outer membrane protein OmpATb (Rv0899) was able to stimulate IFN-γ release in whole blood cultures of 85% of M. bovis infected cattle but in none of the uninfected control animals. Importantly, a significant portion of these animals did not respond to an ESAT-6/CFP10 peptide cocktail suggesting that OmpATb would be an ideal candidate antigen to complement the ESAT-6/CFP-10 peptide cocktail to increase the Se to a calculated 96% with a Sp of 100%. A low number of animals were used in this study and therefore further studies need to be conducted to confirm the preliminary results. Nevertheless these data demonstrate that it is feasible to combine several antigens in a cocktail in order to increase the Se of the IFN-γ test using these additional antigens (Schiller et al., 2009a). The median Sp value for the PPD-based IFN-γ test in these studies differs from the published median Sp of 96.6% (Rua-Domenech et al., 2006). This discrepancy is likely to be due to the fact that the selections of herds in some of the studies summarised here are biased in such a way that bovine TB negative animals originated from TB free ‘problematic’ herds. Some of the selected animals had positive skin tests or IFN-γ test without TB infection being diagnosed

Sensitivity

Specificity

Reference

%

n

%

n

76 39 89 88 98 78 88 77 78 49 80 80 89 95 69 68 36 85 91 97 85 37 77 91 85 50 77 96 100

131 131 131 51 51 68 68 74 74 74 74 74 74 74 56 56 56 56 58 58 58 30 30 30 26 18 26 26 26

99 95 92 99 85 100 92 93 93 94 93 92 93 74 91 94 89 97 93 93 93 100 100 100 100 100 100 100 70

128 128 128 85 85 25 25 72 72 72 72 72 72 72 56 56 56 56 55 55 55 10 10 10 7 20 7 7 7

Pollock et al., 2000

Buddle et al., 2001 Vordermeier et al., 2001 Buddle et al., 2003a

Aagaard et al., 2006

Cockle et al., 2006 Sidders et al., 2008

Schiller et al., 2009a

in the respective herds. Therefore, these reactions have been considered as false-positives and these animals were selected because they allowed investigations into more specific antigens (Buddle et al., 2003a; Schiller et al., 2009a). 3.2.1. BCG vaccine and DIVA diagnostics Despite all the efforts to eradicate bovine TB, the disease has become difficult to control and re-emerged in several countries because of the role of wildlife as reservoirs of tuberculous mycobacteria among other factors. Encouraging results from vaccine trials in New Zealand with the attenuated M. bovis strain BCG (Buddle et al., 1995) have led to proposal of using BCG in vaccination control strategies for bovine TB. However, since vaccination with BCG compromises the use of the skin test as a primary test for bovine TB control in trade and eradication programmes, alternative DIVA diagnostic tests are required before BCG could be licensed and approved for use in cattle (Krebs, 1997). Several antigen cocktails were recently assessed for their potential use as DIVA diagnostic reagents. Of particular interest are peptide cocktails based on ESAT-6 and CFP-10 which were evaluated as stimulation antigens in the BOVIGAM® assay and did not induce IFN-γ responses in BCG-vaccinated cattle. In contrast, stimulation of blood from BCG-vaccinated animals with PPD resulted in up to 80% of the animals giving significant responses in the BOVIGAM® assay (Sidders et al., 2008; Vordermeier et al., 2001). Further potential candidate antigens that have been tested for their DIVA capabilities are Rv3615c (Sidders et al., 2008) and Rv3020c (Jones et al., 2010) both of which showed promising results when used as antigens in the BOVIGAM® assay as well as for skin testing (Jones et al., 2012; Whelan et al., 2010a). 3.2.2. Field studies with specific antigens Several recent field studies reported promising results using specific antigens in the IFN-γ blood test for the diagnosis of bovine TB. A field study in Mexico on 303 animals from bovine TB infected as well as uninfected herds revealed a higher Se and Sp of ESAT-6/

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CFP-10 protein cocktail compared to PPD using both the in vitro BOVIGAM® assay and the SCIT test. In addition, the protein cocktail did not induce IFN-γ responses in TB-free herds infected with MAP (Flores-Villalva et al., 2012). A field study conducted in 23 cattle from a herd with natural M. bovis infection in Spain investigated the responses to a cocktail of ESAT-6/CFP-10/Rv3615c presented either as peptides or recombinant proteins in comparison to PPD in the IFN-γ and in the intradermal test. Highest responder frequencies in the intradermal test were found with PPD (60.9%) and the protein cocktail (60.9%) whereas only 47.8% of the animals responded to the peptide cocktail. The IFN-γ test detected 60.9% of the animals with PPD and slightly lower responder frequencies with peptide (52.2%) and protein cocktails (47.8%) although the differences between these groups were not significant (Casal et al., 2012). A second generation of the BOVIGAM® test which is now marketed under the brand name BOVIGAM® 2G (Prionics, Switzerland) has been validated with defined peptide cocktails based on ESAT-6/CFP-10 (PC-EC, Prionics, Switzerland), and ESAT-6/CFP-10/ Rv3615c and three additional antigens (PC-HP, Prionics, Switzerland). Field studies to assess the diagnostic Sp of PC-HP with BOVIGAM® 2G were conducted in France in a total of 390 TB-free cattle. The resulting Sp using tuberculin PPD was determined in 84% (81–87, 95% CI) whereas the peptide cocktail PC-HP gave rise to a significantly higher Sp of 94% (92–96, 95% CI). Se was analysed in 201 cattle from a TB-infected herd in Ireland with TB prevalence of 16%–31%, and it was found that the Se with PC-HP (85%; 64–96, 95% CI) did not significantly differ from PPD (89%; 75–97, 95% CI). These results suggest that peptide cocktails have the advantage of giving higher Sp and similar Se compared to the PPD-based IFN-γ test (Schroeder et al., 2012). Furthermore, the peptide cocktails PC-EC and PC-HP were evaluated in cattle experimentally infected with three different strains of M. bovis. Both peptide cocktails, PC-EC and PC-HP elicited robust responses as early as 3 weeks following infection and responses to specific antigens did not significantly differ from the responses to PPD. In addition, data obtained with the BOVIGAM ® and the BOVIGAM® 2G kits were compared and the results indicated that the two tests show equivalent performance (Bass et al., 2013). 4. Serodiagnosis of bovine TB Currently, the most commonly diagnostic tools for bovine TB are based on the measurement of the delayed hypersensitivity reaction (Monaghan et al., 1994) and the IFN-γ production (Gormley et al.,

2006; Schiller et al., 2009b; Wood and Rothel, 1994) using antigens of M. bovis and M. avium. Serological tests to detect humoral response may also constitute an alternative for screening herds of livestock and wild animals for M. bovis infections. The combination of methods based on the cellular response against M. bovis, together with serological test could increase the level of detection of the pathogen and help to control bovine TB (Schiller et al., 2010). However, humoral immune response against M. bovis is very complex. The titre of antibodies (Ab) changes significantly during the infection and they are mainly produced in advances stages of TB infection (Pollock and Neill, 2002), although an early antibody response has also been reported in experimentally infected cattle (Waters et al., 2006c). Several antigens have been described as potential diagnostic targets (e.g. ESAT-6, CFP-10 and MPB70), although Ab response is mainly triggered against MPB83 in domestic livestock and wild animals (Amadori et al., 2002; Lyashchenko et al., 2008; McNair et al., 2001; Wiker, 2009). Responses against MPB83, is detected earlier in the course of experimental infections, observing an increase in the Ab response at 3–4 weeks post infection (O’Loan et al., 1994; Waters et al., 2006a, 2010). On the other hand, antibodies against ESAT-6 and MPB70 were detected 12 weeks (Lyashchenko et al., 1998) and 20 months (Fifis et al., 1992) after experimental infection respectively. Therefore, the highest and earlier response triggered against MPB83 protein suggests that this protein is a good candidate as diagnostic antigen for the development of serological tests. Enzyme-linked immunosorbent assay (ELISA) has advantages over the methods traditionally used. It permits to test many samples in a short time, it is simple, rapid and inexpensive and allows the standardisation of the technique in the different laboratories (Cho et al., 2007). Besides, especially in the case of wild animals, it is very important to use a test based on a single sampling. The ELISA has been extensively used to evaluate the humoral response against Mycobacterium bovis in a number of animal species. Initially, PPD were used as antigen; however its Se was low (Amadori et al., 1998; Lilenbaum et al., 1999a). The use of native or recombinant specific antigens such as ESAT-6, MPB70 or MPB83, in indirect or competition ELISAs, also resulted in relatively low values of Se and Sp (Waters et al., 2011), thus, great efforts have focused on finding new antigens that would help to improve these results. Several studies report the use of chimeric proteins formed by the combination of multiple antigens, resulting in Se and Sp values over 65% and 95% respectively (Liu et al., 2007; Souza et al., 2012; Waters et al., 2011) (Table 2). Indirect ELISA tests based on bovine PPD have also been developed and validated for wild animals. Sera from European wild

Table 2 Performance of several serological assays used for diagnosis of tuberculosis in cattle in the last 10 years. Assay

Antigens

Latex bead agglutination assay (LBAA) ELISA LBAA ELISA

ESAT-6 ESAT-6, MPB70

320/155

ELISA Enfer multiplex assay Anigen lateral flow assay Antigen printing

CMP70 N/A MPB70 ESAT-6 CFP-10 MPB83 MPB83 N/A ESAT-6, MPB70, MPB83 ESAT-6, CFP-10, MPB83

62/3 522/1489 214/79 522/1489 522/1489 522/1489 90/895 96/93 107/362 107/161

SeraLyte-Mbv Enfer multiplex assay Recombinant proteins-ELISA Chembio Lateral flow assay

Number of animals tested (infected/non-infected)* 59/10

Sensitivity* (%)

Specificity* (%)

Reference

95.7 97.1 94.8 98.6

100 94.2 92.6 98.5

Koo et al., 2004

84 93.1 83.6 40.6 82.6 78.5 89 77–86.5 67.3–83.2 45

100 98.4 83 86.6 69.7 99.1 98 77.6–100 86.5–95 52.5

Cho et al., 2007 Whelan et al., 2008

Koo et al., 2005

Green et al., 2009 Whelan et al., 2010a Souza et al., 2012 Bermúdez et al., 2012

* In several studies de infection status was only based on the results of other ante-mortem diagnostic assays (it was not confirmed by bacteriology) and therefore it can be considered as apparent sensitivity and specificity. N/A: not available.

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board were absorbed with Mycobacterium phlei suspension to remove non-specific anti-Mycobacterium spp. antibodies, showing a Se of 72% and a Sp of 96% (Aurtenetxe et al., 2008; Fernández et al., 2009). Several studies have shown that the response to M. bovis is not constant since the different species respond to different antigens. Because of that, several proteins may be required to achieve higher Se. In this line, a multi-antigen print immunoassay (MAPIA) has been developed to characterise the antibody response. This test consists of a cocktail of antigens applied to nitrocellulose membranes in narrow bands. The strips are incubated with serum samples and then is followed by immunodetection using standard chromogenic methods (Lyashchenko et al., 2000, 2008; Veerasami et al., 2012). Other researchers have developed and assayed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody directed to more than 20 antigens in a single well in a 96well plate format that has been commercialised as Enferplex TB (Whelan et al., 2008, 2010b, 2011). Based on previous studies, the Se and Sp yielded using both kind of assays was not significantly different from that obtained when using tests based on a single antigen (Table 2). Lateral-flow (LF) rapid tests based on immunochromatografic techniques have also been developed and evaluated for different species, and are commercially available. The LF devices offer many advantages since they are easy to perform, stable at room temperature and do not require high logistical demands. However, the Se of these tests ranges from 45% to 100% depending on the species under study (Buddle et al., 2010; Greenwald et al., 2009; Lyashchenko et al., 2008; Michel and Simoes, 2009). To avoid the complexity of the development of a multi-antigen system and the possible loss of Sp, several systems based on a single antigen have been developed. The SeraLyte-Mbv system based on advanced chemiluminiscence chemistry and optics for the highly sensitive detection of antibodies (Green et al., 2009), uses MPB83 as antigen and reported high Se and Sp. In a similar way, a novel serologic technique based on MPB83 recombinant antigen has been recently reported and assessed in a M. bovis naturally infected alpaca herd in Spain. This new assay used the MPB83 protein as antigen on the plate and as enzyme-conjugated antigen for the antibodies detection, for this reason, the test has been called double recognition ELISA (DR-ELISA), because the antibodies present in the sample have to recognise twice the specific antigen (Bezos et al., 2013). This assay offers many advantages with regard to other ELISA tests. On one hand, it is able to recognise not only IgGs but also other immunoglobulin classes, such as IgM (Venteo et al., 2012) allowing for early detection of the infection. On the other hand, it is a multispecies assay since it uses enzyme-conjugated antigen instead of enzyme-conjugated species-specific secondary antibody and therefore, it can be used with sera from any animal infected by M. bovis. Moreover, it is highly specific due to the double recognition of the MPB83 protein. These characteristics render DR-ELISA a potentially valuable tool for early detection of antibodies against M. bovis and subsequent diagnosis of the TB infection although it has not been assayed in cattle. Other techniques as the latex bead agglutination assay based on the detection of antibodies against one antigen (ESAT6) have been tested in order to differentiate between TB- and PTBinfected animals and showing adequate Se and Sp although the number of samples was low (Koo et al., 2004). An increase of the Se of the serological assays due to an increase of the optical density caused by a recent intradermal tuberculin test (booster effect) has been suggested when sera samples taken in a short interval after the PPD injection are used. The booster effect was firstly reported in goats naturally infected with M. bovis (Gutiérrez et al., 1998) and in studies using experimentally infected cattle (Hanna et al., 1992; Harboe et al., 1990; Koo et al., 2005; Palmer et al., 2006; Waters et al., 2006b). Nevertheless, to our knowledge, few studies have been performed to demonstrate the effect

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in naturally infected cattle (Waters et al., 2011). Therefore, further studies testing different sampling times after PPD injection should be carried out in naturally infected cattle to confirm an increase of the Se of the serological assays based on this booster effect and to evaluate the best time after PPD injection to collect the sera samples. Assays based on detection of specific Ab might show a high potential for the diagnosis of bovine TB although new tests with improved Se and Sp under field conditions have to be developed and validated. These diagnostic assays would complement the current techniques based on the cell-mediated immune response to improve the control and eradication of bovine TB. Indeed, they could be a good approach for the diagnosis of TB in wild animals which, in certain cases, are important reservoirs of the pathogen and contribute to spreading the infection. 5. Conclusions Diagnosis of bovine TB has certain limitations associated to the immunological response against the infection and to the accuracy of the current diagnostic tools that are more evident at the final steps of the eradication process. The intradermal tuberculin test has demonstrated to be an adequate diagnostic tool at herd level, and several countries have achieved the eradication based on this test. The inclusion of the IFN-γ assay for use in infected herds in parallel with the intradermal test has demonstrated to be an adequate methodology to maximise the detection of infected cattle and to accelerate the eradication process. The use of both assays is the most realistic approach for diagnosis of bovine TB in the near future and, therefore, more studies should be performed to further improve them. Diagnosis based on detection of specific Abs could be ancillary in any case since the Se achieved by the current assays under field conditions is, in general, lower than that achieved using the official diagnostic assays. The performance of the serological assays is mainly affected by the irregular and, in general, late pattern of antibody production reported in the humoral response against TB. Nevertheless, the difficulties to achieve the eradication of bovine TB in certain regions or countries should not be focused only to the limitations of the current diagnostic assays or to the epidemiological conditions (presence of wildlife or infection with other non-tuberculous mycobacteria). In this line of thought, other factors related to the practises of all stakeholders involved (private and government veterinarians, managers and herd owners) should be addressed, evaluated and improved if necessary. Acknowledgement This research was funded by the European Project FP7-KBBE2007-1 “Strategies for the eradication of bovine tuberculosis (TBSTEP).” References Álvarez, J., Pérez, A., Bezos, J., Marqués, S., Grau, A., Saéz, J.L., Mínguez, O., de Juan, L., Domínguez, L., 2012. Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach. Veterinary Microbiology 155, 38–43. Aagaard, C., Govaerts, M., Meikle, V., Vallecillo, A.J., Gutierrez-Pabello, J.A., Suarez-Guemes, F., McNair, J., Cataldi, A., Espitia, C., Andersen, P., Pollock, J.M., 2006. Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis. ESAT6-CFP10 mixture shows optimal sensitivity and specificity. Journal of Clinical Microbiology 44, 4326–4335. Aagaard, C., Govaerts, M., Meikle, V., Guitiperrez Pabello, J.A., McNair, J., Andersen, P., Guemes, F.S., Pollock, J., Espitia, C., Cataldi, A., 2010. Detection of bovine tuberculosis in herds with different disease prevalence and influence of paratuberculosis infection on PPDB and ESAT-6/CFP10 specificity. Preventive Veterinary Medicine 96, 161–169. Amadori, M., Tameni, S., Scaccaglia, P., Cavirani, S., Archetti, I.L., Giandomenico, R.Q., 1998. Antibody tests for identification of Mycobacterium bovis-infected bovine herds. Journal of Clinical Microbiology 36, 566–568.

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